A Chimeric SERM-histone deacetylase inhibitor approach to breast cancer therapy

Article abstract of DOI:10.1002/cmdc.201300270

Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four typea I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3a μM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.

Full text of DOI:10.1002/cmdc.201300270

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DOI: 10.1002/cmdc.201300270
A Chimeric SERM–Histone Deacetylase Inhibitor Approach
to Breast Cancer Therapy
Hitisha K. Patel, Marton I. Siklos, Hazem Abdelkarim, Emma L. Mendonca, Aditya Vaidya,
Pavel A. Petukhov, and Gregory R. J. Thatcher*[a]
Dedicated to the memory of Victor Fung, Ph.D., NCI Program Officer and Scientific Review Officer of the Cancer Etiology study section
of CSR/NIH, for his wisdom, compassion, integrity, and his extraordinary contribution to the career development of many investigators
during his own distinguished career.
Breast cancer remains a significant cause of death in women,
and few therapeutic options exist for estrogen receptor nega-
tive (ER(ꢀ)) cancers. Epigenetic reactivation of target genes
using histone deacetylase (HDAC) inhibitors has been pro-
posed in ER(ꢀ) cancers to resensitize to therapy using selec-
tive estrogen receptor modulators (SERMs) that are effective in
ER(+) cancer treatment. Based upon preliminary studies in
ER(+) and ER(ꢀ) breast cancer cells treated with combinations
of HDAC inhibitors and SERMs, hybrid drugs, termed SERM-
ostats, were designed with computational guidance. Assay for
inhibition of four type I HDAC isoforms and antagonism of es-
trogenic activity in two cell lines yielded a SERMostat with 1–
3 mm potency across all targets. The superior hybrid caused
significant cell death in ER(ꢀ) human breast cancer cells and
elicited cell death at the same concentration as the parent
SERM in combination treatment and at an earlier time point.
Introduction
Breast cancer is the second leading cause of cancer death in
women. The chance of developing invasive breast cancer at
some point in a woman’s life remains at 12%. About 70–80%
of breast cancers are estrogen receptor positive (ER(+)), as-
sessed as such because of the presence of ERa that mediates
proliferation in response to estrogen. Classical estrogen signal-
ing is mediated by two isoforms acting as liganded nuclear re-
ceptors: ERa and ERb.[1] Ligand binding causes a conformation-
al change leading to receptor dimerization and binding to
transcriptional elements in DNA which precedes recruitment of
transcriptional coregulators.[2] Tamoxifen, a first line therapy for
patients with ER(+) breast cancer, is sometimes referred to as:
(1) an antiestrogen, since it opposes the actions of estrogen in
the breast; and (2) the prototypical selective estrogen receptor
modulator (SERM), because it can also act as an agonist at the
ER, notably in the uterus. Unfortunately, tamoxifen therapy is
ineffective for ER(ꢀ) and triple-negative breast (TNB) cancers,
and roughly half of ER(+) tumors are insensitive or lose re-
sponse to continued tamoxifen therapy and gain resistance.[3]
Current treatment options for ER(ꢀ), TNB, and tamoxifen-
resistant breast cancers are limited to cytotoxic chemotherapy.
A proposed therapeutic approach is the use of histone deace-
tylase (HDAC) inhibitors for various cancers, which may func-
tion in part via epigenetic modulation of ER signaling.[4] This
approach stems from the crucial role of acetylation in regulat-
ing gene transcription and the importance of HDACs in sup-
pressing transcription of ER-dependent gene products and of
ER itself in ER(ꢀ) breast cancer.[5] Treatment of ER(ꢀ) breast
cancer cells with DNA methyltransferase (DNMT) and HDAC in-
hibitors has been reported to induce re-expression of ERa
while simultaneously sensitizing these cells to the actions of
antiestrogens.[6] The presence of HDAC1 in a transcriptional
corepressor complex with DNMTs was found to be associated
with a silenced ERa promoter in ER(ꢀ) cells and was reported
to be unsilenced upon HDAC inhibitor treatment, leading to
activation of ERa gene expression.[7] Studies on ER(ꢀ) cells
using HDAC inhibitors (panabinostat or scriptaid) in combina-
tion with tamoxifen, reported enhanced antiproliferative
actions and upregulation of ERa.[8]
In ER(+) cells, knockdown of HDAC1 and HDAC inhibition
by either trichostatin A (TSA), butyric acid, or valproic acid
(VPA), have independently been reported to decrease ERa
levels.[9] Several papers have shifted focus from ERa, suggest-
ing that increasing levels of ERb in ER(+) cells is most impor-
tant in relation to the combined interactions of HDAC inhibi-
tors with SERMs and antiestrogens.[10] The combination of an
HDAC inhibitor (TSA, VPA, or vorinostat) with tamoxifen, or the
newer generation SERM, raloxifene, was reported to provide
antiproliferative or apoptotic actions in ER(+) cell lines.[9b,10b,11]
Other researchers have argued against the central importance
of ER in inhibition of breast cancer cell growth;[9a,b,12] however,
regardless of the exact mechanism, the strength of the preclin-
ical data has supported clinical trials on tamoxifen/panobino-
[a] H. K. Patel,+ M. I. Siklos,+ H. Abdelkarim, E. L. Mendonca, A. Vaidya,
Dr. P. A. Petukhov, Dr. G. R. J. Thatcher
Department of Medicinal Chemistry and Pharmacognosy
University of Illinois College of Pharmacy, UIC
833 S. Wood St., Chicago, IL 60612-7231 (USA)
E-mail: thatcher@uic.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300270.
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stat and tamoxifen/vorinostat combination therapy in TNB and
endocrine-resistant breast cancer, respectively.[13]
do report antiproliferative actions on combination treatment
of ER(+) cells.[14]
Estrogen signaling requires the displacement of proteins
from corepressor complexes, including HDACs, and the recruit-
ment of coactivator proteins to transcription complexes con-
taining liganded ER. Substantial evidence supports the direct
interaction of HDACs with corepressor proteins and ER in si-
lenced nuclear transcription complexes in the cell nucleus, as,
for example, in interaction of HDAC1 with the activation func-
tion 2 (AF2) and DNA binding domain (DBD) of ERa.[14] Because
combination SERM+HDAC inhibitor therapy requires both
drugs to be in close proximity in the cell nucleus, the concept
of an HDAC inhibitor and SERM hybrid is justified. We report
the first proof of concept in ER(ꢀ) breast cancer cell lines.
Drug design was based around a benzothiophene SERM scaf-
fold; hence, we term these hybrid HDAC inhibitors SERMostats.
HDACs are divided into four families: class I (HDAC1, 2, 3
and 8), class II (HDAC4, 5, 6, 7, 9 and 10), class III (sirtuins) and
class IV (HDAC11).[17] The presence of HDAC2 and HDAC3 on
promoter regions of ER target genes was identified after ad-
ministration of 4-hydroxytamoxifen (4OHT) to 5’-azadeoxycyti-
dine/TSA pretreated cells: HDAC2 as a part of the Mi2/NuRD
corepressor complex; and HDAC3, a part of the NCoR com-
plex.[18] Inhibition of HDAC2 activity by siRNA also showed re-
duced ERa and progesterone receptor (PR) levels in ER(+)
cells.[9b] The vorinostat/tamoxifen phase 2 clinical trial reported
response to combination therapy in subjects with a higher
level of HDAC2.[13] All class I HDACs, with the exception of
HDAC8, have been implicated as mediators of estrogen signal-
ing in both ER(+) and ER(ꢀ) breast cancer. Less evidence
exists for the importance of class II HDACs, although
(1) HDAC4 was reported to be recruited to target gene pro-
moters as part of corepressor complexes after administration
of tamoxifen and raloxifene,[19] (2) inhibition of HDAC6 has
been reported to downregulate ERa levels by cytoplasmic
mechanisms,[9b,20] and (3) HDAC7 is present on pS2 gene pro-
moter regions.[9d] Class III and IV HDACs have been least stud-
ied in relation to breast cancer.
Results and Discussion
Drug design rationale: HDAC and ER(ꢀ) breast cancer
Endocrine therapy, targeting estrogen signaling through aro-
matase inhibitors or antiestrogens, such as, fulvestrant and ta-
moxifen, is the cornerstone of breast cancer therapy. However,
treatment options for ER(ꢀ) and tamoxifen-resistant breast
cancers are limited to cytotoxic chemotherapeutics. An alterna-
tive therapeutic approach that has been studied in various
cancers is the use of HDAC inhibitors. The mechanistic ration-
ale for combination therapy with antiestrogens in ER(ꢀ) breast
cancer is based upon the use of epigenetic modulators, includ-
ing DNA methyltransferase (DNMT) and histone deacetylase
(HDAC) inhibitors, to induce re-expression of ER in ER(ꢀ)
cancer and thus restore susceptibility to antiestrogen thera-
py.[6,15] There is support for this hypothesis, for example: (1) an
aberrant methylated ERa promoter is found in more than 25%
of ER(ꢀ) breast cancers, whereas in ER(+) tumors, ER promot-
er methylation was not observed;[16] and (2) the presence of
HDAC1 in a transcriptional co-repressor complex with DNMTs
is associated with a silenced ERa promoter in ER(ꢀ) cells.[9b] It
is uncertain if all ER(ꢀ) breast cancers would be resensitized to
antiestrogen therapy through epigenetic upregulation of ERa;
however, enhanced antiproliferative effects of antiestrogens
have been correlated with increased ERa in response to HDAC
inhibition.[6b,7,8,15]
Substantial preclinical data, summarized above, and ongoing
clinical trials on combination therapies justify study of a hybrid
molecule delivering both antiestrogenic SERM activity and
HDAC inhibition (SERMostat) to the ER transcriptional complex
in the cell nucleus, the target for both pharmacophores.
Design of a SERMostat simultaneously binding ER and HDAC
has inherent difficulties and is not supported by structural in-
formation defining the required geometry. On the other hand,
we have shown that a benzothiophene SERM can be used to
transport and localize a cargo to the nucleus of ER(+) breast
cancer cells.[21] The SERMostat was designed wherein both
SERM and HDAC inhibitor pharmacophores are targeted to the
nucleus. We have previously explored extensive modifications
of the benzothiophene SERMs, arzoxifene and raloxifene, dem-
onstrating that antiestrogenic activity and ER binding can be
maintained with substantial chemical modifications to the C3
side chain.[21a,22] The active metabolite of arzoxifene, desmethy-
larzoxifene (DMA), differs from raloxifene by one C atom and
retains the 2-phenylbenzo[b]thiophen-6-ol core. Therefore, dec-
oration of 2-phenylbenzo[b]thiophen-6-ol with a hydroxamate
side chain would be predicted to yield a SERMostat. Vorinostat
(SAHA) and hydroxamate 1 represent control HDAC inhibitors
without actions at the ER. The slow-binding HDAC inhibitor,
entinostat, was the inspiration for design of 10, incorporating
an o-hydroxybenzamide moiety, expected to provide activity
against HDAC1/3.[23] Both approaches incorporate Zn-binding
groups to impart inhibition of class I HDACs (Figure 1a).[22b,24]
Candidate SERMostats were docked to crystal structures of
HDAC2 and ERa to select candidates for synthesis. The HDAC2
isoform was prioritized because of data from clinical trials on
vorinostat/tamoxifen combination therapy.[13] After energy min-
imization in MOE, docking with the crystal structure of HDAC2
(PDB code: 3MAX),[25] and scoring the obtained binding poses
Obviously, the same resensitization rationale for ER(ꢀ) can-
cers cannot be applied to mammary tumor cells that already
express ERa, but do not respond to antiestrogen therapy. De-
creased levels of ERa reported in response to inhibition of
HDAC activity,[9] would attenuate estrogen-dependent prolifer-
ation; however, repressed ERa expression on overexpression of
HDAC1 in ER(+) cells was reported and observed to be re-
stored by administration of trichostatin A (TSA).[14] An alternate
hypothesis for use of HDAC inhibitors in combination in ER(+)
cancer cells has been proposed based upon upregulation of
ERb by HDAC inhibition and antiapoptotic actions of antiestro-
gens mediated by ERb.[10a,12] However, other mechanisms have
been proposed independent of ERb.[9a] Nevertheless, studies
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noalkanoic acid esters through
its chloride to obtain esters 5a
and 5b, which then were deme-
thylated with BBr3 resulting in
the loss of the methyl ester and
the formation of acids 6a and
6b. The acids were esterified
with methanol and HCl to the
methyl esters 7a and 7b before
the final conversion to hydroxa-
mic acids 8a and 8b with excess
NH2OH.[29] Acid 4 was converted
to the acid chloride using SOCl2
and coupled with 3-amino-4-me-
thoxybiphenyl (synthesized ac-
cording to literature proce-
dures)[30] to obtain amide 9. This
compound could be demethylat-
ed with BBr3 to yield o-hydroxy-
benzamide 10 (see Scheme 1).
Friedel–Crafts acylation of the
common starting material 2 with
4-iodobenzoyl chloride afforded
ketone 11 in 60% yield
(Scheme 2).[31] Sonogashira cou-
pling with ethynyltrimethylsilane
and deprotection of the ob-
tained TMS-protected alkyne 12
with KF then demethylation with
BBr3 yielded alkyne 13a.[28]
Figure 1. a) General design of SERMostat hybrid molecules. b) Overlay of raloxifene (blue) and 15b (magenta)
docked to the crystal structure of ERa (PDB code: 1ERR). c) Overlay of SAHA (magenta) and 15b (blue) docked to
the crystal structure of HDAC2 (PDB code: 3MAX). d) Overlay of the N-(2-aminophenyl)benzamide ligand LLX (ma-
genta) and 15b (blue) docked to the crystal structure of HDAC2 (PDB code: 3MAX).[26]
Through
copper(I)-catalysed
Huisgen [3+2] cycloaddition
with w-azidoalkanoic acid esters,
using an HDAC inhibitor training set,[25] binding affinities were
calculated using the Amber 12 forcefield. All hydroxamate li-
gands were confirmed to bind to HDAC2 in silico in a similar
binding orientation to SAHA (Figure 1c). The highest HDAC2
binding affinity values were calculated for triazoles 15a and
15b with the amide-linked hydroxamate 8a of similar affinity
and 8b marginally lower by 1.7 kcalmolꢀ1. Benzamide 10 was
predicted to be a weaker ligand by 6.3 kcalmolꢀ1 compared to
15b (Figure 1d). A similar approach was taken for ERa binding
using the raloxifene cocrystal structure (PDB code: 1ERR; raloxi-
fene cocrystal; Figure 1b).[26] Hybrids 15a and 15b were calcu-
lated to have the highest binding affinity, with 8a, 8b, and 10
having lower affinities by 1–5 kcalmolꢀ1.
this intermediate was used to obtain triazoles 14a and 14b,
which could be converted into the SERMostats, 15a and 15b,
with excess NH2OH in MeOH.[32]
Inhibition of HDACs and antagonism at the ER
The HDAC inhibitory activity for class I HDACs was evaluated
for the synthesized hybrids (Table 1). Both 1 and SAHA are
broad spectrum class I HDAC inhibitors, although 1 demon-
strates selectivity for HDAC3 over 1, 2, and 8. Attachment of an
alkyl hydroxamate chain to the benzothiophene core of DMA/
raloxifene via a simple amide linker led to broad spectrum
type I HDAC inhibitors, generally with sub-micromolar IC50
values. Extension of the 3-benzothiophene side chain to in-
clude more structural components of raloxifene linked via a tri-
azole to the alkyl hydroxamate led to some loss of activity, in
particular towards HDAC8. However, 15b retained reasonable
activity towards HDAC1, 2 and 3. Benzamide derivative 10
demonstrated measurable, time-dependent inhibition, as ex-
pected for this chemotype, for HDAC1 and 3, but as predicted
by computational studies, activity was weak.
Synthesis
The synthesis of the amide-linked SERMostat candidates 8a
and 8b was accomplished from commercially available 6-me-
thoxy-2-(4-methoxyphenyl)benzothiophene (2), which was bro-
minated with bromine in hot chloroform to yield 3-bromoben-
zothiophene 3 in 79% yield (see Scheme 1).[27] Bromine–lithium
exchange using nBuLi in anhydrous tetrahydrofuran (THF) at
ꢀ788C followed by quenching with solid CO2 afforded acid 4
after aqueous workup.[28] This acid could be coupled to w-ami-
The ability of SERMostats to engage the ER target in a cellu-
lar environment was studied using transfection of an estrogen
responsive element (ERE)-luciferase reporter in MCF-7 breast
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Scheme 2. Reagents and conditions: a) 4-iodobenzoyl chloride, AlCl3, CH2Cl2,
RT; b) Me3SiCCH, (Ph3P)2PdCl2, CuI, Et3N, THF, RT; c) BBr3, CH2Cl2, 08C; d) KF,
MeOH, H2O, 208C; e) ethyl-5-azidopentanoate or methyl-6-azidohexanoate,
CuSO4, sodium ascorbate, THF, H2O; f) NH2OH, MeOH, RT.
this assay (Figure 2b). In addition to confirming the potency of
15b in a second cell system, it was seen as important to pro-
vide an indication that the antiestrogenic activity of benzothio-
phene SERMs in endometrial tissues was retained by 15b.
Scheme 1. Reagents and conditions: a) Br2, CHCl3, 508C; b) 1) nBuLi, THF,
ꢀ788C, 2) CO2; c) SOCl2, toluene, reflux; d) H2N(CH2)4COOMe or H2N-
(CH2)5COOMe, Et3N, CH2Cl2, 08C; e) BBr3, CH2Cl2, 0!208C; f) MeOH, HCl,
reflux; g) NH2OH, MeOH, 0!208C; h) 3-amino-4-methoxybiphenyl, Et3N,
CH2Cl2, 08C; i) BBr3, CH2Cl2, 0!208C.
Activity of combinations in breast cancer cells
Preliminary experiments were carried out on the effect of com-
bination treatments in ER(ꢀ) and ER(+) breast cancer cell
lines, BT-20 and MCF-7, respectively. To provide comparison
with previous reports, the active metabolite of tamoxifen,
4OHT, was studied in combination with SAHA showing modest
but significantly increased cell death after 72 h of combination
treatment in ER(+) cells, but not ER(ꢀ) cells (Supporting Infor-
mation, figure 1). In comparison, Bicaku et al. reported approxi-
mately 10% increase in apoptosis of MCF-7 cells after 48 h
treatment with 4-OHT (10 mm) in combination with SAHA over
SAHA alone.[9b] The bioactivity of tamoxifen differs from later
generation SERMs, such as, raloxifene and arzoxifene, in several
important ways, including differing gene profiles and tissue-
selective estrogenicity, most notably in uterine proliferation,
which is a serious safety concern for tamoxifen.[33]
cancer cells. Propyl pyrazole triol (PPT) and diarylpropiolnitrile
(DPN), as ERa-selective and ERb-selective agonists, respectively,
illustrate the assay as indicative of classical ERa-mediated
gene-transcriptional activity (Figure 2a). The extended side
chain of all hybrids prepared is predictive of antiestrogenic as
opposed to agonist activity, and indeed, 8b and 15b showed
concentration-dependent inhibition of estrogenic response.
Other candidate SERMostats showed no significant activity
>25 mm. Upregulation of alkaline phosphatase in Ishikawa
human endometrial cells is a second standard assay for ERa ac-
tivity. The IC50 value of 15b was determined as (2.9ꢁ0.3) mm in
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Given the observations
in combination treatment
of ER(ꢀ) cells, more de-
tailed comparison was
made with SAHA and
Table 1. IC50 values for inhibition of recombinant HDAC isoforms.
Compd
HDAC1 [mm][a] HDAC2 [mm][a] HDAC3 [mm][a] HDAC8 [mm][a]
Vorinostat[b]
0.0224ꢁ0.0047 0.200ꢁ0.014 0.027ꢁ0.001
0.44ꢁ0.02
1 (1 mm) in the two ER(ꢀ)
cell lines, BT-20 and MDA-
MB-231. In combination
with DMA (10 mm), after
72 h incubation, the com-
bination treatment signif-
icantly increased cell
death in both cell lines
(Figure 4). Significant up-
regulation of ER mRNA
and re-
1
0.33ꢁ0.01
1.50ꢁ0.02
2.44ꢁ0.02
0.008ꢁ0.0004 0.480ꢁ0.009
8a
8b
0.596ꢁ0.051
0.168ꢁ0.013
0.214ꢁ0.006
0.719ꢁ0.063
0.400ꢁ0.036
expression of the protein
was reported only after
48 h administration with
TSA.[6b] In another study,
administration of HDAC
inhibitor caused a time-
dependent increase in ER
0.665ꢁ0.052 0.128ꢁ0.007
expression,
suggesting
that the re-expression of
ER by HDAC inhibitors
parallels the cytotoxic
effect of the combinatio-
n.[8a,34] However, other
possible delayed mecha-
nisms of toxicity have
been proposed, for exam-
15a
8.39ꢁ0.30
19.2ꢁ0.65
5.74ꢁ0.53
23.5ꢁ1.5
ple,
through
growth
15b
1.03ꢁ0.08
1.82ꢁ0.11
0.748ꢁ0.034 29.9ꢁ1.3
factor downregulation, or
reduction in NFkB tran-
scription. Our observa-
tions in ER(ꢀ) breast
cancer cells after combi-
nation treatments with
n.d. (5 min)[c]
21–34% (3 h)[c] n.d. (3 h)[c]
n.d. (24 h)[c]
n.d. (5 min)[c] n.d. (5 min)[c]
n.d. (5 min)[c]
0–12% (3 h)[c] n.d. (3 h)[c]
DMA
supported
the
10
design of
hybrid
a
SERMostat
based
upon
a DMA core with a hy-
droxamate side chain.
[a] Data are the mean ꢁSD of 2–4 replicates. [b] Taken from ref. [29]. [c] Inhibition (%) measured after pre-incubation
for different times shown in brackets; n.d.=inhibition not detectable.
Activity of hybrids in
breast cancer cells
Therefore, cell culture experiments were repeated with DMA
in combination with either of one of the three HDAC inhibi-
tors: SAHA, MS-275 or 1 (Figure 3). DMA (10 mm) was toxic to-
wards MCF-7 cells after a 72 h treatment, and although the
combination was significantly more effective than HDAC inhibi-
tor alone for SAHA and 1, the combination effect relative to
DMA alone was not significant. In contrast, ER(ꢀ) cells were
less sensitive to both HDAC inhibitors and DMA. Further, the
cytotoxicity of DMA combinations with SAHA and 1 were sig-
nificantly greater than for DMA alone.
The four hydroxamate and one benzamide candidate SERMo-
stats were assayed in MDA-MB-231 cells to measure the influ-
ence of combining the two pharmacophores in a hybrid mole-
cule (Figure 5). Only 15b showed cytotoxicity towards this
ER(ꢀ) cell line, compatible with this being the only candidate
hybrid that demonstrated both HDAC inhibitory and antiestro-
genic activity, that is, SERMostat properties. The concentration
of the SERMostat required to cause 50% cell death of ER(ꢀ)
breast cancer cells was 5–10-fold higher than the IC50 values
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bination therapy in clinical trials led to the design of
candidate hybrid molecules, SERMostats, that had
dual actions as class I HDAC inhibitors and antiestro-
gens. Preliminary experiments were carried out with
combinations of classical HDAC inhibitors and anties-
trogens/SERMs in ER(+) and ER(ꢀ) cell lines. Since it
was planned to design a SERMostat based upon
a benzothiophene scaffold, comparison with combi-
nation treatments including the benzothiophene
SERM, desmethylarzoxifene (DMA), were prioritized.
Contradictory literature reports have appeared on
the efficacy of combination treatment with HDAC in-
hibitors and antiestrogens in ER(+) cells.[12,36] In our
studies, at the concentration of the three HDAC in-
hibitors tested in combination with DMA, only in
Figure 2. Antiestrogenic activity of SERMostat hybrids. a) Concentration dependent anti-
estrogenic activity of SERMostats 8b and 15b tested in an ERE-luciferase reporter assay.
b) The antiestrogenic activity of SERMostat 15b was confirmed in endometrial cells
tested in the alkaline phosphatase assay, IC50 =2.9ꢁ0.3 mm. Data are the mean ꢁSEM
from three cell passages.
Figure 3. Decreased cell viability measured in ER(ꢀ) cells on treatment with
combinations of HDAC inhibitor and DMA after 72 h treatment. HDACi
(SAHA, 1, and entinostat (MS-2-75)) were screened in combination with and
without DMA in a) ER(ꢀ) BT20, and b) ER(+) MCF7 cells. **p<0.001 com-
pared to DMA treatment. Data are the mean ꢁSEM from three cell passages,
analyzed by one way ANOVA with Dunnett’s post test.
Figure 4. Time dependence of cell death caused by treatment with HDAC
inhibitors (SAHA or 1) and DMA in combination and alone, observed in two
ER(ꢀ) cell lines, a,b) BT20, and c,d) MDA-MB-231. Combination treatment
!
(
) led to significant cell death at 72 h, in contrast to treatment with SAHA
measured for the target proteins, HDAC and ERa. Literature
data on combinations of antiestrogens with HDAC inhibitors
also report that cytotoxicity requires concentrations of anties-
trogens (1 mm,[8,11a] 2.5 mm,[15] 10 mm[9b,10b,18,35]) that are higher
than the measured antiestrogen IC50 value at ERa, although in
these examples the cytotoxic concentrations were 1000-fold
higher.
~
!
&
alone ( ) and DMA alone ( ) (a, c). Combination treatment ( ) led to signifi-
cant cell death at 72 h, in contrast to treatment with HDAC inhibitor 1 ( )
&
&
and DMA alone ( ) (b, d). Data are the mean ꢁSEM from three cell passages
analyzed by one way ANOVA with Dunnett’s post test: **p<0.001 compared
*
to DMSO control ( ).
The SERMostat was further compared with combination
treatments using raloxifene as the antiestrogen. At 72 h, cyto-
toxicity was similar for hybrid and combination treatments. In-
terestingly, it was observed that the hybrid molecule was effi-
cacious at causing cell death at 48 h, in contrast to the combi-
nation treatment. Furthermore, cell death at 48 and 72 h was
observed to be significant and dependent on hybrid drug con-
centration.
ER(ꢀ) cells was a significant increase in cell death observed.
This combination effect was seen in two cell lines and with
two different HDAC inhibitors.
Five candidate SERMostats were designed, synthesized, and
assayed for inhibition of HDAC1, 2, 3, and 8. One SERMostat
proved to be antiestrogenic in cell culture and to inhibit
HDAC1, 2, and 3, with IC50 values of 1–3 mm. The extent of cell
killing of ER(ꢀ) breast cancer cells by the SERMostat was
equivalent to combination treatments with 4OHT, raloxifene, or
DMA, despite these SERMs having antiestrogenic potency
higher by 2–3 orders of magnitude. Moreover, the hybrid SER-
Mostat caused significant cell death at an earlier time point
than combination treatments. A number of interesting ap-
Conclusions
The need for therapeutic targeting of ER(ꢀ) breast cancer and
the current use of HDAC inhibitors and antiestrogens as a com-
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3-Bromo-6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene
(3):[27,28] A solution of 6-methoxy-2-(4-methoxyphenyl)benzo[b]thio-
phene (2; 0.880 g, 3.26 mmol) in CHCl3 (35 mL) was heated to
508C until full dissolution, and a solution of elemental Br2 (0.518 g,
3.24 mmol) in CHCl3 (5 mL) was added dropwise over 5 min while
keeping the temperature constant at 508C, then heating was con-
tinued for 5 min, and the yellow solution was left to cool. The
beige crystals that had separated were recrystallized from CH2Cl2/
hexane to give compound 3 as white crystals (0.897 g, 79%):
1H NMR (CDCl3): d=7.73 (d, 1H, Ar-H, J=8 Hz), 7.70 (d, 2H, 2 Ar-H,
J=8 Hz), 7.28 (s, 1H, Ar-H), 7.09 (d, 1H, Ar-H, J=8 Hz), 7.02 (d, 2H,
Ar-H, J=8 Hz), 3.99 (3H, s, OCH3), 3.91 ppm (3H, s, OCH3); 13C NMR
(CDCl3): d=159.42, 157.77, 138.30, 134.95, 132.89, 130.36 (2C),
125.20, 123.78, 114.65, 113.63 (2C), 104.32, 103.25, 55.32,
54.97 ppm.
6-Methoxy-2-(4-methoxyphenyl)benzo[b]thiophene-3-carboxylic
acid (4):[27] A solution of 3 (1.770 g, 5.07 mmol) in anhyd THF
(35 mL) was cooled to ꢀ788C in a dry ice/acetone bath and treat-
ed dropwise with 1.6m nBuLi in hexanes (3.6 mL, 5.76 mmol). After
the addition was complete, the solution set up to a suspension of
the lithiated compound, and stirring at ꢀ788C was continued for
2 h. The reaction mixture was quenched with solid CO2 (5.0 g,
113.6 mmol) and stirred overnight, allowing the temperature to
rise to 208C. 1m NaOH was cautiously added to the white suspen-
sion until all the solid had gone into solution. This aqueous solu-
tion was washed with CH2Cl2 (2ꢁ), and the washes were discarded.
The solution was acidified to <pH 2 with 37% HCl, and the prod-
uct extracted with EtOAc (4ꢁ50 mL). The organic extracts were
washed with H2O and brine, dried over Na2SO4, filtered and con-
centration in vacuo to give the crude material, which was washed
with hexane to give compound 4 as an off-white powder (1.068 g,
67%): 1H NMR (CDCl3): d=8.27 (1H, d, J=8 Hz), 7.49 (2H, d, J=
8 Hz), 7.27 (1H, s, Ar-H), 7.07 (1H, d, J=8 Hz, Ar-H), 6.95 ppm (2H,
d, J=8 Hz, 2 Ar-H); 13C NMR (CDCl3): d=165.69, 159.53, 156.97,
149.37, 139.25, 132.42, 130.40 (2C), 125.93, 125.01, 114.55, 113.08
(2C), 103.60, 55.08, 54.81 ppm.
Figure 5. Cell death in response to treatment with hybrid molecules, combi-
nations, and HDAC inhibitor and antiestrogen components assayed in MDA-
!
~
&
MB-231 ER(ꢀ) cells. a) Of the hybrid molecules (8a: ; 8b: ; 10: ; 15a: ꢀ;
*
15b: ; 10 mm) assayed at 24, 48 and 72 h, only SERMostat 15b caused sig-
*
nificant cell death. b) Comparison of SERMostat 15b ( ) with combination
*
treatment (+) and component treatment with 1 (N) or raloxifene ( ).
c) Dose response (0.1 mm, 1 mm, 10 mm, 25 mm) for SERMostat 15b at 24, 48
and 72 h. ***p<0.001 compared to DMSO control. Data are the mean ꢁSD
from three cell passages analyzed by one way ANOVA with Tukey’s post
test.
proaches to hybrid HDAC inhibitors have been reported, in-
cluding chimeric nuclear receptor ligands,[37] and a separate
hybrid approach to breast cancer.[38] This is the first report of
a SERMostat approach with efficacy in ER(ꢀ) breast cancer
cells and serves as a proof of concept.
Methyl 7-(6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene-3-
carboxamido)heptanoate (5a): A suspension of acid 4 (0.690 g,
2.20 mmol) in anhyd toluene (15 mL) was refluxed with SOCl2
(2.0 mL) for 2 h, then the solvents were evaporated and the green-
ish-brown crystals of the acid chloride were taken up in CH2Cl2 and
added dropwise to a solution of the HCl salt of methyl 6-amino-
heptanoate (0.510 g, 2.61 mmol) and Et3N (3 mL) in anhyd CH2Cl2
(25 mL) under Ar at 08C. The reaction mixture was stirred for 24 h,
washed with H2O and brine, dried on Na2SO4, filtered and concen-
trated. The residual oil was purified by column chromatography on
silica using hexane/EtOAc (7:3 v/v) as eluent affording compound
5a as a slightly yellowish oil that set up as crystals (0.746 g, 75%):
1H NMR (CDCl3): d=7.98 (1H, d, J=8.8 Hz), 7.50 (2H, d, J=8.4 Hz),
7.26 (2H, s), 7.04 (1H, dd, J1 =2.0 Hz, J2 =8.8 Hz), 6.97 (2H, d, J=
8.4 Hz), 5.55 (1H, b, NH), 3.89 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.67
(3H, s, OCH3), 3.32 (2H, m), 2.29 (2H, m), 1.57 (2H, m), 1.40 (2H,
m), 1.26 (2H, m), 1.16 ppm (2H, m); 13C NMR (CDCl3): d=174.07,
165.22, 160.17, 157.70, 140.78, 139.96, 133.06, 130.45 (2C), 127.62,
125.46, 124.55, 114.82, 114.29 (2C), 104.29, 55.59, 55.37, 51.45,
39.53, 33.90, 29.04, 28.73, 26.48, 24.72 ppm; HRMS-ESI: m/z
[M+Na]+ calcd for C25H29NO5S: 478.16590, found: 478.1657.
Experimental Section
Synthetic procedures
All solvents, chemicals, and reagents were purchased from Sigma–
Aldrich or Fisher Scientific unless specified otherwise. Unless stated
otherwise, all reactions were carried out under an atmosphere of
dry argon in oven-dried glassware. Indicated reaction temperatures
refer to those of the reaction bath, while room temperature (RT) is
noted as 238C. CH2Cl2 was distilled over CaH2, and tetrahydrofuran
(THF) distilled over Na(s). All other solvents were of anhydrous
quality and used as received. Isolated reaction products were typi-
cally dried under high vacuum in the presence of phosphorus
pentoxide. Analytical thin-layer chromatography (TLC) was per-
formed with glass-backed silica plates (5ꢁ20 cm, 60 ꢂ, 250 mm).
Visualization was accomplished using a 254 nm UV lamp. 1H and
13C NMR spectra were recorded on either a Bruker Avance 400 MHz
spectrometer or Bruker DPX 400 MHz spectrophotometer. Chemical
shifts are reported in ppm with tetramethylsilane as standard. Data
are reported as follows: chemical shift, number of protons, multi-
plicity (s=singlet, d=doublet, dd=doublet of doublet, t=triplet,
q=quartet, b=broad, m=multiplet, abq=ab quartet), and cou-
pling constants. High-resolution mass spectral data were collected
on a Shimadzu Q-TOF 6500.
Methyl 6-(6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene-3-
carboxamido)hexanoate (5b):
A suspension of 4 (0.260 g,
0.83 mmol) in anhyd toluene (15 mL) was heated to reflux with
SOCl2 (1.0 mL) for 2 h. The solvents were evaporated, and the acid
chloride was taken up in CH2Cl2 and added dropwise to a solution
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of the HCl salt of methyl 6-aminohexanoate (0.366 g, 2.02 mmol)
and Et3N (1.0 mL) in anhyd CH2Cl2 (7.5 mL) under Ar at 08C. The re-
action mixture was stirred for 24 h, washed with H2O and brine,
dried on Na2SO4, filtered and concentrated. The residual oil was pu-
rified by column chromatography on silica using gradient elution
hexane/CH2Cl2 (2:8 v/v)!CH2Cl2!CH2Cl2/MeOH (20:1 v/v) to afford
7.20 (1H, d=2.1 Hz), 6.93 (1H, dd, J1 =8.8 Hz, J2 =2.1 Hz), 6.87 (2H,
d, J=8.8 Hz), 3.66 (3H, s), 3.28 (2H, t, J=7.5 Hz), 2.27 (2H, t, J=
7.8 Hz), 1.56 (2H, m), 1.41 (2H, m), 1.14 ppm (2H, m); 13C NMR
(CDCl3): d=178.37, 170.24, 161.32, 158.35, 144.332, 143.46, 135.73,
133.72, 127.84, 127.30, 119.23, 118.45, 110.40, 99.60, 55.06, 42.94,
37.29, 32.10, 29.77, 27.94 ppm.
1
compound 5b as brownish crystals (0.300 g, 82%): H NMR (CDCl3):
Methyl 7-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-
carboxamido)heptanoate (7b): Acid 6b (0.235 g, 0.57 mmol) was
dissolved in a solution of MeOH (15 mL) and CH3COCl (0.9 mL) and
held at reflux overnight. After evaporation of the solvent, the re-
maining brown oil was purified by column chromatography using
hexane/EtOAc (1:2 v/v) as eluent, yielding compound 7a as a color-
less oil as the main fraction that set up as crystals (0.146 g, 60%):
1H NMR (CDCl3): d=7.78 (1H, d, J=8.8 Hz), 7.32 (2H, d, J=8.5 Hz),
7.15 (1H, d, J=2.2 Hz), 6.90 (1H, dd, J1 =8.8 Hz, J2 =2.2 Hz), 6.83
(2H, d, J=8.5 Hz), 3.64 (3H, s, OCH3), 3.23 (2H, t, CH2, J=6.8 Hz),
3.06 (3H, b), 2.25 (2H, t, J=7.5 Hz), 1.50 (2H, m), 1.32 (2H, m), 1.24
(2H, m), 1.05 ppm (2H, m); 13C NMR (CDCl3): d=208.49 (COO),
175.16 (CO), 166.12, 157.73, 154.74, 140.89, 139.89, 132.18, 130.32
(2C), 129.92, 124.27, 124.05, 115.75 (2C), 114.92, 106.84, 51.63,
39.44, 33.89, 30.78, 28.74, 26.43, 24.61 ppm.
d=7.99 (1H, d, J=8.5 Hz), 7.51 (2H, d, J=8 Hz), 7.28 (2H, s), 7.05
(1H, dd, J1 =2.2 Hz, J2 =8.8 Hz), 6.98 (2H, d, J=8.5 Hz), 5.63 (1H, b,
NH), 3.89 (3H, s, OCH3), 3.87 (3H, s, OCH3), 3.67 (3H, s, OCH3), 3.33
(2H, m), 2.26 (2H, m), 1.58 (2H, m), 1.41 (2H, m), 1.18 ppm (2H,
m); 13C NMR (CDCl3): d=198.82, 173.94, 165.26, 160.22, 157.73,
140.85, 139.99, 133.10, 130.51 (2C), 127.65, 125.48, 124.57, 114.85,
114.32 (2C), 104.32, 55.62, 55.40, 51.50, 39.41, 33.80, 28.96, 26.34,
24.50 ppm.
6-(6-Hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carboxa-
mido)hexanoic acid (6a): A solution of 5a (0.231 g, 0.52 mmol) in
anhyd CH2Cl2 (7 mL) was placed under a blanket of Ar and cooled
to 08C. A 1m solution of BBr3 in CH2Cl2 (2.5 mL, 2.5 mmol) was
added dropwise, and stirring was continued for 5 h letting the so-
lution attain 208C. The orange-brown solution with yellow precipi-
tate was diluted with EtOAc (40 mL), and the product was extract-
ed into saturated NaHCO3 solution (4ꢁ20 mL). The organic phase
was colorless after the extractions. The combined aqueous layers
were washed with EtOAc, and the solution was adjusted pH 1 by
cautious addition of 5m HCl and extracted with EtOAc (4ꢁ30 mL).
The pooled organic solutions were dried on Na2SO4, filtered and
concentrated in vacuo to obtain compound 6a as a brownish clear
glass (0.151 g, 72%): 1H NMR (CDCl3): d=7.71 (1H, d, J=8.7 Hz),
7.32 (2H, d, J=8.4 Hz), 7.15 (1H, d, J=1.8 Hz), 6.90 (1H, dd, J1 =
8.7 Hz, J2 =1.8 Hz), 6.83 (2H, d, J=8.4 Hz), 3.36 Hz (3H, s), 3.24 (2H,
m), 2.21 (2H, t, J=7.5 Hz), 1.51 (2H, m), 1.39 (2H, m), 1.12 ppm
(2H, m); 13C NMR (CDCl3): d=176.58, 166.85, 166.77, 157.70,
154.74, 140.82, 139.88, 132.15, 130.15 (2C), 126.81, 126.77, 124.30,
123.70, 115.70 (2C), 114.90, 106.88, 49.60, 39.56, 39.43, 33.79, 28.52,
26.22, 24.40 ppm.
6-Hydroxy-N-(6-(hydroxyamino)-6-oxohexyl)-2-(4-hydroxyphen-
yl)benzo[b]thiophene-3-carboxamide (8a):
A
suspension of
NH2OH·HCl (4.418 g, 63.6 mmol) in anhyd MeOH (35 mL) was
cooled to 08C under Ar, and a solution of 85% KOH (4.206 g,
63.7 mmol) in MeOH was added slowly with good stirring. Stirring
and cooling were maintained for 5 min, then the solution was fil-
tered to remove precipitated KCl. To the filtrate a solution of ester
7a (0.155 g, 0.37 mmol) in MeOH was added dropwise with cool-
ing to 08C under Ar. After 45 min, the ester had disappeared as in-
dicated by TLC. Stirring was continued for 15 min and the clear yel-
lowish solution was poured into crushed ice (200 g). The solution
was adjusted to pH 7 with acetic acid, and the now colorless solu-
tion was extracted with EtOAc (4ꢁ100 mL). The extracts were
dried on Na2SO4, filtered and concentrated in vacuo, yielding com-
1
pound 8a as an off-white gum (0.152 g, 98%): H NMR (CDCl3): d=
6-(6-Hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-carboxa-
mido)heptanoic acid (6b): A solution of 5b (0.629 g, 1.38 mmol)
in anhyd CH2Cl2 (15 mL) was placed under a blanket of Ar and
cooled to 08C. A 1m solution of BBr3 in CH2Cl2 (5.5 mL, 5.5 mmol)
was added dropwise, and stirring was continued for 20 h letting
the solution warm to 208C. The colorless solution with yellow pre-
cipitate was quenched by the dropwise addition of saturated
NaHCO3 solution, acidified to <pH 2 with 37% HCl and extracted
with EtOAc (4ꢁ30 mL). The combined organic solutions were
washed with brine, dried on Na2SO4, filtered concentrated in
vacuo. After gradient elution on silica gel using CHCl3!CHCl3/8%
MeOH in 4 steps, compound 6b was obtained as yellowish viscous
7.77 (1H, d, J=8.8 Hz), 7.37 (3H, m), 7.20 (1H, d, J=1.1 Hz), 6.94
(1H, dd, J1 =8.6 Hz, J2 =1.1 Hz), 6.88 (2H, d, J=8.3 Hz), 6.44 (1H, b,
NH), 3.24 (2H, m), 2.02 (2H, m, CH2), 1.55 (2H, m, CH2), 1.37 (2H,
m), 1.26 (1H, m), 1.08 ppm (2H, m); 13C NMR (CDCl3): d=174.76,
170.16, 161.17, 158.37, 144.40, 143.50, 135.63, 133.85, 130.53,
127.96, 127.38, 119.32, 118.46, 110.40, 42.83, 36.12, 32.06, 29.76,
28.64 ppm; HRMS-ESI: m/z [M+H]+ calcd for C21H22N2O5S:
415.1322, found: 415.1329.
6-Hydroxy-N-(7-(hydroxyamino)-7-oxoheptyl)-2-(4-hydroxyphe-
nyl)benzo[b]thiophene-3-carboxamide (8b):
NH2OH·HCl (4.422 g, 63.6 mmol) in anhyd MeOH (35 mL) was
cooled to 08C under Ar, and solution of KOH (4.183 g,
A suspension of
a
1
oil (0.263 g, 46%): H NMR (CDCl3): d=7.77 (1H, d, J=8.7 Hz), 7.37
74.6 mmol) in MeOH was added slowly with good stirring. Stirring
was maintained for 5 min, then the solution was filtered. To the fil-
trate consisting of a solution of NH2OH in MeOH, a solution of 7b
(0.140 g, 0.34 mmol) in MeOH was added dropwise with cooling to
08C under Ar. Stirring was continued for 24 h allowing the temper-
ature to rise to 208C. The clear yellowish solution was poured into
crushed ice (200 g), and the solution was adjusted to pH 7 with
acetic acid. The now colorless solution was extracted with EtOAc
(4ꢁ100 mL). The extracts were dried on Na2SO4, filtered and con-
centrated in vacuo, yielding an off-white gum (105 mg). This was
dissolved in acetone containing a few drops of MeOH and treated
with Et2O and then hexane until a precipitate was obtained. After
cooling to ꢀ408C for 15 min, the suspension was filtered. The pro-
cedure was repeated, and the filtrate concentrated in vacuo. Hy-
(2H, d, J=8.5 Hz), 7.20 (1H, d, J=2.0 Hz), 6.94 (1H, dd, J1 =2.2 Hz,
J2 =8.8 Hz), 6.88 (2H, d, J=8.5 Hz), 3.29 (m, 2H, CH2), 2.28 (2H, m,
CH2), 1.56 (2H, m), 1.39 (2H, m), 1.27 (2H, m), 1.14 ppm (2H, m);
13C NMR (CDCl3): d=166.89, 158.06, 155.10, 141.09, 140.21, 132.47,
130.49 (2C), 127.18, 124.58, 124.10, 116.00 (2C), 115.17, 107.11,
39.81, 34.26, 28.99, 28.96, 26.72, 24.95 ppm.
Methyl 6-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-3-
carboxamido)hexanoate (7a): Acid 6a (0.292 g, 0.73 mmol) was
held at reflux in a solution of MeOH (15 mL) and CH3COCl (1 mL)
overnight, the solvents were evaporated, and the crude oily prod-
uct purified on a column of silica using hexane/EtOAc (1:2 v/v) as
eluent. The ester 7a was obtained as a colorless oil (0.159 g, 53%):
1H NMR (CDCl3): d=7.76 (1H, d, J=8.8 Hz), 7.36 (2H, d, J=8.4 Hz),
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droxamate 8b was obtained as a nearly colorless oil (42.2 mg,
30%): 1H NMR (CDCl3): d=7.67 (1H, d, Ar-H), 7.26 (1H, d, J=
8.0 Hz), 7.25 (1H, d, J=8.0 Hz), 7.10 (1H, s), 6.84 (1H, d, J=8.2 Hz),
6.79 (2H, d, J=8.1 Hz), 6.25 (1H, b, NH), 3.15 (2H, m), 1.97 (2H, m),
1.42 ppm (2H, m); 13C NMR (CDCl3): d=193.28, 158.49, 155.99,
143.80, 139.78, 137.35, 132.41, 132.28, 130.61, 129.95, 126.74,
123.98, 116.06, 107.57, 84.72, 83.11 ppm; HRMS-ESI: m/z [M+H]+
calcd for C22H24N2O5S: 429.1479, found: 429.1484.
H), 7.34 (1H, d, J=2.3 Hz), 7.28 (2H, d, J=8.8 Hz), 7.01 (1H, dd,
J1 =8.9 Hz, J2 =2.4 Hz), 6.76 (2H, d, J=8.7 Hz), 3.90 (3H, s, OCH3),
3.78 ppm (3H, s, OCH3); 13C NMR (CDCl3): d=193.50, 160.02,
157.79, 144.60, 140.08, 137.57, 136.85, 133.61, 131.18, 130.47,
129.63, 125.67, 124.08, 115.00, 114.11, 104.51, 101.19, 55.63,
55.31 ppm.
[6-Methoxy-2-(4-methoxyphenyl)benzo[b]thiophen-3-yl][4-[(tri-
methylsilyl)ethynyl]phenyl] methanone (12): To a degassed solu-
tion of 11 (7.06 g, 14.01 mmol) in anhyd THF (100 mL) and Et3N
(20 mL) was added (Ph3P)2PdCl2 (0.101 g, 0.14 mmol) and CuI
(0.086 g, 0.45 mmol) followed by ethynyltrimethylsilane (5.3 mL,
37.5 mmol), and the solution was stirred at RT for 3 days. The re-
sulting dark suspension was filtered, concentrated in vacuo and
purified on a silica column using hexane/EtOAc (3:1 v/v) as eluent,
yielding protected alkyne 12 as a brownish solid (5.1 g, 77%):
1H NMR (CDCl3): d=7.72 (2H, d, J=8.4 Hz, Ar-H), 7.65 (1H, d, J=
8.9 Hz, Ar-H), 7.33–7.37 (3H, m), 7.31 (2H, d, J=8.8 Hz), 7.00 (1H,
dd, J1 =8.9 Hz, J2 =2.4 Hz), 6.74 (2H, d, J=8.8 Hz), 3.88 (3H, s,
OCH3), 3.75 (3H, s, OCH3), 0.26 ppm (9H, s, SiMe3); 13C NMR (CDCl3):
d=193.41, 159.94, 157.77, 144.45, 140.11, 136.95, 133.73, 131.80,
130.46, 129.86, 129.65, 127.75, 125.75, 124.13, 114.97, 114.09,
104.50, 104.20, 55.60, 55.22, 0.28 ppm.
6-Methoxy-N-(4-methoxy-[1,1’-biphenyl]-3-yl)-2-(4-methoxyphe-
nyl)benzo[b]thiophene-3-carboxamide (9): A suspension of 4
(0.441 g, 1.40 mmol) in anhyd toluene (20 mL) was treated with
SOCl2 (1.3 mL) and held at reflux for 3 h. After 0.5 h, a clear solution
was obtained. The solvents were evaporated, and evaporation was
repeated with the addition of fresh toluene to drive off residual
traces of SOCl2. The acid chloride that was obtained as a brown
solid in quantitative yield was taken up in CH2Cl2 (4 mL) and was
added dropwise into a solution of 4-methoxy-(1,1’-biphenyl)-3-
amine (0.711 g, 3.57 mmol) and Et3N (1.5 mL) in anhyd CH2Cl2
(6 mL) held at 08C under Ar. The reaction was stirred for 3 days at
208C, diluted with CH2Cl2 (15 mL), washed with 0.5m NaOH, 0.5m
HCl, brine and finally dried on Na2SO4, filtered and concentrated in
vacuo. The brown oil was purified by column chromatography on
a silica column using hexane/EtOAc (7:3 v/v) as eluent. Compound
9 was obtained as a light amber foam (0.40 g, 58%): 1H NMR
(CDCl3): d=8.94 (b, NH), 8.26 (d, 1H, J=8.2 Hz), 8.00 (1H, s), 7.68
(d, 2H, J=6.8 Hz), 7.57 (d, 2H, J=6.8 Hz), 7.45 (d, 2H, J=6.8 Hz),
7.35 (d, 1H, J=6.5 Hz), 7.30 (m, 2H), 7.10 (d, 1H, J=8.8 Hz), 6.97
(2H, d, J=6.8 Hz), 6.83 (1H, d, J=8.0 Hz), 3.91 (3H, s), 3.84 (3H, s),
3.57 ppm (3H, s); 13C NMR (CDCl3): d=162.31 (CO), 160.01, 157.41,
147.07, 142.24, 140.36, 139.63, 133.78, 132.81, 130.57 (2C), 128.29
(2C), 127.66, 127.06, 126.90 (2C), 126.63, 124.82, 124.68, 121.72,
118.04, 114.62, 113.99 (2C), 109.66, 103.89, 55.22, 55.12, 54.98 ppm.
[6-Hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophen-3-yl][4-[(trime-
thylsilyl)ethynyl]phenyl] methanone (13a): A solution of protect-
ed alkyne 12 (3.704 g, 7.85 mmol) in anhyd CH2Cl2 (60 mL) was
cooled to 08C in an ice bath under inert gas atmosphere, and
a 1m solution of BBr3 in CH2Cl2 (36 mL) was added dropwise. The
solution turned dark and was stirred for 20 h, poured cautiously
into a mixture of saturated NaHCO3 solution (150 mL) and ice
(100 g) and extracted with EtOAc (3ꢁ100 mL) after stirring for
20 min. The combined organic solutions were washed with H2O
and brine, then concentrated in vacuo to a dark oil, which was pu-
rified on a column of silica using CH2Cl2/hexane (8:2 v/v) as eluent
to give protected alkyne 13a as a yellow solid (2.580 g, 74%). The
compound was deprotected without characterization.
6-Hydroxy-N-[4-hydroxy-(1,1’-biphenyl)-3-yl]-2-(hydroxyphenyl)-
benzo[b]thiophene-3-carboxamide (10): A solution of 9 (0.32 g,
0.65 mmol) in anhyd CH2Cl2 (3 mL) was cooled to 08C under Ar
and treated dropwise with a 1m BBr3 solution in CH2Cl2 (4.4 mL,
4.4 mmol). The brownish yellow suspension was stirred for 18 h
while letting the temperature rise slowly to 208C. The reaction mix-
ture was quenched by the dropwise addition of saturated NaHCO3
solution and extracted with EtOAc (3ꢁ20 mL). The pooled organic
solutions were washed with brine, dried on Na2SO4, filtered and
concentrated in vacuo. Purification by column chromatography on
silica using hexane/EtOAc (6:4 v/v) as eluent afforded compound
10 as a light beige solid (0.160 g, 54%): 1H NMR ([D6]DMSO): d=
9.89 (1H, bs, OH), 9.82 (1H, bs, OH), 9.76 (1H, s, OH), 9.27 (1H, s,
NH), 8.08 (1H, d, J=2.1 Hz), 7.77 (1H, d=8.7 Hz), 7.57 (d, 2H, J=
7.4 Hz), 7.45 (4H, m), 7.31 (3H, m), 6.85–6.96 (2H, m), 6.84 ppm
(2H, d, J=8.6 Hz); 13C NMR ([D6]DMSO): d=163.85, 158.13, 155.40,
148.20, 140.11, 139.09, 131.81, 129.91, 128.97, 127.18, 126.73,
126.14, 123.78, 123.57, 120.91, 116.16, 115.84, 107.02 ppm; HRMS-
ESI: m/z [M+H]+ calcd for C27H19NO4S: 454.11076, found 454.1122.
(4-Ethynylphenyl)[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thio-
phen-3-yl]methanone (13b):
A
solution of 13a (2.570 g,
5.81 mmol) in MeOH (50 mL) was treated with a solution of
KF·2H2O (1.797 g, 19.10 mmol) in H2O (15 mL), then stirred at 208C
for 4 days. After extractive workup with CH2Cl2 and H2O and
column chromatography using hexane/EtOAc (6:4 v/v) as eluent,
compound 13a was obtained as dark yellow solid (1.959 g, 91%):
1H NMR ([D6]DMSO): d=9.81 (1H, s, OH), 9.75 (1H, s, OH), 7.63 (2H,
d, J=8.4 Hz, Ar-H), 7.43 (1H, d, J=8.4 Hz), 7.42 (1H, d, J=8.4 Hz),
7.36 (1H, d, J=2.1 Hz), 7.12 (2H, d, J=8.6 Hz), 6.89 (2H, dd, J1 =
8.8 Hz, J2 =2.2 Hz), 6.64 (2H, d, J=8.6 Hz), 4.45 ppm (1H, s, CH);
13C NMR ([D6]DMSO): d=193.28, 158.49, 155.99, 143.80, 139.78,
137.35, 132.41, 132.28, 130.61, 129.95, 126.74, 123.98, 116.06,
107.57, 84.72, 83.11, 49.04 ppm; HRMS-ESI: m/z [M+H]+ calcd for
C23H14O3S: 371.0742, found: 371.0735.
Ethyl 5-(4-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene-
3-carbonyl)phenyl)-1H-1,2,3 triazol-1-yl)pentanoate (14a): A so-
lution of alkyne 13b (0.340 g, 0.92 mmol), ethyl-5-azidopentanoa-
te[32c] (0.274 g, 1.60 mmol), CuSO4 (7.4 mg, 46 mmol), ascorbic acid
(29 mg, 165 mmol) in THF (3 mL) and H2O (2 mL) was stirred at RT
for 24 h, at which point some unreacted alkyne was detected by
TLC. After stirring was continued for 20 h at 508C, the reaction
mixture was extracted with CH2Cl2 (3ꢁ5 mL). The organic solutions
were washed with H2O, dried on Na2SO4, filtered and concentrated
in vacuo. After column chromatography on silica (6:4 v/v hexane/
EtOAc), compound 14a was obtained as a bright canary yellow
(4-Iodophenyl)(6-methoxy-2-(4-methoxyphenyl)benzo[b]thio-
phen-3-yl)methanone (11):[31] Anhyd AlCl3 (4.83 g, 36.2 mmol) was
added in three portions to a suspension of compound 2 (6.5 g,
24.0 mmol) and 4-iodobenzoyl chloride (6.5 g, 24.0 mmol) in anhyd
CH2Cl2 (100 mL), and the deep red solution was stirred for 24 h at
RT, poured into ice water (400 mL) and extracted with CH2Cl2 4ꢁ
80 mL). The pooled organic extracts were washed with H2O and
brine, concentrated in vacuo, and the crude product was purified
by column chromatography using hexane/EtOAc (8:2 v/v) as
eluent. Compound 11 was obtained as a yellow solid (7.24 g, 60%):
1H NMR (CDCl3): d=7.63 (3H, m, Ar-H), 7.47 (2H, d, J=8.5 Hz, Ar-
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1
solid (0.239 g, 50%): H NMR ([D6]DMSO): d=9.79 (1H, s, OH), 9.69
added, and the red reaction mixture was stirred for 24 h at RT.
After the addition of ice (300 g), the solution was adjusted to pH 5
with 1m HCl, and the light yellow solution was extracted with
EtOAc (3ꢁ80 mL). The pooled extracts were washed with 0.5m
HCl, H2O and brine, dried over Na2SO4, filtered and concentrated in
vacuo, leaving the crude product as an oil (0.441 g). Purification by
gradient elution on a column of silica gel using CH2Cl2/MeOH
(10%!15%!20% v/v) as eluent afforded compound 15b as
(1H, s, OH), 8.67 (1H, s, triazole H), 7.82 (2H, d, J=8.5 Hz, Ar-H),
7.74 (2H, d, J=8.5 Hz, Ar-H), 7.38 (1H, d, J=8.8 Hz), 7.36 (1H, d,
J=2.1 Hz), 7.16 (1H, d, J=2.1 Hz), 7.12 (2H, d, J=8.6 Hz), 6.88 (2H,
dd, J1 =8.8 Hz, J2 =2.2 Hz), 6.64 (2H, d, J=8.6 Hz), 4.40 (2H, t, J=
6.6 Hz, CH2), 4.02 (2H, q, J=7.2 Hz, CH2), 2.33 (2H, t, J=6.6 Hz,
CH2), 1.86 (2H, m, CH2), 1.52 (2H, m, CH2), 1.15 ppm (3H, t, J=
7.2 Hz, CH3); 13C NMR ([D6]DMSO): d=173.00, 158.64, 155.93,
145.55, 139.78, 135.81, 132.59, 130.67, 130.46, 129.69, 125.45,
124.14, 123.93, 123.15, 116.10, 115.76, 107.58, 60.20, 49.71, 33.16,
29.32, 21.80, 14.52 ppm.
1
a yellow oil that set up as crystals (0.371 g, 68%): H NMR (CDCl3):
d=10.33 (1H, s, OH), 9.81 (1H, s, OH), 9.72 (1H, s, NH), 8.67 (1H, s,
Ar-H), 7.83 (2H, d, J=8.4 Hz), 7.75 (2H, d, J=8.4 Hz, Ar-H), 7.39
(1H, d, J=8.8 Hz), 7.37 (1H, d, J=2.1 Hz), 7.17 (2H, d, J=8.6 Hz),
6.89 (1H, dd, J1 =8.8 Hz, J2 =2.2 Hz), 6.66 (2H, d, J=8.6 Hz), 4.38
(2H, t, J=7.2 Hz), 4.10 (2H, q, J=5.2 Hz), 1.94 (2H, t, J=7.3 Hz,
CH2), 1.85 (2H, m, CH2), 1.53 (2H, m, CH2), 1.23 ppm (2H, m, CH2);
13C NMR (CDCl3): d=193.56, 169.37, 158.37, 155.94, 145.54, 142.73,
139.79, 136.46, 135.85, 132.60, 130.67, 129.69, 125.42, 124.15,
123.93, 123.07, 116.10, 115.76, 107.58, 49.93, 49.04, 32.45, 29.69,
25.84, 24.90 ppm. HRMS-ESI: m/z [M+H]+ calcd for C29H26N4O5S:
543.1702, found: 543.1718.
Methyl
6-(4-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thio-
phene-3-carbonyl)phenyl)-1H-1,2,3-triazol-1-yl)hexanoate (14b):
A solution of alkyne 13b (0.549 g, 1.61 mmol), methyl 6-azidohexa-
noate (0.493 g, 2.88 mmol), CuSO4 (0.044 g, 0.28 mmol) and ascor-
bic acid (0.181 g, 1.03 mmol) in THF (6 mL) and H2O (1 mL) was
stirred at RT for 24 h. After the addition of water (10 mL), the solu-
tion was extracted with EtOAc (3ꢁ8 mL). The organic solutions
were combined, washed with H2O, dried on Na2SO4, filtered and
concentrated in vacuo. After column chromatography (6:4 v/v
hexane/EtOAc), ester 14b was obtained as a yellow gum (0.623 g,
1
76%): H NMR (CDCl3): d=9.80 (1H, s, OH), 9.70 (1H, s, OH), 8.67
Biology
(1H, s, NH), 7.82 (2H, d, J=8.5 Hz), 7.75 (2H, d, J=8.5 Hz, Ar-H),
7.39 (1H, d, J=8.8 Hz), 7.37 (1H, d, J=2.1 Hz), 7.17 (2H, d, J=
8.6 Hz), 6.89 (1H, dd, J1 =8.8 Hz, J2 =2.2 Hz), 6.66 (2H, d, J=8.6 Hz),
4.38 (2H, t, J=7.2 Hz), 4.10 (2H, q, J=5.2 Hz), 1.94 (2H, t, J=
7.3 Hz, CH2), 1.85 (2H, m, CH2), 1.53 (2H, m, CH2), 1.23 ppm (2H, m,
CH2); 13C NMR (CDCl3): d=193.56, 169.37, 158.37, 155.94, 145.54,
142.73, 139.79, 136.46, 135.85, 132.60, 130.67, 129.69, 125.42,
124.15, 123.93, 123.07, 116.10, 115.76, 107.58, 49.93, 49.04, 32.45,
29.69, 25.84, 24.90 ppm.
Cell lines and cell culture conditions: MCF-7, BT20 and MDA-MB-231
cells were obtained from American Type Culture Collection (Mana-
ssas, VA, USA). MCF-7 were cultured in phenol red containing RPMI
1640 media supplemented with 10% fetal bovine serum, 1% gluta-
max, 1% non-essential amino acids, insulin (6 mgmLꢀ1), 1% antibi-
otic-antimycotic and 5% CO2 at 378C. Treatment media was pre-
pared by supplementing phenol red-free RPMI 1640 media with
charcoal-dextran treated fetal bovine serum whereas other supple-
ments remained the same. BT20 and MDA-MB-231 cells were cul-
tured in phenol red-containing DMEM media supplemented with
10% fetal bovine serum and 1% penicillin-streptomycin and were
maintained in 5% CO2 at 378C. Treatment media for both cell lines
was prepared by supplementing phenol red-free DMEM media
with 10% charcoal-dextran treated fetal bovine serum and 1%
penicillin-streptomycin.
N-Hydroxy-5-(4-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thio-
phene-3-carbonyl)phenyl)-1H-1,2,3-triazol-1-yl)pentanamide
(15a):[29] A solution of hydroxylamine freebase in anhyd MeOH was
generated from 85% KOH (4.90 g, 74.2 mmol) and NH2OH·HCl
(5.16 g, 74.2 mmol) in MeOH (100 mL), and the solution was fil-
tered free of the formed KCl.
A solution of 14a (0.142 g,
0.26 mmol) was added, and the red reaction mixture was stirred
for 24 h at RT. The solution was poured into ice water (300 mL),
the solution was adjusted to pH 2 with 1m HCl, and the light
yellow solution was extracted with EtOAc (3ꢁ80 mL). The pooled
extracts were washed with brine then dried over Na2SO4, filtered
and concentrated in vacuo. The crude product was purified by gra-
dient elution on a column of silica gel using CH2Cl2/MeOH (10%!
15%!20% v/v) as eluent, affording compound 15a as bright
canary yellow crystals (0.099 g, 72%): 1H NMR (CDCl3): d=10.37
(1H, s, OH), 9.82 (1H, s, OH), 9.75 (1H, b, NH), 8.67 (1H, s, Ar-H),
7.83 (2H, d, J=8.5 Hz), 7.75 (2H, d, J=8.5 Hz, Ar-H), 7.39 (1H, d,
J=8.8 Hz), 7.36 (1H, d, J=2.1 Hz), 7.17 (2H, d, J=8.6 Hz), 6.89 (1H,
dd, J1 =8.7 Hz, J2 =2.2 Hz), 6.65 (2H, d, J=8.6 Hz), 4.39 (2H, t, J=
7.2 Hz), 4.10 (2H, d, J=5.2 Hz), 1.99 (2H, t, J=7.3 Hz, CH2), 1.83
(2H, m, CH2), 1.48 ppm (2H, m, CH2); 13C NMR (CDCl3): d=193.55,
169.37, 158.38, 155.95, 145.53, 142.75, 139.78, 136.47, 135.82,
132.59, 130.67, 129.68, 125.42, 124.14, 123.92, 123.14, 116.10,
115.77, 107.58, 49.76, 49.03, 31.98, 29.57, 22.52 ppm; HRMS-ESI:
m/z [M+H]+ calcd for C28H24N4O5S: 529.1546, found: 529.1555.
Cytotoxicity assay: The CellTiter 96 AQueous One Solution Cell Prolif-
eration Assay (MTS) was purchased from Promega. BT20 and MDA-
MB-231 cells were plated at a density of 5ꢁ103 cells/well in 96-well
plates overnight. Cells were treated with test compounds on the
next day, and cell viability was measured at 24, 48 and 72 h using
the MTS reagent (Promega) according to the manufacturer’s in-
structions. MCF-7 cells were plated at a density of 1ꢁ104 cells/well
in 96-well plates and the above procedure was repeated. Absorb-
ance at 490 nm was measured using the Synergy H4 Hybrid Multi-
Mode Microplate Reader (Bio-Tek). Data is representative of three
individual experiments.
HDAC activity assay: Carried out as described in published detailed
experimental procedures using recombinant HDAC1, 2, 3 and
8.[29,39] Stock solutions of human recombinant HDAC1, 2 and 3 (BPS
Bioscience) and HDAC8 (purified from E. coli) were prepared in
assay buffer 1 (25 mm Tris-HCl, pH 8.0, 137 mm NaCl, 2.7 mm KCl,
1 mm MgCl2, and 1 mgmLꢀ1 BSA). Serial dilutions of the probes
were made in assay buffer 2 (25 mm Tris-HCl, pH 8.0, 137 mm NaCl,
2.7 mm KCl, 1 mm MgCl2). Enzyme was added to the probes in 96-
well opaque half-area microplates (Corning) and preincubated for
different preincubation times. It was observed that the Z-factor for
the assays remained above 0.7 up to 3 h for HDAC1, 3 and 8 and
up to 24 h for HDAC2, hence these preincubation times were
chosen for the assays. After preincubation, HDAC fluorescent sub-
N-Hydroxy-6-(4-(4-(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thio-
phene-3-carbonyl)phenyl)-1H-1,2,3-triazol-1-yl)hexanamide
(15b): A solution of hydroxylamine in anhyd MeOH was generated
from 85% KOH (5.96 g, 90.3 mmol) and NH2OH·HCl (5.91 g,
85.0 mmol) in MeOH (150 mL), and the solution was filtered free of
the formed KCl. A solution of ester 14b (0.545 g, 1.01 mmol) was
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strate Boc-l-Lys(Ac)-AMC (Chem-Impex) in case of HDAC1, 2 and 3
and BML-KI-178 (Biomol Inc.) in case of HDAC8 was added, and the
mixture incubated for 35 min (HDAC1, 3, 8) or 60 min (HDAC2) at
RT. The reaction was quenched with trypsin (1 mgmLꢀ1) and tri-
chostatin A (5 mm) in assay buffer 1 (25 mm Tris-HCl, pH 8.0,
137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2) and further incubated at
RT for 35 min. Plates were read on the Synergy microplate reader
at excitation wavelength 360 nm and emission wavelength
460 nm. The IC50 values were determined using the GraphPad
Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA).
training set consisting of 20 molecules with known HDAC2 inhibi-
tion was used to benchmark the obtained scores.[25] The same ap-
proach was applied to ERa binding using crystal structure of
PDB code 1ERR.[26]
Acknowledgements
The work was supported in part by the US National Institutes of
Health (NIH) (grant no. R01 CA102590 (GT) and R01 CA131970
(PP)). Molecular modeling was in part conducted using the UCSF
Chimera package from the Resource for Biocomputing, Visualiza-
tion, and Informatics at the University of California, San Francis-
co (supported by US NIH P41 RR001081). Huali Dong (University
of Illinois at Chicago (UIC), USA) is acknowledged for her assis-
tance with the Ishikawa assay.
Ishikawa assay: The procedure described previously was used to
run this assay.[22b] Briefly, Ishikawa cells (5ꢁ104 cells/mL) were
plated in 96-well plates and incubated overnight in estrogen-free
media. Cells were treated with the hybrid molecules with 17b es-
tradiol and the appropriate controls at 378C for 4 days. Alkaline
phosphatase enzyme activity was measured by reading the libera-
tion of p-nitrophenol from Mp-nitrophenylphosphate at 340 nm
every 15 s for 16–20 readings using the Synergy microplate reader.
The maximum slope of the lines generated by the kinetic readings
was calculated using a Kinecalc computer program (Bio-Tek Instru-
ment). The reduction in percent induction as compared to DMSO
control was used to obtain antiestrogenic activity according to
Equation (1):
Keywords: breast cancer
· estrogen receptors · HDAC
inhibitors · histone deacetylases · SERMs
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½1ꢀ½ðslopesampleꢀslopecellsÞ=ðslopeestrogenꢀslopecellsÞꢂꢂ ꢃ 100
ð1Þ
Inhibition of 17b estradiol-induced luciferase activity: Antiestrogenic
activity for ERa was measured using MCF7 cells, which were kept
in stripped media three days prior to treatment. Cells were plated
(4ꢁ105 cells) in 12-well plates and were transfected with pERE-luci-
ferase plasmid (3 mg), which contains three copies of the Xenopus
laevis vitellogenin A2 ERE upstream of firefly luciferase. To normal-
ize for cell viability and transfection efficiency, pRL-TK plasmid
(1 mg; Promega, Madison, WI, USA) containing a cDNA encoding
Renilla luciferase was co-transfected along with the ERE plasmid.
Transfection was performed overnight using Lipofectamine 2000
(Invitrogen), in Opti-MEM media according to the manufacturer’s
instructions. Cells were treated with test compounds, and the luci-
ferase activity was measured in cell lysates using the Dual Lucifer-
ase assay system (Promega) with FLUOstar OPTIMA (BMG LABTECH,
Durham, NC). Antiestrogenic activity of the active hybrid molecule
was studied by co-treating the hybrid molecule with E2 to observe
inhibition of E2 response. Data is representative of three individual
experiments reported as the relative luciferase percentage with
1 nm E2 treatment set as 100% and initial luciferase activity values
were calculated by dividing the firefly luciferase (ERE) reading by
the Renilla luciferase (pRL-TK) reading.
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Molecular modeling
Coordinates of HDAC2 and ERa protein structures were download-
ed from the protein data bank (PDB).[26,40] Visual inspection of the
crystal structures of PDB codes 1ERR and 3MAX was performed
using Chimera.[41] Conserved water molecules near the binding site
were included and allowed to be toggled on and off during dock-
ing. MOE was used to prepare ligands and for protonation of the
water oxygen atoms after fixing of protonation states of the pro-
tein. After energy minimization in MOE, the docking of 10 confor-
mations per molecule to the crystal structure of HDAC2 (PDB code:
3MAX)[25] was completed, scoring the obtained binding poses with
MOE using standard parameters and calculating binding affinities
[12] L. Hodges-Gallagher, C. D. Valentine, S. El Bader, P. J. Kushner, Breast
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for the highest-scoring poses using the Amber 12 forcefield.[42]
A
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[17] O. Witt, H. E. Deubzer, T. Milde, I. Oehme, Cancer Lett. 2009, 277, 8–21.
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Received: June 15, 2013
Published online on && &&, 0000
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ChemMedChem 0000, 00, 1 – 13
&12
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These are not the final page numbers!

FULL PAPERS
Two in one: The combination of HDAC
inhibition and selective estrogen recep-
tor modulation caused time-dependent
killing of breast cancer cells. A hybrid
molecule, acting as HDAC inhibitor and
selective estrogen receptor modulator
(SERM), has potential, but can both
activities be retained in killing ER(ꢀ)
breast cancer cells?
H. K. Patel, M. I. Siklos, H. Abdelkarim,
E. L. Mendonca, A. Vaidya, P. A. Petukhov,
G. R. J. Thatcher*
&& – &&
A Chimeric SERM–Histone Deacetylase
Inhibitor Approach to Breast Cancer
Therapy
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 13
&13
&
ÞÞ
These are not the final page numbers!