Synthesis and structure-activity relationships of novel hybrid ferrocenyl compounds based on a bicyclic core skeleton for breast cancer therapy

Article abstract of DOI:10.1016/j.bmc.2016.05.019

Breast cancer is the most frequent cancer in women worldwide, and incidence is increasing year by year. Although current selective estrogen receptor modulators (SERMs) have clear advantages in the treatment of hormone-responsive breast cancer, they are ineffective for ER(-). In this study, we describe the design and synthesis of a series of dual-acting estrogen receptor (ER) and histone deacetylase (HDAC) inhibitors with incorporation of the ferrocenyl moiety, leading to novel hybrid ferrocenyl complexes (FcOBHS-HDACis) for breast cancer therapy. It is worth to note that these ferrocenyl conjugates could not only potently inhibit HDACs and the proliferation of ERα positive (ER(+)) breast cancer cells (MCF-7), but also show significant antiproliferative effect on ER(-) breast cancer cells (MDA-MB-231). Thus, the FcOBHS-HDACi conjugates represent a novel approach to the development of efficiently dual-acting agents for treatment of breast cancer.

Full text of DOI:10.1016/j.bmc.2016.05.019

Accepted Manuscript
Synthesis and structure-activity relationships of novel hybrid ferrocenyl com-
pounds based on a bicyclic core skeleton for breast cancer therapy
Changhao Li, Chu Tang, Zhiye Hu, Chenxi Zhao, Chenlu Li, Silong Zhang,
Chune Dong, Hai-Bing Zhou, Jian Huang
PII:
S0968-0896(16)30343-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.05.019
BMC 13003
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 April 2016
12 May 2016
12 May 2016
Please cite this article as: Li, C., Tang, C., Hu, Z., Zhao, C., Li, C., Zhang, S., Dong, C., Zhou, H-B., Huang, J.,
Synthesis and structure-activity relationships of novel hybrid ferrocenyl compounds based on a bicyclic core
skeleton for breast cancer therapy, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.05.019
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Synthesis and structure-activity relationships of novel hybrid ferrocenyl
compounds based on a bicyclic core skeleton for breast cancer therapy
a
b
Changhao Li , Chu Tang , Zhiye Hub, Chenxi Zhao a, Chenlu Li b, Silong Zhang b, Chune Dong b, Hai-
Bing Zhou b,*, Jian Huang a,*
a College of Life Sciences, Wuhan University, Wuhan 430072, China
bHubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan 430071, China; Key Laboratory of Combinatorial
Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education; Wuhan University School of Pharmaceutical Sciences,
Wuhan 430071, China
*Correspondence to: Hai-Bing Zhou, Tel 862768759586, zhouhb@whu.edu.cn; Jian Huang, Tel 86276875358, jianhuang@whu.edu.cn.
A R T C L E I N F O
A B S T R A C T
Article history:
Received
Breast cancer is the most frequent cancer in women worldwide, and incidence is increasing year
by year. Although current selective estrogen receptor modulators (SERMs) have clear advantages
in the treatment of hormone-responsive breast cancer, they are ineffective for ER(-). In this study,
we describe the design and synthesis of a series of dual-acting estrogen receptor (ER) and histone
deacetylase (HDAC) inhibitors with incorporation of the ferrocenyl moiety, leading to novel
hybrid ferrocenyl complexes (FcOBHS-HDACis) for breast cancer therapy. It is worth to note that
these ferrocenyl conjugates could not only
Received in revised from
Accepted
Available online
Keywords:
Breast cancer
SERMs
positive (ER(+)) breast cancer cells (MCF-7), but also show significant antiproliferative effect on
ER(-) breast cancer cells (MDA-MB-231). Thus, the FcOBHS-HDACi conjugates represent a
novel approach to the development of efficiently dual-acting agents for treatment of breast cancer.
HDACi
Ferrocenyl
associated with uterine hypertrophy and an increased risk of
1. Introduction
Breast cancer is the most frequent cancer in women worldwide,
and incidence is increasing year by year. Approximately 15% of
women are diagnosed with breast cancer in the reproductive age,1
and it is the leading cause of cancer death in women between 35
and 55 years of age.2, 3 Estrogen receptors (ERs, comprising of
two subtypes ER and ER ) and endogenous estrogens play
essential roles in the growth of breast tumors.4 Some selective
estrogen receptor modulators (SERMs) such as tamoxifen or
raloxifene, which act at the breast as anti-estrogens, are widely
used in the clinic for the treatment of this disease.5-7 Although
certain SERMs are useful for the treatment of breast cancer,
unfortunately, an undesired side effect severely limits their
therapeutical use. For instance, tamoxifen is the first clinically
used SERM for treatment of breast cancer,8, 9 however, due to the
partial estrogenic activity on the endometrium, the clinical use is
endometrial cancer; in addition, tamoxifen therapy is ineffective
for ER(-) breast cancer.10-12 It is thus evident that there is a need
for new ER ligands that are more effective in ER(-) breast cancer.
To address the need of disease-modifying drugs for breast cancer,
in recent years, new strategies have been devised in medicinal
chemistry, one that has received significant attention is to
develop bivalent agents through combining two bioactive drugs
into a single molecule.
In the field of breast cancer, this strategy involved linking a
potent ER ligand to a second component, such as an antimitotic
agent (E2-paclitaxel conjugate 1),13 antimetabolite agent (E2-
nucleoside conjugate 2),14 alkylating agent (E2-chlorambucil
conjugate 3),15 steroid sulfatase agent (Estrone 3-O-sulfamate
4),16 tubulin agent (dual ER-tubulin agent 5),17 and aromatase
agent (dual ER-aromatase agent 6) etc.18-21 Despite important
1

progress in this area, there is still room for improvement
regarding dual-acting hybrids, because many bioactive hybrids
show extremely low ER binding affinity or lack target-tissue
selectivity.22 Therefore, numerous efforts have been done to
develop new bioactive hybrid compounds with higher ER binding
affinity, target-tissue selectivity and lower inherent toxicity.
In another aspect, histone deacetylases (HDACs) have recently
emerged as important targets for cancer therapy, including breast
cancer.21, 23 In particular, HDAC may be a potential target for
HDAC inhibition could be a promising approach in breast cancer
therapy. Indeed, recent observations have shown that dual-acting
ER ligand-HDACi conjugates have an improved in vitro
therapeutic index and potent anti-cancer activity;26-29 yet, they
retain some disadvantages. For instance, most benzothiophene
scaffold-based hybrid HDAC inhibitors (e.g., compound 7,
Figure 2) show little effect on ER transcriptional activity,29
ethynylestradiol-HDACi conjugates (e.g., compound 8, Figure 2)
retain the
therapeutic intervention in the treatment of ER negative breast
and Tam-HDACi conjugates (e.g., compound 9, Figure 2) are
toxic to the healthy cells.26
cancers.24,
These evidences suggest simultaneous ER and
25
Figure 1 Currently referenced dual-acting ER ligand conjugates
As part of our long-term interest in the development of ligands
for the ERs having novel structures and activities, we have
prepared a novel series of three-dimensional ER ligands based on
7-oxabicyclo[2.2.1]hept-5-ene core, in which OBHS (5,6-bis-(4-
hydroxyphenyl)-7-oxabicyclo[2.2.1]hept-5-ene-2-sulfonic acid
phenyl ester, compound 10, Figure 2) was one of the most
promising antagonist compounds.30-35 Recently, our research
group has chosen HDACi as the second component and
synthesized dual-acting OBHS-HDACi conjugates.35 Two of
these bifunctional conjugates (e.g. compounds 11 and 12, Figure
2) exhibited good ER binding affinity,
normal cells. It should be emphasized that HDACi unit of
conjugates possessing the suberic acid group rather than
hydroxamate exhibits greater activity.35, 36 Unfortunately, these
bifunctional hybrid OBHS-HDACi conjugates showed very weak
anti-proliferative effect on ER(-) breast cancer (MDA-MB-231,
IC50 > 50 M). Our aim now is to extend therapeutic effectives of
these OBHS-HDACi conjugates that would produce a powerful
antiproliferative effect on both ER(+) and ER(-) breast cancer
cells.
In recent years, bioorganometallic chemistry has attracted
active interest in medicinal chemistry, and numerous classes of
organometallic compounds have been found application in drug
design. Specifically, ferrocene (Fc) has generated much interest
activity and significant anti-
cancer cells (MCF-7); moreover, they had no toxicity towards

in cancer therapeutics literature. Ferrocenyl derivatives usually
are not toxicity and lipophilic, and have good redox properties.37-
ferrocenyl group into the OBHS-HDACi skeleton. First, we
introduced the ferrocenyl group on the phenyl ring of sulfonate
unit (Series I); the second approach involved substituting one
phenol ring on C5 or C6 with ferrocenyl group (Series II).
Subsequently, the biological activity of these FcOBHS-HDACi
conjugates was studied. It has been observed that these dual-
acting conjugates showed moderate binding affinity for ERs and
strong inhibition activity towards HDACs. Several molecules
retain great activity of the parent compound against ER(+) breast
cancer cells (MCF-7), and more importantly, could effectively
inhibit the proliferation of ER(-) breast cancer cells (MDA-MB-
231). While chain length of HDACi unit is a major factor for
their inhibitory activities, and the conjugates presenting that a
shorter rather than longer chain length tend to be more active,
especially those with suberic acid unit. These results suggest that
conjugation of ferrocenyl group to estrogen modulators could
improve therapeutic activity.
39
Several ferrocenyl compounds display interesting antitumor
activity, and the hydroxyferrocifen, the mostly studied that
selectively targets breast cancer and shows
a
strong
antiproliferative effect on both ER(+) and ER(-) breast cancer
cells.40 Furthermore, there has been a number of examples of
other ferrocene containing compounds that also exhibit great
activity against ER(-) breast cancer cells.41-43 At the same time,
our group has reported that incorporation of the ferrocenyl unit
into OBHS can lead to OBHS complexes (termed FcOBHS)
showing significant antiproliferative effect on both ER(+) and
ER(-) breast cancer cells.32
In order to extend therapeutic effectives of OBHS-HDACi
conjugates above for both ER(+) and ER(-) breast cancer cells, in
this paper, we report a new family of ferrocene complexes
(termed FcOBHS-HDACi conjugate, Figure 3) based on OBHS-
HDACi core scaffold, with two strategies to introduce the
Figure 2 Structures of dual-acting ER ligand-HDACi conjugates
3

Figure 3 Design of dual-acting FcOBHS-HDACi conjugates
by three steps: firstly, the Fc 20 reacted with 3-methoxybenzoyl
2. Results and Discussion
chloride 21a, 4-methoxybenzoyl chloride 21b or 4-
methoxybenzyl alcohol 21c in the presence of different acid
catalysts (AlCl3 for 22a-b, CF3COOH for 22c) to afford the
corresponding ferrocenyl substituted products 22a-c. Subsequent
O-demethylation with boron tribromide afforded phenolic
products 23a-c, which were then reacted with 2-
chloroethanesulfonyl chloride in the presence of Et3N gave the
ferrocenyl dienophiles 24a-c (Scheme 2A).32
2.1. Chemical Synthesis
All designed FcOBHS-HDACi conjugates were prepared by a
Diels-Alder cycloaddition of furan derivatives 19a-c or 3-
ferrocenyl-4-(4-hydroxyph-enyl) furan 29 with various
dienophiles (Schemes 1-3). The important intermediates, furan
derivatives 19a-c and 29, were prepared according to our
previously reported methodology (Schemes 1 and 3).32, 35 The
synthesis of ferrocenyl vinyl sulfonates 24a-c was accomplished
Scheme 1 Synthesis of furan derivatives 19a-c. Reagents and conditions: (a) NBS (1.2 equiv.), p-TsOH (0.2 equiv.), CHCl3, rt, 12 h; (b)
4-aminophenylacetic acid, Et3N, CH3CN, rt, 12 h; (c) NaH, DMSO, rt, 3 h; (d) BBr3, CH2Cl2, -20 °C, 12 h; (e) DIBAL-H, THF, -78 °C, 8
h; (f) pimelic anhydride for 19a; suberic anhydride for 19b; azelaic anhydride for 19c, THF, rt, 2 h.

Scheme 2 Synthesis of dienophiles 24a-c and 28. Reagents and conditions: (a) AlCl3 for 22a-b; CF3COOH for 22c, CH2Cl2, 0 °C, 1 h; (b)
BBr3, CH2Cl2, -20 °C, 12 h; (c) 2-chloroethanesulfonyl chloride, Et3N, CH2Cl2, 0 °C, 12 h; (d) acetic anhydride, 150 °C, 1 h; (e) 4-
aminophenol, THF, rt, 1 h; (f) 2-chloroethanesulfonyl chloride, Et3N, THF, 0 °C, 12 h.
The vinyl sulfonate 28 was prepared following a previously
reported route (Scheme 2B).35 We used suberic acid 25 as the
starting material, which was treated with acetic anhydride to give
suberic anhydride 26.44 Then, suberic anhydride 26 was
condensed with 4-aminophenol to generate compound 27, which
was subsequently reacted with 2-chloroethanesulfonyl chloride to
yield the vinyl sulfonate 28. The synthesis of FcOBHS-HDACi
conjugates was carried out successfully to give the first three
conjugates (31a-c, linker length, n = 6, Scheme 3A, Series I) by a
Diels-Alder reaction of furans with vinyl sulfonates, all of them
exhibited moderate-to-excellent binding affinity and nanomolar
inhibition of HDAC (see below); moreover, during our work on
OBHS-core ER ligands, we found that the size of the substituents
on phenyl ring of the OBHS sulfonate moiety were important
determinants of the binding affinity and selectivity of these
compounds. Therefore, we wondered whether the chain length of
HDACi unit was a critical variable in optimizing the positioning
of ligand for ER or of zinc binding group (ZBG) for HDAC
inhibition profiles. Thus, the success of the suberic acid
conjugates prompted us to explore the biological effects of chain-
length variation, and as a result, the pimelic (30a-c, linker length
n = 5, Scheme 3A, Series I) and azelaic acid analogs (32a-c,
linker length n = 7, Scheme 3A, Series I) were prepared,
respectively. However, we found that the suberic acid conjugate
had higher affinity than pimelic and azelaic acid analogs in favor
of ER ; additionally, the suberic acid conjugate also had superior
HDAC1 inhibition activity relative to the other two series (30 and
32). These results illustrate that the suberic acid unit (linker
length n = 6) was essential for optimal biological activity.
Therefore, when the HDACi unit was appended onto the phenyl
ring of sulfonate moiety of OBHS (Scheme 3B, Series II),
additional SAR investigations were also focused on the suberic
acid derivative 33 rather than pimelic and azelaic acid analogs.
However, we found 33 still showed poor binding affinity for both
ERs (see below).
The Diels-Alder reaction of vinyl sulfonates with furans (19a-c
or 29) went very smoothly, and the yields of the products were
generally good. Also, it is noteworthy that high stereoselectivity
was observed in the reaction of furans with dienophiles; as we
described previously,30 the exo products predominated in the
Diels-Alder reaction. It should be noted that the conjugates 30a-c,
31a-c, 32a-c, and 33 were generated by the Diels-Alder reactions
of vinyl sulfonates with the unsymmetrical furans, therefore,
which were studied as a mixture of regioisomers that, despite our
best efforts, could not be separated, the results were summarized
in Table 1.
5

Scheme 3 Synthesis of FcOBHS-HDACi conjugates 30a-c, 31a-c, 32a-c, and 33
2.2. FcOBHS-HDACi Conjugates Exhibit Binding Affinity
effect on binding affinity of conjugates for ER : when ferroceny-
formyl group is moved from C3 to C4-position of the phenyl ring,
results in an obvious increase in binding affinity for ER (Table 2,
30a vs 30b; 31a vs 31b; 32a vs 32b). For example, 31b shows a
5-fold affinity increase compared to 31a. However, when the
carbonyl group of ferroceny-formyl is reduced to a methylene
group, a progressive decrease of binding affinity for ER was
observed (Table 2, 30b vs 30c; 31b vs 31c). These results
illustrate that ferroceny-formyl at C4-position of the phenyl ring
The binding affinities of the FcOBHS-HDACi conjugates for
both ER and ER were determined by a competitive
fluorometric receptor-binding assay and are summarized in Table
2;35 the Ki values of the FcOBHS-HDACi conjugates are also
summarized in Table 2. These affinities are presented as relative
binding affinity (RBA) values, where estradiol has an affinity of
100%.
As a global observation, it is notable first that most of the
conjugates (30a-c, 31b-c, 32b-c, 33) show ER subtype selectivity
for ER of at least 1.5-fold in terms of RBA / ratio. Secondly,
chain length has significant effects on the binding affinity of
conjugates in Series I. We observed that the series of 31 that have
a spacer length of n of 6 (n = 6) seemingly demonstrate a better
ER affinity than the other two series (30, n = 5 and 32, n = 7).
In fact, the suberic acid compound 31b has the highest ER
affinity of all conjugates tested. The RBA values of this
compound are 3.28 and 0.37 for ER and ER , respectively, and
it has an ER / selectivity as high as 8.86 (Table 2, entry 5).
Compared to the parent OBHS-HDACi conjugate 11 (RBA
in sulfonate unit can enhance binding affinity for ER
the
position of the ferroceny group has little effect on binding affinity
for ER . For example, when ferroceny-formyl group is moved
from C3 to C4-position of the phenyl sulfonate unit, pimelic acid
conjugate 30b shows a 1.9-fold increase over 30a (Table 2,
entries 1 vs 2); yet, suberic acid analogue 31b results in a
dramatically decreased binding affinity for ER (Table 2, entries
4 vs 5). In further attempts to increase the RBA, the suberic acid
group was moved from the phenolic ring to the phenyl sulfonate
moiety (compound 33). This led, however, to a loss of binding
affinity for both ERs, with an RBA value not exceeding 1 (entry
10). Compared to the parent OBHS-HDACi conjugate 12 (RBA
values were 2.55 for ER , 3.35 for ER ,
/
was 0.76),35
values were 12.4 for ER , 0.44 for ER ,
/
was 28),35
compound 31b showed higher binding affinity for ER and
subtype selectivity. We are also aware of the position of the
ferrocene on the phenyl group of sulfonate unit has significant
compound 33 showed about 31-fold weaker binding affinity for
ER and 18-fold weaker subtype selectivity.

Table 1 Diels-Alder Reaction of Furans 19, 29 and Dienophiles 24a-c and 28
b
aThe conversion was calculated accounting for the recovered furans 19a-c or 29. Isolated yield by column chromatography purification
based on furans 19a-c or 29.
7

Table 2 Relative Binding Affinity (RBA) of FcOBHS-HDACi Conjugates for ER and ER
estradiol
aRelative Binding Affinity (RBA) values are determined by competitive flourometric binding assays and are expressed as IC50
/
IC50 compound × 100 (RBA, estradiol = 100%). The values are reported as the mean ± standard deviation (mean ± SD) of three independent
experiments. bKi values of each conjugate for each receptor were obtained from the RBA values by the formula Ki =(100/RBA) × Kd. The
Kd
see Experimental Section.

that it can convert from an agonist to antagonist. For example, 3-
2.3. Transcription Activation Assays
Various FcOBHS-HDACi conjugates were tested by an ER-
responsive luciferase reporter gene assays for their ability to
stimulate the transcriptional activities of
ferroceny-formyl and ferroceny-benzyl compounds (31a and 31c)
are sti
agonists despite reduced efficacy, compared to
their corresponding pimelic acid analogues (Table 3, entries 1 vs
4, 3 vs 6); whereas 4-ferroceny-formyl compound 31b profiles as
a full antagonist (entries 2 vs 5), which shows at least 3-fold
-estradiol (E2). Luciferase assays were conducted in
HEK293T cells transfected with a widely used 3 × ERE-
luciferase reporter. These results are summarized in Table 3.
As shown in this table, most of FcOBHS-HDACi conjugates
agonists, and compounds 30a-b (Table 3,
more efficacious as
full antagonist than the parent OBHS-
HDACi conjugate 11. Interestingly, when the chain length of
HDACi unit was changed from the pimelic acid (n = 5) to azelaic
entries 1 vs 2) even exhibit full agonist activities. In fact, several
acid (n
=
7), 3-ferroceny-formyl and ferroceny-benzyl
compounds act as potent and nearly complete
agonists. In contrast, OBHS-HDACi conjugates display a wide
range of activities at hese results demonstrate that very
-selective
compounds (32a-b) display agonist activity for
31a profiles as a superagonist on
; especially,
1.1 times greater than that of estradiol (Table 3, entry 7); yet, the
4-ferroceny-formyl compound 32c profiles as a full antagonist
(Table 3, entry 9). However, compound 32c with decreased
binding affinity (Table 2, entries 5 vs 9), also shows decreased
agonist, compare to suberic acid
small changes to 7-oxabicyclic[2.2.1]hept-5-ene (OBHS)-core
ligand structure can have drastic effects on the transcription
activity.
In addition to this interesting activity, most of conjugates were
found to act as
agonist. However, the effects of increasing
conjugate 31b (Table 3, entries 5 vs 9). Here again, it is clear that
the optimal chain length of HDACi unit is n = 6. Furthermore,
introduction of the suberic acid to the phenyl sulfonate moiety
(compound 33, entry 10) also exhibits the typical ER -selective
full antagonist property, and shows about 2-fold more efficacious
than the parent OBHS-HDACi conjugate 12. Thus, the ability of
compounds bearing OBHS scaffold to stimulate ER activity
does not correlate with their relative binding affinities for ER .
chain length of HDACi unit were very evident. Compounds 30a-
c, which bearing the pimelic acid (n = 5) group on phenol ring,
acting as ER full agonist with nanomolar (nM) range EC50s. In
the case of compound 31, however, replacing pimelic acid (n = 5)
of compounds 30 with suberic acid (n = 6) have obvious effects
on the transcriptional activity of the ER . This suggests that ER
is more sensitive to OBHS-core ligand structure than
Table 3 Effects of FcOBHS-HDACi Conjugates on the Transcriptional Activities of Estrogen Receptor and
Agonist Modea Antagonist Modeb
EC50
Eff (%
EC50
Eff (%
E2)
IC50
Eff (%
E2)c
IC50
Eff (%
E2)
entry
1
cmpd
30a
30b
30c
E2)
0.27
0.99
0.78
1.70
-
89 ± 2
76 ± 1
77 ± 8
46 ± 2
-4 ± 1
2.32
1.11
1.57
0.75
0.35
0.36
2.41
-
92 ± 1
89 ± 5
36 ± 7
13 ± 2
24 ± 3
17 ± 1
26 ± 3
-11 ± 1
37 ± 4
-3 ± 1
0 ± 1
-
118 ± 3
123 ± 20
97 ± 16
95 ± 6
-
105 ± 1
101 ± 4
96 ± 2
73 ± 1
74 ± 3
89 ± 1
100 ± 6
86 ± 7
75 ± 11
102 ± 2
-16 ± 2
2
-
-
3
-
-
4
-
1.10
-
-
31a
31b
31c
5
4 ± 4
-
6
0.38
1.17
1.50
-
52 ± 6
112 ± 13
58 ± 11
-10 ± 2
-29 ± 1
60 ± 2
144 ± 8
118 ± 4
106 ± 2
27 ± 1
-
7
-
-
32a
32b
32c
8
-
-
9
2.03
-
2.38
0.57
0.014
-
-
10
11
-
14 ± 1
33
0.095
-
70 ± 12
0.581
OBHS
aLuciferase activity was measured in HEK293T cells transfected with 3 × ERE-driven luciferase reporter and expression vectors
9

encoding E
-5 M) of the compounds. EC50 and standard deviation
(mean ± SD), shown as a percentage of 10-8
-estradiol (E2), were determined. IC50 and standard deviation (mean ± SD) were
b
determined in the percentage of 10-8
-estradiol (E2
cERs have considerable basal activity in HEK293T cells;
compounds with inverse agonist activity are given negative efficacy values. Omitted EC50 or IC50 values were too high to be determined
accurately.
hydroxyphenyl group of FcOBHS-HDACi conjugate 31b also
engages in the crucial hydrogen bonding with Glu353, which
simulates the interaction of the A-ring of steroidal estrogens in
2.4. Structural Analysis of the Origin of Enhanced Antagonist
Character of FcOBHS-HDACi Conjugates
The ability of the FcOBHS-HDACi conjugates to act as
antagonists on ER is not surprising because it is known that
most of OBHS-core ligands profiles as antagonists. We are
surprised, however, by the position of HDACi unit in dual-
targeting FcOBHS-core ligands-HDACi conjugates. To
understand why suberic acid compound is still able to exhibit full
antagonist activity in different location of OBHS core, we have
used molecular modeling to examine how some of these
ER
suberic acid side chain can directly interfere
with helix 12 (Figure 4C), which is consistent with the action
model of 4-hydroxytamoxifen (Figure 4D). Thus, FcOBHS-
HDACi conjugate represents a novel mechanism to generate a
full
gonist which is distinct from that of OBHS-core
ligands.
2.5. Conjugates Exhibit Potent HDAC Inhibition Activity
We tested all of FcOBHS-HDACi conjugates for HDAC
inhibition activity against HDAC1 and HDAC6, respectively
(Table 4). Although overexpression of HDAC1, HDAC6 and
HDAC8 have been linked to breast cancer,46 HDAC1 and
HDAC6 have been found to be more critical.26, 29 As shown by
the data in Table 4, the HDAC inhibition activity was very much
dependent on the length of the acid chain of FcOBHS-HDACi
conjugates. In general, Series I suberic acid conjugates (31a-c,
linker length n = 6) strongly inhibit HDAC1 (with nanomolar
range IC50s), are demonstrated to be highly selective for HDAC1,
having modest or no active against HDAC6; actually, suberic
conjugates fit into the ligand binding pocket of
Crystal structure of the
4).
OBHS
shows that one p-hydroxyphenyl group mimics the role of the A-
ring phenol of E2, engaging in strong hydrogen bonding with
residue Glu353 of helix 3; while another p-hydroxyphenyl group
forms a hydrogen bond with residue Thr347 in helix 3. The non-
poplar phenyl sulfonate group in OBHS extends between helices
8 and 11, and which makes strong steric clashes with helix 11,
displacing residue His524, which engages in hydrogen bonding
interaction with E2 (PDB ID: 1ERE). Displacement of Helix 11
indirectly modulates the conformation of the critical helix 12 and
transcriptional activity (Figure 4A), a mechanism we have termed
acid conjugate 31b shows the most potent anti-HDAC1 (IC50
=
197 nM) activity; yet, the parent OBHS-HDACi conjugate 11 is
essentially inactive for either of the HDACs.35 However, either
shorter (30a-c, n = 5) or longer chain lengths (32a-c, n = 7) result
in reduced potency, specifically, the seven methylene-linked
compounds 31b-c are inactive against HDAC1; additionally, both
pimelic acid (n = 5) and azelaic acid (n = 7) conjugates are not
selective for HDACs. Interestingly, when suberic acid group is
appended onto the phenyl ring of sulfonate moiety, compound 33
also displayed good inhibition preference for HDACs with the
nanomolar range (Table 4, entry 10), which is 56-fold more
potent than the parent OBHS-HDACi conjugate 12 in HDAC6
inhibition (with IC50 value of 8.34 M for HDAC6). 35
Consistent with this model, OBHS-HDACi conjugate (Figure
4B) can similarly form a hydrogen bond between the p-
hydroxyphenyl group and helix 3 residue Glu353, and the
sulfonate side chain makes clash with helix 11; interestingly,
suberic acid side chain mimics the binding orientation of 4-
hydroxytamoxifen, however, which does not directly interact
with any of helix 12 residues. Instead, the suberic acid side chain
forms hydrogen bond contacts with helix 3 (Ser 341, Leu 345,
and Leu 346) which can provide subtle shifts in helix 3 that
destabilize helix 12, thus giving OBHS-HDACi
antagonist activity. Similar to OBHS-HDACi conjugate 11, the p-

Figure 4 Model of FcOBHS-HDACi conjugates bound to
OBHS-HDACi conjugate and 4-
30
hydroxytamoxifen. (A) Crystal structure of the
oxabicyclic heptane sulfonate (OBHS) bound
.
OBHS
H-bonds to the conserved Glu 353 on helix 3. The phenyl sulfonate binds between helices 8 and 11. (B) Computer-developed model of
OBHS-HDAC conjugate 11 bound to -bonding to Arg 394, Ser 341, Leu 345, Leu 346 and Glu 353, and the
sulfonate moiety extending between helices 8 and 11.35 (C) Computer-developed model of 31b forms hydrogen bonds with Glu 353, and
the suberic acid side chain makes a distinct hydrogen bonding interaction with H12, which directly displaces helix 12; moreover, the
ferrocenyl group clashes with helix 3. (D) Crystal structure of the
4-hydroxytamoxifen (4OHT, PDB: 3ERT).
4-OHT forms hydrogen bonds with Glu 353 and Arg 394, the side chain displaces helix 12.45
than 4OHT (Table 5, entries 7 vs 14), against MDA-MB-231
cells; moreover, they retain great activity of their parent
compounds against MCF-7 cells. This observation indicates that
antiproliferative activity of these FcOBHS-HDACi conjugates
against MDA-MB-231 cells derives mainly from ferrocenyl
group. While antiproliferative activities of these dual-acting
conjugates 30-33 are very dependent on the linker length, and
also closely matched their anti-HDAC activities. We noticed that
compounds 30a-c, with the shortest linker of five methylenes,
also were effective in inhibiting the MDA-MB-231 cells, but they
showed weaker inhibition for MCF-7 cells than the
corresponding six-methylene compound (Table 5, entries 3 vs 6;
4 vs 7). Surprisingly, compounds 32b-c with a spacer length of
seven methylenes show the least antiproliferative activities
against MDA-MB-231 and DU 145 cells; actually, they are
essentially inactive, an observation which agrees with its poor
2.6. Whole Cell Antiproliferative Activity
To evaluate their anticancer activity, all dual-acting conjugates
were screened against MCF-7 (hormone-dependent breast cancer
cells), MDA-MB-231 (hormone-independent breast cancer cells),
DU-145 (hormone-refractory, metastatic prostate cancer cells), as
well as one control cell line, healthy kindney epithelial cells
(VERO), and the results are summarized in Table 5.
A closer analysis of the cell growth inhibition data in Table 5
reveals that introduction of ferrocenyl group on phenyl ring of
sulfonate unit of OBHS-HDACi conjugates shows significant
antiproliferative effect on MDA-MB-231 breast cancer cells
(Table 5, entries 2 vs 6; 2 vs 7; 2 vs 8), unlike the parent OBHS-
HDACi conjugates 11 and 12, which show a weaker activity
(IC50 > 50.0 M). Specifically, 31b stands out among these
FcOBHS-HDACi conjugates because it shows the best
antiproliferative activity, which is about 1.2-fold more potent
11

Table 4 IC50 values of conjugates for inhibition of HDAC1 and HDAC6a
aValues are the means of a minimum of three experiments. bND = inhibition not detectable.

HDAC inhibition profile. However, when suberic acid group is
appended onto the phenyl ring of sulfonate moiety, compound 33
still possesses potent antiproliferative activities both ER(+) and
ER(-) breast cancer cells (Table 5, entry 12). These results
suggest that inhibition of HDAC also can enhance potency
against MDA-MB-231 cells.
against ER dependent cancers.
Additionally, all FcOBHS-HDACi conjugates are nontoxic to
healthy VERO cells, while SAHA and 4OHT showed
considerable toxicity. Comparing the activity of conjugates (30c,
31a-c, and 33), which showed significant antiproliferative effects
on both ER(+) and ER(-) breast cancer cells, with control drugs
SAHA and tamoxifen on VERO, 4OHT had the smallest in vitro
therapeutic index (IVTI), while our conjugates show greater
IVTIs (Table 6).
Another interesting aspect of antiproliferative activity is that
most conjugates showed significant antiproliferative effects on
DU 145 cells, which only express ER . This result suggests that
these FcOBHS-HDACi conjugates may have potential benefit
a
Table 5 Whole cell antiproliferative activity (IC50
MCF-7 MDA-MB-231
20.9 ± 1.00
>50b
Entry
1
Cmpd
OBHS
12
DU-145
>50
VERO
>50
2
19.1 ± 1.09
49.1 ± 0.85
41.4 ± 1.66
22.8 ± 2.04
26.4 ± 0.97
14.8 ± 0.35
24.5 ± 0.56
>50
>50
34.8 ± 2.91
17.0 ± 0.21
30.3 ± 1.21
35.6 ± 1.05
25.5 ± 0.80
25.3 ± 0.30
34.2 ± 1.66
25.3 ± 0.12
>50
>50
3
18.4 ± 0.12
18.9 ± 2.54
21.1 ± 0.22
18.8 ± 0.62
15.5 ± 0.49
20.1 ± 0.19
17.1 ± 1.12
>50
>50
30a
4
>50
30b
5
>50
30c
6
>50
31a
7
>50
31b
8
>50
31c
9
>50
32a
10
11
12
13
14
>50
>50
32b
44.1 ± 0.59
15.6 ± 0.34
2.5 ± 0.33
15.6 ± 1.77
>50
>50
>50
32c
17.1 ± 0.33
1.8 ± 0.07
18.8 ± 0.57
21.2 ± 2.01
1.2 ± 0.07
15.3 ± 4.42
>50
33
4.1 ± 0.19
SAHA
15.1 ± 5.21
4OHT
b
aIC50 values are an average of at least three independent experiments ± standard deviation (mean ± SD). IC50 not determinable up to
highest concentrations tested.
Table 6 In vitro therapeutic index (IVTI) of FcOBHS-HDACi conjugates 30c, 31a-c, and 33
Entry
Compound
30c
IVTIa
> 2.19
> 1.89
> 3.37
> 2.04
> 3.20
1.64
IVTIb
> 2.36
> 2.65
> 3.22
> 2.48
> 2.92
2.27
1
2
3
4
5
6
7
31a
31b
31c
33
SAHA
4OHT
0.97
0.80
aIVTI = IC50(VERO) / IC50(MCF-7); bIVTI = IC50(VERO) / IC50(MDA-MB-231).
13

analytical thin-layer chromatography (TLC). Visualization was
3. Conclusions
1
Breast cancer is a multifactorial disease involving multiple
cross-talks between signaling networks. The estrogen receptor
(ER) plays a predominant role in breast cancer growth, and which
is regarded as important pharmaceutical target for the treatment
of breast cancer.47 Similarly, HDACs inhibitors exert antitumor
effects by altering gene transcription and disrupting mitosis. To
further improve the biological response of OBHS-HDACi
conjugates, particularly against ER(-) breast cancer cells, we have
created dual-acting FcOBHS-HDACi conjugates. A subset of
these conjugates exhibited good binding affinity and excellent
achieved by UV light (254 nm). H NMR and 13C NMR spectra
were measured on a Bruker Biospin AV400 (400 MHz)
instrument. Chemical shifts are reported in parts per million (ppm)
and are referenced to either tetramethylsilane or the solvent.
4.1.2. General procedure for Diels-Alder reaction (30a-c, 31a-
c, 32a-c, and 33). Furan 19 or 29 (0.7 mmol) and dienophile
(24a-c or 28, 0.84 mmol) were in a round flask, and the reaction
mixture was stirred at 90 °C for 12 h. The crude product was
purified by silica gel column chromatography (Dichloromethane-
MeOH, 60 : 1~30 : 1).
4.1.2.1. 7-((4-(3-(4-Hydroxyphenyl)-6-((3-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-7-oxo-heptanoic Acid (30a). Red soild, 95% yield, mp
affinity output showed that they are largely
-selective.
Docking analyses of FcOBHS-HDACi conjugate reveals a novel
mechanism of antagonism via suberic acid group substitutions on
the bicyclic core that directly obstructs helix 12, unlike OBHS-
HDACi conjugate, which indirectly modulates the critical switch
helix 12, by interactions with helix 3; moreover, these conjugates
strongly inhibit HDAC1 and HDAC6. Excitingly, they show
anticancer activity that is selectively more potent against MDA-
MB-231 compared to MCF-7 or DU 145, and all conjugates are
nontoxic to health VERO cells. While linker length of HDACi
unit is a critical factor for activity, and conjugates with a shorter
rather than longer chain length tend to be more active, especially
those with suberic acid derivatives. Actually, the most promising
compound of this study is suberic acid derivative 31b, which has
the highest binding affinity for ER
1
154-156 °C; H NMR (400 MHz, Acetone-d6) 9.34 (s, 1H),
8.01 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.2 Hz, 2H),
7.55 (m, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 8.8 Hz, 2H),
7.27 (t, J = 8.8 Hz, 2H), 6.86 (t, J = 8.4 Hz, 2H), 5.78 (s, 1H),
5.51 (s, 1H), 4.89 (s, 2H), 4.66 (s, 2H), 4.24 (s, 5H), 3.98 (m, 1H),
2.52 (m, 1H), 2.40 (m, 3H), 2.29 (t, J = 6.8 Hz, 2H), 1.71 (t, J =
6.8 Hz, 2H), 1.62 (t, J = 7.2 Hz, 2H), 1.41 (m, 2H). 13C NMR
(100 MHz, Acetone-d6) 197.45, 175.10, 172.50, 158.66, 158.59,
149.98, 143.61, 142.30, 141.97, 140.15, 139.23, 137.53, 131.12,
130.19, 129.80, 128.98, 128.59, 127.58, 125.99. 124.61, 122.98,
120.40, 116.56, 85.19, 83.82, 78.71, 73.91, 72.16, 71.17, 62.11,
37.64, 34.18, 31.68, 31.47, 26.02, 25.42; HRMS (ESI) calcd for
C42H3854FeNO9S [M - H]-, 786.6624; found 786.6621.
4.1.2.2. 7-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-7-oxo-heptanoic Acid (30b). Red soild, 93% yield, mp
antagonist activity, which also show the most potent anti-HDAC1
(IC50 = 197 nM) activity and against both ER(+) and ER(-) breast
cancer cells. In summary, we have demonstrated that conjugation
of ferrocenyl group to anticancer agents is a viable strategy to
improve the biological response of the latter.
1
158-161 °C; H NMR (400 MHz, Acetone-d6) 9.31 (s, 1H),
8.00 (d, J = 8.0 Hz, 2H), 7. 67 (d, J = 8.4 Hz, 1H), 7.63 (d, J =
8.4 Hz, 1H), 7.43 (t, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 1H),
7.32 (t, J = 7.6 Hz, 2H), 7.26 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0
Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 5.76 (s, 1H), 5.50 (s, 1H), 4.84
(s, 2H), 4.66 (s, 2H), 4.23 (s, 5H), 3.95 (m, 1H), 2.51 (m, 1H),
2.39 (m, 3H), 2.32 (t, J = 7.2 Hz, 2H), 1.70 (m, 2H), 1.62 (t, J =
7.2 Hz, 2H), 1.41 (m, 2H). 13C NMR (100 MHz, Acetone-d6)
197.40, 174.90, 172.30, 158.70, 158.56, 152.60, 143.55, 142.00,
140.35, 140.21, 139.32, 137.54, 130.92, 130.34, 129.78, 129.12,
128.47, 127.94, 123.87. 123.03, 120.29, 120.10, 116.71, 116.51,
85.28, 83.73, 78.93, 73.66, 72.17, 71.07, 61.81, 37.61, 34.12,
31.57, 31.36, 25.98, 25.41; HRMS (ESI) calcd for
4. Experimental Section
4.1. Chemistry
4.1.1. General
Unless otherwise noted, starting materials were purchased
from commercial suppliers and were used without further
purification. Tetrahydrofuran (THF) was dried over Na and
distilled prior to use. Dichloromethane and triethylamine were
dried over CaH2 and distilled prior to use. Glassware was oven-
dried, assembled while hot, and cooled under an inert atmosphere.
All reactions were performed under an argon atmosphere unless
otherwise specified. Reaction progress was monitored using

C42H3854FeNO9S [M - H]-, 786.6624; found 786.6621.
(m, 1H), 2.50 (m, 1H), 2.39 (m, 2H), 2.31 (m, 2H), 1.68 (m, 2H),
1.61 (t, J = 6.0 Hz, 2H), 1.36 (m, 4H). 13C NMR (100 MHz,
Acetone-d6) 197.58, 175.19, 172.59, 158.73, 158.59, 152.59,
141.97, 139.25, 137.50, 130.95, 130.35, 129.72, 129.12, 128.51,
128.00, 124.64, 123.84, 123.09, 123.06, 120.39, 120.20, 116.75,
116.56, 85.22, 83.70, 78.88, 73.74, 72.21, 71.11, 61.84, 37.77,
34.28, 31.59, 31.38, 29.68, 26.16, 25.66; HRMS (ESI) calcd for
C43H4054FeNO9S [M - H]-, 800.1829; found 800.1825.
4.1.2.3. 7-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenyl benzyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-7-oxo-heptanoic Acid (30c). Red soild, 94% yield, mp
1
147-150 °C; H NMR (400 MHz, Acetone-d6) 9.14 (s, 1H),
7.53 (t, J = 8.8 Hz, 2H), 7. 17 (d, J = 7.6 Hz, 1H), 7.15 (d, J = 7.2
Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.05
(d, J = 8.8 Hz, 2H), 7.00 (d, J = 7.2 Hz, 1H), 6.99 (d, J = 7.2 Hz,
1H), 6.71 (d, J = 8.8 Hz, 1H), 6.68 (d, J = 8.8 Hz, 1H), 5.53 (s,
1H), 5.31 (t, J = 3.2 Hz, 1H), 3.97 (s, 7H), 3.92 (s, 2H), 3.64 (m,
1H), 3.60 (m, 2H), 2.27 (m, 3H), 2.17 (m, 3H), 1.58 (t, J = 7.6 Hz,
2H), 1.46 (t, J = 7.6 Hz, 2H), 1.30 (m, 2H). 13C NMR (100 MHz,
Acetone-d6) 174.83, 172.18, 158.56, 158.42, 148.63, 142.13,
141.90, 140.21, 140.09, 139.24, 137.59, 130.60, 130.29, 129.11,
128.46, 127.95, 124.71, 123.90, 122.84, 120.23, 116.40, 88.54,
85.22, 83.64, 69.43, 69.36, 68.39, 61.21, 37.55, 35.90, 34.08,
31.51, 31.31, 25.98, 25.39; HRMS (ESI) calcd for
C42H4054FeNO8S [M - H]-, 772.1824; found 772.1820.
4.1.2.6. 8-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenyl benzyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-8-oxo-octanoic Acid (31c). Red soild, 92% yield, mp
1
153-155 °C; H NMR (400 MHz, Acetone-d6) 9.31 (s, 1H),
7.67 (t, J = 8.8 Hz, 2H), 7.30 (m, 2H), 7.23 (t, J = 7.6 Hz, 2H),
7.18 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 7.6 Hz, 1H), 7.12 (d, J = 7.6
Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 5.67
(s, 1H), 5.44 (s, 1H), 4.15 (s, 7H), 4.10 (s, 2H), 3.76 (m, 1H),
3.65 (s, 2H), 2.40 (m, 3H), 2.29 (m, 3H), 1.70 (t, J = 6.0 Hz, 2H),
1.59 (m, 2H), 1.37 (m, 4H). 13C NMR (100 MHz, Acetone-d6)
175.13, 172.50, 158.66, 158.52, 148.61, 142.11, 139.26, 137.56,
130.63, 130.61, 130.30, 129.66, 129.11, 128.47, 124.69, 123.89,
122.90, 122.80, 120.39, 120.15, 116.72, 116.51, 85.25, 83.64,
69.56, 69.47, 68.51, 61.25, 55.04, 37.77, 35.90, 31.52, 31.32,
26.18, 25.63; HRMS (ESI) calcd for C43H4254FeNO8S [M - H]-,
786.1976; found 786.1951.
4.1.2.4. 8-((4-(3-(4-Hydroxyphenyl)-6-((3-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-8-oxo-octanoic Acid (31a). Red soild, 93% yield, mp
1
162-164 °C; H NMR (400 MHz, Acetone-d6) 9.27 (s, 1H),
8.00 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.2 Hz, 2H),
7.55 (m, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 2H),
7.24 (t, J = 8.4 Hz, 2H), 6.83 (t, J = 8.4 Hz, 2H), 5.76 (s, 1H),
5.50 (s, 1H), 4.86 (s, 2H), 4.66 (s, 2H), 4.25 (s, 5H), 3.93 (m, 1H),
2.52 (m, 1H), 2.38 (t, J = 7.2 Hz, 3H), 2.29 (t, J = 7.2 Hz, 2H),
1.69 (t, J = 6.8 Hz, 2H), 1.61 (t, J = 6.4 Hz, 2H), 1.37 (m, 4H).
4.1.2.7. 9-((4-(3-(4-Hydroxyphenyl)-6-((3-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-9-oxo-nonanoic Acid (32a). Red soild, 93% yield, mp
1
165-167 °C; H NMR (400 MHz, Acetone-d6) 9.30 (s, 1H),
13C NMR (100 MHz, Acetone-d6)
197.30, 175.06, 172.44,
8.00 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 2H),
7.56 (m, 2H), 7.50 (t, J = 8.0 Hz, 1H), 7.35 (t, J = 8.8 Hz, 2H),
7.30 (d, J = 7.2 Hz, 1H), 7.27 (t, J = 8.8 Hz, 2H), 6.85 (t, J = 8.8
Hz, 2H), 5.77 (s, 1H), 5.51 (t, J = 3.6 Hz, 1H), 4.86 (s, 2H), 4.66
(s, 2H), 4.25 (s, 5H), 3.99 (m, 1H), 2.52 (m, 1H), 2.39 (m, 3H),
2.29 (m, 2H), 1.68 (t, J = 7.2 Hz, 3H), 1.58 (t, J = 6.8 Hz, 3H),
1.34 (m, 4H). 13C NMR (100 MHz, Acetone-d6) 197.38, 175.10,
172.54, 158.61, 158.55, 149.99, 143.59, 142.30, 141.99, 142.30,
141.99, 140.24, 139.21, 137.56, 131.10, 130.19, 129.80, 128.97,
128.59, 127.56, 125.97, 124.65, 122.98, 120.37, 116.54, 85.20,
83.81, 78.73, 73.87, 72.11, 71.15, 61.97, 37.81, 37.78, 34.27,
31.68, 31.47, 29.73, 26.27, 25.65; HRMS (ESI) calcd for
C44H4254FeNO9S [M - H]-, 814.1930; found 814.1928.
158.62, 158.56, 150.01, 143.57, 142.32, 142.02, 140.26, 139.20,
137.57, 131.08, 129.77, 128.57, 127.53, 125.95, 124.65, 123.94,
123.01, 122.95, 120.32, 85.19, 83.77, 78.74, 73.83, 72.08, 71.07,
61.94, 55.92, 37.74, 34.21, 31.45, 26.15, 25.54, 29.63; HRMS
(ESI) calcd for C43H4054FeNO9S [M - H]-, 800.1829; found
800.1825.
4.1.2.5. 8-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-8-oxo-octanoic Acid (31b). Red soild, 96% yield, mp
1
160-163 °C; H NMR (400 MHz, Acetone-d6) 9.34 (s, 1H),
7.97 (d, J = 8.4 Hz, 2H), 7.64 (m, 2H), 7.41 (t, J = 7.6 Hz, 2H),
7.34 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0
Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 5.76
(s, 1H), 5.50 (s, 1H), 4.84 (s, 2H), 4.65 (s, 2H), 4.23 (s, 5H), 3.96
4.1.2.8. 9-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenylformyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
15

amino)-9-oxo-nonanoic Acid (32b). Red soild, 96% yield, mp
30.67, 29.62, 26.06, 25.52; HRMS (ESI) calcd for
C36H3654FeNO8S [M - H]-, 696.1593; found 696.1589.
1
162-165 °C; H NMR (400 MHz, Acetone-d6) 9.29 (s, 1H),
8.00 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.8
Hz, 1H), 7.43 (t, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.32 (t,
J = 8.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H),
6.83 (d, J = 8.4 Hz, 1H), 5.77 (s, 1H), 5.50 (s, 1H), 4.84 (s, 2H),
4.65 (s, 2H), 4.23 (s, 5H), 3.95 (m, 1H), 2.51 (m, 1H), 2.39 (m,
3H), 2.31 (t, J = 7.6 Hz, 2H), 1.68 (m, 3H), 1.59 (t, J = 6.4 Hz,
3H), 1.33 (m, 4H). 13C NMR (100 MHz, Acetone-d6) 197.50,
175.11, 172.56, 158.67, 158.53, 152.60, 143.56, 141.63, 139.30,
137.53, 130.94, 130.36, 129.72, 129.13, 128.51, 124.68, 123.89,
123.05, 116.72, 116.53, 85.23, 83.75, 78.91, 73.70, 72.20, 71.09,
62.03, 37.81, 34.27, 31.58, 31.37, 29.84, 29.73, 26.26, 25.65;
HRMS (ESI) calcd for C44H4254FeNO9S [M - H]-, 814.1930;
found 814.1928.
4.2. Estrogen Receptor Binding Affinity
Relative binding affinities were determined by a competitive
fluorometric binding assay as previously described. Briefly, 40
nM fluorescence tracer (coumestrol, Sigma-Aldrich, MO) and 0.8
were diluted in 100 mM potassium phosphate buffer (pH 7.4),
containing 100 g/mL bovine gamma globulin (Sigma-Aldrich,
MO). Incubations were for 2 h at room temperature (25 °C).
Fluorescence polarization values were then measured. The
binding affinities are expressed as relative binding affinity (RBA)
-estradiol set to 100%. The values
given are the average ± range of two independent determinations.
IC50 values were calculated according to equations described
previously.35
4.1.2.9. 9-((4-(3-(4-Hydroxyphenyl)-6-((4-ferrocenyl benzyl)
phenoxysulfonyl)-7-oxabicyclo[2.2.1]hept-2-en-2-yl)phenyl)
amino)-9-oxo-nonanoic Acid (32c). Red soild, 95% yield, mp
4.3. Gene Transcriptional Activity
The human embryonic kidney cell lines, HEK 293T, was
1
155-158 °C; H NMR (400 MHz, Acetone-d6) 9.25 (s, 1H),
7.65 (t, J = 8.8 Hz, 2H), 7.30 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 7.6
Hz, 1H), 7.25 (t, J = 8.4 Hz, 2H), 7.18 (t, J = 8.4 Hz, 2H), 7.14 (d,
J = 8.4 Hz, 2H), 6.85 (d, J = 8.8 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H),
5.67 (s, 1H), 5.45 (t, J = 3.6 Hz, 1H), 4.11 (s, 7H), 4.06 (s, 2H),
3.77 (m, 1H), 3.69 (m, 2H), 2.43 (m, 3H), 2.31 (t, J = 7.6 Hz, 3H),
1.68 (t, J = 6.4 Hz, 3H), 1.59 (m, 3H), 1.34 (m, 4H). 13C NMR
(100 MHz, Acetone-d6) 174.95, 172.34, 158.54, 158.41, 148.58,
143.42, 142.24, 142.13, 141.90, 139.24, 137.59, 130.60, 130.30,
129.66, 129.12, 128.47, 124.73, 123.92, 122.79, 120.24, 116.41,
88.56, 85.22, 83.60, 69.44, 69.37, 68.40, 61.32, 37.74, 35.91,
34.21, 31.52, 31.30, 29.83, 26.26, 25.64; HRMS (ESI) calcd for
C44H4454FeNO8S [M - H]-, 800.2138; found 800.2137.
(Gibco by Invitrogen Corp., CA) with 10% fetal bovine serum
(FBS) (Hylcone by Thermo Scientific, UT). Cells were plated in
phenol red-free DMEM with 10% FBS. HEK 293T cells were
transfected with 25 L mixture per well, containing 300 ng of 3 ×
ERE-
125 mM calcium chloride (GuoYao, China) and 12.5 L 2 × HBS.
The next day, the cells were treated with increasing doses of ER
ligands diluted in phenol red-free DMEM with 10% FBS. After
24h, luciferase activity was measured using Dual-Luciferase
Reporter Assay System (Promega, MI) according to the
4.4. HDAC Activity Assay
4.1.2.10. 8-(4-(((5-(4-Hydroxyphenyl)-6-ferroceny-7-oxabicy-
clo[2.2.1]hept-5-en-2-yl)sulfonyl)oxy)phenylamino)-8- oxooc-
In vitro HDAC activity was measured using Fluorogenic
HDAC6 Assay Kit (BPS Bioscience, CA) according to the
1
tanoic Acid (33). Red soild, 93% yield, mp 162-164 °C; H
NMR (400 MHz, Acetone-d6) 9.32 (s, 1H, -NH2), 8.37 (s, 1H, -
OH), 7.74 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.35 (d, J
= 8.4 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.56 (s, 1H), 5.39 (s, 1H),
4.35 (s, 2H), 4.28 (s, 2H), 4.18 (s, 5H), 3.82 (m, 1H), 2.42 (m,
1H), 2.36 (t, J = 6.8 Hz, 2H), 2.28 (m, 2H), 2.22 (m, 1H), 1.69 (t,
J = 6.0 Hz, 2H), 1.59 (t, J = 6.8 Hz, 2H), 1.35 (m, 2H). 13C NMR
(100 MHz, Acetone-d6) 174.85, 172.27, 158.29, 145.65, 141.33,
139.52, 137.13, 130.10, 125.06, 123.46, 121.12, 116.26, 85.21,
83.89, 77.05, 70.06, 69.76, 67.63, 62.11, 37.62, 34.18, 33.01,
prepared in DMSO and were diluted in HDAC assay buffer to
different concentration. The enzymatic reactions were conducted
in duplicate at 37 °C for 30 min in a 50 L mixture containing
HDAC assay buffer, 5 g of BSA, HDAC substrate, HDAC
enzyme (human recombinant HDAC1, HDAC6), and various
concentrations of tested compound. Then, 50 L of 2 × HDAC
Developer was added to each well and the plate was incubated at
room temperature for 15 min. Fluorescence values were
measured at an excitation of 350 nm and an emission of 440 nm

using SpectraMax M2 microplate reader. IC50 values were
calculated according to the following equation using Origin
software: Y = Fb + (Ft Fb)/(1+ 10[ (LogIC50-X)×Hillslope]), where Y =
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/
References and Notes
fluorescence value, Fb = minimum fluorescence value, Ft
maximum fluorescence value, X = Log [inhibitor].
4.5. Cell Culture and Cell Viability Assay
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Synthesis and structure-activity relationships of novel hybrid ferrocenyl
compounds based on a bicyclic core skeleton for breast cancer therapy
a
b
Changhao Li , Chu Tang , Zhiye Hu b, Chenxi Zhao a, Chenlu Li b, Silong Zhang b, Chune Dong b, Hai-Bing
Zhou b,*, Jian Huang a,*
Incorporation of the ferrocenyl group into OBHS-HDACi skeleton could lead to FcOBHS-HDACi complexes, which
show significant antiproliferative effect on both ER(+) and ER(-) breast cancer cells.
19