Incorporation of histone deacetylase inhibitory activity into the core of tamoxifen – A new hybrid design paradigm

Article abstract of DOI:10.1016/j.bmc.2018.07.026

Hybrid antiestrogen/histone deacetylase (HDAC) inhibitors were designed by appending zinc binding groups to the 4-hydroxystilbene core of 4-hydroxytamoxifen. The resulting hybrids were fully bifunctional, and displayed high nanomolar to low micromolar IC<

Full text of DOI:10.1016/j.bmc.2018.07.026

Accepted Manuscript
Incorporation of histone deacetylase inhibitory activity into the core of tamox-
ifen - a new hybrid design paradigm
Anthony F. Palermo, Marine Diennet, Mohamed El Ezzy, Benjamin M.
Williams, David Cotnoir-White, Sylvie Mader, James L. Gleason
PII:
S0968-0896(18)30433-4
https://doi.org/10.1016/j.bmc.2018.07.026
BMC 14463
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
23 March 2018
5 July 2018
14 July 2018
Please cite this article as: Palermo, A.F., Diennet, M., El Ezzy, M., Williams, B.M., Cotnoir-White, D., Mader, S.,
Gleason, J.L., Incorporation of histone deacetylase inhibitory activity into the core of tamoxifen - a new hybrid
design paradigm, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.026
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Incorporation of histone deacetylase
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inhibitory activity into the core of tamoxifen
-
a new hybrid design paradigm
Anthony F. Palermo, Marine Diennet, Mohamed El Ezzy, Benjamin M. Williams, David Cotnoir-White, Sylvie
Mader* and James L. Gleason*
Department of Chemistry, McGill University, 801 Sherbrooke W., Montreal, QC, Canada, H3A 0B8

Bioorganic & Medicinal Chemistry
Incorporation of histone deacetylase inhibitory activity into the core of tamoxifen - a
new hybrid design paradigm.
1
,5
2,5
2
1
Anthony F. Palermo, Marine Diennet, Mohamed El Ezzy, Benjamin M. Williams, David Cotnoir-
2
,2,3,4
,1
White, Sylvie Mader*
and James L. Gleason*
1
Department of Chemistry, McGill University, 801 Sherbrooke W., Montreal, QC, Canada, H3A 0B8
Institute for Research in Immunology and Cancer, Pavillon Marcelle-Coutu, Université de Montréal, 2950 chemin de Polytechnique, Montréal, QC, Canada,
2
H3T 1J4
3
Biochemistry Department, Pavillon Roger-Gaudry, Université de Montréal, 2900 Bd Edouard Montpetit, Montréal, QC, Canada, H3T 1J4
Centre de Recherche du CHUM, Université de Montréal, Montréal, QC, Canada, H2X 0A9
These authors contributed equally to this manuscript.
4
5
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Hybrid antiestrogen / histone deacetylase (HDAC) inhibitors were designed by appending zinc
binding groups to the 4-hydroxystilbene core of 4-hydroxytamoxifen. The resulting hybrids
were fully bifunctional, and displayed high nanomolar to low micromolar IC50 values against
both the estrogen receptor  (ER) and HDACs in vitro and in cell-based assays. The hybrids
were antiproliferative against ER+ MCF-7 breast cancer cells, with hybrid 28b possessing an
improved activity profile compared to either 4-hydroxytamoxifen or SAHA. Hybrid 28b
displayed gene expression patterns that reflected both ER and HDAC inhibition.
Available online
Keywords:
Keyword_1
Keyword_2
Keyword_3
Keyword_4
Keyword_5
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
effects on estrogen signaling. In breast, it antagonizes estrogen-
induced growth, while it has agonist activity for expression of
estrogen target genes in uterine cells. Tamoxifen itself has low
affinity for ERs and acts mainly as a prodrug. It is oxidized in
vivo to several active metabolites, including 4-hydroxytamoxifen
Estrogens, mainly 17-estradiol (E2, 1, Fig 1), are the primary
6-9
hormones responsible for the development of female secondary
sexual characteristics, including normal growth of the mammary
gland. E2 genomic signalling occurs mainly through estrogen
1
(
4-OHT, 3) and endoxifen, which have potent antiproliferative
receptor- and - (ER and ER), members of the nuclear2
receptor superfamily of ligand-activated transcription factors.
Binding of E2 to ERs results in a conformational change that
involves the folding of helix 12 (H12) over the ligand binding
pocket (LBP), which induces receptor binding to DNA at
estrogen response elements (EREs) located in the regulatory
regions of target genes, release of transcriptional co-repressors,
and the recruitment of co-activators and transcription machinery.
10, 11
activities in ER+ breast cancer cells in vitro.
Tamoxifen is
used in first line endocrine therapy of all stages of ER+ breast
tumors, especially in pre-menopausal women as aromatase
inhibitors have demonstrated superior efficacy in the post-
12
menopausal setting. Tamoxifen has an overall clinical response
rate of about 50%, although it is less effective in metastatic
12,13,12, 14
cases.
Unfortunately, relapse in patients with primary
tumors can occur years after treatment, suggesting incomplete
eradication of tumor cells and benefit from extension of
Expression of ER, which is observed in about 70% of breast
1
5
hormonal therapy to 10 years instead of five. A second class of
antiestrogens called pure antiestrogens or selective estrogen
receptor down-regulators (SERDs) are devoid of the partial
agonist activity of tamoxifen in the uterus and possess the ability
to induce SUMOylation, ubiquitination, and degradation of
tumors, mediates the growth-stimulatory effects of estrogens on
3-5
these tumors. Efforts to inhibit E2-mediated tumor growth have
led to the development of ER antagonists as therapeutic tools for
ER+ breast cancer. The most commonly employed antiestrogen
(AE) has been tamoxifen (2), which is classified as a selective
16-18
ER.
The SERD fulvestrant (5) has proven beneficial as a
estrogen receptor modulator (SERM) for its tissue-specific

second line therapy for patients that have previously undergone
1
9, 20
hormonal treatment.
Figure 2 Structures of antiestrogen / HDACi hybrids.
Figure 1. Structures of antiestrogens and HDAC inhibitors.
2. Hybrid Design and Synthesis
The steroidal, 4-hydroxystilbene, or 2-arylbenzothiophene
cores of antiestrogens mainly provide affinity for the LBP. We
have observed with vitamin D/HDACi hybrids that groups that
Histone deacetylases (HDACs) function as transcriptional co-
regulators, modulating in combination with histone acetyl
transferases the acetylation state of histones and the accessibility
provide HDACi function can be accommodated by the LBP of
21
41-44
of DNA in chromatin. In addition, HDACs are also known to
deacetylate non-genomic targets such as tubulin, HSP90, and
the vitamin D receptor (VDR).
Given the similarity between
nuclear receptor binding pockets we therefore postulated that it
might be possible to incorporate HDACi function into the core of
an antiestrogen without significantly affecting affinity for ER,
allowing the antiestrogenic side-chain to remain unmodified and
22
p53. HDACs are overexpressed in many cancers, including
23, 24
breast cancer.
Several HDAC inhibitors (HDACi’s) are
clinically approved for blood cancer indications and have been
investigated in combination with other agents for use in solid
4
5
retain full functionality.
2
5,26,27
tumors, including breast cancer.
The prototype of this class
The phenol of 4-OHT mimics the A-ring phenol of E2,
is suberoylanilide hydroxamic acid (SAHA, 6, Fig 1), which has
46
2
8
forming hydrogen bonds to Glu353 and Arg394. While E2
possesses a second hydroxyl group in the D-ring that engages in
been approved for treatment of cutaneous T-cell lymphoma.
47
Several studies have shown a combinatorial effect of
HDACi’s and antiestrogens in breast cancer. Tamoxifen
exhibited cooperativity with several HDACi’s to inhibit growth
a hydrogen bond with His524, the remaining aromatic ring in 4-
OHT remains unoxidized and thus appeared to be a potential
position to incorporate polar functionality - indeed raloxifene
places a second phenolic OH in this vicinity. Additionally, while
many residues lining the ER binding pocket show little
positional variation among X-ray crystal structures of various
estrogens and antiestrogens, His524 is mobile and can
accommodate different positioning of hydroxyl groups, as in
29, 30
of ER+ MCF-7 breast cancer in vitro and in vivo.
Other
studies have shown combinatorial effects of antiestrogens and
3
1-33
HDACi’s in both ER+ and ER- breast cancer cell lines.
Moreover, the combination of tamoxifen and SAHA was shown
in a phase II study to have a 40% clinical benefit for patients with
3
4
48
49
ER+ tumors that had progressed during endocrine therapy.
raloxifene, and bulkier groups as in 2-arylindole antagonists.
We sought to exploit this flexibility by developing hybrids which
attach HDACi function to the B-ring of 4-OHT. The potential
advantage of this design is that it would not require alteration of
the side-chain that is essential for antiestrogen function.
Moreover, metabolic inactivation of the HDACi unit would not
be expected to alter the antiestrogenic character of the molecules.
Based on the synergy between antiestrogens and HDACi’s,
several groups including ours have investigated hybrid structures
3
5-
that combine both biochemical activities in a single molecule.
39
Our previous work incorporated HDACi function in the side-
35
chain of fulvestrant (7, Fig 2). Other hybrids have also
incorporated HDACi function in the side-chains of raloxifene (8)
37-39
and tamoxifen (9).
While all these hybrids possessed
The hybrids were prepared using two separate routes. Hybrid
antiproliferative activity, they were generally less potent than
standard monotherapies. For example, fulvestrant hybrid 7
displayed antiproliferative activity in both ER+ MCF-7 cells and
in ER- MDA-MB-231 cells, but was less potent than 4-OHT (in
BMW-275 (16) was prepared using a McMurry cross-coupling
50
strategy. Mono-alkylation of symmetrical benzophenone 10
followed by acylation with pivaloyl chloride provided ketone 12
in 50% yield. McMurry cross-coupling with 4’-
hydroxypropiophenone provided alkene 13 as a 7:1 E/Z mixture.
Triflation under standard conditions and then palladium-
catalyzed carboxylation afforded 15 in 55% yield over 2 steps.
Finally, treatment of the methyl ester with hydroxylamine and
KOH afforded hydroxamic acid 16 in 45% yield.
35
MCF-7) or SAHA (in MDA-MB-231).
The side-chains of fulvestrant, 4-OHT, and raloxifene are
responsible for their antagonist action by preventing the proper
folding of H12 over the LBP and thus interfering with the
recruitment of transcription cofactors. In SERDs such as
fulvestrant, the long hydrophobic side chain can interact with the
The remaining hybrids were prepared via a three-component,
4
0
51
coactivator binding groove, a capacity that correlates with
nickel-catalyzed alkyne/Grignard/halide coupling. Treatment of
induction of ER modifications and complete transcriptional
aryl butyne 18 with an appropriately substituted aryl Grignard
and aryl iodide in the presence of NiCl •6H O afforded alkene 19
17
suppression. Thus the incorporation of polar zinc binding
groups at the end of the side-chain might alter the ability of
SERDs to induce ER degradation.
2
2
as a single alkene stereoisomer. Unfortunately, unlike tamoxifen,
the alkene in 19, and its derivatives, is highly prone to
isomerization, particularly under acidic conditions including
purification by silica gel chromatography. For instance, simple
removal of the TBS group in 19 with NaOH in methanol

followed by workup and silica gel chromatography afforded 20
as a 1:1 E/Z mixture. This propensity to isomerize presumably
arises from the additional electron donating groups on the aryl
rings not present in the parent tamoxifen. We thus proceeded
with the 1:1 mixture and separated alkene isomers by HPLC
below basal levels (Figure 3A). Titration curves in the presence
of E2 showed that hybrids 23, 28a, and 28b all fully inhibited
ER function (Figure 3B). While none of these hybrids were
more potent than 4-OHT, only a minimal loss of potency was
52
4
-(TBSO)-PhMgBr
Me2N
LDA; EtI
4-(Me2N(CH2)2O)-PhI
OH
O
THF/HMPA
2 2
NiCl •6H O
THF, PhMe, 60 °C
BnO
BnO
1) Cl(CH2)2NMe2·HCl
Cs2CO3, DMF, 90 °C
-78 - 0 °C
1
7
93%
18
2
) PivCl, NaH
THF, 0 °C to reflux
O
O
HO
RO
11 R = H, 51%
R = Piv, 98%
Me2N
Me2N
10
O
O
12
O
1
) NaH, Et3N
Br(CH ) CO Me
2 4 2
OR
O
O
X
4
TiCl4, Zn, THF
10 °C to reflux
2
3
) H2, Pd/C, MeOH
) NH2OH, KOH
-
HO
BnO
RO
Me2N
19 R = TBS, E/Z >99:1
R = H, E/Z = 1:1
21 R = Bn, X = OMe, 37%
22 R = H, X = OMe, 99%
23 R = H, X = NHOH, 57%
NaOH
MeOH:THF
78%
O
Me2N
O
2
0
1
2
) Tf2O, Et3N, DCM, -30 °C
) Pd(OAc)2, dppp, CO,
Et3N, DMF/MeOH, 70 °C
R
OH
1
) Tf2O, Et3N
3
. NH2OH, KOH,
THF/MeOH/H2O
0 °C to rt
2) CH2CHBF3K
PdCl2(dppf), Et3N, nPrOH
PivO
PivO
14
15
16
R = OTf, 73%
R = CO2Me, 76%
R = C(O)NHOH, 45%
13 27%, 7:1 E/Z
Me2N
Me2N
O
O
Scheme 1 Synthesis of hybrid 16 using a McMurry cross-
coupling strategy.
X
n CO2Me
CO2Me
n
Grubbs' II, pTSA
CH2Cl2, 40 °C
BnO
BnO
upon completion of the syntheses. Treatment of 20 with NaH and
methyl 5-bromopentanoate followed by hydrogenolytic cleavage
of the benzyl protecting group and hydroxamate formation, as
above, afforded AFP-277 (23) in 21% yield over three steps.
2
6a n = 0, 71%
2
4
X = OTf, 83%
26b n = 2, 74%
25 X = CH=CH2, 81%
1
2
) H2, Pd/C, MeOH
) NH2OH, KOH
Alternatively, triflation of 20 followed by Suzuki-Miyaura
cross-coupling afforded styrene 25 in excellent yield. Cross
metathesis with either methyl acrylate or methyl 4-pentenoate
using Grubbs’ second-generation catalyst proceeded cleanly to
afford alkenes 26a/b in good yield. Subsequent treatment with
Me2N
Me2N
O
O
O
OH
X
N
H
n
O
2
H /Pd-C resulted in alkene hydrogenation and hydrogenolysis of
the benzyl protecting group. Finally, treatment with
29
HO
HO
hydroxylamine afforded hybrids AFP-345 (28a) and AFP-477
2
2
2
7a X = OMe, n = 0, 87%
7b X = OMe, n = 2, 85%
8a X = NHOH, n = 0, 57%
(28b) in 35% and 21% yield, respectively, over three steps.
Finally, hybrid AFP-458 (29) bearing a cinnamate unit could be
prepared in an analogous sequence to 27a by using a para-
methoxybenzyl protecting group to avoid the need for
hydrogenolysis conditions (see Supporting Information).
28b X = NHOH, n = 2, 34%
Scheme 2. Synthesis of hybrids 23 and 27a/b using a
nickel-catalyzed three-component cross-coupling strategy.
3
. Biochemical Analysis
observed, with 23 and 28b maintaining sub-micromolar potency.
Hybrid 16 also fully inhibited ER, but unlike hybrids 23 and
The antiestrogenic activity of the hybrids was first assessed
2
8a/b, 16 was less potent than 4-OHT (see Supporting
using a bioluminescence resonance energy transfer (BRET) assay
used previously to characterize our fulvestrant hybrids (Figure
Information). In contrast, cinnamyl hydroxamic acid 29
displayed only partial inhibition of cofactor recruitment by ER
in the presence of E2 at the highest concentration tested. These
results clearly demonstrate that it is possible to incorporate
HDACi function into the B-ring of 4-OHT while maintaining
ER antagonist function but that efficacy is structure-dependent.
35
3). This assay measures recruitment of a coactivator (SRC1)
receptor-interacting domain fused to a YFP by ER fused to
Renilla Luciferase (RLucII) via energy transfer between the two
luminescent proteins in live transfected HEK293T cells. Thus,
the BRET assay reflects the activity of the receptor in live cells in
real time, avoiding effects on receptor expression levels caused
by HDACi activity in luciferase assays. As expected, the agonist
E2 (5 nM) increased net BRET values. The hybrids were initially
assessed at 10 µM in the absence and presence of 5 nM E2
HDACi activity was assessed in vitro using a standard
53
fluorometric assay. Initial screening was carried out against
HDAC6, a class IIa HDAC (Table 1). Hybrids 23, 28a/b, and 29
were within one order of magnitude potency of SAHA, with low
µM or high nM IC50 value. Hybrid 28b was the most potent with
an IC50 of 300 nM. Hybrids 23, 28a/b and 29 were further
screened against HDAC3, an example of a Class I HDAC. All
hybrids were again effective, with 28b being the most potent
(Figure 3A). All hybrids displayed antiestrogenic behaviour, with
28b most closely approaching the effectiveness of 4-OHT in
suppressing SRC1 recruitment in the presence of E2.
Importantly, in the absence of E2, all hybrids were devoid of
partial agonist activity, in every case suppressing fluorescence

a
Table 1. HDACi activity of hybrids
Compound
HDAC6 (µM)
HDAC3 (µM)
0.110
6
0.060
23
1.78
2.10
28a
0.484
2.01
28b
0.300
0.734
29
0.519
6.72
a) Determined in a fluorometric assay using N-Ac-Leu-
Gly-(-N-Ac)-Lys-AMC as substrate.
between day 4 and 7. Furthermore, 4-OHT is cytotoxic at 5-10
µM resulting in full loss of cell viability. SAHA is less
antiproliferative at sub-micromolar concentrations, but inhibited
cell survival more efficiently than 4-OHT in the micromolar
range.
All hybrids tested had antiproliferative effects in the
nanomolar range, with 23, 28a, and 28b approximating the effect
of 4-OHT and being more antiproliferative than SAHA over both
4
and 7-day treatment. The antiproliferative effect in that
concentration range was weakest at 7 days for 29, potentially
Figure 3. Antiestrogenic activity of AE-HDACi hybrid in the
presence and absence of E2 in BRET assays. A) HEK293T cells were co-
transfected with a constant amount of ERα-RLucII and/or YFP-SRC1.
After 48 h, cells were treated with E2 (5 nM) and/or 4-OHT or
AE/HDACi hybrids (10 μM) for 1 h. Transfer of energy between ERα-
RLucII and YFP-SRC1 was measured in a BRET assay. B) Dose
response curves were performed in HEK293T cells co-transfected with a
constant amount of ERα-RLucII and YFP-SRC1. After 48 h, cells were
treated with E2 (5 nM) and/or increasing amounts of 4-OHT and
AE/HDACi hybrids for 1 h. A-B: Graphs represent the mean +/- SEM
of data from 2 independent biological replicates.
with an IC50 of 734 nM - again within an order of magnitude of
SAHA. Hybrid 16, which displayed only partial antiestrogenic
activity, was also a poor HDACi (see Supporting Table S1),
presumably due to the lack of a linker between the tamoxifen
core and the hydroxamic acid. These assays clearly establish that
attachment of short chain hydroxamic acids directly to the 4-
OHT core is capable of producing viable, potent HDAC
inhibitors.
Figure 4. Antiproliferative activity in MCF-7. Cell proliferation
assay in MCF-7 cells treated for 4 (top) and 7 (middle) days with either
4
-OHT, SAHA, 23, 28a, 28b, or 29. All hybrids tested except for 29
have an anti-proliferative effect similar to that of 4-OHT at sub-
micromolar concentrations. 28b is more potent than 4-OHT or SAHA
alone in triggering cytotoxicity. Relative MCF-7 growth was calculated
by dividing the luminescent signal at day 7 over that at day 0. Values
represent the means of 2 independent experiments and error bars are the
SEM.
With the bifunctionality of the hybrids established, the
antiproliferative and cytotoxic activity of 23, 28a/b, and 29 were
tested in CellTiter-Glo cell viability assays. In ER+ MCF-7 cells,
4
-OHT displays antiproliferative effects at low concentrations
relative to untreated cells (Figure 4). These effects are more
marked at day 7, with cells being essentially growth-arrested

reflecting its reduced potency at suppressing ER activation.
Hybrids 23, 28b, and, to a lesser extent 28a, displayed cytotoxic
activity in the micromolar range at lower concentrations than 4-
OHT, likely reflecting their incorporated HDACi activity. 28b
displayed the lowest IC50 for cytotoxicity among all hybrids, in
keeping with its superior HDACi activity (Supporting Table S2)
The resulting bimodal antiproliferative profile of 28b is thus
improved with respect to either 4-OHT or SAHA alone,
particularly for 28b in the high nM to low M concentration
range over both 4 and 7 days. This is notable as 28b was less
potent than either 4-OHT or SAHA in single-target assays and
thus highlights the potential combinatorial effects of the hybrid
structures.
HDAC target proteins and on ER and HDAC target genes in
cells. Western blotting of MCF-7 cells treated with 28b or SAHA
both showed dose-dependent hyperacetylation of histone H4 and
tubulin, consistent with its HDACi functionality (Figure 6).
SAHA was slightly more potent, with effects being observed at 1
µM vs. 3 µM for 28b. As expected, 4-OHT had no significant
effect on acetylation of either histone H4 or tubulin. In addition,
Western analysis also revealed a decrease in ER protein levels
upon treatment by either SAHA or 28b (Figure 6), consistent
with previous reports that treatment with HDACi’s suppresses
54-57
both ER RNA and protein levels.
Assessment of gene expression levels in MCF-7 by RT-qPCR
also showed regulation patterns consistent with both ER
antagonism and HDACi activity. At 5 M, 28b, like SAHA but
not 4-OHT, suppressed ESR1 mRNA levels (Figure 7, top panel),
consistent with the loss of ER protein levels described above.
Accordingly, expression of estrogen target genes TFF1, GREB1,
and MYC was suppressed by 28b in a manner similar to both
SAHA and 4-OHT (Figure 7, top panel). Hybrid 28b also
induced expression of SREBF1, CTGF , and CDKN1A, which are
We also assessed the efficacy of 28b in ER– cell lines. In
triple negative MDA-MB-231 cells, neither 28b nor 4-OHT
showed antiproliferative activity at nanomolar concentrations, in
keeping with the lack of ER-dependency for cell proliferation.
28b displayed cytotoxicity in the micromolar range with an IC50
intermediate between that of SAHA and 4-OHT over a 7-day
course of treatment (Figure 5 and Supporting Table S3). The
lower potency of 28b relative to SAHA is in keeping with its
lower HDACi potency. Similar behaviour of 28b, i.e. cytotoxic
activity in the micromolar range intermediate between that of
SAHA and 4-OHT, was observed in ER– MCF-10A cells (see
Supporting Figure S3).
58-60
induced by acetylation or HDACi treatment
but only mildly
affected by 4-OHT (Figure 7, bottom panel), supporting the bi-
functionality of the molecule. Finally, expression of several
proliferative genes including E2F1, MKI67, MYBL2, CCND1,
and CDC6 was suppressed by 28b as well as either 4-OHT or
SAHA, with similar or intermediate efficacies, in keeping with
its anti-proliferative activity in MCF-7 cells (Figure 7, top panel).
Figure 5. Antiproliferative activity in MDA-MB-231 cells. Cells were
treated for 7 days with either 4-OHT, SAHA, or 28b. Relative cell
proliferation was calculated by dividing the luminescent signal at day 7 over
that at day 0. Values represent the means of 3 independent experiments and
error bars are the SEM.
The high potency of 28b in the BRET and HDACi assays, and
its effectiveness in the antiproliferative assays spurred a more in-
depth examination of its properties, including its effects on
Figure 7. Hybrid 28b regulates both ER target genes and SAHA
responsive genes. MCF-7 cells were treated for 24 h with either 4-OHT,
SAHA, or 28b (5 μM). Indicated genes were tested by RT-qPCR. Expression
levels were normalized to those of the RPLP0, TBP and YWHAZ house-
keeping genes. Values represent the means of 2 independent experiments and
error bars are the SEM. *p-values where calculated with a Holm- Šidák t-test.
Figure 6. HDACi activity in MCF-7 cells. Cells were treated for 8 h
with different concentrations of 4-OHT, SAHA or 28b. Acetylation of
histones H4 and of α-tubulin in the presence of different doses of SAHA,
4
-OHT or 28b was analyzed by Western blotting with antibodies against
the corresponding acetylated proteins. ERα protein levels were also
assessed. Results are representative of 2 experiments. Upper and lower
Western blots (separated by the dashed line) are from two different gels
loaded from identical samples.

4. Computer Docking and Discussion
The data above clearly show that while all hybrids were
bifunctional to some extent, 28b displayed
a superior
combination of ER antagonist, HDACi and antiproliferative
activity. The ability of hybrids 23, 28a, and 28b to act as
effective antagonists for the ER suggests that the ER LBP can
accommodate the additional HDACi functionality in the portion
of the pocket where the D-ring of E2 binds. To examine potential
binding modes, we docked the hybrids in ER crystal structures
4
6, 49
from its complexes with 4-OHT (PDB: 3ERT)
arylindole (PDB: 2IOG) using FITTED,
and a larger 2-
a docking platform
that has performed well in other nuclear receptor ligand hybrid
61, 62
35, 63, 64
studies.
None of the hybrids docked well into the 4-OHT
crystal structure - the phenol in the hybrids did not overlap with
that found in 4-OHT and docking scores were quite low. In
contrast, the expanded pocket in the 2-arylindole/ER structure
easily accommodated the hybrids. The structure of 28b (Figure 8)
shows the hydroxamate side chain occupying a space that is
present in the 2-arylindole/ER crystal structure but not in the 4-
OHT/ER structure. While these docking solutions are crystal
structure-dependent, in combination with the experimental data
they suggest that the ER is sufficiently flexible to adapt to the
hybrid ligands.
Figure 9. Comparison of docking solutions of 28b (left) and 16 (right) to
HDAC 6 (PDB:5EDU) showing improved coordination to zinc by 28b. Zinc
coordinated to His651, Asp 649 and Asp 742 in lower left.
bearing a branched chain are accommodated without losing VDR
6
9
agonist function and with only minor losses in potency.
Compared to our prior hybrid 9,
35
hybrid 28b displays an
excellent combination of antiestrogenicity and HDACi activity
that leads to an improved antiproliferative profile; the molecule is
as effective as 4-OHT at low concentration and more cytotoxic
th
than either SAHA or OHT, even though it is only 1/10 as potent
towards HDAC3 and 6. Future studies will examine the activity
profile of these hybrids using in vivo models, including their
uterotrophic activity, and further explore their modes of
interaction with ER in structural studies.
5. Experimental Section
Unless otherwise stated, reactions were conducted under an
argon atmosphere and glassware was oven dried prior to use.
Tetrahydrofuran and diethyl ether were purified by distillation
from sodium under
a
nitrogen atmosphere. Toluene,
dichloromethane and triethylamine were purified by distillation
from calcium hydride under nitrogen atmosphere. Deuterated
chloroform was stored over activated 4 Å molecular sieves. All
commercial reagents and solvents were used as purchased
without further purification. Thin-layer chromatography (TLC)
was carried out on glass-backed Ultrapure silica TLC plates
Figure 8. AFP-477 (28b) docked to the crystal structure of ERα derived
from its complex with a 2-arylindole (PDB: 2IOG). The hydroxamate chain
occupies a space (lower left) that is not present in the x-ray structure of 4-
OHT bound to ERα (PDB 3ERT).
(
extra hard layer, 60 Å, thickness: 250 µm, saturated with F-254
indicator). Flash column chromatography was carried out on 230-
00 mesh silica gel (Silicycle) using reagent grade solvents.
Infrared (IR) spectra were obtained using Nicolet Avatar 360 FT-
4
65
We also docked the hybrids to HDAC6 (PDB: 5EDU).
-
1
While hybrids 23, 28a/b, and 29 all easily docked with tight
coordination of the hydroxamic acid to the active site zinc, the
proximity of the aromatic ring in hybrid 16 prevented the
hydroxamic acid from fully entering the active site (Figure 9).
This was consistent with the relatively poor potency of 26 and
suggests that a minimum linker of at least 2 atoms is necessary
for efficient binding.
IR infrared spectrophotometer and data are reported in cm .
Proton and carbon nuclear magnetic resonance spectra were
obtained on Varian 300, 400, and 500 or Bruker 400 and 500
MHz spectrometers. Chemical shifts (δ) were internally
referenced to the residual proton resonance CDCl (δ 7.26 ppm),
3
CD OD (δ 3.31 ppm), (CD ) SO (δ 2.50 ppm). Coupling
3
3 2
constants (J) are reported in Hertz (Hz). HPLC Analysis was
performed using a Waters ALLIANCE instrument (e2695 with
The hybrids described here represent a new design motif for
bifunctional antiestrogens. Most prior examples have focused on
incorporating secondary functionality in the side-chain of
2489 UV detector and 3100 mass spectrometer). HRMS were
obtained by Dr. Nadim Saadeh or Dr Alexander S. Wahba at
McGill University Department of Chemistry
35,
antiestrogens, including HDACi and cytotoxic functionalities.
37, 38, 66-68
The results herein suggest that the LBP of ER is
sufficiently pliable to accommodate larger groups while
maintaining both high affinity and antagonist behavior. Similar
plasticity has been observed in the VDR where Gemini ligands
(
4-(2-(Dimethylamino)ethoxy)phenyl)(4-
hydroxyphenyl)methanone (11): Cs CO (8.55 g, 26.3 mmol,
.0 eq.) was added to a solution of 4,4’-hydroxybenzophenone 10
2
3
4

(
1.41 g, 6.56 mmol, 1.0 eq.) in dry DMF (15 mL) at room
dropwise to a solution of 13 (99.8 mg, 0.206 mmol, 1.0 eq.) in
CH Cl (5 mL) at -30°C. The reaction mixture was stirred at -
temperature. The mixture was heated to reflux and stirred for 10
min, at which point the solution was cooled to room temperature
and
hydrochloride (945 mg, 6.56 mmol, 1.0 eq.) was added portion-
wise. The reaction was then heated to 80°C and stirred for 4 h,
whereupon it was cooled to room temperature, quenched with
saturated ammonium chloride solution (10 mL) and extracted
with ethyl acetate (3 x 25 mL). The crude product was purified
2
2
30°C for 2 h, whereupon additional triethylamine (68.6 μL, 0.492
mmol) and triflic anhydride (82.6 μL, 0.492 mmol) were added
and the solution was stirred at -30°C for another 3 h. The reaction
was quenched with distilled water (5 mL) and extracted with
ethyl acetate (3 x 10 mL). The combined organic layers were
dried, filtered and concentrated. Purification by column
N,N-dimethyl-2-chloro-N,N-dimethylethylamine
2 2
chromatography eluting with 5% MeOH in CH Cl afforded 14
2 2
by column chromatography eluting with 10% MeOH in CH Cl
to afford 11 as a white solid (960 mg, 3.37 mmol) in 51% yield.
as a bright yellow product (93.8 mg, 0.151 mmol) in 73% yield
as a 5:1 mixture of E/Z isomers. H NMR (500 MHz, CD OD) δ
3
1
The spectroscopic data of the product is in agreement with that
reported in the literature.
7.26 (t, J = 9.0 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 8.6
Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 3.99
70
(
2
t, J = 5.4 Hz, 2H), 2.76 (t, J = 5.3 Hz, 2H), 2.51 (q, J = 7.6 Hz,
H), 2.34 (s, 6H), 1.36 (s, 9H), 0.94 (t, J = 7.5 Hz, 3H). C NMR
4
-(4-(2-(Dimethylamino)ethoxy)benzoyl)phenyl
pivalate
13
(
12): NaH (60% in mineral oil, 200.3 mg, 5.01 mmol, 1.5 eq.)
(126 MHz, CD OD) δ 177.3, 157.1, 153.2, 150.1, 147.9, 143.2,
3
was added to a solution of 11 (952 mg, 3.34 mmol, 1.0 eq.) in
THF (15 mL) and the resulting bright yellow suspension was
stirred for 15 min. The reaction mixture was cooled to 0 °C and
pivaloyl chloride (493 µL, 4.01 mmol, 1.2 eq.) was added
dropwise. The reaction was warmed to room temperature and
stirred for 1 h, whereupon it was quenched with distilled water
1
1
40.5, 140.1, 139.2, 134.9, 131.6, 131.5, 130.0, 121.0, 120.4,
14.0, 113.3, 64.7, 57.5, 44.2, 38.7, 28.2, 26.0, 12.3. HRMS calc.
+
for C32
H
37NO
6
SF
3
(M+H) : 620.2288. Found: 620.2284.
(E/Z)-Methyl-4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-
(4-(pivaloyloxy) phenyl)but-1-en-2-yl)benzoate (15): In a
flame-dried Schlenk bomb, triethylamine (17.5 μL, 0.125 mmol,
3.0 eq.) was added dropwise to a solution of 14 (25.9 mg, 41.8
(
10 mL) and extracted with ethyl acetate (3 x 25 mL). The
organic layers were dried over anhydrous sodium sulfate, filtered
and concentrated to afford 12 as a white solid (1.22 g, 3.27
μmol, 1.0 eq.) in DMF (2 mL) at room temperature. Pd(OAc)
2
1
mmol) in 98% yield. H NMR (400 MHz, CDCl
3
) δ 7.80 (dd, J =
(3.8 mg, 17 μmol, 0.40 eq.), 1,3-bis(diphenylphosphino)propane
(5.2 mg, 13 μmol, 0.30 eq.) and MeOH (1.5 mL) were added
sequentially to the solution and the flask was charged with 4 atm.
carbon monoxide. The reaction mixture was heated to 70 °C and
stirred overnight (18 h), after which it was cooled to room
temperature and the carbon monoxide was vented. The mixture
was diluted with distilled water (5 mL), extracted with ethyl
acetate (3 x 10 mL) and rinsed with brine (4 mL). The organic
layers were dried over anhydrous sodium sulfate, filtered and
concentrated to yield an orange oil. Purification by column
8
2
6
1
4
1
1
.8, 3.0 Hz, 4H), 7.17 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 8.9 Hz,
H), 4.14 (t, J = 5.7 Hz, 2H), 2.76 (t, J = 5.7 Hz, 2H), 2.35 (s,
H), 1.38 (s, 9H). C NMR (126 MHz, CDCl ) δ 194.5, 176.7,
3
62.4, 154.0, 135.5, 132.5, 131.3, 130.2, 121.4, 114.1, 66.0, 58.0,
5.8, 39.2, 27.1. IR (film)= 3661, 2973, 2873, 2822, 2773, 1751,
650, 1599, 1508, 1479, 1460, 1304, 1276, 1253, 1202, 1161,
13
104, 1029, 928, 898, 846, 762. HRMS calc. for C22H28NO
4
+
(M+H) : 370.2001. Found: 370.2013.
(E/Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-2-(4-
chromatography eluting with 3% MeOH in CH
as a clear, colorless oil (16.8 mg, 31.7 μmol) in 76% yield as a
:1 mixture of E/Z isomers (note: some 1,3-bis(diphenyl-
phosphino)propane co-eluted with 15 and yield is calculated from
2 2
Cl afforded 15
hydroxyphenyl)but-1-en-1-yl)phenyl pivalate (13): Titanium
IV) chloride (1.76 mL, 16.1 mmol, 4.0 eq.) was added to a
(
7
suspension of zinc dust (2.11 g, 32.2 mmol, 8.0 eq.) in THF (12
mL) at 0 °C. The resulting deep brown slurry was stirred at reflux
for 3h, whereupon the reaction mixture was cooled to 0 °C and
ketone 12 (1.49 g, 4.02 mmol, 1.0 eq.) and 4’-
hydroxypropiophenone (1.82 g, 12.1 mmol, 3.0 eq.) in THF (20
mL) were added simultaneously to the solution. The reaction
mixture was stirred in the dark at reflux for 3 h, then cooled to 0
1
HNMR analysis). H NMR (400 MHz, CD
2
2
3
OD) δ 7.87 – 7.76 (m,
H), 7.33 – 7.19 (m, 4H), 7.14 – 7.02 (m, 2H), 6.79 (dd, J = 8.6,
.0 Hz, 2H), 6.68 – 6.56 (m, 2H), 3.95 (q, J = 3.6 Hz, 2H), 3.85
(d, J = 1.8 Hz, 3H), 2.78 – 2.66 (m, 2H), 2.62 – 2.45 (m, 2H),
2
7
1
1
2
.31 (d, J = 2.0 Hz, 6H), 1.37 (d, J = 1.9 Hz, 8H), 0.93 (td, J =
.4, 1.9 Hz, 3H). C NMR (126 MHz, CD OD) δ 177.3, 167.1,
3
57.0, 149.1, 147.8, 141.0, 140.6, 138.9, 135.1, 131.6, 131.0,
13
°
2 3
C and quenched with 10% K CO (50 mL). The mixture was
filtered and the filtrate was extracted with ethyl acetate (3 x 50
mL). The organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. The crude product was purified
29.7, 128.8, 127.7, 120.9, 113.3, 64.6, 57.5, 51.0, 45.0, 38.7,
+
8.1, 26.0, 12.4. HRMS calc. for C33
H
40NO
5
(M+H) : 530.2901.
Found: 530.2904
by column chromatography eluting with 8-16% MeOH in CH
2 2
Cl
to afford 13 as a beige foam (534 mg, 1.1 mmol) in 28% yield as
(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-1-(4-
hydroxyphenyl)but- 1-en-2-yl)-N-hydroxybenzamide (16):
Hydroxylamine (50% w/w in H O, 500 eq.) was added to a
2
solution of methyl ester 15 (15.8 mg, 34.9 μmol, 1.0 eq.) in 5:1
THF:MeOH (1.8 mL). 3M KOH (58.2 μL, 0.175 mmol, 5.0 eq.)
was then added dropwise at 0 °C and the mixture was warmed to
room temperature and stirred until reaction completion. The
reaction mixture was subsequently neutralized with 2M HCl and
crude product was concentrated under reduced pressure to afford
1
a 7:1 mixture of E/Z isomers. H NMR (500 MHz, CD
.21 (d, J = 8.6 Hz, 2H), 7.02 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.7
Hz, 2H), 6.78 (d, J = 8.9 Hz, 2H), 6.59 (d, J = 8.4 Hz, 4H), 3.97
3
OD) δ
7
(
2
t, J = 5.4 Hz, 2H), 2.73 (t, J = 5.4 Hz, 2H), 2.42 (q, J = 7.4 Hz,
H), 2.32 (s, 6H), 1.35 (s, 9H), 0.91 (t, J = 7.4 Hz, 3H). C NMR
13
(126 MHz, CD
3
OD) δ 177.3, 156.6, 155.5, 149.7, 141.6, 141.5,
1
6
1
1
36.7, 135.9, 133.0, 131.6, 130.5, 130.1, 120.8, 114.4, 113.1,
4.7, 57.6, 44.3, 38.7, 28.4, 26.1, 12.6. film ν = 3449, 2966,
750, 1607, 1508, 1478, 1397, 1275, 1240, 1197, 1164, 1116,
a
brown residue. Purification by reverse-phase column
chromatography eluting with 10-90% MeOH in H O afforded
product 16 as an orange residue (6.6 mg, 21.1μmol) in 45% yield
note: some 1,3-bis(diphenyl-phosphino)propane once more co-
+
029, 831, 590. HRMS calc. for C31
H
38NO
4
(M+H) : 488.2795.
2
Found: 488.2782.
(
(E/Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-2-(4-
eluted with 16 and yield is calculated from HNMR analysis).
Further purification by preparatory HPLC eluting with 26-40%
(((trifluoromethyl) sulfonyl)oxy)phenyl)but-1-en-1-yl)phenyl
pivalate (14): Triethylamine (34.3 μL, 0.246 mmol, 1.2 eq.) and
triflic anhydride (41.3 μL, 0.246 mmol, 1.2 eq.) were added
2
MeCN in H O with 0.1% formic acid afforded the formate salt of
the product as the Z isomer uniquely. Analytical HPLC (C18, 5%

1
to 100% MeCN in H
NMR (400 MHz, DMSO-d
8.2 Hz, 2H), 6.96 (d, J = 8.3 Hz, 2H), 6.73 (d, J = 8.5 Hz, 2H),
.69 (d, J = 8.3 Hz, 2H), 6.58 (d, J = 8.3 Hz, 2H), 3.87 (t, J = 4.7
Hz, 2H), 2.41 (d, J = 8.0 Hz, 2H), 2.13 (s, 6H), 0.81 (t, J = 7.4
2
O) indicated the product was 94% pure. H
aqueous was extracted with EtOAc (3 x 10 mL), dried over
Na SO filtered, and concentrated to a brown residue.
Purification by silica gel column chromatography using a 6.5%
MeOH in CH Cl isocratic solvent system gave 21 as an orange
oil (117 mg, 0.19 mmol) in 37% yield as a 1:1 mixture of E:Z
6
) δ 7.54 (d, J = 8.2 Hz, 2H), 7.14 (d, J
2
4
,
=
6
2
2
1
3
1
Hz, 3H). C NMR (75 MHz, DMSO-d
6
) δ 157.0, 156.7, 145.9,
39.6, 139.2, 137.8, 135.6, 134.1, 131.9, 130.5, 129.9, 126.9,
15.4, 113.9, 112.9, 65.9, 58.1, 45.9, 28.8, 13.8. HRMS calc. for
3
isomers. H NMR (500 MHz, CDCl ) δ 7.56 – 7.25 (m, 12H),
1
1
C
7.18 (dd, J = 8.5, 5.2 Hz, 3H), 7.05 (dd, J = 8.6, 4.0 Hz, 3H),
6.98 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.82 (dd, J =
8.7, 6.4 Hz, 4H), 6.73 (d, J = 3.4 Hz, 2H), 6.72 – 6.65 (m, 3H),
+
27 31
H N
2
O
4
(M+H) : 447.2278. Found: 447.2293.
6
5
2
7
1
.61 (d, J = 8.7 Hz, 2H), 5.09 (s, 2H), 4.96 (s, 2H), 4.14 (t, J =
.8 Hz, 2H), 4.00 (t, J = 5.7 Hz, 2H), 3.94 (t, J = 5.6 Hz, 3H),
.82 (t, J = 5.6 Hz, 2H), 2.74 (t, J = 5.6 Hz, 2H), 2.49 (dd, J =
(
Z)-2-(4-(1-(4-(Benzyloxy)phenyl)-2-(4-((tert-
butyldimethylsilyl)oxy)phenyl)but-1-en-1-yl)phenoxy)-N,N-
dimethylethan-1-amine (19): To a solution of 18 (1.0 g, 4.23
mmol, 1.0 eq.), 2-(4-iodophenoxy)-N,N-dimethylethan-1-amine
.4, 3.8 Hz, 3H), 2.42 (d, J = 4.7 Hz, 9H), 2.37 (s, 6H), 1.94 –
13
.76 (m, 8H), 1.01 – 0.89 (m, 6H). C NMR (126 MHz, CD
3
OD)
(
2 2
1.48 g, 5.08 mmol, 1.2 eq.), and NiCl ·6H O (10.1 mg, 0.042
δ 173.9, 157.5, 157.4, 157.1, 156.7, 156.5, 140.6, 137.3, 137.1,
mmol, 1 mol%) in PhMe (17 mL) was added slowly (4-((tert-
butyldimethylsilyl)oxy)phenyl)-magnesium bromide (0.80 M
solution in THF) (6.35 mL, 5.08 mmol, 1.2 eq.). The reaction
was then heated to 50 °C and stirred for 24 h. The reaction was
cooled, quenched with 1 mL H
plug eluting with EtOAc. Purification by silica gel column
chromatography using a 1-9% MeOH in CH Cl solvent gradient
gave 19 as a brown oil (786 mg, 1.31 mmol) in 31% yield as the
pure Z isomer. H NMR (400 MHz, CDCl
H), 7.46 – 7.40 (m, 3H), 7.39 – 7.34 (m, 1H), 7.19 (d, J = 8.7
Hz, 2H), 6.99 (d, J = 8.6 Hz, 4H), 6.80 (d, J = 8.7 Hz, 2H), 6.69
d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.8 Hz, 2H), 5.11 (s, 2H), 3.99 (t,
J = 5.8 Hz, 2H), 2.71 (t, J = 5.8 Hz, 2H), 2.50 (q, J = 7.3 Hz,
H), 2.35 (s, 7H), 1.01 (s, 15H), 0.21 (s, 6H). C NMR (126
MHz, CDCl ) δ 157.5, 156.6, 153.8, 140.7, 137.3, 137.1, 136.8,
36.1, 135.6, 132.0, 130.7,, 128.6, 128.0, 127.6, 119.6, 114.3,
13.3, 70.1, 65.6, 58.3, 45.9, 28.9, 25.8, 18.3, 13.7, -4.4. HRMS
1
1
1
6
3
36.8, 136.7, 136.4, 136.3, 134.7, 134.7, 132.0, 131.9, 130.7,
30.6, 130.4, 128.6, 128.5, 128.0, 127.9, 127.6, 127.5, 127.4,
14.4, 114.1, 114.0, 113.9, 113.8, 113.7, 113.4, 113.3, 70.0, 69.8,
7.2, 65.7, 65.5, 60.3, 58.3, 58.2, 53.5, 51.5, 45.8, 45.7, 34.0,
2
O and filtered through a silica
3.8, 33.7, 30.9, 29.0, 28.8, 21.7, 14.3, 13.8, 13.7. HRMS calc.
+
for C39
H
46NO
5
(M+H) : 608.3370. Found: 608.3382.
2
2
General Procedure A: Hydrogenations of benzyl esters
and olefins: To a solution of starting material (1.0 eq.) in MeOH
(0.1 M) was added 10 wt% of 10% Pd/C. The reaction was
1
3
) δ 7.52 – 7.47 (m,
2
2
placed under an atmosphere of H using a balloon and a vent to
(
purge the flask of all air. The reaction was stirred for 24 h upon
which it was vented, filtered through Celite, and concentrated
directly.
13
2
3
(E/Z)-5-(4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-1-(4-
1
1
hydroxyphenyl)but-1-en-2-yl)phenoxy)pentanoate (22):
+
Prepared from 21 (82 mg, 0.13 mmol, 1.0 eq.) according to
General Procedure A. The product 22 was isolated as a bright
yellow oil (71 mg, 0.13 mmol) in quantitative yield as a 1:1
calc. for C39
3
H50NO Si (M+H) : 608.3554. Found: 608.3567.
(E/Z)-4-(1-(4-(Benzyloxy)phenyl)-1-(4-(2-
1
(
dimethylamino)ethoxy)phenyl)but-1-en-2-yl)phenol (20): To
mixture of E:Z isomers and used without further purification. H
a solution of 19 (875 mg, 1.44 mmol, 1.0 eq.) in MeOH (14 mL)
was added crushed NaOH (570 mg, 14.25 mmol, 10 eq.) and the
reaction was stirred overnight at room temperature. The reaction
3
NMR (400 MHz, CD OD) δ 7.13 (d, J = 8.7 Hz, 1H), 7.07 – 6.98
(m, 3H), 6.94 (d, J = 8.7 Hz, 2H), 6.84 – 6.64 (m, 5H), 6.61 (d, J
= 8.9 Hz, 1H), 6.44 (d, J = 8.7 Hz, 1H), 4.16 (dd, J = 5.9, 4.9 Hz,
2H), 4.05 – 3.84 (m, 3H), 2.88 (q, J = 5.2 Hz, 1H), 2.80 (t, J =
5.5 Hz, 1H), 2.53 – 2.34 (m, 10H), 1.79 (ddt, J = 6.8, 4.6, 2.9 Hz,
was then quenched with 5 mL H
vacuo and the aqueous was extracted with EtOAc (3 x 15 mL),
dried over Na SO , filtered, and concentrated to a brown oil.
Purification by silica gel column chromatography using a 0-8%
MeOH in CH Cl solvent gradient gave 20 as a brown oil that
2
O. MeOH was removed in
13
2
4
3
5H), 0.98 – 0.88 (m, 6H). C NMR (126 MHz, CDCl ) δ174.2,
174.1, 157.1, 157.0, 156.3, 155.5, 154.5, 140.1, 140.0, 137.5,
136.9, 136.4, 135.5, 135.3, 134.9, 132.1, 131.9, 130.8, 130.7,
130.6, 130.5, 115.3, 115.2, 114.7, 114.6, 114.0, 113.9, 113.8,
113.2, 67.2, 65.9, 64.9, 64.5, 58.1, 58.0, 53.5, 51.6, 45.4, 45.3,
2
2
foamed while drying under vacuum (557 mg, 1.13 mmol) in 78%
1
yield as a 1:1 mixture of E:Z isomers. H NMR (400 MHz,
CDCl
7
6
3
) δ 7.48 (d, J = 7.3 Hz, 2H), 7.44 – 7.27 (m, 3H), 7.18 –
.09 (m, 2H), 7.03 – 6.89 (m, 4H), 6.79 (dd, J = 9.0, 2.4 Hz, 2H),
.66 – 6.56 (m, 4H), 5.11 (s, 2H), 4.02 (t, J = 5.4 Hz, 2H), 2.79
33.8, 33.7, 29.0, 28.9, 28.8, 28.7, 21.7, 15.2, 14.3, 13.8. HRMS
+
calc. for C32
H
40NO
5
(M+H) : 518.2901. Found: 518.2909.
General Procedure B: Hydroxamic acid formation from
methyl esters: To a solution of methyl ester (1.0 eq.) in 5:1
THF:MeOH at 0 °C was added hydroxylamine (50% w/w in
(
s, 2H), 2.51 – 2.39 (q, 2H), 2.37 (s, 6H), 0.93 (t, J = 7.4 Hz, 3H).
C NMR (126 MHz, CDCl ) δ 157.5, 157.3, 156.7, 156.3, 154.3,
3
1
3
1
1
1
7
54.0, 140.8, 140.6, 137.1, 136.8, 136.7, 136.4, 136.3, 134.7,
34.3, 132.0, 131.9, 130.9, 130.6, 128.6, 128.5, 128.5, 128.0,
27.9, 127.6, 127.6, 115.0, 114.9, 114.3, 114.1, 113.7, 113.4,
H
(
2
O) (500 eq.) followed by dropwise addition of cold 3M KOH
7.0 eq.). The resulting mixture was warmed to room temperature
and stirred for 24 h. The reaction was neutralized with 3M HCl,
extracted three times with EtOAc, dried over Na SO , filtered,
0.0, 69.8, 65.5, 65.0, 58.2, 58.0, 45.7, 45.4, 28.9, 13.7. HRMS
+
2
4
calc. for C33
H
36NO
3
(M+H) : 494.2690. Found: 494.2699.
and concentrated in vacuo. The crude mixture was purified by
Methyl
(E/Z)-5-(4-(1-(4-(benzyloxy)phenyl)-1-(4-(2-
reverse phase column chromatography using a 10-100% MeOH
(dimethylamino)ethoxy)phenyl)but-1-en-2-
2
in H O gradient. Concentration of the purified fractions in vacuo
yl)phenoxy)pentanoate (21): To a solution of 20 (244 mg, 0.52
mmol, 1.0 eq.) in THF (3 mL) was added NaH (60% dispersion
in mineral oil) (25 mg, 0.62 mmol, 1.2 eq.) followed by freshly
distilled Et N (87 μL, 0.62 mmol, 1.2 eq) and methyl γ-bromo
3
valerate (112 μL, 0.78 mmol, 1.5 eq.) which had first been passed
followed by lyophilization of residual H
purified products.
2
O overnight yielded the
(Z)-5-(4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-1-(4-
hydroxyphenyl)but-1-en-2-yl)phenoxy)-N-
hydroxypentanamide (23): Prepared from 22 (70 mg, 0.14
mmol, 1.0 eq.) according to General Procedure B. Reverse phase
purification yielded 23 as an off white solid (41 mg, 0.06 mmol)
in 57% yield as a 1:1 mixture of E:Z isomers. Preparatory reverse
through a plug of basic alumina. The reaction was heated to
50 °C and stirred for 5 h, then cooled to room temperature and
quenched with 5 mL H O. THF was removed in vacuo and the
2

phase HPLC purification using a 12-54% MeCN in H
and lyophilization of the desired fractions yielded the Z isomer of
2
O gradient
113.7, 113.5, 113.4, 113.0, 70.1, 69.8, 65.9, 65.7, 58.4, 58.3,
+
45.9, 28.9, 13.7. HRMS calc. for C35
Found: 504.2906.
2
H38NO (M+H) : 504.2897.
2
5
3 as a fluffy, amorphous, white solid. Analytical HPLC (C18,
% to 100% MeCN in H O) indicated the product was 96% pure.
OD) δ 7.01 (dd, J = 7.9, 4.5 Hz, 5H),
2
1
Methyl
(E/Z)-3-(4-((Z)-1-(4-(benzyloxy)phenyl)-1-(4-(2-
H NMR (400 MHz, CD
3
(dimethylamino)ethoxy)phenyl)but-1-en-2-yl)phenyl)acrylate
6
3
–
.86 – 6.75 (m, 5H), 6.68 (dd, J = 25.8, 8.3 Hz, 6H), 4.11 (s, 2H),
.94 (s, 2H), 3.22 – 3.05 (m, 3H), 2.65 (d, J = 13.3 Hz, 7H), 2.54
(26a): To a solution of 25 (155 mg, 0.31 mmol, 1.0 eq.) in
1
3
2 2
CH Cl (1 mL) was added anhydrous PTSA (58 mg, 0.34 mmol,
2.39 (m, 4H), 1.79 (s, 6H), 0.92 (td, J = 7.4, 3.1 Hz, 3H).
OD) δ 157.3, 156.9, 154.9, 140.2, 137.6,
37.4, 134.8, 134.6, 131.7, 131.6, 130.5, 130.3, 130.2, 128.6,
14.5, 113.9, 113.8, 113.5, 113.1, 63.4, 57.0, 43.4, 28.3, 12.6.
C
1.1 eq.). The reaction was stirred at room temperature for 10
NMR (101 MHz, CD
1
1
3
minutes until the complete dissolution of PTSA and then was
concentrated in vacuo. To the dry residue under argon was added
methyl acrylate (281 μL, 3.1 mmol, 10 eq.), and Grubbs’ gen. 2
catalyst (13.2 mg, 0.02 mmol, 5 mol%) in CH Cl (0.5 mL). The
2 2
reaction was heated to 40 °C and stirred for 24 h and then cooled
+
HRMS calc. for C31
19.2858.
39
H N
2
O
5
(M+H) : 519.2853. Found:
5
(
E/Z)-4-(1-(4-(Benzyloxy)phenyl)-1-(4-(2-
dimethylamino)ethoxy)phenyl)but-1-en-2-yl)phenyl
trifluoromethanesulfonate (24): To a solution of 20 (700 mg,
.42 mmol, 1.0 eq) in CH Cl (28 mL) at -40 °C was added
freshly distilled Et N (297 μL, 2.13 mmol, 1.5 eq.) followed by
dropwise addition of freshly distilled Tf O (238 μL, 1.42 mmol,
.0 eq.). The reaction was stirred for 1 hour and then quenched
with 200 μL ethylenediamine followed by 30 mL H O. After
warming to room temperature, the reaction was extracted with
CH Cl (3 x 25 mL), dried over Na SO , filtered, and
to room temperature. To the reaction was again added Grubbs’
gen. 2 catalyst (13.2 mg, 0.02 mmol, 5 mol%) in CH Cl (0.5
2 2
mL) and the reaction was heated to 40 °C and stirred overnight.
The reaction was cooled to room temperature, quenched with
(
1
2
2
3
saturated NaHCO
Purification by silica gel column chromatography using a 0-8%
MeOH in CH Cl gradient yielded 26a as a dark brown/green oil
(123 mg, 0.22 mmol) in 71% yield as a 1:1 mixture of E:Z
3 2 2
(5 mL) and extracted with CH Cl (3 x 5 mL).
2
1
2
2
2
1
3
isomers. H NMR (400 MHz, CDCl ) δ 7.65 (dd, J = 16.0, 3.0
2
2
2
4
Hz, 2H), 7.51 – 7.45 (m, 2H), 7.45 – 7.29 (m, 12H), 7.22 – 7.13
(m, 6H), 6.98 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.79
(dd, J = 8.7, 5.8 Hz, 3H), 6.66 (d, J = 8.8 Hz, 2H), 6.59 (d, J =
8.8 Hz, 2H), 6.39 (dd, J = 16.0, 2.3 Hz, 2H), 5.10 (s, 2H), 4.95 (s,
2H), 4.12 (t, J = 5.8 Hz, 2H), 3.97 (t, J = 5.7 Hz, 2H), 3.82 (s,
6H), 2.80 (t, J = 5.7 Hz, 2H), 2.70 (t, J = 5.7 Hz, 2H), 2.53 (qd, J
= 7.4, 3.6 Hz, 4H), 2.40 (s, 6H), 2.34 (s, 6H), 0.96 (td, J = 7.4,
concentrated to a foamy yellow oil. Purification by silica gel
column chromatography using a 0-8% solvent gradient yielded
4 as a yellow oil (737 mg, 1.18 mmol) in 83% yield as a 1:1
mixture of E:Z isomers. H NMR (400 MHz, CDCl
.31 (m, 12H), 7.24 – 7.12 (m, 6H), 7.09 (dd, J = 8.8, 1.8 Hz,
H), 6.99 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.79 –
.67 (m, 4H), 6.65 (d, J = 8.8 Hz, 2H), 6.59 (d, J = 8.8 Hz, 2H),
.10 (s, 2H), 4.95 (s, 2H), 4.11 (t, J = 5.8 Hz, 2H), 3.96 (t, J = 5.8
Hz, 2H), 2.77 (t, J = 5.7 Hz, 2H), 2.68 (t, J = 5.8 Hz, 2H), 2.52
dq, J = 7.9, 4.0 Hz, 3H), 2.38 (s, 7H), 2.32 (s, 6H), 1.01 – 0.88
t, 6H). C NMR (126 MHz, CDCl
43.4, 139.5, 136.9, 135.9, 131.9, 131.9, 131.4, 130.5, 130.5,
28.6, 128.5, 128.0, 127.9, 127.6, 127.5, 120.7, 114.4, 114.2,
13.9, 113.6, 70.1, 69.9, 66.0, 65.8, 58.4, 58.3, 46.0, 45.9, 28.8,
3.6. LRMS calc. for C34
26.2.
2
1
3
) δ 7.54 –
7
4
6
5
13
3
1.1 Hz, 6H). C NMR (126 MHz, CDCl ) δ 167.6, 157.7, 157.0,
145.4, 144.8, 140.2, 138.9, 137.0, 136.3, 136.1, 135.8, 135.6,
132.0, 131.9, 130.6, 130.3, 128.6, 128.5, 128.0, 127.9, 127.8,
127.7, 127.6, 127.5, 116.9, 114.4, 114.1, 113.8, 113.5, 70.1, 69.8,
(
(
13
3
) δ 157.8, 157.1, 147.6,
65.9, 65.7, 58.3, 51.6, 45.9, 28.8, 13.7. HRMS calc. for
+
1
1
1
1
6
C
37
H40NO
4
(M+H) : 562.2952. Found: 562.2965.
Methyl
(E/Z)-3-(4-(1-(4-(2-(dimethylamino)ethoxy)
+
phenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenyl)propanoate
27a): Prepared from 26a (123 mg, 0.22 mmol, 1.0 eq.)
35 3
H F NO
5
S (M+H) : 626.22. Found:
(
according to General Procedure A. 27a was isolated as a pale
(
E/Z)-2-(4-(1-(4-(Benzyloxy)phenyl)-2-(4-vinylphenyl)but-
yellow oil (90 mg, 0.19 mmol) that was used without further
purification in 87% yield as a 1:1 mixture of E:Z isomers. H
1
1
-en-1-yl)phenoxy)-N,N-dimethylethan-1-amine (25): To a
solution of 24 (380 mg, 0.61 mmol, 1.0 eq.) in nPrOH (12 mL)
3
NMR (500 MHz, CDCl ) δ 7.09 (d, J = 8.6 Hz, 2H), 7.07 – 6.92
was added potassium vinyltrifluoroborate (97.8 mg, 0.73, 1.2
(m, 10H), 6.80 (d, J = 7.9 Hz, 3H), 6.75 – 6.61 (m, 6H), 6.47 (d,
J = 8.2 Hz, 2H), 6.39 – 6.32 (m, 2H), 4.10 (t, J = 5.6 Hz, 2H),
3.94 (t, J = 5.6 Hz, 2H), 3.67 (d, J = 4.0 Hz, 6H), 2.93 – 2.86 (m,
4H), 2.84 (t, J = 5.5 Hz, 2H), 2.75 (t, J = 5.6 Hz, 2H), 2.61 (td, J
= 7.9, 5.5 Hz, 4H), 2.48 (dd, J = 11.1, 7.4 Hz, 6H), 2.41 (s, 6H),
eq.), PdCl
distilled Et
heated to 100 °C and stirred for 24 h. After cooling to room
temperature, the reaction was quenched with 5 mL H O,
extracted with EtOAc (3 x 15 mL), dried over Na SO , filtered,
and concentrated to a dark yellow oil. Purification by silica gel
column chromatography using a 0-8% MeOH in CH Cl solvent
2
(dppf) (22.3 mg, 0.031 mmol, 5 mol%). and freshly
3
N (85 μL, 0.61 mmol, 1.0 eq). The reaction was
2
1
3
2
4
2.36 (s, 6H), 0.93 (td, J = 7.4, 4.3 Hz, 6H). C NMR (126 MHz,
CDCl ) δ 173.6, 173.6, 157.2, 156.4, 155.8, 154.8, 140.8, 140.7,
3
2
2
140.2, 140.1, 138.1, 138.0, 137.8, 136.7, 136.2, 135.1, 134.8,
132.1, 131.9, 130.6, 130.5, 129.9, 129.8, 127.7, 115.4, 114.7,
113.8, 113.1, 64.9, 64.5, 58.1, 53.5, 51.6, 45.6, 45.4, 45.3, 35.7,
gradient yielded 25 as a yellow oil (250 mg, 0.50 mmol) in 81%
1
yield as a 1:1 mixture of E:Z isomers. H NMR (400 MHz,
CDCl
3
) δ 7.52 – 7.45 (m, 2H), 7.45 – 7.29 (m, 10H), 7.27 – 7.22
30.7, 30.6, 30.4, 29.0, 28.9, 27.0, 26.9, 26.6, 26.5, 26.4, 26.3,
+
(m, 3H), 7.18 (dd, J = 8.6, 6.2 Hz, 4H), 7.11 (dd, J = 8.3, 3.5 Hz,
25.3, 13.8, 13.7. HRMS calc. for C30
Found: 474.2636.
H36NO
4
(M+H) : 474.2639.
3H), 6.99 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.83 (dd,
J = 8.9, 7.1 Hz, 3H), 6.67 (d, J = 8.8 Hz, 3H), 6.61 (d, J = 8.8 Hz,
(
Z)-3-(4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-1-(4-
2
2
2
5
1
H), 5.72 (ddd, J = 17.6, 3.0, 1.0 Hz, 2H), 5.22 (ddd, J = 10.8,
.6, 1.0 Hz, 2H), 5.11 (s, 2H), 4.96 (s, 2H), 4.13 (t, J = 5.8 Hz,
H), 3.98 (t, J = 5.8 Hz, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.71 (t, J =
.7 Hz, 2H), 2.52 (qd, J = 7.4, 3.6 Hz, 4H), 2.37 (d, J = 30.1 Hz,
hydroxyphenyl)but-1-en-2-yl)phenyl)-N-
hydroxypropanamide (28a): Prepared from 27a (90 mg, 0.19
mmol, 1.0 eq.) according to General Procedure B. The product
was isolated as an amorphous white solid (51 mg, 0.11 mmol) in
7% yield as a 1:1 mixture of E:Z isomers. Subsequent
preparatory reverse phase HPLC purification using a 12-54%
MeCN in H O gradient and lyophilization of the desired fractions
yielded the Z isomer of 28a as a fluffy, amorphous, white solid.
13
3
2H), 0.97 (td, J = 7.5, 1.4 Hz, 6H). C NMR (126 MHz, CDCl )
5
δ 157.6, 156.8, 142.4, 142.3, 140.7, 138.0, 137.1, 136.8, 136.7,
1
1
1
36.6, 136.4, 136.1, 135.9, 135.1, 132.0, 131.9, 131.7, 130.6,
30.4, 129.9, 128.6, 128.5, 128.3, 128.0, 127.9, 127.6, 127.5,
27.4, 126.2, 126.1, 125.8, 115.4, 114.9, 114.4, 114.1, 114.0,
2

+
Analytical HPLC (C18, 5% to 100% MeCN/H
2
O) indicated the
product was 96% pure H NMR (500 MHz, CDCl ) δ 8.57 (s,
H), 7.02 (d, J = 8.5 Hz, 6H), 6.84 – 6.71 (m, 4H), 6.60 (d, J =
7.4 Hz, 4H). HRMS calc. for C31
Found: 503.2912.
39 2
H N O4 (M+H) : 503.2904.
1
3
3
8
2
Cell lines and reagents:
.8 Hz, 2H), 4.02 (t, J = 5.4 Hz, 2H), 2.86 (t, J = 7.7 Hz, 2H),
.80 (t, J = 5.4 Hz, 2H), 2.52 – 2.44 (m, 3H), 2.39 (s, 6H), 0.91
Cell lines were purchased from the American Type Culture
Collection (ATCC) and maintained in a humidified 37°C, 5%
CO incubator. MCF-7 cells were cultured at 37°C in alpha
+
(t, J = 7.4 Hz, 3H). HRMS calc. for C29
H
35
N
2
O
4
(M+H) :
475.2591. Found: 475.2594.
2
modification of Eagle’s medium (αMEM, Wisent) supplemented
Methyl (E/Z)-5-(4-((Z)-1-(4-(benzyloxy)phenyl)-1-(4-(2-
with 10% Fetal Bovine Serum (FBS, Sigma), 2 mM L-glutamine
and 100 UI/mL penicillin-streptomycin (Wisent). MCF-10A cells
were maintained in Dulbecco’s modified Eagle’s F-12 media
(
dimethylamino)ethoxy)phenyl)but-1-en-2-yl)phenyl)pent-4-
enoate (26b): 26b was prepared using an identical procedure as
6a, using a solution of 25 (148 mg, 0.29 mmol, 1.0 eq.) in
CH Cl (1 mL), anhydrous PTSA (56 mg, 0.32 mmol, 1.1 eq.),
2
(DMEM F-12, Wisent) without calcium chloride and
2
2
supplemented with 10 % FBS, 10 ng / ml epidermal growth
factor (EGF), 10 μg / ml insulin, 0,5 μg / ml hydrocortisone, 100
ng / ml cholera enterotoxin (Sigma) and 100 UI/mL penicillin-
streptomycin. MDA-MB-231 cells were cultured in DMEM
methyl 4-pentenoate (336 mg, 2.9 mmol, 10eq.), and two
additions of Grubbs’ gen. 2 catalyst (12.5 mg, 0.015 mmol, 5
mol%) in CH
chromatography using a 0-8% MeOH in CH
6b as a dark brown/green oil (129 mg, 0.22 mmol) in 74% yield
2
Cl
2
(0.5 mL). Purification by silica gel column
2
Cl gradient yielded
2
(Wisent) supplemented with 5 % FBS and 100 UI/mL penicillin-
2
1
streptomycin. HEK293T cells were maintained in DMEM
supplemented with 10% FBS and 100 UI/mL penicillin-
streptomycin.
as a 1:1 mixture of E:Z isomers. H NMR (500 MHz, CDCl
3
) δ
.48 (d, J = 7.0 Hz, 2H), 7.46 – 7.30 (m, 9H), 7.24 – 7.10 (m,
H), 7.06 (td, J = 6.0, 3.1 Hz, 4H), 6.97 (d, J = 8.6 Hz, 3H), 6.91
7
7
(
d, J = 8.6 Hz, 2H), 6.80 (ddd, J = 8.0, 6.4, 1.6 Hz, 4H), 6.71 –
The transfection reagent polyethylenimine (PEI) was ordered
from Polysciences, Inc. 17β-Estradiol, 4-hydroxytamoxifen (4-
OHT) and suberoylanilide hydroxamic acid (SAHA) were
purchased from Sigma, Tocris and Cayman Chemical Company
respectively.
6
4
3
.62 (m, 2H), 6.59 (d, J = 8.7 Hz, 2H), 5.09 (s, 2H), 4.95 (s, 1H),
.12 (t, J = 5.8 Hz, 2H), 3.97 (t, J = 6.0 Hz, 2H), 3.74 (s, 2H),
.71 (s, 3H), 3.29 – 3.22 (m, 1H), 2.78 (s, 2H), 2.69 (s, 2H), 2.51
(td, J = 9.4, 8.7, 3.8 Hz, 6H), 2.39 (s, 6H), 2.33 (s, 6H), 2.00 –
1
.79 (m, 6H), 1.45 (t, J = 10.4 Hz, 2H), 0.94 (t, J = 7.3 Hz, 6H).
C NMR (126 MHz, CDCl ) δ 132.0, 131.9, 130.6, 129.9, 128.6,
3
28.5, 128.0, 127.6, 127.5, 126.1, 125.6, 120.8, 119.2, 114.3,
13
Rabbit polyclonal anti-acetyl-histone H4 (06-598) and rabbit
monoclonal ERα, clone 60C (04-820) were purchased from EMD
Millipore. Mouse monoclonal anti-acetyl-tubulin α (ab24610)
was ordered from Abcam. Mouse monoclonal anti-β-actin, clone
AC-15 (A5441) was obtained from Sigma. Horseradish
peroxidase (HRP)-conjugated secondary antibodies were
obtained from Jackson Laboratory. Polyvinylidene difluoride
(PVDF) membranes were purchased from EMD Millipore.
Enhanced chemiluminescence (ECL) detection reagents were
ordered from Bio-Rad.
1
1
2
14.1, 113.7, 113.4, 70.0, 69.8, 53.4, 51.6, 45.9, 27.0, 26.9, 26.4,
+
6.2, 13.7. HRMS calc. for C39
H
44NO
4
(M+H) : 590.3265.
Found: 590.3260.
Methyl
(E/Z)-5-(4-(1-(4-(2-(dimethylamino)ethoxy)
phenyl)-1-(4-hydroxyphenyl)but-1-en-2-yl)phenyl)pentanoate
(27b): Prepared from 26b (127 mg, 0.22 mmol, 1.0 eq.)
according to General Procedure A. 27b was isolated as a pale
yellow oil (94 mg, 0.19 mmol) that was used without further
purification in 85% yield as a 1:1 mixture of E:Z isomers. H
NMR (500 MHz, CDCl
7
6
3
1
Cell transfection: For BRET assays, HEK293T cells were
maintained in DMEM (Wisent) supplemented with 10% FBS,
3
) δ 7.21 – 7.03 (m, 5H), 7.03 – 6.89 (m,
100 UI/mL penicillin/streptomycin, and cultured at 37°C. Before
H), 6.79 (dd, J = 10.8, 8.5 Hz, 4H), 6.75 – 6.60 (m, 4H), 6.53 –
.43 (m, 2H), 6.43 – 6.34 (m, 1H), 4.10 (td, J = 5.7, 1.8 Hz, 2H),
.94 (t, J = 5.6 Hz, 2H), 3.69 (s, 4H), 2.85 – 2.76 (m, 2H), 2.72
each experiment, cells were switched for 48 h in DMEM without
phenol red, supplemented with 10% charcoal-dextran-treated
FBS, 100 UI/mL penicillin/streptomycin and 4 mM L-glutamine.
On the following day, cells were co-transfected with an
expression vector for Renilla Luciferase II conjugated to the C-
terminus of human ERα (pcDNA3.1-ERα-RLucII; 30 ng/million
cells), either alone (for background evaluation) or together with
an expression vector for YFP (Topaz) fused to aa 625-1050 of
human NCOA1/SRC1 (pcDNA3.1-NCOA1-eYFP, 1.2 g) or for
two copies of YFP (Topaz) fused at the N- and C-termini of a
tandem repeat of an LXXLL motif derived from NCOA2 (L
peptide: KHKILHRLLQDSS) and of the glucocorticoid receptor
(t, J = 5.6 Hz, 2H), 2.62 – 2.43 (m, 6H), 2.43 – 2.36 (m, 5H),
2
1
.35 – 2.24 (m, 4H), 2.00 – 1.80 (m, 6H), 1.71 – 1.53 (m, 5H),
13
.44 (t, J = 9.4 Hz, 3H), 1.02 – 0.83 (m, 6H). C NMR (126
) δ 140.8, 140.6, 132.1, 131.9, 130.7, 130.6, 130.5,
29.7, 129.7, 129.6, 127.9, 127.8, 127.8, 126.3, 115.2, 114.4,
MHz, CDCl
3
1
1
3
13.9, 113.1, 58.3, 53.4, 51.6, 51.5, 45.7, 45.5, 35.5, 35.0, 34.2,
4.0, 33.4, 30.7, 27.0, 26.9, 26.3, 26.1, 24.6, 13.7. HRMS calc.
+
for C32
H
40NO
4
(M+H) : 502.2952. Found: 502.2948.
(Z)-5-(4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-1-(4-
hydroxyphenyl)but-1-en-2-yl)phenyl)-N-
nuclear
localization
signal
(N
peptide:
hydroxypentanamide (28b): Prepared from 27b (93 mg, 0.19
mmol, 1.0 eq.) according to General Procedure B. Purification by
reverse phase column chromatography yielded 28b as a white
solid (33 mg, 0.06 mmol) in 34% yield as a 1:1 mixture of E:Z
isomers. Subsequent preparatory reverse phase HPLC
DRAHSTPPKNKRNVRDPKDRAHSTPPKNKRNVRDPK)
(vector pYFP-L2N2-YFP; 1.5 g/million cells). The amount of
DNA was complemented to a total of 1.7 µg using pcDNA 3.1
(empty vector) in 75 l of PBS/million cells. Transient
transfections were performed using PEI (dissolved in water and
heated up to 80°C). PEI (3 µg of linear PEI and 1 µg of
polybranched PEI for each µg of DNA diluted in PBS) was
mixed with DNA (1V/V, 150 l total) and left for 15-20 min at
room temperature. Cells (1 million in 850 l) were added to the
PEI:DNA mixture and were seeded (50,000 cells per well) in 96-
well white plates (Costar, Corning). 48 h later, HEK293T cells
were treated with hormones in triplicates. The medium was
2
purification using a 12-54% MeCN in H O gradient and
lyophilisation of the desired fractions yielded the Z isomer of 28b
as a fluffy, amorphous, white solid. Analytical HPLC (C18, 5%
1
to 100% MeCN/H
2
O) indicated the product was >99% pure H
NMR (500 MHz, CD
3
OD) δ 7.07 – 6.94 (m, 6H), 6.83 – 6.73 (m,
H), 6.62 (d, J = 8.4 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 3.28 (s,
4
2
2
H), 2.76 (s, 6H), 2.58 (t, J = 6.8 Hz, 2H), 2.49 (q, J = 7.4 Hz,
H), 2.10 (d, J = 7.5 Hz, 2H), 1.70 – 1.47 (m, 5H), 0.94 (t, J =

aspirated and replaced by PBS supplemented with hormones 1 h
before BRET assays.
Cheng−Prusoff equation [K
assumption of a standard fast-on−fast-off mechanism of
i
m
= IC50/(1+[S]/K )] with the
inhibition.
BRET Assays: Coelenterazine
Technology) was added to each well to a final concentration of
0 µM. Readings were then collected using a MITHRAS LB940
Berthold Technology) multidetector plate reader, allowing the
H
(Coel-h, Nanolight
Cell proliferation assay: MCF-7 cells were plated at 300
cells per well in αMEM supplemented with 5% FBS (v/v), 2 mM
L-glutamine, 100 UI/mL penicillin and 100 µg / mL streptomycin
in 384-well plates, (Corning® Costar®, Sigma). MCF-10A and
MDA-MB-231 were seeded at 200 cells / well in the same media.
Cells were treated every 2 days with different concentrations of
either 4-OHT, SAHA, or hybrids with media replenishment after
4 days. After 7 days of treatment, cell proliferation was measured
using the CellTiter-Glo® luminescent assay (Promega) following
the manufacturer’s instructions. Acquisition of luminescence was
performed using the Synergy NEO microplate reader (BioTek).
Results were analyzed using GraphPad Prism software.
1
(
sequential integration of the signals detected in the 485/20 nm
and 530/25 nm windows, for luciferase and YFP light emissions,
respectively. The BRET signal was determined by calculating the
ratio of the light intensity emitted by the YFP fusion over the
light intensity emitted by the Luc fusion. The values were
corrected by subtracting the background BRET signal detected
when the Luc fusion construct was expressed alone. For BRET
titration experiments, BRET ratios were expressed as a function
of the [acceptor]/[donor] expression ratio (YFP/Luc). Total
fluorescence and luminescence were used as a relative measure
of total expression of the acceptor and donor proteins,
Western Blotting: MCF-7 cells were plated at 0.6 million
cells per 6 cm Petri dish in αMEM supplemented with 5% FBS
respectively. Total fluorescence was determined with
a
(v/v), 2 mM L-glutamine, 100 UI penicillin and 100 µg/mL
FlexStation II microplate reader (Molecular Devices) using an
excitation filter at 485/9 nm and an emission filter at 538/18 nm.
Total luminescence was measured in the MITHRAS LB940 plate
reader 3 min after the addition of Coel-h (10 μM, Nanolight
Technology) in the absence of emission filter. IC50 values were
calculated with GraphPad from 2 independent experiments
streptomycin. Cells were treated for 8 h and harvested in protein
extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM
EDTA, 2% SDS, 0.5% Triton X-100, 1% NP-40). Proteins were
loaded on SDS-PAGE gel (14%, 50 µg protein/lane) and
transferred to a PVDF membrane, then blotted overnight with
primary antibodies targeting either acetyl-tubulin, acetyl-histone
H4, ERα or β-actin.
(standard errors lower than 5%).
In Vitro HDAC Assays: HDAC3−“NCoR1” and HDAC6
were purchased from Cayman Chemicals and used without
further purification. The HDAC assay buffer consisted of 50 mM
2
Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl , and bovine
serum albumin (0.5 mg/mL), pH was adjusted to 8 using 6 M
NaOH and 1 M HCl as needed. Trypsin [25 mg/mL, from
porcine pancreas, in 0.9% sodium chloride] was from Sigma
Aldrich. Stock solutions of inhibitors and substrate were obtained
by dissolution in DMSO and addition of HDAC assay buffer to
afford solutions containing 1.7 % v/v DMSO. Serial dilution
using HDAC buffer contacting 1.7 % v/v DMSO was used to
obtain all requisite inhibitors and substrate solutions.
Reverse transcription and Real-Time quantitative PCR:
MCF-7 cells were seeded at 0.2 million cells per well of a 6-well
plate and grown for 24 h in αMEM supplemented with 5% FBS
(v/v), 2 mM L-glutamine, 100 UI penicillin and 100 µg/mL
streptomycin. After 24 h of treatment with either 4-OHT, SAHA
or 28b (5 µM), cells were collected in QIAzol reagent (Qiagen)
and RNA extraction was performed following manufacturer’s
instructions. Total RNA (2 µg) was reverse transcribed using the
RevertAid H first minus strand cDNA synthesis kit (Fermentas).
cDNA was diluted 10 times in water and used for RT-qPCR.
Relative gene expression levels were evaluated using the
Universal Probe Library (Roche). Amplification levels were
detected with the Viia7 Real-Time PCR system (Life
Technologies). All reactions have been performed in triplicates in
two independent experiments. The ΔΔCT method was used to
evaluate relative gene expression. Housekeeping genes (RPLP0,
TBP and YWHAZ) were used as endogenous controls. For
specific primers and probes used for RT-qPCR, see Supporting
Information.
For inhibition of recombinant human HDAC3 and HDAC6,
dose−response experiments with internal controls were
performed in black low-binding Nunc 96-well microtiter plates.
Dilution series (8 concentrations) were prepared in HDAC assay
buffer with 1.7 % v/v DMSO. The appropriate dilution of
inhibitor (10 μL of 5 times the desired final concentration) was
added to each well followed by HDAC assay buffer (25 μL)
containing substrate [Ac-Leu-Gly-Lys(Ac)-AMC, 40 or 30 μM
for HDAC 3 and 80 or 60 μM for HDAC 6]. Finally, a solution
of the appropriate HDAC (15 μL) was added [HDAC3, 10
ng/well; HDAC 6, 60 ng/well] and the plate incubated at 37 °C
for 30 min with mechanical shaking (270 rpm). Then trypsin (50
μL, 0.4 mg/mL) was added and the assay developed for 30 min at
room temperature with mechanical shaking (50 rpm).
Fluorescence measurements were then taken on a Molecular
Devices SpectraMax i3x plate reader with excitation at 360/9 nm
nm and detecting emission at 460 nm/15 nm. Each assay was
performed in triplicate at two different substrate concentrations.
Baseline fluorescence emission was accounted for using blanks,
run in triplicate, containing substrate (25 μL), HDAC assay
buffer (15 μL), HDAC assay buffer with 1.7 % v/v DMSO (10
μL), and trypsin (50 μL). Fluorescence emission was normalized
using controls, run in triplicate, containing substrate (25 μL),
HDAC (15 μL), HDAC assay buffer with 1.7 % v/v DMSO (10
μL), and trypsin (50 μL). The data were analyzed by nonlinear
regression with GraphPad Prism to afford IC50 values from the
6. Acknowledgments
The authors thank the Canadian Institutes of Health Research
for funding. S.M. holds the CIBC breast cancer research chair at
Université de Montréal. A.F.P. thanks CIHR for a postgraduate
fellowship. B.M.W. thanks NSERC for
a
postgraduate
fellowship, M.D. and M.E.E. thank the Molecular Biology
training program at Université de Montréal for studentships.
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