Efficient new constructs against triple negative breast cancer cells: Synthesis and preliminary biological study of ferrocifen-SAHA hybrids and related species

Article abstract of DOI:10.1039/c3dt51917a

Chemotherapeutic agents combining several active groups within a single molecule can modulate multiple cellular pathways and, thus, exhibit higher efficacy than single-target drugs. In this study, six new hybrid compounds combining tamoxifen (TAM) or ferrocifen (FcTAM) structural motifs with suberoylanilide hydroxamic acid (SAHA) were synthesised and evaluated. Antiproliferative activity was first explored in cancer cell lines. Combining FcTAM and SAHA structural motifs to form the unprecedented FcTAM-SAHA hybrid molecule led to an increased cytotoxicity (IC₅₀ = 0.7 μM) in triple-negative MDA-MB-231 breast cancer cells when compared to FcTAM or SAHA alone (IC₅₀ = 2.6 μM and 3.6 μM, respectively), while the organic hybrid analogue TAM-SAHA was far less cytotoxic (IC₅₀ = 8.6 μM). In hormone-dependent MCF-7 breast cancer cells, FcTAM-SAHA was more active (IC₅₀ = 2.0 μM) than FcTAM (IC₅₀ = 4.4 μM) and TAM-SAHA (IC₅₀ > 10 μM), but less toxic than SAHA (IC ₅₀ = 1.0 μM). Surprisingly, FcTAM-PSA, an N¹- phenylsuberamide derivative, also possessed strong antiproliferative activity (IC₅₀ = 0.5 μM and 1.8 μM in MDA-MB-231 and MCF-7 cells, respectively). Subsequent biochemical studies indicate that estrogen receptor alpha (ERα) and histone deacetylases (HDAC) are not the main targets of the hybrid compounds for their antiproliferative effect. Interestingly, both organometallic compounds were able to induce p21^waf1/cip1 gene expression in MCF-7 breast cancer cells in accordance with their antiproliferative activity.

Full text of DOI:10.1039/c3dt51917a

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Efficient new constructs against triple negative breast
cancer cells: synthesis and preliminary biological study
of ferrocifen–SAHA hybrids and related species
Cite this: Dalton Trans., 2013, 42, 15489
José de Jesús Cázares Marinero,a,b Marion Lapierre,c,d,e,f Vincent Cavaillès,*c,d,e,f
Rénette Saint-Fort,a,b Anne Vessières,a,b Siden Top*a,b and Gérard Jaouena,b
Chemotherapeutic agents combining several active groups within a single molecule can modulate mul-
tiple cellular pathways and, thus, exhibit higher efficacy than single-target drugs. In this study, six new
hybrid compounds combining tamoxifen (TAM) or ferrocifen (FcTAM) structural motifs with suberoyl-
anilide hydroxamic acid (SAHA) were synthesised and evaluated. Antiproliferative activity was first explored
in cancer cell lines. Combining FcTAM and SAHA structural motifs to form the unprecedented FcTAM–
SAHA hybrid molecule led to an increased cytotoxicity (IC50 = 0.7 μM) in triple-negative MDA-MB-231
breast cancer cells when compared to FcTAM or SAHA alone (IC50 = 2.6 μM and 3.6 μM, respectively),
while the organic hybrid analogue TAM–SAHA was far less cytotoxic (IC50 = 8.6 μM). In hormone-depen-
dent MCF-7 breast cancer cells, FcTAM–SAHA was more active (IC50 = 2.0 μM) than FcTAM (IC50 = 4.4 μM)
and TAM–SAHA (IC50 > 10 μM), but less toxic than SAHA (IC50 = 1.0 μM). Surprisingly, FcTAM–PSA, an
N1-phenylsuberamide derivative, also possessed strong antiproliferative activity (IC50 = 0.5 μM and 1.8 μM
in MDA-MB-231 and MCF-7 cells, respectively). Subsequent biochemical studies indicate that estrogen
receptor alpha (ERα) and histone deacetylases (HDAC) are not the main targets of the hybrid compounds
for their antiproliferative effect. Interestingly, both organometallic compounds were able to induce
p21waf1/cip1 gene expression in MCF-7 breast cancer cells in accordance with their antiproliferative
activity.
Received 15th July 2013,
Accepted 22nd August 2013
DOI: 10.1039/c3dt51917a
www.rsc.org/dalton
derivatives with potent antiproliferative activities, particularly
Introduction
against breast cancer cells.7–10 Replacement of the β-phenyl
Organometallic chemical biology has recently been defined as group in tamoxifen (TAM) – the primary antitumor drug cur-
a new field of research.1,2 In this context, organometallic com- rently used to treat hormone-dependent breast cancer – or in
pounds, which are complexes containing at least one metal– its active metabolite, hydroxytamoxifen (OHTAM), with a ferro-
carbon covalent bond, have also been recognised – in favorable cenyl group generates the organometallic compounds ferro-
situations – as strong candidates for use as anticancer cifen (FcTAM) and hydroxyferrocifen (FcOHTAM) (Chart 1).11 It
drugs.3,4 They offer a wide range of possibilities for the design is worthwhile to note that FcOHTAM significantly inhibits pro-
of new classes of medicinal compounds with novel mecha- liferation in both hormone-dependent MCF-7 and hormone-
nisms of action compared to conventional drugs.5,6 Our group
previously described the synthesis of the first organometallic
aChimie ParisTech, Laboratoire Charles Friedel, 11, rue Pierre et Marie Curie,
F75231 Paris cedex, France
bCNRS, UMR 7223, Paris, France. For medicinal chemistry (S.T.).
E-mail: siden-top@chimie-paristech.fr; Fax: (+33)143260061; Tel: (+33)144276699
cIRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298,
France
dINSERM, U896, Montpellier, F-34298, France
eUniversité Montpellier1, Montpellier, F-34298, France
fCRLC Val d’Aurelle Paul Lamarque, Montpellier, F-34298, France. For molecular
biology (V.C.). E-mail: vincent.cavailles@inserm.fr; Fax: (+33)467613787;
Tel: (+33)467612405
Chart 1 Chemical structure of ferrocifen (FcTAM), hydroxyferrocifen,
(FcOHTAM), tamoxifen (TAM), hydroxytamoxifen (OHTAM), suberoylanilide
hydroxamic acid (SAHA), N1-phenylsuberamide (PSA), and 8-oxo-8-(phenyl-
amino)octanoic acid (OPOA).
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independent MDA-MB-231 breast cancer cells but has no effect against breast cancer cells. In an effort to improve SAHA
on normal cells.12 As a triple-negative breast cancer (TNBC) HDACi activity, to enhance its cytotoxicity and to better under-
cell line, MDA-MB-231 lacks expression of estrogen receptor stand the structural requirements for the design of new drugs,
(ER), progesterone receptor (PR) and human epidermal growth research has been directed towards the synthesis of analogous
factor receptor 2 (HER2), making it more aggressive than other compounds with subtle modifications to its cap, linker and
cancer subtypes since no available molecularly targeted agents binding group (Chart 1).34 The phenyl group has been modi-
are effective.13,14
fied by the presence of heterocycles,35,36 nuclear receptor ago-
We have postulated that the ferrocenyl group (Fc) acts as a nists,37 phenyl substituents,38 platinum agents,39 and most
redox antenna for phenol oxidation via an intramolecular recently with the incorporation of the ferrocene moiety.40 Simi-
mechanism producing cytotoxic species under mild oxidizing larly, previous studies have also been conducted on the effect
conditions.15 This hypothesis is supported by results obtained of substituents,41 the chain length, and the presence of stereo-
using other ferrocenyl analogues comprising modifications at genic centers.42 Finally, the binding group has been replaced
different structural levels: the alkyl chain,16 the organometallic by different functional groups, including oximes43 and sulfur
moiety,17,18 the phenol position,19 conjugation,20 and aromatic derivatives.44
substituents.21 Moreover, the proposed hypothesis is consist-
ent with the fact that cancer cells possess different redox pro- able to synergize with other drugs to improve antitumor
perties compared to healthy cells.22,23 activity.45–50 One example of an active hybrid compound is tri-
Both in vitro and in vivo studies have shown that SAHA is
On the other hand, recent research has reflected a keen inter- ciferol, which combines an HDACi motif with 1α,25-dihydroxy-
est in the study of compounds that carry out their therapeutic vitamin-D3 (1,25D).37 Hence, incorporation of the SAHA
effects by enzyme inhibition mechanisms. Histone deacetylase pharmacophore into selected agents may produce new hybrid
inhibitors (HDACi) are an important class of epigenetic drugs. bifunctional drugs with improved efficacy. In order to evaluate
Specifically, they inhibit the activity of histone deacetylases the impact of the structural combinations of selected pharma-
(HDACs), which catalyse the deacetylation of histones. Histone cophores with the FcTAM molecule and to investigate their
deacetylation silences gene expression by inducing DNA to biological responses, we felt it would be interesting to prepare
adopt a closed conformation with histones, restricting its an FcTAM–SAHA hybrid and its corresponding organic ana-
access to the transcription factors.24,25 In addition to their logue TAM–SAHA by replacing the 3-(dimethylamino)propan-1-
effects on histones, HDACi favor hyperacetylation of non- oxy group of FcTAM or TAM with the 8-hydroxyamino-8-oxo-
histone targets, such as transcription factors and other pro- octanamido group of SAHA (Chart 2). In addition to FcTAM–
teins involved in cell cycle progression. They promote DNA SAHA and TAM–SAHA, hybrids derived from two SAHA-type
repair, cell differentiation, arrest of uncontrolled growth and molecules, N1-phenylsuberamide (PSA) and 8-oxo-8-(phenyl-
cancer cell death.26,27 Based on their chemical structure, amino)octanoic acid (OPOA), bearing primary amide (CONH2)
HDACi can be classified into six main groups.28 Among them, and carboxylic acid (COOH) functions, respectively, were also
hydroxamic acids have been the most widely studied agents, prepared (Charts 1 and 2).
and suberoylanilide hydroxamic acid (SAHA, Chart 1) is a
In total, six new hybrid compounds corresponding to three
model compound. SAHA was validated in 2007 as a new thera- ferrocenyl (FcTAM-) and phenyl (TAM-) couples were syn-
peutic option for the treatment of cutaneous T cell lymphoma. thesised. Some biological properties associated with each part
The mechanism of action involves binding of its hydroxamate of the hybrids were then tested: (i) the antiproliferative effect
group to the zinc cation (Zn2+) located in the HDAC cavity.29
on cancer cells, which is very well documented for the FcTAM
HDAC inhibition represents a potentially exploitable mech- family, (ii) the interaction with estrogen receptor ERα, which is
anism to design new antitumor agents with the goal of extend- associated with the TAM and OHTAM skeleton and (iii) the
ing their therapeutic spectra to other cancer types, such as ability to inhibit histone deacetylase activity to increase p21
breast cancer. For instance, tetrahydroisoquinoline-based mRNA expression, which is commonly observed using SAHA
hydroxamic acid derivatives,30 N-hydroxy-7-(2-naphthylthio)- alone, was investigated for all hybrids to determine any possible
heptanomide31 and SAHA,32,33 were observed to be active synergistic effects.
Chart 2 Design of the newly synthesised hybrid molecules.
15490 | Dalton Trans., 2013, 42, 15489–15501
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reaction of 2, 3 or 4 with 5 resulted in similar yields (50%) of
carboxylic acid FcTAM–OPOA. Chloride 3 was more suitable for
the synthesis of carboxylic acid FcTAM–OPOA due to its ease of
Results and discussion
Synthesis
Suberic anhydride 2 was obtained in good yields from the formation and rapid reaction with aniline 5. Under the same
thermal reaction of suberic acid with acetic anhydride.51 Sub- reaction conditions, the organic carboxylic acid TAM–OPOA was
eroyl chloride 3 was obtained using modified published obtained from 6 in 51% yield. In both cases, undesirable for-
methods,52,53 in which oxalyl chloride and dimethylformamide mation of bisanilides 7 and 8 (20–50%) from the reaction with
(DMF) were used as catalysts for the chlorination reaction. Car- 2, 3 and 4 were observed. To obtain the ferrocenyl hybrid com-
bonate 4 was produced in good yield following a modified lit- pound FcTAM–SAHA and the organic hybrid compound TAM–
erature method54 by the reaction of ethyl chloroformate SAHA, carboxylic acids FcTAM–OPOA and TAM–OPOA were first
(ClCO2Et) with suberic acid 1 in the presence of triethylamine activated by reaction with ClCO2Et and Et3N. Addition of freshly
(Et3N) (Scheme 1).
prepared hydroxylamine (NH2OH) to the activated carboxylic
4-(2-Ferrocenyl-1-phenylbut-1-en-1-yl)aniline 5 and 4-(1,2- acids FcTAM–OPOA and TAM–OPOA produced FcTAM–SAHA
diphenylbut-1-en-1-yl)aniline 6 were obtained as a mixture of and TAM–SAHA, respectively, each in 30% yield. In a similar
Z and E isomers (Z/E ratio = 85/15 for 5 and 95/5 for 6) reaction, primary amides FcTAM–PSA and TAM–PSA were syn-
by McMurry cross coupling reactions between 4-amino- thesised by addition of sodium amide (NaNH2) in excess to the
benzophenone and the corresponding ketone as previously activated carboxylic acids FcTAM–OPOA and TAM–OPOA.
described,55,56 N1-[4-(2-ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]-
All compounds were obtained as a mixture of Z and E
N8-hydroxysuberamide FcTAM–SAHA can be prepared from the isomers that could not be separated by flash chromatography.
reaction of the aniline 5 with one of the activated forms of 1, Identification of E and Z isomers was performed by 2D NMR
such as suberic anhydride 2, suberoyl chloride 3 or suberoyl experiments. Proportions of E and Z isomers are summarised
ethylcarbonate 4 (Scheme 2). The nucleophilic substitution in Table 1. A large excess of the Z isomer, similar to that of 5
and 6, was observed in all mixtures (Table 1). Stability of the
1
compounds was assessed by H NMR, which indicated that all
compounds were stable in DMSO-d6 at room temperature for
at least 10 days. During this period, no isomerization was
observed. The organic carboxylic acid OPOA and the organic
amide PSA (Chart 1), corresponding to the simplest SAHA ana-
logues, were also synthesised for comparison.
Biological evaluation
1. Antiproliferative effect. The antiproliferative effect of
10 µM of the compounds was first evaluated in three cancer
Scheme 1 Synthesis of suberic acid derivatives. Reagents and conditions:
cell lines, including MDA-MB-231 and MCF-7 breast cancer
cells, which are the archetypes of hormone-independent and
(i) acetic anhydride, 160 °C, 2 h, 99%. (ii) (COCl)2, DMF, CH2Cl2, 1 h, 92%.
(iii) ClCO2Et, Et3N, THF, 30 min, rt, 95%.
Scheme 2 Synthesis of hybrid compounds. Reagents and conditions: (i) THF, 48–55 °C, 1 h with 2; rt, 15 min with 3; rt, 1 h with 4, 50% and 51% yield for FcTAM–
OPOA and TAM–OPOA, respectively. (ii) THF, ClCO2Et, Et3N, 10 min, 0 °C and then NH2OH·HCl, KOH, MeOH, 15 min, 0 °C to rt, 30% for FcTAM–SAHA and TAM–
SAHA. (iii) THF, ClCO2Et, Et3N, 10 min, 0 °C then NaNH2, rt, 30 min, 45% and 40% yield for FcTAM–PSA and TAM–PSA, respectively.
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Table 1 Z/E isomer proportions (%) of organometallic and organic compounds
Table 3 IC50 values (µM) of selected compounds in MDA-MB-231 and MCF-7
breast cancer cell lines after 72 ha
Compound
Z
E
Compound
Z
E
Compound
MDA-MB-231
MCF-7
FcTAM–SAHA
FcTAM–PSA
FcTAM–OPOA
94
82
86
06
18
14
TAM–SAHA
TAM–PSA
TAM–OPOA
89
92
91
11
08
09
SAHA
FcTAM
FcTAM–SAHA
TAM–SAHA
FcTAM–PSA
TAM–PSA
3.6 0.5
2.6 0.7
0.7 0.1
8.6 0.8
0.5 0.1
25.9 4.9
1.0 0.2
4.4 0.9
2.0 0.1
>10
1.8 0.9
>10
Table 2 Cell growth inhibition (%) using 10 µM of each compound in three
cancer cells lines after 72 ha and lipophilicity (log Po/w
a Data are the means standard deviation (SD) of two independent
experiments performed in duplicate.
)
analogues (IC50 = 8.6 µM for TAM–SAHA and 25.9 µM for
TAM–PSA) was confirmed. These IC50 values of FcTAM–SAHA
and FcTAM–PSA have been mentioned by us for the sake of
comparison with other ferrocenyl complexes bearing succinic
and adipic chains.58 Interestingly, these results suggest a
synergistic effect between FcTAM and SAHA or PSA. IC50 values
of FcTAM–SAHA and FcTAM–PSA were four to seven times
lower than those of their parent molecules (IC50 = 0.5 µM or
0.7 µM for FcTAM–PSA and FcTAM–SAHA vs. 2.6 µM or 3.6 µM
for FcTAM and SAHA, respectively). Such a synergistic effect
was not observed with organic TAM hybrids.
Cell lines
Compound
R
X
MDA-MB-231 MCF-7 PC-3
log Po/w
SAHA
H
A
B
H
A
B
H
A
NHOH 90
NHOH 83
NHOH 58
1
1
3
3
1
4
3
6
1
87
75
43
41
76
36
32
59
32
1
2
4
5
4
4
3
1
7
77
47
1
3
1.7
6.8
FcTAM–SAHA
TAM–SAHA
PSA
FcTAM–PSA
TAM–PSA
OPOA
49 10 5.8
NH2
NH2
NH2
OH
OH
OH
21
92
52
7
57
41
23
37
<5
26
26
<5
9
3
2.1
6.6
5.6
3.7
7.5
6.5
2
3
FcTAM–OPOA
TAM–OPOA
B
Superiority of organometallic hybrid compounds over
organic derivatives was also observed in hormone-dependent
MCF-7 breast cancer cells. However, antiproliferative effects
were still lower compared to those in MDA-MB-231 cells. This
may be due to the estrogenic effects expressed by these hybrid
compounds on MCF-7 cells at low concentrations which
slightly counteract their antiproliferative activity. This point
will be further discussed.
Regarding the lipophilicity of the compounds (Table 2, log
Po/w), the ferrocene derivatives were, as expected, more lipophilic
than their corresponding hybrid organic analogues. Both series
were much more lipophilic than SAHA and its PSA and OPOA
analogues. In terms of functionality, the order of lipophilici-
ties is: carboxylic acid > hydroxamic acid > amide, except for
amide PSA, which was slightly more lipophilic than SAHA.
2. Analysis of the effects on estrogen signaling. Part of the
antiproliferative activity of the compounds in MCF-7 cells, the
archetype of estrogen receptor positive (ER+) breast cancer
cells, may be related to an anti-estrogenic effect. Thus, the
expression of the latter was studied via three different evalu-
ations: (i) the determination of their relative binding affinity
(RBA) for the alpha form of the estrogen receptor (ERα), (ii) the
regulation of estrogen signalling, and (iii) the effect of com-
pounds at low concentration on the growth of MCF-7 cells.
2.1 Relative binding affinity (RBA) for ERα. RBA of the six
a Data are the means standard deviation (SD) of two independent
experiments performed in triplicate.
hormone-dependent breast cancer cells, respectively, and one
hormone-independent prostate cancer cell line, PC-3 (Table 2).
All experiments were performed using mixtures of both
isomers, taking into account that Z isomers comprised more
than 80%.
At 10 µM, all hybrid compounds showed significant anti-
proliferative effect on breast cancer cells with a slightly greater
effect against MDA-MB-231 (ER−) cells than MCF-7 (ER+) cells.
SAHA was also very effective on MDA-MB-231 cells, while PSA
and OPOA, differing only by the functional group, were signifi-
cantly less active against these cell lines. The antiproliferative
effects of the ferrocenyl hybrids FcTAM–SAHA, FcTAM–PSA
and FcTAM–OPOA were always greater than those of their
corresponding organic phenyl hybrids (TAM–SAHA, TAM–PSA
and TAM–OPOA). Cytotoxicities of SAHA and PSA hybrids were
quite similar and significantly higher than those of OPOA
derivatives. In PC-3 cells, all compounds showed lesser
efficiency but following a similar trend. Interestingly, findings
of the current study are not consistent with our previous study
using a series of ferrocenophane compounds, which exhibited
similar antiproliferative effects in MDA-MB-231 and PC-3 hybrids for ERα was measured and summarised in Table 4.
cells.57
IC50 values for MDA-MB-231 and MCF-7 breast cancer
TAM–SAHA, TAM–PSA and TAM–OPOA organic derivatives
exhibited high affinity for ERα, ranging from 21.7 to 25%.
cells were determined for the most active compounds, and the These values are higher than would be expected for com-
results are summarised in Table 3. The superior antiproliferative pounds lacking the 4-hydroxy group. The latter is considered
essential for receptor–ligand interactions, such as in OHTAM.
effect of ferrocenyl hybrids (IC50 = 0.7 µM for FcTAM–SAHA
and 0.5 µM for FcTAM–PSA) over their corresponding organic RBA values for FcTAM–SAHA, FcTAM–PSA and FcTAM–OPOA
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Table 4 Relative binding affinity (RBA) of compounds for ERα
Compounds
RBA (%)
β-Estradiol (E2)
TAM–SAHA
TAM–PSA
TAM–OPOA
FcTAM–SAHA
FcTAM–PSA
FcTAM–OPOA
100
21.7
25.0
22.3
9.5
6.9
4.1
Fig. 2 Effect of 10 nM 17β-estradiol (E2), OHTAM and hybrid compounds on
the growth of MCF-7 cells after five days of culture. The mean of three separate
experiments is reported.
ferrocene derivatives were lower than the organic compounds;
however, they were reasonably well-recognized by the receptor
with RBA values of 9.5%, 6.9% and 4.1%, respectively. The
lower affinity of organometallic compounds compared to that
of their organic analogues was previously reported for this
class of organometallic species.11 The difference in affinity
between the carboxylic acid, hydroxamic acid and amide func-
tions became more pronounced in the ferrocene series, where
the affinity followed the order: hydroxamic acid > amide > carb-
oxylic acid.
2.2 Regulation of estrogen signaling. The effects of the two
hybrid hydroxamic acids, FcTAM–SAHA and TAM–SAHA, and
the two hybrid amides, FcTAM–PSA and TAM–PSA, on ERα
transcriptional activity were investigated. To do so, we used
stably transfected bioluminescent reporter HELN-ERα cells,
which are derived from HeLa cells stably expressing full-length
ERα and an ERE-driven luciferase plasmid.59 As shown in
Fig. 1, strong anti-estrogenic activity was observed for 10 nM
OHTAM[3], while a moderate effect was only observed at
higher concentrations of the four hybrid compounds. This
result confirms that the strong antiproliferative effects of ferro-
cene complexes are not related to their anti-estrogenic activity,
and ERα is not their principal target for its cytotoxicity.
respectively. A significant estrogenic effect was induced by all
hybrid compounds (Fig. 2). Ferrocenyl compounds FcTAM–
SAHA and FcTAM–PSA showed similar effects as an organic
compound TAM–PSA, while TAM–SAHA possessed slightly
lower estrogenic activity. Thus, as previously observed, the
presence of a TAM-like structure is not systematically associ-
ated with an anti-estrogenic effect, and RBA values do not
correlate with estrogenicity of molecules. Eventually, this
estrogenic effect could explain the increase of IC50 values from
MDA-MB-231 to MCF-7 (vide supra).
3. Effect on histone deacetylase activity. The ability of
different compounds to act as histone deacetylase inhibitors
(HDACi) was then measured experimentally using a fluorescent
assay based on the deacetylation reaction of a substrate
bearing an acetylated lysine side chain (BML-AK500 kit from
Enzo Life Sciences). 10 µM trichostatin A (TSA) was used as a
control. As expected, the results show that SAHA inhibited
HDAC activity (Fig. 3). In contrast, its organic analogue, amide
PSA, was inactive. The latter proves that hydroxamic function
is important for HDAC inhibition. Both hybrid hydroxamic
acids, FcTAM–SAHA and TAM–SAHA, showed significant enzy-
matic inhibition; however, this activity was lower than that of
SAHA. Similar to PSA, TAM–PSA and FcTAM–PSA amides,
which both lack hydroxamic function, were not active. This
2.3 Estrogenic and anti-estrogenic effects in MCF-7 cells. The
estrogenic and anti-estrogenic effects of compounds were
tested at a low concentration (10 nM) on hormone-dependent
MCF-7 breast cancer cells. 17β-Estradiol (E2) and OHTAM were
used as references for estrogenic and anti-estrogenic activities,
Fig. 3 Effect of hydroxamic acids (SAHA, FcTAM–SAHA, TAM–SAHA), amides
(PSA, FcTAM–PSA, TAM–PSA), TAM[3] and FcTAM at 0.1, 1, 10, and 100 µM on
HDAC activity. The background activity is denoted by (−). 10 µM trichostatin A
(TSA) was used as a control.
Fig. 1 Effect of hybrid hydroxamic acids (FcTAM–SAHA and TAM–SAHA) and
amides (FcTAM–PSA and TAM–PSA), as compared to OHTAM[3], on ERα
activity in HELN-ERα cells.
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was also the case for TAM[3] and FcTAM, which had no effect compared to FcTAM–SAHA, while organic amide TAM–PSA
on HDAC activity. Therefore, only hydroxamic compounds displayed a low effect similar to PSA.
inhibited HDAC activity. Interestingly, contrary to the remark-
Similar to SAHA, FcTAM–SAHA and FcTAM–PSA ferrocene
able difference in the antiproliferative effects of FcTAM–SAHA derivatives are also able to induce p21 gene expression. Since
and TAM–SAHA, both organometallic and organic compounds amide FcTAM–PSA had no HDACi activity, ferrocene derivatives
exhibited similar HDAC inhibition activity, indicating that and SAHA may not follow the same mechanism to increase
HDAC enzymes are not their principal target.
p21 gene expression. Consequently, the high activity of this
In this context, it is well documented that the HDACi type of organometallic complexes may be attributed to the
activity of SAHA is related to the interaction of hydroxamate specific properties of the ferrocenyl antenna. A possible expla-
with Zn2+ in the enzyme pocket of HDAC.17 In addition, it is nation for upregulation of p21 mRNA induced by ferrocene
also known that hydroxamic acids are used in the extraction of derivatives is that the conjugated metallocene may induce pro-
certain metals, such as iron and zinc.60–62 Furthermore, hydrox- duction of reactive oxygen species.69,70 One of the transcrip-
amic acids are considered to be siderophores because of their tional targets of redox status is the tumour suppressor gene
powerful chelating capacity and transport of Fe3+ in microbial p53,71 which is known to be a major inducer of p21 gene
metabolism,63 and they are easily revealed by qualitative colori- expression.72 As such, ferrocifen derivatives may be able to
metric tests with ferric salts.64 Therefore, we evaluated the che- differentially impact the p53 signaling pathway. Preliminary
lating ability of hydroxamic acids FcTAM–SAHA, TAM–SAHA results indicate that the ferrocene derivative FcTAM–PSA is
and SAHA by reaction with Fe3+. The colourless THF solutions able to upregulate the expression of other p53 target mRNAs
of SAHA and TAM–SAHA, as well as the orange solution of (namely PIG and PUMA73) in MCF-7 breast cancer cells.
FcTAM–SAHA, immediately turned dark brown upon the
addition of FeCl3. This colour change was not observed with
amides and carboxylic acids. As such, only the hydroxamic
Conclusion
acids FcTAM–SAHA and TAM–SAHA could form chelates with
Fe3+. This observation is consistent with a recent crystallo-
This study describes a new family of hybrid compounds,
graphic study that demonstrated interaction of SAHA with
metals, such as Fe3+ and Zn2+, to form tris- and bis-hydroxa-
mato complexes, respectively.65
which combine select structural motifs of tamoxifen (TAM),
ferrocifen (FcTAM) and SAHA. Results indicate that ferrocene
derivatives are far more active than organic analogues in
triple-negative MDA-MB-231 cells and hormone-dependent
MCF-7 breast cancer cell lines, confirming the importance of
this organometallic moiety in the anticancer activity of the
compounds. The antiproliferative activity of FcTAM can be
improved by replacing its 3-(dimethylamino)propan-1-
oxy group with an 8-hydroxyamino-8-oxooctanamido or an
8-amino-8-oxooctanamido group. FcTAM–SAHA and amide
FcTAM–PSA exhibited better antiproliferative effects than
SAHA against triple-negative MDA-MB-231 breast cancer cells,
but both were observed to be less active than SAHA in
hormone-dependent MCF-7 breast cancer cells. In contrast to
its strong antiproliferative effects, the hybrid amide FcTAM–
PSA was less active than SAHA, TAM–SAHA and FcTAM–SAHA
as an HDAC inhibitor. FcTAM–SAHA and TAM–SAHA exhibit
similar HDACi activity yet display a large difference in their
4. Effect on the expression of p21. Effects on the
expression of an endogenous HDACi target gene, p21waf1/cip1
(CDKN1A), in MCF-7 breast cancer cells were investigated for
the hydroxamic acids SAHA, TAM–SAHA and FcTAM–SAHA, as
well as the amides PSA, TAM–PSA and FcTAM–PSA using pre-
viously described methods.66,67 As shown in Fig. 4, the levels
of p21 mRNA increased upon treatment with SAHA, but only
moderately with PSA. The high level of p21 mRNA after SAHA
treatment may be related to its HDAC inhibition activity.68
Thus, low levels of p21 mRNA resulting from treatment with
TAM–SAHA compared to SAHA may be a consequence of its
low HDACi activity (Fig. 3). Interestingly, FcTAM–SAHA led to
increased expression of p21 mRNA compared to TAM–SAHA,
while both had similar HDACi activity. Surprisingly, organome-
tallic amide FcTAM–PSA also led to increased p21 mRNA
Fig. 4 Effect of hydroxamic acids and amides on the expression of p21 mRNA in MCF-7 cells.
15494 | Dalton Trans., 2013, 42, 15489–15501
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cytotoxicities, while FcTAM–PSA shows strong toxic effects temperature, all the volatile compounds were evaporated.
against cancer but does not produce HDAC inhibition. This 6.45 g (92%) of desired product 3 were obtained. 1H-NMR
enables us to infer that the antiproliferative activity of such ferro- (300 MHz, CDCl3, ppm): δ 1.38 (m, 4H, CH2), 1.71 (m, 4H,
cenyl compounds is likely due to specific properties of the CH2), 2.89 (t, J = 7.2 Hz, 4H, CH2). 13C-NMR (75 MHz, CDCl3,
organometallic antenna. Anti-estrogenic effects and HDAC ppm): δ 24.7 (CH2), 27.9 (CH2), 46.9 (CH2), 173.7 (CO). IR
inhibition may not play a primary role in the antiproliferative (CH2Cl2, υmax/cm−1): 2943, 2866 (Alkyl C–H stretch), 1797 (CvO
activity of such complexes. In other words, ERα and HDAC are stretch). MS (EI, m/z): 211 [M]+•, 175 [M − Cl]+, 139 [M − Cl −
not the principal targets whereby the ferrocene derivatives HCl]+.
exert their cytotoxic effects as described in this work. Interest-
Suberoyl ethylcarbonate (4). To a solution of 1 (31.4 mmol,
ingly, both organometallic compounds were able to induce 5.473 g) in 60 mL of THF, ClCO2Et (69.2 mmol, 6.6 mL) was
p21 gene expression. The redox properties of ferrocene com- added followed by Et3N (75.5 mmol, 10.5 mL). After 1 h of stir-
pounds and the production of reactive oxygen species, which ring at room temperature, the mixture was filtered, washed
we have already explored on other related series,69 could be the with water and extracted with AcOEt. The organic layer was
key to their activity via a mechanism that may involve the dried with MgSO4, filtered and the solvents were evaporated
modulation of p53 activity. The fact remains that the hybrid under vacuum. 9.5 g (95%) of a yellowish transparent oil were
constructs FcTAM–SAHA and FcTAM–PSA, despite the lack of a obtained. 1H-NMR (300 MHz, (CD3)2CO, ppm): δ 1.32 (t, J = 7.1
phenol function in the molecule as in FcOHTAM,69 are aston- Hz, 6H, CH3), 1.43 (m, 4H, CH2), 1.69 (m, 4H, CH2), 2.55 (t, J =
ishingly active against the subtype of breast cancer cells called 7.3 Hz, 4H, CH2), 4.30 (q, J = 7.1 Hz, 4H, CH2). 13C-NMR
triple negative and whose vital prognosis is bleak. The reasons (75 MHz, (CD3)2CO, ppm): δ 14.2 (CH3), 24.7 (CH2), 28.1 (CH2),
for this efficacy have to be investigated.
33.4 (CH2), 66.3 (CH2), 149.9 (CO), 168.9 (CO).
8-[4-(2-Ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]amino-8-oxo-
octanoic acid (FcTAM–OPOA). A solution of 5 (5.2 mmol,
2.1 g) in 50 mL of CH2Cl2 was added in 10 min into a stirred
solution of 2 (7.7 mmol, 1.2 g) in 20 mL of CH2Cl2. After it was
stirred at 50–55 °C for 1 h, the mixture was cooled at room
temperature, poured into a solution of KOH and acidified with
HCl. The product was extracted with AcOEt, the organic phase
was dried over MgSO4 and filtered. Solvents were evaporated
and the crude product was purified by column chromato-
graphy using AcOEt as an eluent. 1.45 g (50%) of a brownish
solid of FcTAM–OPOA was obtained. With 4, the reaction pro-
ceeded for 1 h as well, but at room temperature, and with 3
the reaction finished in 10 min at room temperature. Z/E ratio:
58/42, mp. 153–154 °C. Z isomer 1H-NMR (300 MHz, (CD3)2SO,
ppm): δ 0.97 (t, J = 7.0 Hz, 3H, CH3), 1.26 (m, 4H, 2CH2), 1.47
(m, 2H, CH2), 1.54 (m, 2H, CH2), 2.16 (m, 2H, CH2), 2.24 (m,
2H, CH2), 2.46 (q, J = 7.0 Hz, 2H, CH2), 3.81 (t, J = 1.9 Hz, 2H,
Cpsubst), 4.09 (t, J = 1.9 Hz, Cpsubst), 4.11 (s, 5H, Cp), 6.94 (d, J =
8.7 Hz, 2H, 2CHAr), 7.10–7.35 (m, 5H, 5CHAr), 7.46 (d, J = 8.7
Hz, 2H, 2CHAr), 9.82 (s, 1H, NH), 11.97 (s, 1H, OH). E isomer
1H-NMR (300 MHz, (CD3)2SO, ppm): δ 0.98 (t, J = 7.0 Hz, 3H,
CH3), 1.26 (m, 4H, 2CH2), 1.47 (m, 2H, CH2), 1.54 (m, 2H,
CH2), 2.16 (m, 2H, CH2), 2.24 (m, 2H, CH2), 2.46 (q, J = 7.0 Hz,
2H, CH2), 3.75 (t, J = 1.9 Hz, 2H, Cpsubst), 4.06 (t, J = 1.9 Hz,
2H, Cpsubst), 4.10 (s, 5H, Cp), 7.02 (d, J = 8.7 Hz, 2H, 2CHAr),
7.10–7.35 (m, 5H, 5CHAr), 7.53 (d, J = 8.7 Hz, 2H, 2CHAr), 9.85
(s, 1H, NH), 11.97 (s, 1H, OH). 13C-NMR (75 MHz, (CD3)2CO,
ppm): δ 15.9 (CH3), 25.5 (CH2), 26.1 (CH2), 28.4 (CH2), 29.5
(CH2), 29.6 (CH2), 34.1 (CH2), 37.7 (CH2), 68.9 (Cpsubst), 69.9
(Cp), 70.0 (Cpsubst), 87.2 (Cpipso), 119.7 (CAr), 127.0 (CAr), 129.1
(CAr), 130.4 (CAr), 130.8 (CAr), 138.0 (CvC), 138.4 (CvC), 138.8
(CAr), 140.5 (CAr), 145.7 (CAr), 171.9 (CvO), 174.7 (CvO). IR
Experimental section
Chemical procedures
General considerations. THF was distilled from Na/benzo-
phenone under an argon atmosphere and CH2Cl2 was distilled
from P2O5. All reagents and solvents were obtained from com-
mercial suppliers and used without further purification. Thin
layer chromatography was performed on silica gel 60 GF254
.
Column chromatography was performed on silica gel Merck 60
(40–63 µm). All of the products were characterized by conven-
tional techniques. IR spectra were recorded using a Jasco FT/
IR-4100 Fourier transform infrared spectrometer by the KBr
technique and all data are expressed in wave numbers (cm−1).
Melting points were obtained using a Kofler device and are
uncorrected. 1H and 13C NMR spectra were recorded using a
300 MHz Bruker spectrometer and chemical shifts (δ) are
expressed in ppm. The mass spectra were obtained using a
DSQII and ITQ 1100 Thermo Scientific spectrometer for both
the electronic impact (EI) and chemical ionization (CI)
methods and an API 3000 PE Sciex from Applied Biosystems
for the electrospray ionization (ESI) method. A purity of >99%
was confirmed by elemental analysis and analytical reverse
phase HPLC with a column Kromasil C18, 10 µm, L = 25 cm,
D = 4.6 mm using MeOH as an eluent, flow rate = 1 mL min−1
,
λ = 254 nm. Elemental analyses were performed by the Labo-
ratory of Microanalysis at ICSN of CNRS at Gif sur Yvette, France.
FcTAM, OPOA and SAHA were prepared according to literature
procedures.11,38
Procedures and analytical data
Suberoyl chloride (3). A suspension of 1 (28.7 mmol, 5.00 g) (KBr, υmax/cm−1): 3320 (N–H and O–H stretch), 3094, 3043 (aro-
in 50 mL of CH2Cl2 was stirred at room temperature. Oxalyl matic C–H stretch), 2931, 2873 (alkyl C–H stretch), 1705
chloride (63.2 mmol, 8.05 g) was added dropwise and then (OCvO stretch), 1647 (NCvO stretch), 1593 (aromatic CvC
0.5 mL of DMF was added. After 1 h of stirring at room stretch), 1523 (N–H bend), 1400 (C–N stretch). MS (EI, m/z):
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563 [M]+•, 498 [M − Cp]+, 480 [M − H2O − Cp]+. Anal. Calc. for 1.42–1.65 (m, 4H, 2CH2), 2.19 (t, J = 7.3 Hz, 4H, 2CH2), 2.37 (q,
C34H37FeNO3·H2O (%): C, 70.22; H, 6.76; N, 2.41. Found: C, J = 7.3 Hz, 4H, 2CH2), 6.72 (d, J = 8.5 Hz, 4H, 4CHAr), 7.05–7.30
70.12; H, 6.66; N, 2.15. RF: 0.81 (Me2CO). HPLC (RT), 3.44 min.
(m, 20H, 20CHAr), 7.38 (t, J = 7.4 Hz, 4H, 4CHAr), 9.69 (s, 1H,
8-[4-(1,2-Diphenylbut-1-en-1-yl)phenyl]amino-8-oxooctanoic NH). 13C-NMR (75 MHz, (CD3)2SO, ppm): δ 13.4 (CH3), 25.1
acid (FcTAM–OPOA). A solution of 6 (4.0 mmol, 1.19 g) and 2 (CH2), 28.5 (2CH2), 36.3 (CH2), 118.1 (CAr), 126.3 (CAr), 126.8
(10.0 mmol, 1.56 g) in 30 mL of distilled THF was stirred at (CAr), 128.0 (CAr), 128.3 (CAr), 129.0 (CAr), 129.4 (CAr), 130.5
48–50 °C for 1 h. The mixture was cooled at room temperature, (CAr), 138.0 (2CAr), 138.9 (CvC), 141.1 (CvC), 141.7 (CAr),
poured into a solution of KOH and acidified with HCl. The 142.6 (CAr), 171.0 (CvO). IR (KBr, υmax/cm−1): 3271 (N–H
product was extracted with AcOEt, and the organic phase was stretch), 3097, 3047 (aromatic C–H stretch), 2962, 2931, 2862
dried over MgSO4 and filtered. Solvents were evaporated and (alkyl C–H stretch), 1655 (NCvO stretch), 1596 (aromatic CvC
the crude product was purified by column chromatography stretch), 1527 (N–H bend), 1400 (C–N stretch). MS (CI, m/z):
using AcOEt as an eluent. 0.927 g (51%) of a white solid was 754 [MNH4]+, 737 [MH]+. HRMS for C52H53N2O2 [MH]+, calc.:
obtained as the desired product 8. With 4, the reaction pro- 737.4107; found: 737.4110. RF: 0.48 (hexane–AcOEt: 50/50).
ceeded for 1 h at room temperature. With 3, the reaction fin- HPLC (RT) 3.28 min (Macherey-Nagel C18, 5 micron, 4.6 ×
ished in 10 min at room temperature. Z/E ratio: 91/9, mp: 150 mm).
169–170 °C. Z isomer 1H-NMR (300 MHz, (CD3)2SO, ppm): δ
N1-[4-(2-Ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]-N8-hydroxy-
0.83 (t, J = 7.2 Hz, 3H, CH3), 1.23 (m, 4H, 2CH2), 1.47 (m, 4H, suberamide (FcTAM–SAHA). A solution of NH2OH·HCl
2CH2), 2.12–2.20 (m, 4H, 2CH2), 2.36 (q, J = 7.2 Hz, 2H, CH2), (4.2 mmol, 0.293 g) in 5 mL of MeOH was added to a stirred
6.71 (d, J = 8.7 Hz, 2H, 2CHAr), 7.08–7.29 (m, 10H, 10CHAr), solution of KOH (4.2 mmol, 0.236 g) in 5 mL of MeOH at 0 °C.
7.34 (t, J = 7.5 Hz, 2H, 2CHAr), 9.69 (s, 1H, NH), 11.93 (s, 1H, After it was stirred for 15 min, the precipitate was removed and
OH). 13C-NMR (75 MHz, (CD3)2CO, ppm): δ 13.9 (CH3), 25.5 the filtrate was placed in a flask. In another flask, to a solution
(CH2), 26.1 (CH2), 29.5 (CH2), 29.6 (CH2), 29.7 (CH2), 34.2 of FcTAM–OPOA (1.4 mmol, 0.80 g) in 15 mL of anhydrous
(CH2), 37.7 (CH2), 119.0 (CAr), 127.1 (CAr), 127.6 (CAr), 128.9 THF, cooled to 0 °C, ClCO2Et (2.1 mmol, 0.2 mL) and Et3N
(CAr), 129.2 (CAr), 130.2 (CAr), 130.6 (CAr), 131.8 (CAr), 138.5 (2.5 mmol, 0.35 mL) were added and the mixture was stirred
(CAr), 138.8 (CAr), 139.6 (CvC), 142.6 (CvC), 143.3 (CAr), 144.6 for 10 min and filtered. The filtrate was added to the freshly
(CAr), 171.8 (CvO), 174.7 (CvO). IR (KBr, υmax/cm−1): 3336 (N– prepared solution of NH2OH in MeOH. The resulting mixture
H and O–H stretch), 3060 (aromatic C–H stretch), 2931, 2862 was stirred at room temperature for 15 min. After that, water
(alkyl C–H stretch), 1705 (OCvO stretch), 1643 (NCvO was added, the mixture was slightly acidified with HCl and the
stretch), 1597 (aromatic CvC stretch), 1531 (N–H bend), 1408 product was extracted with AcOEt; the organic phase was dried
(C–N stretch). MS (CI, m/z): 473 [MNH4]+, 456 [MH]+. Anal. over MgSO4 and filtered. The solvents were evaporated and the
Calc. for C30H33NO3 (%): C, 79.09; H, 7.30; N, 3.07. Found: C, crude product was purified by column chromatography using
78.60; H, 7.32; N, 3.12. RF: 0.73 (Me2CO). HPLC (RT), 2.90 min.
AcOEt–petroleum ether as an eluent. 0.246 g (30%) of FcTAM–
N1,N8-Bis[4-(2-ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]suber- SAHA was obtained. Z/E isomer ratio: 94/6, mp: 142–144 °C. Z
amide (7). This compound is the byproduct of the reaction to isomer 1H-NMR (300 MHz, (CD3)2SO, ppm): δ 0.97 (t, J = 7.4
obtain FcTAM–OPOA. mp: 208–210 °C. 1H-NMR (300 MHz, Hz, 3H, CH3), 1.24 (m, 4H, 2CH2), 1.46 (m, 2H, CH2), 1.53 (m,
(CD3)2SO, ppm): δ 0.99 (t, J = 7.4 Hz, 6H, 2CH3), 1.27–1.41 (m, 2H, CH2), 1.91 (t, J = 7.4 Hz, 2H, CH2), 2.24 (t, J = 7.4 Hz, 2H,
4H, 2CH2), 1.50–1.68 (m, 4H, 2CH2), 2.20–2.37 (m, 4H, 2CH2), CH2), 2.46 (q, J = 7.4 Hz, 2H, CH2), 3.81 (t, J = 1.9 Hz, 2H,
2.40–2.60 (q, J = 7.4, 4H, 2CH2), 3.83 (t, J = 1.9 Hz, 4H, Cpsubst), Cpsubst), 4.09 (t, J = 1.9 Hz, 2H, Cpsubst), 4.10 (s, 5H, Cp), 6.94
4.10 (t, J = 1.9 Hz, 4H, Cpsubst), 4.12 (s, 10H, 2Cp), 6.96 (d, J = (d, J = 8.6 Hz, 2H, 2CHAr), 7.18–7.23 (m, 3H, 3CHAr), 7.32 (t, J =
8.4 Hz, 4H, 4CHAr), 7.06–7.25 (m, 6H, 6CHAr), 7.34 (t, J = 7.4 7.4 Hz, 2H, 2CHAr), 7.46 (d, J = 8.6 Hz, 2H, 2CHAr), 8.65 (s, 1H,
Hz, 4H, 4CHAr) 7.49 (d, J = 8.4 Hz, 4H, 4CHAr), 9.84 (s, 1H, NH), 9.82 (s, 1H, NH), 10.31 (s, 1H, OH). E isomer 1H-NMR
NH). 13C-NMR (75 MHz, (CD3)2SO, ppm): δ 15.4 (CH3), 25.1 (300 MHz, (CD3)2SO, ppm): δ 0.97 (t, J = 7.4 Hz, 3H, CH3), 1.24
(CH2), 27.1 (CH2), 28.5 (CH2), 36.3 (CH2), 68.0 (Cpsubst), 68.8 (m, 4H, 2CH2), 1.46 (m, 2H, CH2), 1.53 (m, 2H, CH2), 1.91 (t,
(Cpsubst), 69.1 (Cp), 85.5 (Cpipso), 119.0 (CAr), 126.2 (CAr), 128.4 J = 7.4 Hz, 2H, CH2), 2.24 (t, J = 7.4 Hz, 2H, CH2), 2.46 (q,
(CAr), 128.8 (CAr), 129.5 (CAr), 136.6 (CvC), 137.0 (CvC), 137.6 J = 7.4 Hz, 2H, CH2), 3.75 (t, J = 1.9 Hz, 2H, Cpsubst), 4.06 (t,
(CAr), 139.1 (CAr), 144.4 (CAr), 171.1 (CvO). IR (KBr, υmax
/
J = 1.9 Hz, 2H, Cpsubst), 4.10 (s, 5H, Cp), 6.94 (d, J = 8.6 Hz, 2H,
cm−1): 3398, 3290 (N–H stretch), 3093, 3040 (aromatic C–H 2CHAr), 7.18–7.23 (m, 3H, 3CHAr), 7.32 (t, J = 7.4 Hz, 2H,
stretch), 2931, 2866 (alkyl C–H stretch), 1662 (NCvO stretch), 2CHAr), 7.46 (d, J = 8.6 Hz, 2H, 2CHAr), 8.65 (s, 1H, NH), 9.82
1593 (aromatic CvC stretch), 1520 (N–H bend), 1400 (C–N (s, 1H, NH), 10.31 (s, 1H, OH). 13C-NMR (100 MHz, (CD3)2SO,
stretch). MS (ESI, m/z): 953 [MH]+. HRMS for C60H60Fe2N2O2 ppm): δ 15.7 (CH3), 25.9 (CH2), 26.0 (CH2), 28.3 (CH2), 29.3
[M]+, calc.: 952.3354; found: 952.3379. RF: 0.58 (hexane–AcOEt: (CH2), 29.5(CH2), 33.0 (CH2), 37.5 (CH2), 68.8 (Cpsubst), 69.8
50/50). HPLC (RT), 5.00 min.
(Cp), 69.9 (Cpsubst), 87.1 (Cpipso), 119.6 (CAr), 126.8 (CAr), 129.0
N1,N8-Bis[4-(1,2-diphenylbut-1-en-1-yl)phenyl]suberamide (CAr), 129.9 (CAr), 130.7 (CAr), 137.9 (CvC), 138.3 (CvC), 138.7
(8). This compound is the byproduct of the reaction to obtain (CAr), 140.4 (CAr), 145.5 (CAr), 170.6 (CvO), 171.7 (CvO). IR
TAM–OPOA. mp: 212–214 °C. 1H-NMR (300 MHz, (CD3)2SO, (KBr, υmax/cm−1): 3266 (N–H and O–H stretch), 3095, 3035 (aro-
ppm): δ 0.85 (t, J = 7.3 Hz, 6H, 2CH3), 1.18–1.36 (m, 4H, 2CH2), matic C–H stretch), 2927, 2862 (alkyl C–H stretch), 1655
15496 | Dalton Trans., 2013, 42, 15489–15501
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(NCvO stretch), 1593 (aromatic CvC stretch), 1519 (N–H CH3), 1.25 (m, 4H, 2CH2), 1.45 (m, 2H, CH2), 1.54 (m, 2H,
bend), 1400 (C–N stretch). MS (EI, m/z): 578 [M]+•, 562 [M − CH2), 2.00 (t, J = 7.4 Hz, 2H, CH2), 2.25 (t, J = 7.4 Hz, 2H, CH2),
O]+•, 544 [M
− − O −
H2O2]+•, 497 [M Cp]+, 407 2.46 (q, J = 7.4 Hz, 2H, CH2), 3.81 (t, J = 1.9 Hz, 2H, Cpsubst),
[FcEtCvCPhPhNH2]+•. (ESI, MeCN, m/z): 578 [M]+•. Anal. Calc. 4.09 (t, J = 1.9 Hz, 2H, Cpsubst), 4.11 (s, 5H, Cp), 6.67 (s, 1H,
for C34H38FeN2O3·13H2O (%): C, 69.86; H, 6.77; N, 4.79. Found: NH), 6.94 (d, J = 8.7 Hz, 2H, 2CHAr), 7.10–7.25 (m, 4H, 3CHAr
C, 70.02; H, 6.64; N, 4.62. RF: 0.74 (Me2CO). HPLC (RT), and NH), 7.32 (t, J = 7.3 Hz, 2H, 2CHAr), 7.46 (d, J = 8.7 Hz, 2H,
3.70 min.
2CHAr), 9.82 (s, 1H, NH). E isomer 1H-NMR (300 MHz,
N1-[4-(1,2-Diphenylbut-1-en-1-yl)phenyl]-N8-hydroxysuber- (CD3)2SO, ppm): δ 0.97 (t, J = 7.4 Hz, 3H, CH3), 1.25 (m, 4H,
amide (TAM–SAHA). A solution of NH2OH·HCl (4.2 mmol, 2CH2), 1.45 (m, 2H, CH2), 1.54 (m, 2H, CH2), 2.00 (t, J = 7.4 Hz,
0.293 g) in 5 mL of MeOH was added to a stirred solution of 2H, CH2), 2.25 (t, J = 7.4 Hz, 2H, CH2), 2.46 (q, J = 7.4 Hz, 2H,
KOH (4.2 mmol, 0.236 g) in 5 mL of MeOH at 0 °C. After it was CH2), 3.75 (t, J = 1.9 Hz, 2H, Cpsubst), 4.06 (t, J = 1.9 Hz, 2H,
stirred for 15 min, the precipitate was removed and the filtrate Cpsubst), 4.11 (s, 5H, Cp), 6.67 (s, 1H, NH), 7.02 (d, J = 8.7 Hz,
was placed in a flask. In another flask, to a solution of 8 2H, 2CHAr), 7.10–7.25 (m, 4H, 3CHAr and NH), 7.32 (t, J = 7.3
(1.0 mmol, 0.455 g) in 10 mL of anhydrous THF, cooled to Hz, 2H, 2CHAr), 7.53 (d, J = 8.7 Hz, 2H, 2CHAr), 9.85 (s, 1H,
0 °C, ClCO2Et (1.4 mmol, 0.13 mL) and Et3N (1.7 mmol, NH). 13C-NMR (75 MHz, (CD3)2SO, ppm): δ 15.4 (CH3), 24.9
0.24 mL) were added and the mixture was stirred for 10 min (CH2), 25.0 (CH2), 27.0 (CH2), 28.4 (CH2), 28.5 (CH2), 35.1
and filtered. The filtrate was added to the freshly prepared (CH2), 36.3 (CH2), 68.0 (Cpsubst), 68.7 (Cpsubst), 69.0 (Cp), 85.4
solution of NH2OH in MeOH. The resulting mixture was (Cpipso), 118.9 (CAr), 126.1 (CAr), 128.3 (CAr), 128.7 (CAr), 129.4
stirred at room temperature for 15 min. After that, water was (CAr), 136.5 (CvC), 136.9 (CvC), 137.5 (CAr), 139.0 (CAr), 144.3
added, the mixture was slightly acidified with HCl and the (CAr), 171.1 (CvO), 174.2 (CvO). IR (KBr, υmax/cm−1): 3410 (N–
product was extracted with AcOEt; the organic phase was dried H stretch), 3097 (aromatic C–H stretch), 2931, 2862 (alkyl C–H
over MgSO4 and filtered. The solvents were evaporated and the stretch), 1662 (NCvO stretch), 1601 (aromatic CvC stretch),
crude product was purified by column chromatography using 1523 (N–H bend), 1408 (C–N stretch). MS (EI, m/z): 562
AcOEt–petroleum ether as an eluent. 0.141 g (30%) of TAM– [M]+•, 497 [M
− − H2O −
Cp]+, 479 [M Cp]+, 407
SAHA was isolated. Z/E isomer ratio: 89/11, mp. 135–137 °C. Z [FcEtCvCPhPhNH2]+•, 342 [FeCpEtCvCPhPhNH2]. Anal. Calc.
isomer 1H-NMR (300 MHz, (CD3)2SO, ppm): δ 0.83 (t, J = 7.3 for C34H38FeN2O2·H2O (%): C, 70.34; H, 6.94; N, 4.83. Found:
Hz, 3H, CH3), 1.20 (m, 4H, 2CH2), 1.46 (m, 4H, 2CH2), 1.89 (t, C, 70.30; H, 7.00, N, 4.58. RF: 0.70 (Me2CO). HPLC (RT)
J = 7.4 Hz, 2H, CH2), 2.18 (t, J = 7.4 Hz, 2H, CH2), 2.36 (q, J = 3.68 min.
7.3 Hz, 2H, CH2), 6.71 (d, J = 8.5 Hz, 2H, 2CHAr), 7.09–7.29 (m,
N1-[4-(1,2-Diphenylbut-1-en-1-yl)phenyl]suberamide (TAM–
10H, 10CHAr), 7.36 (t, J = 7.2 Hz, 2H, 2CHAr), 8.64 (s, 1H, NH), PSA). To a solution of TAM–OPOA (2.0 mmol, 0.910 g) in
9.69 (s, 1H, NH), 10.30 (s, 1H, OH). 13C-NMR (75 MHz, 15 mL of anhydrous THF, cooled to 0 °C, ClCO2Et (3.0 mmol,
(CD3)2CO, ppm): δ 13.8 (CH3), 26.0 (CH2), 26.1 (CH2), 29.4 0.29 mL) and Et3N (3.4 mmol, 0.47 mL) were added and the
(CH2), 29.5 (CH2), 29.6 (CH2), 33.1 (CH2), 37.6 (CH2), 118.0 mixture was stirred for 10 min. The solid was filtered off and
(CAr), 127.0 (CAr), 127.5 (CAr), 128.8 (CAr), 129.1 (CAr), 130.1 an excess of NaNH2 was added to the filtrate. After 30 min of
(CAr), 130.5 (CAr), 131.7 (CAr), 138.3 (CAr), 138.7 (CAr), 139.5 stirring, 20 mL of water was slowly added. The product was
(CvC), 142.5 (CvC), 143.2 (CAr), 144.5 (CAr), 170.9 (CvO), extracted with AcOEt, and the organic layer was dried over
171.8 (CvO). IR (KBr, υmax/cm−1): 3240 (N–H and O–H MgSO4, filtered and evaporated. The crude was purified by
stretch), 3051 (aromatic C–H stretch), 2931, 2862 (alkyl C–H column chromatography using mixtures of AcOEt and pet-
stretch), 1655 (NCvO stretch), 1597 (aromatic CvC stretch), roleum ether. 0.363 g (40%) of TAM–PSA was isolated. Z/E
1523 (N–H bend), 1400 (C–N stretch). MS (CI, m/z): 488 isomer ratio, 92/8, mp: 178–180 °C. Z isomer 1H-NMR
[MNH4]+, 471 [MH]+. Anal. Calc. for C30H34N2O3·12H2O (%): C, (300 MHz, (CD3)2SO, ppm): δ 0.83 (t, J = 7.4 Hz, 3H, CH3), 1.21
75.13; H, 7.36; N, 5.84. Found: C, 75.06; H, 7.35; N, 4.70. RF: (m, 4H, 2CH2), 1.40–1.51 (m, 4H, 2CH2), 1.98 (t, J = 7.4 Hz, 2H,
0.70 (Me2CO). HPLC (RT), 3.34 min.
CH2), 2.18 (t, J = 7.5 Hz, 2H, CH2), 2.36 (q, J = 7.4 Hz, 2H,
N1-[4-(2-Ferrocenyl-1-phenylbut-1-en-1-yl)phenyl]suberamide CH2), 6.66 (s, 1H, NH), 6.71 (d, J = 8.7 Hz, 2H, 2CHAr),
(FcTAM–PSA). To a solution of Fc-TAM–OPOA (1.4 mmol, 7.09–7.29 (m, 11H, 10CHAr and NH), 7.36 (t, J = 7.3 Hz, 2H,
0.800 g) in 15 mL of anhydrous THF, cooled to 0 °C, ClCO2Et 2CHAr), 9.69 (s, 1H, NH). 13C-NMR (75 MHz, (CD3)2SO, ppm): δ
(2.1 mmol, 0.2 mL) and Et3N (2.5 mmol, 0.35 mL) were added 13.3 (CH3), 24.9 (CH2), 25.0 (CH2), 28.4 (CH2), 28.5 (CH2), 28.6
and the mixture was stirred for 10 min. The solid was filtered (CH2), 35.0 (CH2), 36.3 (CH2), 118.0 (CAr), 126.2 (CAr), 126.7
off and an excess of NaNH2 was added to the filtrate. After (CAr), 127.9 (CAr), 128.2 (CAr), 128.9 (CAr), 129.3 (CAr), 130.4
30 min of stirring, 20 mL of water was slowly added. The (CAr), 137.0 (CAr), 137.2 (CAr), 138.0 (CvC), 141.0 (CvC), 141.7
product was extracted with AcOEt; the organic layer was dried (CAr), 143.0 (CAr), 171.0 (CvO), 174.2 (CvO). IR (KBr, υmax
/
over MgSO4, filtered and evaporated. The crude product was cm−1): 3464, 3278, 3182 (N–H stretch), 3101, 3043 (aromatic
purified by column chromatography using mixtures of AcOEt C–H stretch), 2927, 2858 (alkyl C–H stretch), 1662 (NCvO
and petroleum ether. 0.350 g (45%) of FcTAM–PSA was stretch), 1601 (aromatic CvC stretch), 1523 (N–H bend), 1400
obtained. Z/E isomer ratio: 82/18, mp: 98–100 °C. Z isomer (C–N stretch). MS (CI, m/z): 472 [MNH4]+, 455 [MH]+. Anal.
1H-NMR (300 MHz, (CD3)2SO, ppm): δ 0.97 (t, J = 7.4 Hz, 3H, Calc. for C30H34N2O2·1H2O (%): C, 77.72; H, 7.61; N, 6.04.
2
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Found: C, 77.65; H, 7.60; N, 5.78. RF: 0.68 (Me2CO). HPLC (RT), (CH, CAr), 128.7 (CH, CAr), 129.0 (CH, CAr), 130.1 (CH, CAr),
3.28 min.
130.5 (CH, CAr), 131.2 (CH, CAr), 131.4 (CH, CAr), 132.5 (CH,
solution of OPOA CAr). 136.1, 136.5, 139.6, 142.0, 142.6, 143.2, 144.4, 144.7 and
N1-Phenyloctanediamide (PSA). To
a
(3.0 mmol, 0.747 g, prepared as FcTAM–OPOA and TAM– 159.0 (Cq, C6H4 + 2C6H5 + CvC). (CI, m/z): 386.24 [M + H]+.
OPOA) in 5 mL of anhydrous THF, cooled to 0 °C, ClCO2Et Anal. Calc. For C27H31NO: C, 84.11; H, 8.10; N, 3.63. Found: C,
(4.5 mmol, 0.44 mL) and Et3N (5.1 mmol, 0.71 mL) were added 83.68; H, 8.12; N, 3.66.
and the mixture was stirred for 10 min. The solid was filtered
3-[4-(2-Ferrocenyl-1-phenylbut-1-en-1-yl)phenoxy]-N,N-
off and an excess of NaNH2 was added to the filtrate. After dimethylpropan-1-amine (FcTAM). In a Schlenk tube, (E + Z)-
30 min of stirring, 20 mL of water was slowly added. The 4-(2-ferrocenyl-1-phenylbut-1-en-1-yl)phenol (1 mmol, 408 mg)
product was extracted with AcOEt, and the organic layer was was dissolved in anhydrous DMF (8 mL). NaH (60% in oil,
dried over MgSO4, filtered and evaporated. The product was 1.1 mmol, 44 mg) was added as a powder into the solution
purified by column chromatography using mixtures of AcOEt within 10 min. In another Schlenk tube, Et3N (3 mmol,
and petroleum ether as an eluent. 0.372 g (50%) of the product 303 mg) was added to a suspension of Cl(CH2)3NMe2·HCl
PSA was isolated. mp: 160–161 °C. 1H-NMR (300 MHz, (3 mmol, 474 mg) in 15 mL of THF. After stirring for 30 min,
(CD3)2SO, ppm): δ 1.26 (m, 4H, 2CH2), 1.46 (quint, J = 7.0 Hz, the solution was filtered and concentrated to 1 mL. 2 mL of
2H, CH2), 1.56 (quint, J = 7.1 Hz, 2H, CH2), 2.00 (t, J = 7.4 Hz, DMF was added. This chloroamine solution was added to the
2H, CH2), 2.27 (t, J = 7.3 Hz, 2H, CH2), 6.67 (s, 1H, NH), 6.99 (t, first solution and the mixture was heated at 110 °C with an oil
J = 7.8 Hz, 1H, CHAr), 7.21 (s, 1H, NH), 7.26 (t, J = 7.8 Hz, 2H, bath. After 1 h 30 min of heating, the mixture was allowed to
2CHAr), 7.56 (d, J = 7.8 Hz, 2H, 2CHAr), 9.83 (s, 1H, CONH). cool to room temperature, and then 100 mL of ethyl acetate
13C-NMR (300 MHz, (CD3)2SO, ppm): δ 25.0 (2CH2), 28.5 was added. The solution was washed with 2 × 40 mL of water.
(2CH2), 35.0 (CH2), 36.3 (CH2), 119.0 (CAr), 122.8 (CAr), 128.6 After removal of solvent, the crude product was first purified
(CAr), 139.3 (Cipso), 171.2 (CvO), 174.3 (CvO). IR (KBr, υmax
/
by a silica gel flash column. Compounds were first eluted with
cm−1): 3406, 3309, 3201 (N–H stretch), 3055 (aromatic C–H acetone and then with acetone–Et3N (10 : 1). 350 mg of a pure
stretch), 2939, 2856 (alkyl C–H stretch), 1658 (NCvO), 1604 compound was isolated as an orange oil (70%, Z/E = 50 : 50).
(aromatic CvC stretch), 1527 (N–H bend), 1419 (C–N stretch). 1H NMR (300 MHz, (CD3)2CO, ppm): δ 1.01 and 1.03 (t and t,
MS (EI, m/z): 248 [M]+•, 232 [M − NH2]+, 190 [M − CONH2]+, 93 J = 7.4 Hz, 3H, CH3), 1.89 (m, 2 H, CH2CH2CH2), 2.15 and 2.16
[PhNH2]+•. HRMS for C14H21N2O2 [MH]+, calc.: 249.1603; (s and s, 6 H, N(CH3)2), 2.38 (m, 2 H, CH2CH3), 2.60 (m, 2 H,
found: 249.1596. RF: 0.73 (Me2CO). HPLC (RT), 2.68 min.
CH2CH2CH2N), 4.00 (m, 2 H, OCH2CH2CH2), 3.85 and 3.91
3-[4-(1,2-Diphenylbut-1-en-1-yl)phenoxy]-N,N-dimethylpro- (t and t, J = 1.9 Hz, 2H, Cpsubst), 4.04 and 4.07 (t and t, J = 1.9 Hz,
pan-1-amine (TAM[3]). In a Schlenk tube, 4-(1,2-diphenylbut- 2H, Cpsubst), 4.12 and 4.12 (s and s, 5H, Cp), 6.77–7.34 (m, 9
1-en-1-yl)phenol (1 mmol, 300 mg) was dissolved in anhydrous H, C6H4 + C6H5). 13C NMR (75 MHz, (CD3)2CO, ppm): δ 15.8
DMF (8 mL). NaH (60% in oil, 1.1 mmol, 44 mg) was added as (CH3), 28.3 (CH2), 45.7 (N(CH3)2), 56.9 (CH2CH2CH2N), 66.7
a powder into the solution within 10 min. In another Schlenk (OCH2CH2CH2), 68.8 (Cpsubst), 69.9 (Cp + Cpsubst), 87.3 (Cip,
tube, Et3N (3 mmol, 303 mg) was added to a suspension of Cl- C5H4), 115.0 (CH, CAr), 115.1 (CH, CAr), 126.9 (CH, CAr), 129.0
(CH2)3NMe2·HCl (3 mmol, 474 mg) in 15 mL of THF. After stir- (CH, CAr), 129.1 (CH, CAr), 130.0 (CH, CAr), 131.1 (CH, CAr),
ring for 30 min, the solution was filtered and concentrated to 131.6 (CH, CAr), 137.7, 137.9, 138.0, 138.5, 145.9, 146.1, and
1 mL. After this, 2 mL of DMF was added. This chloroamine 158.6 (Cq, C6H4 + C6H5 + CvC). MS (CI, m/z): 494.20 [M + H]+.
solution was added to the first solution and the mixture was Anal. Calc. for C31H35FeNO: C, 75.45; H, 7.15; N, 2.84. Found:
heated at 110 °C with an oil bath. After 1.5 h of heating, the C, 75.22; H, 7.20; N, 2.62.
mixture was allowed to cool to room temperature, and then
Lipophilicity. Measurements of the octanol/water partition
100 mL of AcOEt was added. The solution was washed with 2 × coefficient (log Po/w) were made by the HPLC technique accord-
40 mL of water. After solvent removal, the crude product was ing to a method described previously.74 Measurement of the
first purified by a silica gel flash column. Compounds were chromatographic capacity factors (k′) for each molecule was
first eluted with acetone and then with acetone–Et3N (10 : 1). done at various concentrations in the range of 95–75% metha-
346 mg of a pure compound was isolated as a colourless oil nol containing 0.25% (v/v) 1-octanol and an aqueous phase
(90%, Z/E or E : Z = 69 : 31). 1H NMR (300 MHz, (CD3)2CO, consisting of 0.15% (v/v) n-decylamine in the buffering
ppm): δ 0.89 (minor) and 0.91 (major) (t and t, 3 H, J = 7.4 Hz, agent MOPS (3-morpholinopropane-1-sulfonic acid, prepared in
CH2CH3), 1.80 (minor) and 1.90 (major) (quin and quin, 2 H, 1-octanol saturated water) adjusted to pH 7.4. These capacity
CH2CH2CH2), 2.12 (minor) and 2.17 (major) (s and s, 6 H, factors (k′) are extrapolated to 100% of the aqueous component
N(CH3)2), 2.29–2.53 (m, 4 H, CH2CH3 + CH2CH2CH2N), 3.88 given the value of k′w. The log Po/w is obtained by the formula
(minor) and 4.05 (t and t, 2 H, J = 6.4 Hz, OCH2CH2CH2), log Po/w = 0.13418 + 0.98452 log k′.
6.55–7.37 (m, 14 H, C6H4 + 2C6H5). 13C NMR (75 MHz,
(CD3)2CO, ppm): δ 13.8 (CH3), 28.3 (CH2), 29.6 (CH2CH2CH2),
Biological methods
45.7 (N(CH3)2), 56.8 (CH2CH2CH2N), 66.5 and 6.7
Relative binding affinity (RBA). Stock solutions (1 mM) of
(OCH2CH2CH2), 114.2 (CH, CAr), 115.0 (CH, CAr), 126.5 (CH, the compounds were prepared in DMSO and kept at 4 °C in
CAr), 126.9 (CH, CAr), 127.4 (CH, CAr), 128.1 (CH, CAr), 128.6 the dark. It has already been proven that the compounds
15498 | Dalton Trans., 2013, 42, 15489–15501
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FcTAM–OPOA, TAM–OPOA,
Paper
FcTAM–SAHA,
TAM–SAHA, added, and the plate was incubated for 1 h at 37 °C. Then the
FcTAM–PSA and TAM–PSA are stable under these conditions. absorbance of each well was measured at 655 nm using a
Serial dilutions in DMSO were prepared just prior to use. RBA Biorad spectrophotometer (microplate reader). The results are
values were measured on ERα from lamb uterine cytosol pur- expressed as the percentage of proteins versus the control.
chased from Pan Vera (Madison, WI, USA). Aliquots (200 µL) of Experiments were performed in triplicate.
ERα in glass tubes were incubated for 3 h at 0 °C with [6,7-3H]-
Luciferase assays. HELN-ERα cells58 were seeded at
a
estradiol (2 nM, specific activity 1.62 TBq mmol−1, NEN Life density of 4 × 104 cells per well on 96-well white opaque tissue
Science, Boston, MA) in the presence of nine concentrations of culture plates (Greiner CellStar, D. Dutscher, Brumath, France)
compounds. At the end of the incubation period, the free and and maintained in 5% DCC–FCS. Compounds were added
bound fractions of the tracer were separated by protamine 20 h later and the cells were incubated with the compounds
sulfate precipitation. The percentage reduction in binding of for 16 h. Experiments were performed in quadruplicate. At the
[3H]-estradiol (Y) was calculated using the logit transformation end of the incubation, the medium was removed and replaced
of Y (logit Y: ln[Y/1 − Y] versus the log of the mass of the com- by 0.3 mM luciferin containing 5% DCC–FCS. The 96-well
peting steroid). The concentration of unlabeled steroid plate was then introduced into a microplate luminometer
required to displace 50% of the bound [3H]-estradiol was calcu- (Microbeta, Wallac) and intact living cell luminescence was
lated for each compound tested and the results are expressed measured for 2 s.
as RBA values. The RBA value of 17β-estradiol is by definition
mRNA quantification. MCF7 human breast cells were grown
equal to 100%.
in steroid-free DMEM/F12 medium supplemented with 3%
IC50 determination. The breast adenocarcinoma cell lines DCC–FCS, 100 U mL−1 penicillin, 100 mg mL−1 streptomycin
MDA-MB-231 and MCF7 were obtained respectively from and 100 mg mL−1 sodium pyruvate during 5 days. Cells were
ATCC and Dr Matthias Kassack (Bonn, Germany). Cells were then stimulated in media containing 3% DCC–FCS with 10 nM
grown in RPMI medium supplemented with 10% fetal calf of estradiol for 1 day. Total RNA was extracted from the cells
serum, in the presence of penicillin, streptomycin and fungi- using a High Pure RNA Isolation kit (Roche) according to the
zone in a 75 cm2 flask under 5% CO2. Cells were plated in 96- manufacturer’s instructions. Total RNA (1 µg) was subjected to
well tissue culture plates in 200 µL medium and treated 24 h reverse-transcription using Superscript II reverse transcriptase
later with 2 µL stock solution of compounds dissolved in (Invitrogen). Quantitative RT-PCR (qPCR) was then performed
DMSO using a Biomek 3000 (Beckman-Coulter). Controls on specific primers for the indicated genes, using LightCycler
received the same volume of DMSO (1% final volume). After 480 SYBR Green I Master (Roche Diagnostics). qPCR was
72 h of exposure, MTS reagent (Promega) was added and incu- carried out in a final volume of 10 μL using 0.25 μL of each
bated for 3 h at 37 °C: the absorbance was monitored at primer (25 μM), 5 μL of the supplied enzyme mix, 3 μL of H2O,
490 nm and results are expressed as the inhibition of cell pro- and 2 μL of the template diluted at 1 : 10. After a 10 min pre-
liferation calculated as the ratio [(1 − (OD490 treated/OD490 incubation at 95 °C, runs corresponded to 35 cycles of 15 s
control) × 100] in triplicate experiments. For IC50 determi- each at 95 °C, 5 s at 60 °C and 15 s at 72 °C. Melting curves of
nation [50% inhibition of cell proliferation], cells were incu- the PCR products were analyzed using the LightCycler software
bated for 72
h following the same protocol with the system to exclude the amplification of unspecific products.
compound concentration range 5 nM to 100 µM in separate Results were normalized to the RS9 housekeeping gene tran-
duplicate experiments.
scripts. Primers are available upon request.
Estrogenic/antiestrogenic effect of compounds on MCF-7
HDACi assay. The HDACi activity was measured using the
cells. Cells were maintained in monolayer culture in DMEM Fluor de Lys® Fluorescent Assay System (Enzo Life Sciences)
with phenol red/Glutamax I, supplemented with 9% decomple- according to the manufacturer’s instructions. Briefly, the Fluor
mented fetal calf serum and 0.9% kanamycin, at 37 °C in a 5% de Lys® Substrate, which comprises an acetylated lysine side
CO2 air humidified incubator. Cells were plated on 24-well chain, was incubated with a nuclear extract prepared from
sterile plates at a density of 3 × 104 cells in 1 mL of DMEM HeLa cells and containing HDAC. Different concentrations of
without phenol red, supplemented with 9% fetal calf serum compounds were added and the reaction mixture was left for
desteroided on dextran charcoal, 0.9% Glutamax I and 0.9% 30 minutes at room temperature. The reaction was enhanced
kanamycin, and were incubated for 24 h. The following day by the addition of the Fluor de Lys® Developer. Deacetylation
(D0), 1 mL of the same medium containing the compounds to of the substrate sensitizes the substrate which produces a
be tested diluted in DMSO was added to the plates (final fluorophore. Arbitrary fluorescence units (AFU) of each sample
volumes of DMSO: 0.1%; 4 wells for each condition). After were quantified using a microtiter-plate reading fluorimeter at
three days (D3), the incubation medium was removed and 460 nm.
2 mL of fresh medium containing the compounds was added.
On 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
Acknowledgements
methanol with methylene blue (2.5 mg mL−1), and then We thank the Agence Nationale de la Recherche for financial
washed thoroughly with water. 2 mL of HCl (0.1 M) was then support (ANR 2010 BLAN 7061 blanc “Mecaferrol”) and the
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Institut National du Cancer (INCa TRANSLA 2011-054). We
also thank the National Council of Science and Technology of
Mexico (CONACyT) for the PhD scholarship for JJCM.
H. Müller-Bunz and G. Jaouen, Dalton Trans., 2009, 4318–
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