Synthesis and structure-activity relationships of the first ferrocenyl-aryl-hydantoin derivatives of the nonsteroidal antiandrogen nilutamide

Article abstract of DOI:10.1021/jm701264d

We present here the first synthesis of organometallic complexes derived from the nonsteroidal antiandrogen nilutamide, bearing a ferrocenyl substituent at position N(1) or at C(5) of the hydantoin ring; for comparison, we also describe the C(5) p-anisyl organic analogue. All of these complexes retain a modest affinity for the androgen receptor. The N-substituted complexes show a weak or moderate antiproliferative effect (IC₅₀ around 68 μM) on hormone-dependent and -independent prostate cancer cells, while the C(5)-substituted compounds exhibit toxicity levels 10 times higher (IC ₅₀ around 5.4 μM). This strong antiproliferative effect is probably due to a structural effect linked to the aromatic character of the ferrocene rather than to its organometallic feature. In addition, it seems connected to a cytotoxic effect rather than an antihormonal one. These results open the way toward a new family of molecules that are active against both hormone-dependent and hormone-independent prostate cancer cells.

Full text of DOI:10.1021/jm701264d

J. Med. Chem. 2008, 51, 1791–1799
1791
Synthesis and Structure–Activity Relationships of the First Ferrocenyl-Aryl-Hydantoin
Derivatives of the Nonsteroidal Antiandrogen Nilutamide
Olivier Payen,† Siden Top,† Anne Vessières,† Emilie Brulé,† Marie-Aude Plamont,† Michael J. McGlinchey,‡
Helge Müller-Bunz,‡ and Gérard Jaouen*,†
Ecole Nationale Supérieure de Chimie de Paris, Laboratoire de Chimie et Biochimie des Complexes Moléculaires, UMR CNRS 7576, 11 rue
Pierre et Marie Curie, 75231 Paris Cedex05, France, and UCD School of Chemistry and Chemical Biology, UniVersity College Dublin,
Belfield, Dublin 4, Ireland
ReceiVed October 7, 2007
We present here the first synthesis of organometallic complexes derived from the nonsteroidal antiandrogen
nilutamide, bearing a ferrocenyl substituent at position N(1) or at C(5) of the hydantoin ring; for comparison,
we also describe the C(5) p-anisyl organic analogue. All of these complexes retain a modest affinity for the
androgen receptor. The N-substituted complexes show a weak or moderate antiproliferative effect (IC50
around 68 µM) on hormone-dependent and -independent prostate cancer cells, while the C(5)-substituted
compounds exhibit toxicity levels 10 times higher (IC50 around 5.4 µM). This strong antiproliferative effect
is probably due to a structural effect linked to the aromatic character of the ferrocene rather than to its
organometallic feature. In addition, it seems connected to a cytotoxic effect rather than an antihormonal
one. These results open the way toward a new family of molecules that are active against both hormone-
dependent and hormone-independent prostate cancer cells.
tamoxifen can lead to ferrocenyl complexes showing a strong
antiproliferative effect on both hormone-dependent and -inde-
pendent breast cancer cells.18,19 However, the presence of a
ferrocenyl unit is not always associated with a cytotoxic effect.20
The IC50 values found for the ferrocenyl complexes covered a
wide range of concentrations varying from 0.5 µM for the most
active ferrocenyl-diphenol-ethene derivatives19 to 160 µM, the
value found for the ferrocene unit alone, or even higher for some
ferrocenyl-estradiol derivatives.20 This prompted us to study the
effect of the introduction of a cytotoxic metal moiety into a
nonsteroidal antiandrogen such as nilutamidesa topic that has
not been previously investigated. We postulated that the
antiandrogenic effect of nilutamide could be preserved, while
the addition of an intrinsic cytotoxic function of the metal
complex could lead to molecules active on both hormone-
dependent and -independent prostate cancers. It has already been
demonstrated that when a hydroxyalkyl or cyanoalkyl group
was anchored to the NH of nilutamide it increased the affinity
of the molecules for the androgen receptor, as shown with the
two compounds from the former pharmaceutical company
Roussel-Uclaf: 4-[4,4-dimethyl-2,5-dioxo-3-(4-hydroxybutyl)-
1-imidazolidinyl]-2-trifluoromethylbenzonitrile (RU 58841)21
and 3-[4-cyano-3-(trifluoromethyl)phenyl]-5,5-dimethyl-2,4-di-
oxo-1-imidazolidineacetonitrile (RU 58642)22 (Chart 1). These
molecules also bear a cyano group instead of a nitro substituent
on the aromatic ring. The replacement of the nitro group
prevents its reduction in vivo, and was also found to give better
results for bicalutamide which contains a cyano group.2
Therefore, we started our study by linking the ferrocenyl unit
to the free NH function of nilutamide derivatives, 1–4 (Chart
2). The second approach involved the introduction of the
ferrocenyl moiety at C(5) of the hydantoin ring. By doing so,
we obtained 5 which is a disubstituted C(5) derivative bearing
a 4-cyano-3-trifluoromethylphenyl moiety instead of a simple
hydrogen. In addition, molecule 6, a purely organic molecule
related to 5, but with a p-anisyl group instead of ferrocenyl,
was also prepared.
1. Introduction
Prostate cancer is the most prevalent cancer and the third
leading cause of cancer death in American men.1 The natural
steroid androgens, namely testosterone, the circulating hormone,
and dihydrotestosterone (DHT), its active metabolite, are
responsible for the development and maintenance of normal
prostatic cells. However, they can also promote the malignant
growth of the prostate gland, and it is known that most of the
prostate cancers are androgen-dependent.2–4 The mechanism of
action of the androgens involves an interaction with a specific
androgen receptor (ARa), and this receptor is postulated to play
a crucial role in the development of prostate cancer.5 The current
treatment for prostate cancer includes a combination of surgery,
radiation, and chemotherapy.6 Clinical trials have been con-
ducted with chemotherapeutic agents, such as docetaxel, mi-
toxantrone, and doxorubicin, in men with hormone refractory
prostate cancer; unfortunately, these agents have limited efficacy
and significant adverse side effects.7 The therapeutic agents also
include nonsteroidal antiandrogens such as nilutamide,8,9
flutamide,6,10–12 and bicalutamide13 (Chart 1). However, after
a period within 1-3 years, most patients relapse with advanced
metastatic disease. The cancer usually becomes hormone
refractory possibly because of mutations of the androgen
receptor,14 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. Recently,
novel candidates arising from artemisine,15 curcumin,16 and
salicylhydrazide17 have been published.
We have previously shown that the incorporation of a
ferrocenyl unit into nonsteroidal antiestrogens such as OH-
* Corresponding authors. Phone: (+33)1-43-26-95-55. Fax: (+33)1-43-
26-00-61. E-mail: gerard-jaouen@enscp.fr, siden-top@enscp.fr
†
Ecole Nationale Supérieure de Chimie de Paris.
University College Dublin.
‡
a Abbreviations; AR, androgen receptor; DHT, dihydrotestosterone; RBA,
relative binding affinity.
10.1021/jm701264d CCC: $40.75
2008 American Chemical Society
Published on Web 02/28/2008

1792 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Payen et al.
Chart 1. Natural Steroidal Androgens and Representative Examples of Nonsteroidal Antiandrogens
Chart 2. Newly Synthesized Molecules
N-ferrocenyl hydantoins 1 and 2 in 80% and 90% yield,
respectively (Scheme 2).
X-ray Crystal Structures of 1 and 2. The X-ray crystal
structures of 1 and 2 are shown in Figures 1 and 2, respectively,
and selected geometric data are also listed. There are evident
similarities between the two structures which differ only by a
single methylene unit. The structural parameters for both
ferrocenyl substituents are within the normal ranges, and the
iron atom is sandwiched almost perfectly centrally between the
two cyclopentadienyl rings. However, the hydantoin and phenyl
rings deviate markedly from coplanarity: in 1 and 2, these
interplanar angles are 43.8(1)° and 35.8(1)°, respectively. As
shown in the space-filling representations depicted in Figure 3,
the additional methylene unit in 2 places the ferrocenyl group
further from the gem-dimethyls at C(5) in the hydantoin ring,
thus allowing the organometallic sandwich fragment a greater
degree of rotational flexibility. The interplanar angles between
the hydantoin ring and the nearer cyclopentadienyl ring
(C17-C21) are 68.8(1)° for 1 and 61.5(1)° for 2.
Scheme 1. Synthesis of the Isocyanatobenzene 12a
Synthesis of C(5)-Disubstituted Nilutamide Derivatives
5 and 6. 5-Ferrocenyl-2,4-imidazolidinedione 13, prepared from
ferrocenecarboxaldehyde,26 was allowed to react with 4-fluoro-
2-trifluoromethylbenzonitrile in the presence of potassium
carbonate (Scheme 3). However, not only did the substitution
occur on the NH situated between the two carbonyl groups, as
in nilutamide, but also at the 5-position of the hydantoin ring,
already bearing the ferrocenyl moiety. The ferrocenyl hydantoin
5 was obtained in 40% yield, even though the steric hindrance
at the 5-position is much greater for this ferrocenyl analogue
than for nilutamide bearing two methyl groups.
a Key: (a) (i) CuCN, DMF, (ii) aq NaCN, 92% yield; (b) COCl2, toluene,
AcOEt, 98%.
The somewhat surprising disubstitution at the 5-position of
the hydantoin ring in 5 prompted us to synthesize an organic
analogue possessing a degree of steric hindrance similar to that
caused by the ferrocenyl unit in 5; this was done to get a realistic
comparison of the steric requirements of the organometallic and
organic compounds. Hence, the ferrocenyl moiety was replaced
by a p-methoxy aromatic ring by mixing an aqueous solution
of potassium cyanide and ammonium carbonate with p-
anisaldehyde, thus forming the hydantoin 1427 (Scheme 4). As
described for 5, the hydantoin 14 was subsequently disubstituted
by treatment with 4-fluoro-2-trifluoromethylbenzonitrile in the
presence of potassium carbonate to form the hydantoin 6.
2.2. Biochemical Studies. The biological properties of
compounds 1–6 were tested and compared with the results
obtained with the nonsteroidal antiandrogens nilutamide and
bicalutamide. The relative binding affinity (RBA) values for
the compounds were assayed in a standard competitive radio-
ligand assay, using full length human recombinant AR and
[3-H]-DHT as a tracer. The RBA values obtained for the
compounds are reported in Table 1.
2. Results and Discussion
2.1. Synthesis. Synthesis of the N-Substituted
Nilutamide Derivatives 1–4. The reaction of the amine 7,
derived from ferrocenecarboxaldehyde,23 with 2-hydroxyisobu-
tyronitrile led to the formation of ferrocenyl derivative 9 in 25%
yield. Similarly, the reaction of amine 8, obtained from the
reduction of cyanomethylferrocene with LiAlH4, delivered the
ferrocenyl derivative 10 in 82% yield. The isocyanatobenzene,
12, was obtained in two steps from 4-bromo-3-trifluoromethy-
laniline (Scheme 1).24 The first step consisted of the aromatic
nucleophilic displacement of bromide from 4-bromo-3-triflu-
oromethyl-aniline, by using copper cyanide, leading to the
formation of 4-cyano-3-trifluoromethylaniline, 11, in 92%
yield.25 The aniline derivative was then converted in excellent
yield (98%) into the isocyanatobenzene 12 by treatment with
phosgene.
The reaction of the isocyanatobenzene, 12, with 9 and 10
furnished the imines 3 and 4, respectively. Finally, these imines
were hydrolyzed with HCl leading to the formation of the

Synthesis of the Nonsteroidal Antiandrogen Nilutamide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1793
Scheme 2. Synthesis of the N-Ferrocenyl Hydantoins 1 and 2a
a Key: (a) 1,2-dichloroethane, NEt3, 0 °C; (b) 2 N HCl, MeOH, reflux.
All of these compounds show an affinity for AR, as they are
able to displace the binding of the [3-H]-DHT to the receptor,
but the RBA values found are low, i.e. less than 1%, for both
the ferrocenyl complexes and nilutamide and bicalutamide. The
value found for nilutamide is in accordance with the value found
in the literature.28 It is interesting to note that compounds 1
and 2 have RBA values for the AR that are distinctly higher
than those of nilutamide (6 and 3 times higher, respectively)
but comparable to that of bicalutamide, while the values obtained
for compounds 5 and 6 are lower, by a factor of 2. The absence
of hydrogen bonding between the nonsteroidal antiandrogens
and amino acids T877 and N705 of the receptor has been
suggested to explain their weak affinity for the receptor.2 The
same authors note that the B ring of the bicalutamide is not
aligned with the mean plane of the molecule, but instead is
positioned almost at a right angle. This situation is similar to
that found in complexes 1 and 2 where, as noted above, the
ferrocenyl group is rotated markedly relative to the other rings
in the molecule. It seems therefore that a well-positioned
aromatic group, whether fluorophenyl or ferrocenyl, can improve
the affinity of the ligand. The effect of the newly synthesized
molecules was then studied on both hormone-independent (PC-
3) and hormone-dependent prostate cancer cells (LNCaP), and
the results obtained in the presence of 10 µM of the different
complexes are displayed in Figures 4 and 5. As expected, on
PC-3 cells the antiandrogen nilutamide, whose action is sup-
posed to be mediated by the AR, has no effect on these cells
without AR. Complexes 3 and 4 show almost no antiproliferative
effect (less than 10%; Table 1), bicalutamide has a weak
antiproliferative effect (15%), the two N-substituted ferrocenyl
complexes 1 and 2 have a significant antiproliferative effect
(37-34% respectively), and the two bulky C(5)-subsituted
compounds, i.e., the ferrocenyl complex 5 and the organic
compound 6, display similar, strong antiproliferative effects (84
and 76%, respectively). The IC50 values were then determined
on PC-3 for the four compounds having an antiproliferative
effect greater than 20% (Table 1). The two N-substituted
ferrocenyl complexes 1 and 2 show IC50 values of 68 and 67
µM, while the two most active compounds, 5 and 6, display
IC50 values equal to 5.4 and 5.6 µM, i.e., values 10 times lower
than the ones found for 1 and 2. An almost identical IC50 value
(7.4 µM) was found for 5 on MDA-MB-231 cells (hormone-
independent breast cancer cells). These values are lower than
the value found, on MDA-MB-231 cells, for the reference drug
cisplatin (12.7 µM).29
The results obtained on the hormone-dependent prostate
cancer cells LNCaP are shown in Figure 5. As expected for
these cells with androgen receptors, the natural androgen DHT
Figure 1. X-ray crystal structure of 1. Selected bond lengths (Å) and angles (deg): Fe-Cent(1) 1.649(2); Fe-Cent(2) 1.644(1); C17-C16 1.497(2);
C16-N1 1.4671(18); N1-C2 1.3467(18); C2-N3 1.4286(18); N3-C4 1.3875(18); C4-C5 1.5313(19); C5-N1 1.4696(18); N3-C6 1.4194(18); C9-C12,
1.446(2); C12-N12 1.144(2); C8-C13, 1.501(2); C2-O1, 1.2120(18); C4-O2, 1.2045(18); C21-C17-C18, 107.63(13); C17-C16-N1 112.66(12);
N1-C2-N3, 107.04(12); C2-N3-C4 110.87(11); N3-C4-C5 107.23(12); C4-C5-N1 101.14(11); C5-N1-C2 113.50(11).

1794 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Payen et al.
is an antihormonal effect mediated by androgen receptors while
the effect of 5 and 6 is connected to an intrinsic cytoxicity of
the molecules.
Finally, the values found for the lipophilicity of the new
molecules are higher than the log Po/w values of bicalutamide
and nilutamide. This is expected as they all possess phenyl or
ferrocenyl substituents which are known to be lipophilic.
3. Discussion
These results raise a number of points, regarding both the
newly synthesized products and the nonsteroidal antiandrogens,
which are good candidates for treatment of prostate cancer at
an early stage (bicalutamide) or postcastration late-stage (ni-
lutamide). We were unable to find literature data on testing of
these compounds on cell lines. This may be because the
compounds reached the market at a time before cell-line testing
was as widespread as it is today. It should also be noted that,
unlike breast cancer, for which several hormone-dependent and
hormone-independent cell lines are available, the choice of
hormone-dependent human prostate cancer cell lines is very
restricted and essentially limited to LNCaP cells. However, it
is known that the androgen receptor expressed by these cells is
a mutated one that has been described as having a modified
response to antiandrogens.30 It is thus plausible that the
proliferative effect observed may be linked to this mutation. In
terms of the newly synthesized products, it can be noted that
the attachment to N(1) of a ferrocenyl substituent, via an
aliphatic chain with one or two links, increases toxicity relative
to that of nilutamide essentially only in the case of the
derivatives of 1 and 2, and not of their precursors 3 and 4. The
two most cytotoxic compounds are 5 and 6 which have bulky
lipophilic substituents attached directly onto C5 of the hydantoin
ring. The IC50 values of these compounds are of the same order
of magnitude as those found for other ferrocenyl complexes, in
particular certain complexes with an sp3 carbon.31 However,
the toxicity of 5 does not appear to be linked to the organo-
metallic feature of the ferrocenyl substituent or to the presence
of an iron atom, since its organic analogue, 6, shows the same
antiproliferative effect. What the two substituents have in
common is their aromaticity, which increases their lipophilicity
compared to the parent compound nilutamide (3.2 for niluta-
mide, 6.5 and 5.7 for 5 and 6; see Table 1). Furthermore, it has
been previously shown in a QSAR (quantitative structure–ac-
tivity relationship study) of phenolic compounds that an increase
in the lipophilicity correlates with an increase in their cytotox-
icity.32 Hence, this may be the source of the antiproliferative
effect observed with our molecules. It should also be noted
that there is a certain analogy between the chemical structure
of compounds 5 and 6 and that of hydantoins that have
recently been reported which are substituted at the C5 position
by aromatics or by electron-rich substituents.33–35 The
aromatic hydantoins show an affinity for the cannabinoid
receptor,34,35 while electron-rich substituents induce activity
for the calcium channels, at the site of verapamil.33 Moreover,
it has been shown that certain ligands of the cannabinoid
receptor can induce apoptosis of the LNCaP and PC-3
prostate cancer cells36,37 and that verapamil is capable of
inhibiting proliferation of LNCaP cells via an interaction at
the level of the potassium channels.38 It would therefore be
Figure 2. X-ray crystal structure of 2. Selected bond lengths (Å) and
angles (deg): Fe-Cent(1), 1.646(1); Fe-Cent(2), 1.645(1); C18-C17
1.492(3); C17-C16 1.528(3); C16-N1 1.468(2); N1-C2 1.334(2); C2-N3
1.431(2); N3-C4 1.388(2); C4-C5 1.528(2); C5-N1 1.468(2); N3-C6
1.417(2); C9-C12, 1.444(2); C12-N12 1.143(2); C8-C13, 1.503(2);
C2-O1, 1.211(2); C4-O2, 1.206(2); C19-C18-C22, 106.83(17);
C18-C17-C16 112.66(15); C17-C16-N1 113.32(15); N1-C2-N3,
107.29(14); C2-N3-C4 110.45(14); N3-C4-C5 107.23(14); C4-C5-N1
101.21(13); C5-N1-C2 113.57(14).
Figure 3. Space-filling views of the ferrocenyl complexes 1 and 2.
Scheme 3. Synthesis of the C(5)-Ferrocenyl Derivative 5a
a Key: (a) K2CO3, DMF, 80 °C, 40% yield.
Scheme 4. Synthesis of the C(5)-Diphenyl Hydantoin
Derivative 6a
a Key: (a) KCN, (NH4)2CO3, H2O/EtOH, 60 °C, 42% yield; (b) 4-fluoro-
2-trifluoromethylbenzonitrile, K2CO3, DMF, 80 °C, 30% yield.
displays a proliferative effect. Bicalutamide shows a rather weak
antiproliferative effect, nilutamide has an unexpected but clear
and reproducible proliferative effect, while 5 and 6 have an
almost identical strong antiproliferative effect. The addition of
DHT reverses the antiproliferative effect of bicalutamide, but
not the effect observed with 5 and 6. This could be explained
by the fact that the modest effect observed with bicalutamide

Synthesis of the Nonsteroidal Antiandrogen Nilutamide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1795
Table 1. RBA for AR, Antiproliferative Effect, and IC50 Values on PC-3 (Hormone-Independent Prostate Cancer Cells) and log Po/w of the
Compounds
a Mean of two experiments ( range. b IC50 values were determined only for compounds having an antiproliferative effect, on PC-3 at 10 µM, higher than
20%. c Fc ) ferrocenyl (η5-C5H4-Fe-C5H5).
Figure 4. Effect of 10 µM of the nonsteroidal antiandrogens nilutamide
(nilu) and bicalutamide (bica) and of the new compounds (1-6) on
the growth of hormone-independent prostate cancer cells PC-3, after 5
days of culture. Nontreated PC-3 cells are used as the control (C). Mean
of two separate experiments (six measurements for each one) ( range.
Figure 5. Effect of 10 nM of the natural androgen DHT and of 10
µM of the nonsteroidal antiandrogens nilutamide (nilu), bicalutamide
(bica) and of 5 and 6 in the absence or presence of 10 nM DHT on the
growth of hormone-dependent prostate cancer cells LNCaP, after 5 days
of culture. Nontreated LNCaP cells are used as the control (C). Mean
of two separate experiments (six measurements for each one) ( range.
worthwhile to investigate further in order to establish whether
our compounds act via one or other of these pathways.
and 6, the most effective in the current study, show an unusual
behavior. Their activity does not occur via the androgen receptor,
even in the case of LNCaP cells. The introduction of a ferrocenyl
group produces the same effect as a purely organic aromatic
moiety. Structures 5 and 6 may act via recognition by other
receptors. Of these, the cannabinoid receptors suggest an
interesting hypothesis, since a higher level of these receptors is
found in the cancerous prostate relative to the healthy organ,37
and structures somewhat analogous to our molecules are active
by this route. Further studies are underway on this series of
molecules to test this particular mechanistic hypothesis. In any
case, the activity of these structures merits further attention in
itself.
Conclusion
A number of reasons have been put forward to justify the
medical use of metallocenes, of which ferrocene is the archetype,
attached to biological nanovectors.39–41 These moieties are small,
rigid, lipophilic, and easily able to cross cell membranes even
after substitution. They are also similar in shape to an aromatic
nucleus, even if their sandwich structure makes them somewhat
thicker. Finally, the ferrocenyl compounds sometimes show an
unusual redox activity in terms of the target. An illustration of
these different behaviors was provided by our study of their
antiproliferative effects on both hormone-dependent and non-
hormone-dependent breast cancer cells. The novel molecules 5

1796 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Payen et al.
124.3 (C7), 129.1 (C11), 135.0 (C10), 153.2 (CO), 168.3 (CdNH);
EI-MS m/z 494 [M+•]. Anal. (C24H21F3FeN4O) H, N. C: calcd 58.32,
found 57.79.
4. Experimental Section
4.1. General Comments. All reactions were carried out 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 chromatography was performed on silica
gel Merck 60 (40–63 µm). Melting points were measured with a
Kofler device. Infrared spectra were recorded on an IR-FT BOMEM
Michelson-100 spectrometer. 1H and 13C NMR spectra were
recorded on a 300 MHz Bruker or a 600 MHz Varian spectrometer;
assignments were made by standard 2-D HSQC and HMBC
techniques. 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 µm, L ) 25 cm, D
) 4.6 mm, eluent: acetonitrile/water 80:20, flow rate ) 1 mL/min,
λ ) 254 nm.
4-(4,4-Dimethyl-3-ferrocenylethyl-5-imino-2-oxo-1-imidazo-
lidinyl)-2-trifluoromethylbenzonitrile (4). A solution of isocyanate
12 (0.716 g, 3.38 mmol) in 1,2-dichloroethane (50 mL) was added
dropwise at 0 °C to a solution of 10 (1.00 g, 3.38 mmol) in 1,2-
dichloroethane (30 mL) and triethylamine (0.3 mL), and the reaction
mixture was stirred at 0 °C for 2 h. The solution was allowed to
warm to room temperature, and the solvent was then evaporated
under reduced pressure. The crude residue was purified by column
chromatography (SiO2; diethyl ether), and 4 was obtained as a
yellow-orange solid (1.10 g, 59% yield). mp 210 °C; IR (KBr) 3283
1
(NH), 2234 (CN), 1725 (CO), 1662 (imine) cm-1; H NMR (300
MHz, CDCl3) δ 1.42 (s, 6H, 2 CH3), 2.76 (m, 2H, CH2), 3.40 (m,
2H, CH2N), 4.10 (s, 4H, C5H4), 4.15 (s, 5H, C5H5), 7.42 (s, 1H,
NdH), 7.92 (d, 1H, H10), 8.08 (d, 1H, H11), 8.24 (s, 1H, H7);
13C NMR (75 MHz, CDCl3) δ 25.4 (2 CH3), 29.5 (CH2), 41.9
(CH2N), 61.4 (C(CH3)2), 67.9, 68.4 (C5H4), 68.8 (C5H5), 84.7 (C5H4,
Cip), 124.4 (C7), 129.3 (C11), 135.3 (C10), 153.4 (CO), 168.3
(dNH); EI-MS m/z 508 [M+•]. Anal. (C25H23F3FeN4O) C, H, N.
3-(4-Cyano-3-trifluoromethylphenyl)-5-(ferrocenyl)-5-(4-cy-
ano-3-trifluoromethylphenyl)imidazolidine-2,4-dione (5). Com-
pound 13 (1.13 g, 3.96 mmol) and potassium carbonate (0.55 g,
3.96 mmol) were added to a solution of 4-fluoro-2-trifluorometh-
ylbenzonitrile (0.500 g, 2.64 mmol) in anhydrous DMF (30 mL).
The reaction mixture was stirred at 80 °C for 4 h. Ethyl acetate
was then added to the solution, which was filtered on Celite. The
filtrate was evaporated to give a crude product which was purified
by chromatography (SiO2; ethyl acetate/petroleum ether 2:3 f 1:2).
Compound 5 was obtained as a yellow orange solid (1.06 g, 40%
yield): mp 200–202 °C; IR (KBr) 3309 (NH), 2238 (CN) 1792
4.2. Synthesis. Compounds 7, 8,42 and 1125 were prepared
following the literature procedures.
4-(4,4-Dimethyl-2,5-dioxo-3-ferrocenylmethyl-1-
imidazolidinyl)-2-trifluoromethylbenzonitrile (1). Aqueous HCl
solution (2 N, 3.2 mL) was added to a solution of imine 3 (0.390
g, 0.79 mmol) in methanol (20 mL). The reaction mixture was
heated under reflux for 2 h. The solution was then cooled to room
temperature and was poured into cold water (100 mL). The mixture
was extracted with ethyl acetate, and the organic phase was dried
on magnesium sulfate, filtered, and evaporated to give 1 as a yellow-
orange solid (0.340 g, 80% yield): mp 173–174 °C; IR (KBr) 2230
(CN), 1777 and 1716 (2 CO) cm-1; H NMR (300 MHz, CDCl3)
1
δ 1.44 (s, 6H, 2 CH3), 4.17 (t, 2H, C5H4, J ) 1.8 Hz), 4.19 (s, 5H,
C5H5), 4.32 (t, 2H, C5H4, J ) 1.8 Hz), 4.41 (s, 2H, CH2N), 7.89
(d, 1H, H10, J ) 8.7 Hz), 7.98 (dd, 1H, H11, J ) 8.7 Hz, J ) 1.8
Hz), 8.14 (d, 1H, H7, J ) 1.8 Hz); 13C NMR (75 MHz, CDCl3) δ
23.7 (2 CH3), 39.8 (CH2N), 61.9 (C(CH3)2), 68.8 (C5H4), 68.9
(C5H5), 70.0 (C5H4), 82.7 (C5H4, Cip), 115.2 (CN), 123.1 (C7), 128.0
(C11), 134.0 (q, C8), 135.3 (C10), 136.5 (C6), 152.7 (CO), 174.8
(CO); EI-MS m/z 495 [M+•]. Anal. (C24H20F3FeN3O2) C, H, N.
4-(4,4-Dimethyl-2,5-dioxo-3-ferrocenylethyl-1-imidazolidinyl)-
2-trifluoromethylbenzonitrile (2). Aqueous HCl solution (2 N,
3.2 mL) was added to a solution of imine 4 (0.620 g, 1.22 mmol)
in methanol (40 mL). The reaction mixture was heated under reflux
for 1 h. The solution was then cooled to room temperature and
was poured into cold water (100 mL). The mixture was extracted
with ethyl acetate, and the organic phase was dried on magnesium
sulfate, filtered, and evaporated giving 2 as a yellow-orange solid
(0.60 g, 89% yield): mp 140–142 °C; IR (KBr) 2230 (CN), 1777
1
and 1739 (2 CO) cm-1; H NMR (600 MHz, acetone-d6) δ 4.24
(s, 5H, C5H5), 4.40 (m, 1H), 4.42 (m, 1H), 4.43 (m, 1H), 4.77
(m,1H) (C5H4), 8.11 (d, 1H, J ) 8.5 Hz, H16), 8.15 (d, 1H J ) 8.5
Hz, H17), 8.16 (s, 1H, H13), 8.29 (d, 1H, J ) 8.5 Hz, H10), 8.32
(d, 1H, J ) 8.5 Hz, H11), 8.34 (s, 1H, H7), 9.19 (s, 1H, NH); 13
C
NMR (150 MHz, acetone-d6) δ 66.74 (C5H4), 67.77 (C5), 67.85
(C5H4), 69.73 (C5H4), 69.81 (C5H5), 70.45 (C5H4), 90.57 (C5H4,
Cipso), 109.00 (q, J ) 2 Hz, C9), 110.37 (q, J ) 2 Hz, C15), 115.67
(C9-CN), 115.71 (C15-CN), 123.32 (q, J ) 273 Hz, C8-CF3),
123.48 (q, J ) 273 Hz, C15-CF3), 124.17 (q, J ) 5 Hz, C7), 125.39
(q, J ) 5 Hz, C13), 129.88 (C11), 131.93 (C17), 132.57 (q, J )
32 Hz, C14), 133.28 (q, J ) 33 Hz, C8), 136.30 (C16), 137.00
(C10), 137.49 (C6), 146.04 (C12), 154.38 (C2), 170.71 (C4); EI-
MS m/z 622 [M+•]. Anal. (C29H16F6FeN4O2) C, H, N.
1
and 1720 (2 CO) cm-1; H NMR (300 MHz, CDCl3) δ 1.43 (s,
3-(4-Cyano-3-trifluoromethylphenyl)-5-(4-methoxyphenyl)-5-
(4-cyano-3-trifluoromethylphenyl)imidazolidine-2,4-dione (6).
Compound 14 (0.310 g, 1.50 mmol), 4-fluoro-2-trifluoromethyl-
benzonitrile (0.600 g, 3.15 mmol), and potassium carbonate (0.430
g, 3.15 mmol) were mixed in distilled DMF (10 mL). The reaction
mixture was stirred at 80 °C for 4 h before it was filtered over a
short pad of Celite. The solvent was evaporated to yield a yellow
oil which was purified by column chromatography (SiO2; ethyl
acetate/petroleum ether 2:3). The second fraction corresponded to
the pure product as a white solid (0.245 g, 30% yield): mp
degradation at 82 °C; IR (KBr) 3305 (NH) 2235 (CN) 1793 and
6H, 2 CH3), 2.79 (m, 2H, CH2), 3.44 (m, 2H, CH2N); 4.11 (s, 4H,
C5H4), 4.15 (s, 5H, C5H5), 7.92 (d, 1H, H10, J ) 8.4 Hz), 8.02
(dd, 1H, H11, J ) 8.4 Hz, J ) 1.8 Hz), 8.17 (d, 1H, H7, J ) 1.8
Hz); 13C NMR (75 MHz, CDCl3) δ 23.5 (2 CH3), 29.3 (CH2), 41.9
(CH2N), 62.0 (C(CH3)2), 67.9, 68.3 (C5H4), 68.8 (C5H5), 84.4 (C5H4,
Cip), 108.3 (C9), 115.1 (CN), 122.0 (q, CF3), 123.0 (C7), 127.9
(C11), 134.0 (q, C8), 135.3 (C10), 136.6 (C6), 152.8 (CO), 174.7
(CO); EI-MS m/z 509 [M+•]. Anal. (C25H22F3FeN3O2) C, H, N.
4-(4,4-Dimethyl-3-ferrocenylmethyl-5-imino-2-oxo-1-imidazo-
lidinyl)-2-trifluoromethylbenzonitrile (3). A solution of isocyanate
12 (1.35 g, 6.35 mmol) in 1,2-dichloroethane (50 mL) was added
dropwise at 0 °C to a solution of 9 (1.79 g, 6.35 mmol) in 1,2-
dichloroethane (30 mL) and triethylamine (0.6 mL). The reaction
mixture was stirred at 0 °C for 2 h. The solution was heated to
room temperature; the solvent was then evaporated under reduced
pressure. The crude residue was purified by column chromatography
(SiO2; diethyl ether), and 3 was obtained as a yellow-orange solid
(1.16 g, 34% yield): mp 220 °C; IR (KBr) 3281 (NH) 2229 (CN)
1738 (CO) cm-1 1H NMR (300 MHz, CDCl3) δ 3.83 (s, 3H,
;
OCH3), 6.92–6.98 (m, 2H, H20 and H22), 7.09 (s, 1H, NH),
7.18–7.23 (m, 2H, H13 and H19), 7.80–7.97 (m, 5H, H13, H16,
H17, H10 and H11), 8.09 (s, 1H, H7); 13C NMR (75 MHz, CDCl3)
δ 55.5 (OCH3), 69.2 (C5), 109.3, 110.7 (C9 and C15), 114.6 (CN),
115.1 (C20 and C22), 120.1 (CF3) 123.3, 125.1 (C7 and C13), 127.7
(C19 and C23), 128.3 (C7), 129.1 (C18), 131.0 (C13), 133.3, 134.1
(C8 and C14, J ) 33.2 Hz), 135.2 (C10), 135.4 (C6), 135.5 (C16),
143.7 (C12), 153.5 (CO), 160.6 (C18), 170.1 (CO); EI-MS m/z 544
[M+•]. Anal. (C26H14F6N4O3) C, H, N.
1723 (CO) 1661 (imine) cm-1 1H NMR (300 MHz, CDCl3) δ
;
1.41 (s, 6H, 2 CH3), 4.15 (t, 2H, C5H4, J ) 1.8 Hz), 4.18 (s, 5H,
C5H5), 4.31 (t, 2H, C5H4, J ) 1.8 Hz), 4.38 (s, 2H, CH2), 7.42 (s,
1H, NdH), 7.92 (d, 1H, H10), 8.08 (s, 1H, H11), 8.24 (d, 1H, H7);
13C NMR (75 MHz, CDCl3) δ 25.6 (2 CH3), 39.7 (CH2), 61.1
(C(CH3)2), 68.6 (C5H4), 68.9 (C5H5), 69.9 (C5H4), 83.3 (C5H4,Cip),
2-Cyano-2-[N-(ferrocenylmethyl)amino]propane (9). The amine
7 (0.500 g, 2.33 mmol) was dissolved in diethyl ether (12 mL).
This solution was added slowly to 2-hydroxyisobutyronitrile (2.7
mL, 31.5 mmol). The mixture was stirred for 3 h at room

Synthesis of the Nonsteroidal Antiandrogen Nilutamide
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1797
temperature, water was added, the mixture was extracted with
diethyl ether, and the organic phase was dried on magnesium sulfate,
filtered, and evaporated to give an orange oil. The product was
purified by filtration on silica gel (diethyl ether/petroleum ether 1:1),
yielding 9 as an orange solid (0.190 g, 25% yield): mp 62 °C; IR
(KBr) 3313 (NH), 2219 (CN) cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.51 (s, 6H, 2 CH3), 3.60 (s, 2H, CH2NH), 4.14 (t, 2H, C5H4, J
was reached. A white solid appeared and was filtered off to give the
pure product (0.86 g, 42% yield): mp 193 °C; H NMR (300 MHz,
acetone-d6) δ 3.66 (s, 3H, OCH3), 5.01 (s, 1H, CH), 6.82 (d, 2H, J )
9.0 Hz, C6H4), 7.22 (d, 2H, J ) 8.7 Hz, C6H4), 7.22 (s, 1H, NH), 9.55
(s, 1H, NH); 13C NMR (75 MHz, acetone-d6) δ 55.6 (OCH3), 62.3
(CH), 114.9, 128.8, 128.9 (C6H4), 157.9 (CdO), 160.8 (C6H4), 174.4
(CdO); CI-MS m/z 491 [MNH4+].
1
) 1.8 Hz), 4.16 (s, 5H, C5H5), 4.21 (t, 2H, C5H4, J ) 1.8 Hz); 13
C
Bicalutamide. Bicalutamide was prepared following the literature
1
NMR (75 MHz, CDCl3) δ 27.6 (2 CH3), 44.5 (CH2NH), 51.8
(C(CH3)2), 68.1, 68.2 (C5H4), 68.6 (C5H5), 86.0 (C5H4 ipso), 123.0
(CN); EI-MS m/z 282 [M+•]. Anal. (C15H18FeN2) C, H, N.
2-Cyano-2-[N-(ferrocenylethyl)amino]propane (10). Amine 8
(1.00 g, 4.36 mmol) was dissolved in diethyl ether (18 mL), and
this solution was added slowly to 2-hydroxyisobutyronitrile (2.5
mL, 27.5 mmol). The reaction mixture was stirred for 3 h at room
temperature, water was added, and the mixture was extracted with
diethyl ether. The organic phase was dried on magnesium sulfate,
filtered, and evaporated to give an orange oil. The product was
purified by filtration on silica gel (diethyl ether/petroleum ether 1:1),
yielding 10 as an orange oil (1.22 g, 82% yield). This oil was
crystallized from pentane to give an orange solid: mp 74–76 °C;
IR (KBr) 3314 (NH) 2218 (CN) cm-1; 1H NMR (300 MHz, CDCl3)
δ 1.46 (s, 6H, 2 CH3), 2.57 (t, 2H, CH2, J ) 7.2 Hz), 2.87 (t, 2H,
CH2NH, J ) 7.2 Hz), 4.08 (t, 2H, C5H4), 4.11 (s, 5H, C5H5), 4.13
(t, 2H, C5H4); 13C NMR (75 MHz, CDCl3) δ 27.5 (2 CH3), 30.2
(CH2), 46.0 (CH2NH), 51.5 (C(CH3)2), 67.7, 68.4 (C5H4), 68.7
(C5H5), 85.7 (C5H4, Cipso), 122.8 (CN); EI-MS m/z 296 [M+•]. Anal.
(C16H20FeN2) C, H. N: calcd 9.46, found 8.83.
procedure:44 mp 190–193 °C; H NMR (300 MHz, acetone-d6):
δ1.54 (s, 3H,), 3.68 (d, 1H, CH2SO2, J ) 14.8 Hz), 4.07 (d, 1H,
CH2SO2, J ) 14.8 Hz), 5.52 (s, 1H, OH), 7.28 and 7.30 (d, d, 1H,
1H, C6H4, J ) 8,8 Hz), 7.96–8.01 (m, 2H, C6H4), 8.02 (d, 1H,
C6H3, J ) 8,9 Hz), 8.21 (dd, 1H, C6H3, J ) 8.9 Hz, J ) 2.1 Hz),
8,41 (d, 1H, C6H3, J ) 2.1 Hz), 9.94 (s, 1H, NH). 13C NMR (75
MHz, DMSO-d6) δ 27.2 (CH3), 63.5 (CH2SO2), 73.2 (C(OH)).
102.0 (C6H3, Cipso), 114.5 (CN), 115.9 and 116.2 (C6H4), 117.5
(C6H3), 121.5 (q, CF3, J ) 273.8 Hz), 122.9 (C6H3), 131.3 and
131,4 (C6H4), 134.8 (q, C6H3), 136.2 (C6H3), 137,1 (C6H4, Cipso),
143.2 ((C6H3, Cipso), 163.9 (C6H4, Cipso), 173.7 (NHCO); EI-MS
m/z 430 [M+•]. Anal. (C18H14F4N2O4S) C, H, N.
4.3. X-ray Crystal Structure Determinations for 1 and 2.
Crystal data were collected using a Bruker SMART APEX CCD
area detector diffractometer and are listed in Table S1 (Supporting
Information). A full sphere of reciprocal space was scanned by ꢀ-ω
scans. Pseudoempirical absorption correction based on redundant
reflections was performed by the program SADABS.45The struc-
tures were solved by direct methods using SHELXS-9746 and
refined by full-matrix least-squares on F2 for all data using
SHELXL-97.47 All hydrogen atoms were located in the difference
Fourier map and allowed to refine freely with isotropic thermal
displacement factors. Anisotropic thermal displacement parameters
were used for all nonhydrogen atoms.
4-Cyano-3-trifluoromethyl-isocyanatobenzene (12). 24 A solu-
tion of 4-cyano-3-trifluoromethylaniline 11 (2.00 g, 10.8 mmol) in
distilled ethyl acetate (15 mL) was added dropwise at 0 °C to a
solution of 20% (m/v) phosgene in toluene (7 mL, 13.0 mmol).
The reaction vessel was set up like a distillation apparatus and was
equipped to trap the HCl formed. After being stirred at 0 °C for 30
min, the reaction mixture was allowed to warm to room temperature
and then was heated under reflux. A part of the solvent was distilled
and was replaced by distilled toluene (20 mL) until the temperature
reached 110 °C. Reflux was continued until HCl evolution subsided.
A precipitate was removed by filtration, and the filtrate was
evaporated under vacuum. The product 12 was obtained as an
orange oil (2.24 g, 98% yield). This oil was directly used for the
Crystallographic data for the structures reported in this paper
have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication nos. CCDC-655743 (1) and
CCDC-655742 (2). Copies of this information may be obtained
free of charge from the Director, CCDC, 12 Union Road, Cam-
bridge CB2 1EZ (Fax: +44-1223-336033; E-mail: deposit@ccdc.
cam.ac.uk or http://www.ccdc.cam.ac.uk).
4.4. Biochemical
Experiments.
a. Purity
of
the
Compounds. The purity of the compounds tested in biochemical
experiments (i.e., 1–6 and bicalutamide) was checked by reversed-
phase HPLC on a Chromasil C18 column (10 µm, L ) 25 cm D )
4.6 mm) eluted with a mixture acetonitrile/water (80/20) and
measurement of the absorbance at 254 nm.
1
next step: IR (CH2Cl2) 2268 (NCO) 2233 (CN) cm-1; H NMR
(300 MHz, CDCl3) δ 7.39 (dd, 1H, H6, J ) 8.3 Hz, J ) 2.2 Hz),
7.50 (d, 1H, H2, J ) 2.2 Hz), 7.82 (d, 1H, H5, J ) 8.3 Hz); 13C
NMR (75 MHz, CDCl3) δ 107.1 (C4), 114.9 (CN), 121.7 (q, CF3,
J ) 265.3 Hz), 123.4 (C2), 126.3 (NCO), 128.4 (C6), 134.2 (C3),
136.3 (C5), 138.7 (C1).
b. Materials. DHT, nilutamide, and protamine sulfate was
obtained from Sigma-Aldrich (France). Stock solutions (1 × 10-3
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) was purchased
from Invitrogen. Fetal calf serum, glutamine and kanamycine were
obtained from Invitrogen. Prostate and breast cancer cells LNCaP,
PC3, and MDA-MB231 cells were from the American type Culture
Collection (ATCC). [1,2-3H]-DHT was purchased from NEN Life
Science Product.
5-(Ferrocenyl)imidazolidine-2,4-dione (13). 26,43 A solution of
formylferrocene (1.00 g, 4.67 mmol) in ethanol (25 mL) was added
to 20 mL of NaHSO3 aqueous solution (0.97 g). In another Schlenk
tube, potassium cyanide (1.37 g, 20 mmol) and ammonium
carbonate (4.04 g, 42 mmol) were dissolved in a mixture of distilled
water (27 mL) and ethanol (5 mL). The first solution was then added
to the second solution. The mixture was heated for 2 h at 60 °C.
The reaction mixture was cooled to room temperature, and ethanol
was removed under vacuum. A yellow solid obtained was filtered
and washed with diethyl ether to give the pure product (0.52 g,
39% yield): mp 252–254 °C; IR (KBr) 3407 and 3203 (2 NH),
c. Determination of the RBA of the Compounds for the
AR. RBA values were measured on AR PanVera (750 pmol)
purchased from Invitrogen. The ARs were aliquoted in 15 µL
fractions and kept in liquid nitrogen 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 µL of the AR solution were incubated in
1
1775 and 1724 (2 CO) cm-1; H NMR (300 MHz, acetone-d6) δ
4.25 (s, 5H, C5H5), 4.28 and 4.47 (2 t, 4H, C5H4, J ) 1.2 Hz), 5.85
(s, 1H, H5), 7.79 (s, 1H, NH), 9.57 (s, 1H, NH);. 13C NMR (75
MHz, acetone-d6) δ 56.7 (C5), 65.3, 66.5, 67.7, 69.1, 84.1 (C5H4),
69.0 (C5H5), 157.4 (C)O), 173.5 (C)O).
5-(4-Methoxyphenyl)imidazolidine-2,4-dione (14). 27 Potassium
cyanide (0.800 g, 0.012 mol) and ammonium carbonate (4.80 g, 0.05
mol) were dissolved in distilled water (27 mL). A solution of
p-anisaldehyde (0.63 mL, 0.01 mol) in absolute ethanol (20 mL) was
then added dropwise at room temperature. The colorless solution was
heated for 4 h at 60 °C and 2 h at 110 °C to eliminate the excess of
ammonium carbonate. The reaction mixture was cooled to 0 °C before
concentrated HCl was added dropwise until pH 2–3 of the solution
3
polypropylene tubes for 3 h 30 at 4 °C with [1,2- H]-DHT (2 ×
10-9M , specific activity 1.6 TBq/mmol) in the presence of nine
concentrations of the compounds to be tested (between 1 × 10-5
M and 6 × 10-7 M) or of non radioactive DHT (between 8 ×
10-8 and 7.5 × 10-10 M). At the end of the incubation period, the
fractions of [3H]-DHT bound to the androgen receptor (Y values)
were precipitated by addition of a 200 µl of a cold solution of

1798 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Payen et al.
(4) Reid, P.; Kantoff, P.; Oh, W. Antiandrogens in prostate cancer. InVest.
New Drugs 1999, 17, 271–284.
(5) Ross, R. K.; Pike, M. C.; Coetzee, G. A.; Reichardt, J. K.; Yu, M. C.;
Feigelson, H.; Stanczyk, F. Z.; Kolonel, L. N.; Henderson, B. E.
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protamine sulfate (1 mg/mL in water). After a 10 min period of
incubation at 4 °C, the precipitates were recovered by filtration on
25 mm circle glass microfiber filters GF/C filters using a Millipore
12 well filtration ramp. The filters were rinsed twice with cold
phosphate buffer and then transferred in 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 concentration 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 ) ln(Y/100 - Y) versus the mass of the
competing complex. The RBA was calculated as follows: RBA of
a compound ) concentration of DHT required to displace 50% of
[3H]-DHT × 100/concentration of the compound required to
displace 50% of [3H]-DHT. The RBA value of DHT is by definition
equal to 100%.
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prostatic cancer: a review. Prostate 1984, 5, 299–311.
d. 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
described by Minick48 and Pomper.49 Measurement of the chro-
matographic capacity factors (k′) for each molecule was done at
various concentrations in the range 95–80% methanol (containing
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 (k′) are extrapolated to 100% of the aqueous
component given the value of kw′. The log Po/w is then obtained by
the formula log Po/w ) 0.13418 + 0.98452 log k′.
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e. Culture Conditions. Cells were maintained in monolayer
culture in DMEM with phenol red/Glutamax I, supplemented with
9% of decomplemented fetal calf serum and 0.9% kanamycine, at
37 °C in a 5% CO2 air humidified incubator. For proliferation
assays, cells were seeded at a density of 15000 to 25000 cells per
millilter in 24-well sterile plates in 1 mL of DMEM without phenol
red, supplemented with 9% of fetal calf 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 (four wells for each product). After 3 days (D3), the
incubation medium was removed, and 2 mL of fresh medium
containing the compounds was added. At different days (D3, D4,
D5, and D6), 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.
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W.; D’Angelo, J. G.; Posner, G. H.; Foster, B. A. Biological
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of new curcumin analogues as potential anti-prostate cancer agents.
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D. I.; Neamati, N. Discovery and preclinical evaluation of a novel
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-independent cancer cell lines. Mol. Cancer Ther. 2005, 4, 1105–1113.
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J.; Huché, M.; Jaouen, G. Synthesis, biochemical properties and
molecular modelling studies of organometallic specific estrogen
receptor modulators (SERMs), the ferrocifens and hydroxyferrocifens:
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Acknowledgment. We thank the Ministère de la Recherche
and the Centre National de la Recherche Scientifique for
financial support, A. Cordaville for technical assistance, the Irish
Council for Science, Engineering and Technology (IRCSET)
for a France-Ireland Exchange, COST D39, and John Grealis
for the 600 MHz NMR spectra.
Supporting Information Available: Numbering schemes for
NMR assignment of compounds 3, 5, and 6, crystallographic data
for compounds 1 and 2, and elemental analyses. This material is
available free of charge via the Internet at http://pubs.acs.org.
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