Development of a Novel Ferrocenyl Histone Deacetylase Inhibitor for Triple-Negative Breast Cancer Therapy

Article abstract of DOI:10.1021/acs.organomet.8b00354

Owing to the lack of target-directed therapies, triple-negative breast cancer (TNBC) is difficult to effectively treat. In this article, a novel organometallic histone deacetylase (HDAC) inhibitor, Fc-SelSA, based on the selenocyanide (SelSA) zinc-binding motif, was synthesized using a ferrocenyl group as the cap to confer activity against TNBC. The synthesized Fc-SelSA was evaluated for bioactivity in vitro and in vivo. An enzymatic assay showed that Fc-SelSA was a potent HDAC inhibitor with a half-maximum inhibitory concentration (IC₅₀) of 14.8 nM. Molecular docking studies of Fc-SelSA with HDAC suggested that the ferrocenyl unit overlaps with the phenyl group of suberoylanilide hydroxamic acid (SAHA) and the amido group of Fc-SelSA can form hydrogen bonds with the D98 and G151 residues of HDAC, but SAHA and SelSA do not show similar interactions. Moreover, Fc-SelSA reactivated the estrogen receptor alpha (ERα) expression, sensitized TNBC cells to the antagonist tamoxifen, and exerted more potent antitumor effects against TNBC MDA-MB-231 tumor xenografts in comparison to SelSA with no obvious side effects. Our results indicate that Fc-SelSA is a potent oral anticancer candidate for HDAC-targeted TNBC therapy and deserves further investigation for clinical application.

Full text of DOI:10.1021/acs.organomet.8b00354

Article
Cite This: Organometallics XXXX, XXX, XXX−XXX
Development of a Novel Ferrocenyl Histone Deacetylase Inhibitor
for Triple-Negative Breast Cancer Therapy
Chu Tang,†,‡,# Yang Du,‡,§,∥,# Qian Liang,‡ Zhen Cheng,*,⊥ and Jie Tian*,†,‡,§,∥
†Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology,
Xidian University, Xi’an, Shaanxi 710126, People’s Republic of China
‡CAS Key Laboratory of Molecular Imaging, The State Key Laboratory of Management and Control for Complex Systems, Institute
of Automation, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
§Beijing Key Laboratory of Molecular Imaging, Beijing 100190, People’s Republic of China
∥University of Chinese Academy of Sciences, Beijing 100080, People’s Republic of China
⊥Molecular Imaging Program at Stanford (MIPS), Department of Radiology, and Bio-X Program, Canary Center at Stanford for
Cancer Early Detection, Stanford University, Stanford, California 94305-5344, United States
S
* Supporting Information
ABSTRACT: Owing to the lack of target-directed therapies,
triple-negative breast cancer (TNBC) is difficult to effectively
treat. In this article, a novel organometallic histone deacetylase
(HDAC) inhibitor, Fc-SelSA, based on the selenocyanide
(SelSA) zinc-binding motif, was synthesized using a ferrocenyl
group as the cap to confer activity against TNBC. The
synthesized Fc-SelSA was evaluated for bioactivity in vitro and
in vivo. An enzymatic assay showed that Fc-SelSA was a
potent HDAC inhibitor with a half-maximum inhibitory
concentration (IC50) of 14.8 nM. Molecular docking studies
of Fc-SelSA with HDAC suggested that the ferrocenyl unit overlaps with the phenyl group of suberoylanilide hydroxamic acid
(SAHA) and the amido group of Fc-SelSA can form hydrogen bonds with the D98 and G151 residues of HDAC, but SAHA
and SelSA do not show similar interactions. Moreover, Fc-SelSA reactivated the estrogen receptor alpha (ERα) expression,
sensitized TNBC cells to the antagonist tamoxifen, and exerted more potent antitumor effects against TNBC MDA-MB-231
tumor xenografts in comparison to SelSA with no obvious side effects. Our results indicate that Fc-SelSA is a potent oral
anticancer candidate for HDAC-targeted TNBC therapy and deserves further investigation for clinical application.
been reported to enhance the therapeutic effects of tamoxifen
through the activation of ERα gene (ESR1) expression.13,14
Therefore, reactivation of the ERα expression by increasing
histone acetylation can be used for the treatment of TNBC.
Currently, a large number of structurally diverse HDACis
have been reported, and many of them are hydroxamic acid
derivatives,15 including the three U.S. Food and Drug
Administration (FDA) approved HDACis SAHA,16 belinostat
(PXD101; Figure 1),17 and panobinostat (LBH-589; Figure
1).18 Although the hydroxamic acid group can interact with the
Zn2+ ion in the active site of HDAC and is frequently
employed as the zinc-binding group (ZBG), one of the major
barriers for the development of hydroxamic acid derivatives as
clinical drugs is their toxicity. They are prone to hydrolysis,
resulting in the formation of potentially mutagenic hydroxyl-
amine;19 in addition, metabolic and pharmacokinetic problems
also seriously hinder their clinical application.20 Therefore, a
number of new and nonhydroxamic acid derived inhibitors
INTRODUCTION
■
In contrast to other subtypes of breast cancer,1,2 triple-negative
breast cancer (TNBC) usually occurs in young women and is
very prone to exacerbation.3 Because of the lack of expression
of estrogen receptors (ERs) and progesterone receptors (PRs),
as well as human epidermal growth factor receptor-2 (HER-2)
in TNBC, traditional targeted therapies such as endocrine
therapy and treatment with aromatase inhibitors are
ineffective.4 Therefore, much effort has been made to identify
new prognostic markers or therapeutic targets to improve the
treatment efficacy against TNBC.5−7
Previous studies have shown that the lack of ERα gene
expression in TNBC is due to epigenetic changes, including
increased deacetylation and methylation.8−10 Accordingly,
histone deacetylase (HDAC) inhibitors (HDACis) such as
suberoylanilide hydroxamic acid (SAHA or vorinostat; Figure
1) and DNA methyltransferase inhibitors such as 5-aza-2′-
deoxycytidine have been successfully used to reactivate the
expression of ERα and simultaneously sensitize TNBC to
treatment with antiestrogen drugs.11,12 A combination of
HDACis such as panobinostat or scriptaid with tamoxifen has
Received: May 25, 2018
© XXXX American Chemical Society
A
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Figure 1. HDAC inhibitors and their pharmacophores.
Scheme 1. Synthesis of Ferrocenyl Derivatives 5 and 7
have been reported to date, including benzamides (entinostat
(MS-275), tacedinaline (CI-994); Figure 1), short-chain fatty
acids (valproic and phenylbutyric acids; Figure 1), cyclic
tetrapeptides (romidepsin (FK-228); Figure 1), and keto
derivatives (trifluoromethyl ketone and α-keto amide; Figure
1).21−23 However, benzamides, short-chain fatty acids, cyclic
tetrapeptides, and keto derivatives often have reduced
potencies in comparison with those of hydroxamic acid
derived inhibitors. Encouragingly, Desai et al.24 have recently
reported that a selenocyanide group containing analogue of
SAHA (named SelSA; Figure 1) seemed to show a reasonable
zinc-binding activity and exhibited a more than 20-fold greater
potency against HDAC in comparison to that of SAHA. While
it is structurally similar to SAHA, the phenyl capping unit of
SelSA makes contacts with the pocket entrance in HDAC and,
if replaced with other hydrophobic aromatic rings, can increase
the HDAC inhibition activity, owing to the so-called “cap-
linker-ZBG” pharmacophore of SAHA.
derivatives can exhibit significant antiproliferative effects on
TNBC cells and act as effective metallodrugs. In addition,
incorporation of the Fc unit into ligands has a potential benefit
of filling the active-site space, which is not possible with
alicyclic or simple planar aromatic systems and can enhance
the ligand-binding affinity.29,30 In this study, we developed a
metal cap based SelSA derivative and examined the effects of a
nonplanar phenyl ring, the Fc unit, on HDAC inhibition and
anticancer activity. Subsequently, reactivation of the ERα
expression, due to anti-HDAC function, and anti-breast-cancer
effects of the synthesized ferrocenyl analogues were further
evaluated.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic procedures for the ferrocenyl
derivatives Fc-SelSA (5) and 7 were performed according to
Scheme 1. Commercially available 6-bromohexanoyl chloride
(1) was used as the starting material; it was treated with
aminoferrocene (2) to obtain a key intermediate (4).
Previous studies from Jaouen’s group25−27 and our group28
have demonstrated that organometallic ferrocene (Fc)
B
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Meanwhile, 1 was allowed to react with Fc (3) in the presence
of AlCl3 to afford another intermediate (6). Subsequently,
intermediates 4 and 6 were further treated with KSeCN in
CH3CN to produce the target selenocyanides Fc-SelSA (5)
and 7, respectively.
In Vitro Enzyme Inhibition. To determine whether the
ferrocenyl moiety is a suitable surface-recognition cap group
for a prototypical selenocyanide HDACi, we initially
synthesized Fc-SelSA (5), in which the cap phenyl ring of
SelSA was replaced with a ferrocenyl unit. Subsequently,
HDAC inhibition activities of SelSA and the Fc-SelSA were
tested in an in vitro fluorescence assay using a HeLa nuclear
extract as the source of HDAC activity. Figure 2 shows that the
the nonplanar ferrocenyl moiety as a cap group was a
successful strategy for the design of HDACis, which allowed
an enhancement of the HDAC inhibition activity. Molecular
docking simulations (see below) further demonstrated that the
ferrocenyl of Fc-SelSA could be better accommodated in the
active-site pocket of HDAC in comparison to the phenyl of
SelSA, which allowed the amido group of Fc-SelSA to form
hydrogen bonds with residues of the active pocket, which
SelSA was not able to do. To evaluate the influence of the
hydrogen-bond interactions, analogue 7, in which the amido
group of Fc-SelSA was replaced with a carbonyl group, was
also tested in the HDAC inhibition assay; however, 7 displayed
a 26-fold lower activity in comparison to that of Fc-SelSA. This
result indicated that the hydrogen-bond interactions of the
amido group were necessary for potent activity.
HDAC Docking Analysis. To understand how Fc-SelSA
binds to HDAC, we performed a docking study with Fc-SelSA
and HDAC (PDB: 1ZZ1) to examine the binding mode, and
the results are shown in Figure 3. Similar to the case for
traditional hydroxamic acid HDACis, such as SAHA, the two
selenocyanide compounds SelSA and Fc-SelSA also adopted an
optimal binding pose in the HDAC active-site pocket (Figure
3A−C), and the selenium atom of the selenocyanide group was
able to coordinate perfectly with the catalytic zinc ion (Zn2+−
Se of SeCN: 2.5 Å for SelSA and 2.2 Å for Fc-SelSA).
Additionally, the selenocyanide group of SelSA and Fc-SelSA is
engaged in the key hydrogen-bond formation with Y312
(Figure 3E,F). This hydrogen bond is critical for stabilizing
inhibitor chelation with the zinc ion in the catalytic domain.
The hydroxamic acid group of the reference compound SAHA
also formed a hydrogen bond with Y312 (Figure 3D). The
selenocyanide group also formed another hydrogen bond, with
D268 (Figure 3E,F), but SAHA did not form this hydrogen
bond. This might explain why the selenocyanide compounds
(SelSA and Fc-SelSA) had stronger HDAC inhibition activities
in comparison to SAHA. More importantly, the ferrocenyl unit
overlapped with the phenyl of SAHA, and the amido group of
Figure 2. Inhibitory activities of Fc-SelSA, SelSA, and derivative 7
against HDAC using a HeLa nuclear extract.
inhibitory activity of Fc-SelSA (IC50 = 14.8 0.07 nM) was
significantly higher than that of the lead compound SelSA
(IC50 = 20.3 1.13 nM). This result indicated that the use of
Figure 3. Docked complexes of SAHA, SelSA, and Fc-SelSA bound to HDAC. (A) Surface representation of the HDAC-SAHA complex. (B)
Surface representation of the HDAC-SelSA complex. (C) Surface representation of the HDAC-Fc-SelSA complex. (D) Docking poses of SAHA in
HDAC that can form hydrogen bonds with residues H142, H143, and Y312. (E) Docking poses of SelSA in HDAC that can form hydrogen bonds
with residues D268 and Y312. (F) Docking poses of Fc-SelSA in HDAC that can form hydrogen bonds with residues D98, G151, D268, and Y312.
The zinc ion is shown as a gray ball; interactions between the selenocyanide (SeCN) group and zinc are shown as red dashed lines. Distances are
provided in Å.
C
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Figure 4. Fc-SelSA activation of the ERα expression and sensitization of MDA-MB-231 cells to E2 and tamoxifen treatment. (A) ESR1 mRNA
expression induced by Fc-SelSA in MDA-MB-231 cells in a dose-dependent manner, as measured by qPCR. (B) ERα protein expression in MDA-
MB-231 cells treated with Fc-SelSA. (C) Relative viability of MDA-MB-231 cells in response to E2 and tamoxifen treatment, with or without Fc-
SelSA. MCF-7 cells were used as a positive control. (D) Relative PGR mRNA expression in MDA-MB-231 cells in response to E2 and tamoxifen
treatment. MCF-7 cells were used as a positive control. Legend: (*) P < 0.05.
Fc-SelSA can form hydrogen bonds with the D98 and G151
residues (Figure 3F), whereas SAHA and SelSA did not have
similar hydrogen-bonding interactions with D98 and G151.
Thus, Fc-SelSA displayed the most potent anti-HDAC activity
among these three HDACis.
therapeutic applications for TNBC through reactivation of the
ERα expression and sensitization of cells to endocrine therapy.
Antiproliferative Activity of Fc-SelSA in Vitro. To
investigate the cell-type selectivity and anticancer potency of
Fc-SelSA, compounds Fc-SelSA and 7 were screened against
the two breast cancer cell lines MCF-7 (hormone-dependent
breast cancer) and MDA-MB-231 (TNBC) by the MTS assay
using SelSA as a positive control. As expected, Fc-SelSA was
much more potent than SelSA against both MCF-7 and MDA-
MB-231 cells (Table 1). In particular, Fc-SelSA (CC50 = 0.17
Reactivation of ERα Expression. One efficient strategy
for TNBC-targeted therapy is to change TNBC into ERα
positive by reactivation of the ESR1 gene expression. Previous
studies have shown that HDACis can reactivate ERα
expression in TNBC.11,12 We therefore investigated the effects
of Fc-SelSA on the expression of ERα mRNA and protein in
MDA-MB-231 cells using qPCR and Western blot assays,
respectively. As shown in Figure 4A, Fc-SelSA treatment
resulted in a significantly increased expression of ESR1 mRNA
(P < 0.05) in comparison with the control. Importantly,
expression of the ERα protein was also observed in MDA-MB-
231 cells after treatment with Fc-SelSA (Figure 4B). We
further examined whether this effect can render the TNBC
responsive to the ER ligand estradiol (E2) or the ER antagonist
tamoxifen. The results indicated that ERα-positive MCF-7 cells
showed good responses to both E2 and tamoxifen, whereas the
cell viability as well as the expression of the PGR gene, an ERα-
responsive downstream gene, was unaffected by E2 and
tamoxifen in MDA-MB-231 cells without Fc-SelSA treatment
(Figure 4C,D). In contrast, treatment with Fc-SelSA resulted
in a significant change in the MDA-MB-231 cell viability and
PGR expression, which was similar to those of MCF-7 cells in
response to these two compounds (E2 and tamoxifen). These
results suggest that Fc-SelSA possesses the potential for
Table 1. Antiproliferative Activities of SelSA and Ferrocenyl
Derivatives
a
CC50 (μM)
compound
MCF-7
MDA-MB-231
MCF-10A
Vero
b
SelSA
Fc-SelSA
7
1.36 0.11
1.18 0.34
5.46 0.49
0.64 0.06
0.17 0.03
2.04 0.27
>100
>100
>100
>100
>100
>100
a
CC50 values are the mean
standard deviation of at least three
b
independent experiments. The highest concentration tested.
0.07 μM; the dose−response curves are shown in Figure S1)
inhibited the MDA-MB-231 cell growth 4-fold more potently
than SelSA and showed a 7-fold greater selectivity for MDA-
MB-231 cells over MCF-7 cells. However, the replacement of
the amido group of Fc-SelSA with a carbonyl group
(compound 7) resulted in at least a 4-fold decrease of its
antiproliferative activity against both MCF-7 and MDA-MB-
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Table 2. Acute Toxicity of Fc-SelSA in Healthy Mice
mortality
a
dose (mg/kg)
no. of mice
1 h
5 h
4 days
5−21 days
total mortality
survival (%) on day 21
LD50 (mg/kg)
605.6
895
716
573
458.4
366.7
8
8
8
8
8
1
0
0
0
0
2
1
0
0
0
5
5
3
1
0
0
0
0
0
0
8
6
3
1
0
0
25.0
62.5
87.5
100
a
The 95% confidence limits: 541.14−677.94 mg/kg.
Figure 5. Antitumor effects of Fc-SelSA and SelSA on MDA-MB-231 breast tumor xenograft mouse model. (A) Average tumor volumes in
xenograft mice after different treatments. (B) Average tumor weights in xenograft mice after different treatments. (C) Tumors dissected at the end
of experiment. (D) Average body weight changes in xenograft mice over a period of 21 days with different treatments. Legend: (**) P < 0.01 vs the
control; (#) P < 0.05, the Fc-SelSA treatment group vs the SelSA group.
231 cells and in a 3-fold weaker cell-type selectivity, which
agrees with the poor HDAC inhibition profile of 7. These
above results indicate that the antiproliferative activity of the
ferrocenyl compounds Fc-SelSA and 7 against MDA-MB-231
cells is related to the inhibition of HDAC, and the introduction
of the ferrocenyl group can enhance potency against MDA-
MB-231 cells.
Since the cytotoxicity is important for an anticancer
compound, we further tested Fc-SelSA, 7, and SelSA against
the noncancer cells MCF-10A (human normal breast epithelial
cells) and Vero (healthy kidney epithelial cells). Our study has
indicated that the FDA-approved SAHA showed considerable
toxicity to the healthy cells (Vero), while our selenocyanide
compounds (Fc-SelSA, 7, and SelSA) were nontoxic to healthy
cells (MCF-10A and Vero).
Safety Profile of Fc-SelSA in Healthy Mice. Since Fc-
SelSA showed significant antiproliferative effects in vitro, its
therapeutic efficacy was studied in vivo. First, an acute toxicity
test was performed to evaluate the safety of Fc-SelSA in
healthy mice. Adult BALB/c mice (n = 8) were treated with
Fc-SelSA at 895, 716, 573, 458.4, and 366.7 mg/kg by oral
gavage, and the survival of these mice was continuously
monitored for 21 days after treatment. The oral treatment with
366.7 mg/kg Fc-SelSA did not cause any death, whereas all the
mice were killed at a dose of 895 mg/kg. The survival rates
were 87.5%, 62.5%, and 25% at doses of 458.4, 573, and 716
mg/kg, respectively (Table 2). On the basis of these data, the
median lethal dose (LD50) value of Fc-SelSA was calculated to
be 605.6 mg/kg.
In Vivo Anticancer Activity. To evaluate the in vivo
anticancer activity of Fc-SelSA, it was orally administered at 20
mg/kg daily for 21 days, and SelSA was administered at the
same dose as a positive control. As shown in Figure 5,
treatment with SelSA and Fc-SelSA significantly reduced the
tumor volumes in the MDA-MB-231 tumor-bearing mouse
model (P < 0.01 vs control; Figure 5A); the tumor volume in
the Fc-SelSA-treated mice was smaller than that in the mice
administered SelSA (P < 0.05; Figure 5A). Moreover, Fc-SelSA
treatment significantly reduced the weight of MDA-MB-231
tumor xenografts by 58.6% (P < 0.01 vs control), which was
superior to the effect observed in the SelSA group (41.9%
reduction vs control; P < 0.01 vs control; Figure 5B,C).
Additionally, the body weights in the tumor-bearing mice
treated with Fc-SelSA and SelSA were not significantly
affected, and no adverse side effects were observed during
the treatment period (Figure 5D). Taken together, these
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Synthesis of 5-Ferrocenylcarbamoylpentyl Selenocyanide
(Fc-SelSA, 5). To a solution of bromo amide 4 (1.475 g, 3.9 mmol),
in dry acetonitrile (25 mL), was added KSeCN (0.682 g, 4.7 mmol).
The reaction solution was stirred at room temperature for 24 h under
an Ar atmosphere. The solvent was removed by rotary evaporation to
yield the crude product, which was purified by silica gel column
chromatography (petroleum ether/EtOAc, 3/2). The Fc-SelSA
product was isolated (1.232 g, 78.3% yield) as a red solid. 1H
NMR (400 MHz, CD3OD): δ 4.62 (s, 2H), 4.15 (s, 5H), 4.00 (s,
2H), 3.59 (t, J = 7.2 Hz, 2H), 2.28 (t, J = 6.8 Hz, 2H), 1.78−1.82 (m,
2H), 1.68−1.72 (m, 2H), 1.51−1.56 (m, 2H). 13C NMR (100 MHz,
CD3OD): δ 172.78, 102.79, 94.37, 68.75, 64.07, 61.18, 35.92, 30.60,
29.09, 28.21, 24.86. HRMS (ESI): calcd for C17H2154FeN2OSe [M +
H]+, 402.1664; found, 402.1669.
results highlighted the superior anti-TNBC effects of Fc-SelSA,
in comparison with those of SelSA, with no adverse side
effects. Thus, Fc-SelSA may be used as a novel and potent
HDACi for TNBC therapy.
CONCLUSIONS
■
Due to the lack of targeted therapies, it is difficult to effectively
treat TNBC. HDAC have emerged as effective targets for the
development of anti-TNBC agents because of their association
with ERα expression-reactivating effects. In the present study,
we designed, synthesized, and evaluated a novel metallic
HDACi with a Fc cap (Fc-SelSA) for the treatment of TNBC.
We found that the replacement of the phenyl ring of SelSA
with a nonplanar bioisostere ferrocenyl group resulted in a
more potent anti-HDAC activity of the compound. Sub-
sequent molecular docking analysis showed that the ferrocenyl
group overlaps with the phenyl ring, allowing the amido group
of Fc-SelSA to form hydrogen-bonding interactions with
residues D98 and G151, which SAHA and SelSA were not
able to do. These results indicated that using a three-
dimensional aryl group as a cap is an effective strategy for
the design of HDACis. Moreover, Fc-SelSA treatment can
induce the ERα expression in TNBC and sensitize MDA-MB-
231 cells to conventional endocrine therapy by reactivating
ERα. Furthermore, Fc-SelSA was selectively more potent
against MDA-MB-231 cells in comparison to MCF-7 cells with
no toxicity against normal cells in the in vitro antiproliferation
test. In addition, Fc-SelSA showed a relatively low acute
toxicity in mice, and treatment with Fc-SelSA significantly
inhibited the growth of TNBC in a xenograft mouse model in
comparison to SelSA (P < 0.05). Given its high HDAC-
binding affinity and potent therapeutic effect, Fc-SelSA can
therefore serve as a promising targeting ligand to study HDAC-
related epigenetic changes during tumorigenesis and as an anti-
TNBC therapeutic agent.
Synthesis of 6-Bromo-1-Ferrocenylhexan-1-one (6). To a
solution of 6-bromohexanoyl chloride (1; 4.932 g, 23.1 mmol) and
AlCl3 (9.241 g, 69.3 mmol) in dry CH2Cl2 (60 mL) at 0 °C was
added a solution of Fc (3; 2.141 g, 11.5 mmol) in dry CH2Cl2 (30
mL) dropwise over 20 min under an Ar atmosphere. After the
solution was stirred for 12 h at room temperature, saturated NaHCO3
(50 mL) was added, and the mixture was extracted with CH2Cl2 (3 ×
60 mL). The extracts were dried (anhydrous Na2SO4) and
concentrated. The crude product was purified by silica gel column
chromatography (petroleum ether/EtOAc, 9/1) to yield the bromo
1
intermediate 6 as a red solid (1.282 g, 30.7% yield). H NMR (400
MHz, CDCl3): δ 4.78 (s, 2H), 4.50 (s, 2H), 4.19 (s, 5H), 3.44 (t, J =
6.4 Hz, 2H), 2.72 (t, J = 7.2 Hz, 2H), 1.89−1.96 (m, 2H), 1.70−1.77
(m, 2H), 1.52−1.57 (m, 2H). 13C NMR (100 MHz, CDCl3): δ
204.21, 79.03, 72.23, 69.78, 69.32, 39.40, 33.78, 32.69, 28.08, 23.58.
Synthesis of 5-Ferrocenyl Carbonylpentyl Selenocyanide
(7). The process was performed as for the preparation of Fc-SelSA
using the bromo intermediate 6, instead of 4, to afford selenocyanide
1
7. A red solid was obtained in 82.5% yield. H NMR (400 MHz,
CDCl3): δ 4.78 (s, 2H), 4.50 (s, 2H), 4.19 (s, 5H), 3.10 (t, J = 7.2 Hz,
2H), 2.74 (t, J = 7.2 Hz, 2H), 1.93−2.01 (m, 2H), 1.72−1.79 (m,
2H), 1.50−1.58 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 203.95,
101.58, 78.96, 72.26, 69.78, 69.29, 39.14, 30.74, 29.38, 28.81, 23.47.
HRMS (ESI): calcd for C17H2054FeNOSe [M + H]+, 387.1518; found,
387.1522.
Cell Lines. Breast cancer MCF-7 and MDA-MB-231 cells were
purchased from the American Type Culture Collection (ATCC).
Human normal breast epithelial cells (MCF-10A) and healthy kidney
epithelial cells (VERO) were purchased from the cell bank of the
Chinese Academy of Sciences (Shanghai, People’s Republic of
China). The cells were cultured in Dulbecco Minimum Essential
Medium (DMEM, Hyclone, Thermo Scientific, Waltham, USA)
containing 10% (v/v) fetal bovine serum (FBS, Hyclone, Thermo
Scientific) and 1% (v/v) penicillin/streptomycin. Cells were
maintained at 37 °C 5% in a CO2 incubator.
HDAC Inhibition Assay. The HDAC inhibition activity was
measured using an HDAC assay kit (BPS Bioscience) according to a
previously described method.28,31 Half-maximum inhibitory concen-
tration (IC50) values were calculated from dose−response curves
using Origin software (OriginLab, Inc.).
EXPERIMENTAL SECTION
■
General Reagents and Methods. All chemical reagents and
solvents were purchased from Aldrich, Acros, Aladdin Reagents, and
Alfa Aesar. Dichloromethane and acetonitrile were distilled from
anhydrous CaH2. Unless otherwise noted, all reactions were
conducted under an inert (Ar) atmosphere. The reaction progress
was monitored by analytical thin-layer chromatography under UV
light (254 nm).
1H NMR and 13C NMR spectra were measured on a Bruker
Biospin AV400 instrument (at 400 and 100 MHz, respectively).
Chemical shifts are reported in ppm and are referenced to either
tetramethylsilane or the solvent. The purity of all compounds (>95%)
for biological testing was confirmed by high-performance liquid
chromatography.
Western Blot Analysis. MDA-MB-231 cells were treated with Fc-
SelSA for 48 h and then lysed in RIPA buffer. Total protein was
separated by electrophoresis in Tris-glycine gradient gels and then
analyzed by Western blotting. The primary antibody was anti-ERα
(Pierce; 1/1000 dilution), and the secondary antibody was horse-
radish peroxidase conjugated antigoat IgG or an antirabbit antibody
(Pierce; 1/10000 dilution).
Molecular Modeling. The crystal structure of HDAC (PDB:
1ZZ1) was obtained from the Protein Data Bank,32 and all water
molecules were removed. Crystallographic coordinates of SelSA and
Fc-SelSA were prepared using BioChem Office software. Preparation
of the ligands and protein was performed with AutoDockTools, and
molecular docking experiments were performed using AutoDock
software (version 4.2). The figures were prepared using PyMOL
Synthesis of 7-Bromoheptanoic Acid Ferrocenylamide (4).
To a solution of aminoferrocene were added 2 (0.904 g, 4.5 mmol), in
dry CH2Cl2 (30 mL), cooled to 0 °C, triethylamine (0.688 g, 6.8
mmol), and 6-bromohexanoyl chloride (1; 1.153 g, 5.4 mmol) under
an Ar atmosphere. The reaction solution was warmed to room
temperature and then stirred for 24 h. Subsequently, the reaction
mixture was poured into water and extracted with CH2Cl2 (3 × 30
mL). The extracts were dried (anhydrous Na2SO4) and evaporated.
The crude product was purified by silica gel column chromatography
(petroleum ether/ethyl acetate (EtOAc), 9/1) to yield bromo amide
4 as a red oil (1.509 g, 88.7% yield). 1H NMR (400 MHz, CD3OD): δ
4.61 (s, 2H), 4.15 (s, 5H), 4.00 (s, 2H), 3.60 (t, J = 6.0 Hz, 2H), 2.28
(t, J = 7.2 Hz, 2H), 1.81−1.85 (m, 2H), 1.69−1.73 (m, 2H), 1.53−
1.57 (m, 2H). 13C NMR (100 MHz, CD3OD): δ 172.85, 94.44, 68.75,
64.06, 61.14, 44.26, 36.04, 32.11, 26.16, 24.95.
̈
(Erwin Schrodinger).
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DOI: 10.1021/acs.organomet.8b00354
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ERα Expression Reactivation Assays. To evaluate the effects of
Fc-SelSA on ERα expression, MDA-MB-231 cells were treated with
Fc-SelSA for 3 days, and then ERα mRNA and protein expression was
examined using quantitative real-time PCR (qPCR) and Western
blotting assays, as previously described.19 The PCR primers used were
the ERα ligand-binding domain forward (5′-ATAGGATCCATC-
AAACGCTCTAAGAAG-3′) and reverse (5′-ATACTCGAGGC-
TAGTGGGCGCATGTAG-3′) primers.9
Z161100002616022, the Fundamental Research Funds for the
Central universities (JB161204, JBG161201, XJS16011),
China Postdoctoral Science Foundation funded project
(2017M613084), and Shaanxi Postdoctoral Science Founda-
tion funded project (2017BSHEDZZ91).
REFERENCES
■
To investigate whether Fc-SelSA can enable TNBC cells to
respond to estradiol (E2) and tamoxifen by reactivating the ERα
expression, Fc-SelSA-pretreated MDA-MB-231 cells were treated with
10 nM E2 or 1 μM tamoxifen for 2 days, and then cell viability was
evaluated using a standard MTS assay (Biovision, Inc.). The
expression of the PR-encoding gene (PGR), an ERα target gene,
was also detected using real-time PCR with the following
oligonucleotide primers: forward (5′-AACTGCCCAGCATGTC-
GCCT-3′) and reverse (5′-GGAACGCCCACTGGCTGTGG-3′).9
Cell Proliferation Assay. The antiproliferative effects of SelSA
and Fc-SelSA were evaluated using the MTS assay according to a
previously described method.28,32 CC50 (50% cytotoxic concen-
tration) values were calculated from dose−response curves using
Origin software (OriginLab, Inc.).
Establishment of a Tumor-Bearing Mouse Model. Female
BALB/c mice (6 weeks old, 15−18 g) were purchased from Beijing
Vital River Laboratory Animal Technology Co., Ltd. All animal study
procedures were approved by the Institutional Animal Care and Use
Committee at Peking University (Permit No. 2011-0039). MCF-7
and MDA-MB-231 cells were transplanted subcutaneously in the
axillary region of the mice.
(1) Dent, R.; Trudeau, M.; Pritchard, K. I.; Hanna, W. M.; Kahn, H.
K.; Sawka, C. A.; Lickley, L. A.; Rawlinson, E.; Sun, P.; Narod, S. A.
Triple-negative breast cancer: clinical features and patterns of
recurrence. Clin. Cancer Res. 2007, 13 (15), 4429−4434.
(2) Carey, L.; Winer, E.; Viale, G.; Cameron, D.; Gianni, L. Triple-
negative breast cancer: disease entity or title of convenience? Nat. Rev.
Clin. Oncol. 2010, 7 (12), 683−692.
(3) Narod, S. A. Breast cancer in young women. Nat. Rev. Clin.
Oncol. 2012, 9 (8), 460−470.
(4) Papa, A.; Caruso, D.; Tomao, S.; Rossi, L.; Zaccarelli, E.; Tomao,
F. Triple-negative breast cancer: investigating potential molecular
therapeutic target. Expert Opin. Ther. Targets 2015, 19 (1), 55−75.
(5) Barbie, T. U.; Alexe, G.; Aref, A. R.; Li, S.; Zhu, Z.; Zhang, X.;
Imamura, Y.; Thai, T. C.; Huang, Y.; Bowden, M.; Herndon, J.;
Cohoon, T. J.; Fleming, T.; Tamayo, P.; Mesirov, J. P.; Ogino, S.;
Wong, K. K.; Ellis, M. J.; Hahn, W. C.; Barbie, D. A.; Gillanders, W. E.
Targeting an IKBKE cytokine network impairs triple-negative breast
cancer growth. J. Clin. Invest. 2014, 124 (12), 5411−5423.
(6) Bianchini, G.; Balko, J. M.; Mayer, I. A.; Sanders, M. E.; Gianni,
L. Triple-negative breast cancer: challenges and opportunities of a
heterogeneous disease. Nat. Rev. Clin. Oncol. 2016, 13 (11), 674−690.
(7) Fusella, F.; Seclì, L.; Busso, E.; Krepelova, A.; Moiso, E.; Rocca,
S.; Conti, L.; Annaratone, L.; Rubinetto, C.; Mello-Grand, M.; Singh,
V.; Chiorino, G.; Silengo, L.; Altruda, F.; Turco, E.; Morotti, A.;
Oliviero, S.; Castellano, I.; Cavallo, F.; Provero, P.; Tarone, G.;
Brancaccio, M. The IKK/NF-κB signaling pathway requires Morgana
to drive breast cancer metastasis. Nat. Commun. 2017, 8 (1), 1636.
(8) Zhou, Q.; Atadja, P.; Davidson, N. E. Histone deacetylase
inhibitor LBH589 reactivates silenced estrogen receptor alpha (ER)
gene expression without loss of DNA hypermethylation. Cancer Biol.
Ther. 2007, 6 (1), 64−69.
(9) Li, Y.; Yuan, Y. Y.; Meeran, S. M.; Tollefsbol, T. O. Synergistic
epigenetic reactivation of estrogen receptor-α (ERα) by combined
green tea polyphenol and histone deacetylase inhibitor in ERα-
negative breast cancer cells. Mol. Cancer 2010, 9, 274−285.
(10) Sabnis, G. J.; Goloubeva, O.; Chumsri, S.; Nguyen, N.;
Sukumar, S.; Brodie, A. M. Functional activation of the estrogen
receptor-α and aromatase by the HDAC inhibitor entinostat sensitizes
ER-negative tumors to letrozole. Cancer Res. 2011, 71 (5), 1893−
1903.
Statistics. Data are shown as the means
SD from three
independent experiments. One-way ANOVA with a Tukey test was
performed to compare differences between groups, and a two-tailed
Student’s t test was performed to compare two groups. LegendL (*) P
< 0.05 was considered statistically significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.8b00354.
■
S
Dose−response curves and H NMR and 13C NMR
spectral information for compounds 5 and 7 (PDF)
1
AUTHOR INFORMATION
Corresponding Authors
*Z.C.: e-mail, zcheng@stanford.edu; tel, 650-723-7866; fax,
650-736-7925.
■
*J.T.: e-mail, jie.tian@ia.ac.cn, tian@ieee.org; tel, 8610
62527995; fax, 86 10 62527995; web, http://www.3dmed.net.
ORCID
Zhen Cheng: 0000-0001-8177-9463
Jie Tian: 0000-0002-9523-2271
(11) Yang, X.; Phillips, D. L.; Ferguson, A. T.; Nelson, W. G.;
Herman, J. G.; Davidson, N. E. Synergistic activation of functional
estrogen receptor (ER)-α by DNA methyltransferase and histone
deacetylase inhibition in human ER-α-negative breast cancer cells.
Cancer Res. 2001, 61 (19), 7025−7029.
(12) Khan, S. I.; Aumsuwan, P.; Khan, I. A.; Walker, L. A.;
Dasmahapatra, A. K. Epigenetic events associated with breast cancer
and their prevention by dietary components targeting the epigenome.
Chem. Res. Toxicol. 2012, 25 (1), 61−73.
Author Contributions
#C.T. and Y.D. contributed equally to this work.
Notes
(13) Sharma, D.; Saxena, N. K.; Davidson, N. E.; Vertino, P. M.
Restoration of tamoxifen sensitivity in estrogen receptor-negative
breast cancer cells: tamoxifen-bound reactivated ER recruits
distinctive corepressor complexes. Cancer Res. 2006, 66 (12),
6370−6378.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This paper was supported by the Ministry of Science and
Technology of China under Grant Nos. 2017YFA0205200 and
2016YFC0102000, the National Natural Science Foundation
of China under Grant Nos. 81470083, 81527805, 61231004,
81601548, 81701771, and 81772011, the International
Innovation Team of CAS under Grant No. 20140491524,
Beijing Municipal Science & Technology Commission No.
(14) Giacinti, L.; Giacinti, C.; Gabellini, C.; Rizzuto, E.; Lopez, M.;
Giordano, A. Scriptaid effects on breast cancer cell lines. J. Cell.
Physiol. 2012, 227 (10), 3426−3433.
(15) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. Histone
deacetylase inhibitors: from bench to clinic. J. Med. Chem. 2008, 51
(6), 1505−1529.
G
DOI: 10.1021/acs.organomet.8b00354
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
(16) Duvic, M.; Vu, J. Vorinostat: a new oral histone deacetylase
inhibitor approved for cutaneous T-cell lymphoma. Expert Opin.
Invest. Drugs 2007, 16 (7), 1111−1120.
(17) Garnock-Jones, K. P. Panobinostat: first global approval. Drugs
2015, 75 (6), 695−704.
(18) Poole, R. M. Belinostat: first global approval. Drugs 2014, 74
(13), 1543−1554.
(19) Sanderson, L.; Taylor, G. W.; Aboagye, E. O.; Alao, J. P.;
Latigo, J. R.; Coombes, R. C.; Vigushin, D. M. Plasma
pharmacokinetics and metabolism of the histone deacetylase inhibitor
trichostatin a after intraperitoneal administration to mice. Drug Metab.
Dispos. 2004, 32 (10), 1132−1138.
(20) Mulder, G. J.; Meerman, J. H. Sulfation and glucuronidation as
competing pathways in the metabolism of hydroxamic acids: the role
of N,O-sulfonation in chemical carcinogenesis of aromatic amines.
Environ. Health Perspect. 1983, 49 (4), 27−32.
(21) Bertrand, P. Inside HDAC with HDAC inhibitors. Eur. J. Med.
Chem. 2010, 45, 2095−2116.
(22) Thaler, F.; Mercurio, C. Towards selective inhibition of histone
deacetylase isoforms: what has been achieved, where we are and what
will be next. ChemMedChem 2014, 9 (3), 523−526.
(23) Roche, J.; Bertrand, P. Inside HDACs with more selective
HDAC inhibitors. Eur. J. Med. Chem. 2016, 121, 451−483.
(24) Desai, D.; Salli, U.; Vrana, K. E.; Amin, S. SelSA, selenium
analogs of SAHA as potent histone deacetylase inhibitors. Bioorg. Med.
Chem. Lett. 2010, 20 (6), 2044−2047.
̀
́
(25) Plazuk, D.; Vessieres, A.; Hillard, E. A.; Buriez, O.; Labbe, E.;
Pigeon, P.; Plamont, M. A.; Amatore, C.; Zakrzewski, J.; Jaouen, G. A
[3]ferrocenophane polyphenol showing a remarkable antiproliferative
activity on breast and prostate cancer cell lines. J. Med. Chem. 2009,
52 (15), 4964−4967.
́
̀
(26) Cazares Marinero, J. d. J.; Lapierre, M.; Cavailles, V.; Saint-
Fort, R.; Vessieres, A.; Top, S.; Jaouen, G. Efficient new constructs
̀
against triple negative breast cancer cells: synthesis and preliminary
biological study of ferrocifen-SAHA hybrids and related species.
Dalton Trans. 2013, 42 (43), 15489−15501.
́
̀
(27) Cazares-Marinero, J. d. J.; Top, S.; Vessieres, A.; Jaouen, G.
Synthesis and antiproliferative activity of hydroxyferrocifen hybrids
against triple-negative breast cancer cells. Dalton Trans. 2014, 43 (2),
817−830.
(28) Li, C. H.; Tang, C.; Hu, Z. Y.; Li, C. L.; Zhang, S. L.; Dong, C.;
Zhou, H. B.; Huang, J. Synthesis and structure-activity relationships of
novel hybrid ferrocenyl compounds based on a bicyclic core skeleton
for breast cancer therapy. Bioorg. Med. Chem. 2016, 24 (13), 3062−
3074.
(29) Meggers, E. Targeting proteins with metal complexes. Chem.
Commun. 2009, 7 (9), 1001−1010.
(30) Spencer, J.; Amin, J.; Wang, M.; Packham, G.; Alwi, S. S.;
Tizzard, G. J.; Coles, S. J.; Paranal, R. M.; Bradner, J. E.; Heightman,
T. D. Synthesis and biological evaluation of JAHAs: ferrocene-based
histone deacetylase inhibitors. ACS Med. Chem. Lett. 2011, 2 (5),
358−362.
(31) Tang, C.; Li, C. H.; Zhang, S. L.; Hu, Z.; Wu, J.; Dong, C.;
Huang, J.; Zhou, H. B. Novel bioactive hybrid compound dual
targeting estrogen receptor and histone deacetylase for the treatment
of breast cancer. J. Med. Chem. 2015, 58 (11), 4550−4572.
(32) Gryder, B. E.; Rood, M. K.; Johnson, K. A.; Patil, V.; Raftery, E.
D.; Yao, L. P.; Rice, M.; Azizi, B.; Doyle, D. F.; Oyelere, A. K. Histone
deacetylase inhibitors equipped with estrogen receptor modulation
activity. J. Med. Chem. 2013, 56 (14), 5782−5796.
H
DOI: 10.1021/acs.organomet.8b00354
Organometallics XXXX, XXX, XXX−XXX