Novel Hybrid Conjugates with Dual Suppression of Estrogenic and Inflammatory Activities Display Significantly Improved Potency against Breast Cancer

Article abstract of DOI:10.1021/acs.jmedchem.8b00224

In this work, we developed a small library of novel OBHS-RES hybrid compounds with dual inhibition activities targeting both the estrogen receptor α (ERα) and NF-?B by incorporating resveratrol (RES), a known inhibitor of NF-?B, into a privileged indirect antagonism structural motif (OBHS, oxabicycloheptene sulfonate) of estrogen receptor (ER). The OBHS-RES conjugates could bind well to ER and showed remarkable ERα antagonistic activity, and they also exhibited excellent NO inhibition in macrophage RAW 264.7 cells. Compared with 4-hydroxytamoxifen, some of them showed better antiproliferative efficacy in MCF-7 cell lines with IC₅₀ up to 3.7 μM. In vivo experiments in a MCF-7 breast cancer model in Balb/c nude mice indicated that compound 26a was more potent than tamoxifen. Exploration of the compliancy of the structure against ER specificity utilizing these types of isomeric three-dimensional ligands indicated that one enantiomer had much better biological activity than the other.

Full text of DOI:10.1021/acs.jmedchem.8b00224

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Article
Novel Hybrid Conjugates with Dual Suppression
of Estrogenic and Inflammatory Activities Display
Significantly Improved Potency against Breast Cancer
Wentao Ning, Zhiye Hu, Chu Tang, Lu Yang, Silong Zhang, Chune Dong, Jian Huang, and Hai-Bing Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00224 • Publication Date (Web): 27 Jul 2018
Downloaded from http://pubs.acs.org on July 28, 2018
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Novel Hybrid Conjugates with Dual Suppression of Estrogenic and
Inflammatory Activities Display Significantly Improved Potency against Breast
Cancer
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a†
a†
b
a
a
a
Wentao Ning , Zhiye Hu , Chu Tang , Lu Yang , Silong Zhang , Chune Dong , Jian
c
a
Huang and HaiꢀBing Zhou *
a
State Key Laboratory of Virology, Hubei Province Engineering and Technology
Research Center for Fluorinated Pharmaceuticals, Key Laboratory of Combinatorial
Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Wuhan
University School of Pharmaceutical Sciences, Wuhan 430071, China
b
Engineering Research Center of Molecular and Neuro Imaging, Ministry of
Education, School of Life Science and Technology, Xidian University, Xi’an, Shaanxi
710126, China
c
College of Life Sciences, Wuhan University, Wuhan 430072, China
These two authors contributed equally.
†
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Abstract: In this work, we developed a small library of novel OBHSꢀRES hybrid
compounds with dual inhibition activities targeting both the estrogen receptor α (ERα)
and NFꢀκB by incorporating resveratrol (RES), a known inhibitor of NFꢀκB, into a
privileged indirect antagonism structural motif (OBHS, oxabicycloheptene sulfonate)
of estrogen receptor (ER). The OBHSꢀRES conjugates could bind well to ER and
showed remarkable ERα antagonistic activity, and they also exhibited excellent NO
inhibition in macrophage RAW 264.7 cells. Compared with 4ꢀhydroxytamoxifen,
some of them showed better antiꢀproliferative efficacy in MCFꢀ7 cell lines with IC50
up to 3.7 µM. In vivo experiments in a MCFꢀ7 breast cancer model in Balb/c nude
mice indicated that compound 26a was more potent than tamoxifen. Exploration of
the compliancy of the structure against ER specificity utilizing these types of isomeric
threeꢀdimensional ligands indicated that one enantiomer had much better biological
activity than the other.
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INTRODUCTION:
Among a variety of cancers, the most commonly diagnosed cancer among
1, 2
females is breast cancer.
Though the overall 5ꢀyear relative survival rate has
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improved in developed countries,
the incidence rate remains high, with a
5
continuously younger patient population. At diagnosis, approximately 70% of
patients have ERꢀpositive breast cancer, and ER is well known to be a favorable
6
prognostic factor in breast cancer. The primary function of the ER is to activate gene
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transcription following binding of estrogen to the receptor. As a transcription factor
of the nuclear receptor superfamily, ER can be classified into two isoforms, ERα and
8
, 9
ERβ. ERα has a more profound effect not only on the development and function of
the mammary gland and uterus but also on maintaining metabolic and skeletal
homeostasis, among other effects. By contrast, ERβ has more pronounced effects on
1
0
the central nervous system. Therefore, ERα can serve as an important target for
endocrine therapy of breast cancer.
11
Endocrine therapy is a useful treatment for ERꢀpositive breast cancer patients.
It mainly includes three groups of antiꢀestrogen drugs, selective estrogen receptor
modulators (SERMs), selective estrogen receptor downregulators (SERDs) and
aromatase inhibitors (AIs). SERMs, represented by tamoxifen, are effective in some
1
2
forms of breast cancer, notably ERꢀpositive disease. However, in this clinical
pathological subtype, de novo resistance to estrogen antagonists is encountered; in
addition, endocrine therapyꢀresistant disease typically develops in patients who are
13, 14
initially responsive.
SERDs are more complete ER antagonists; they can degrade
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ERs at high doses, providing more effective treatments, but resistance is still
16
encountered. As an alternative to endocrine therapy, AIs are also effective in this
subtype of breast cancer. However, they also encounter treatment resistance with
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obvious side effects.
Inflammation is an exacerbating factor in cancers and elicits tumor tissue
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infiltration and metastases. In fact, nearly 150 years ago, the relationship between
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inflammation and cancers was noticed. Patients with chronic inflammatory diseases
are at a much higher risk of developing cancer, and inflammation is thought to
20ꢀ22
contribute to approximately 25% of all cancer cases worldwide,
strong relationship between inflammation and carcinogenesis.
suggesting a
Activation of the NFꢀκB transcription family plays a central role in
23ꢀ25
26ꢀ28
inflammation.
A large amount of evidence
has shown that ER/NFꢀκB
interactions are implicated in the progression of breast cancer. In addition, NFꢀκB
29, 30
signaling driving oncogenic transcription programs
may contribute to the
13
development of endocrine therapy resistance. Currently, antitumor agents do not
uniformly provide optimal antiꢀproliferative as well as antiꢀinflammatory effects. This
consideration is particularly pertinent in ERꢀpositive breast cancer, where
antiꢀestrogens, though antiꢀproliferative, generally have poor antiꢀinflammatory
activity, whereas the proꢀproliferative activity of estrogens renders their generally
13
antiꢀinflammatory activity of no benefit in hormoneꢀresponsive disease.
Additionally, estrogen signaling through the ER can also inhibit NFꢀκB activation to
26
exert antiꢀinflammatory activity, but it will also promote cell proliferation.
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Antiꢀproliferative agents that also suppress NFꢀκB signaling could be especially
31ꢀ33
effective as breast cancer endocrine therapeutics.
Therefore, we hoped to develop
a type of novel small molecule having dual inhibition that targeted both ER and
NFꢀκB.
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The natural product resveratrol (3,5,4'ꢀtrihydroxystilbene, RES, 1, Figure 1) has
numerous biological activities, such as acting as a cardioprotective and
neuroprotective agent. In addition, resveratrol is a known inhibitor of NFꢀκB, which is
3
4ꢀ36
used as an antiꢀinflammatory drug.
Although resveratrol is favorable for
intervening in some therapeutically interesting pathways, its low potency limits its
35, 37
utility.
To address this issue, Zhu and his colleagues designed and synthesized a
novel class of resveratrolꢀbased aspirin prodrugs (2, Figure 1) to enhance anticancer
38
activities and thereby attenuate the side effects caused by aspirin. It has also been
reported that sulfateꢀconjugated resveratrol metabolites (3, Figure 1) are less active
39
than resveratrol but retain some degree of antiꢀinflammatory activity. Additionally,
resveratrol can inhibit hypoxia/reoxygenationꢀinduced NFꢀκB activation and attenuate
34
hypoxia/reoxygenationꢀcaused inflammatory responses in alveolar epithelial cells.
In breast cancer treatment, resveratrol can also enhance the efficacy of
40
sorafenibꢀmediated apoptosis in MCFꢀ7 cells.
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Figure 1. Structures of resveratrol, resveratrol conjugates, OBHS and OBHS
conjugates.
In recent years, our group has been engaged in the search for breast cancer drug
candidates, and some potential SERMs with threeꢀdimensional core scaffolds have
been designed and synthesized. OBHS (4, Figure 1), a kind of oxabicycloheptene
compound, has been shown to have the greatest potential, behaving overall as a partial
41
42
antagonist, and this compound had moderate antiꢀinflammatory activity. In some
reports, the introduction of privileged substructure to some molecular scaffolds can
43
increase the binding affinity to receptors and obtain potent derivatives, with some
44, 45
successful examples provided in our previous work.
For example, the
OBHSꢀHDACi conjugate (5, Figure 1), as a good ER ligand, can efficiently
antagonize ERα and inhibit histone deacetylase (HDAC) activity. Another example is
the OBHSꢀFerrocene conjugate (6, Figure 1), which exhibits high binding affinity and
good selectivity for the two ER subtypes and provides potent effects against
ERꢀpositive and ERꢀnegative breast cancer cell lines. In addition, when the sulfonate
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group of OBHS is replaced with sulfonamide, the resulting OBHꢀsulfonamide
(OBHSA) exhibits full antagonist activity with efficacy in ERαꢀpositive MCFꢀ7
46, 47
comparable to that of fulvestrant.
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To increase its antiꢀinflammatory effect, we proposed that the combination of the
RES moiety into the OBHS scaffold could afford a new series of OBHSꢀRES
conjugates that would not only have antiꢀinflammatory activity but also act as a good
ER ligand. Herein, two series of OBHSꢀRES conjugates (Figure 2) were designed via
the DielsꢀAlder reaction. These compounds have an antiꢀinflammatory unit and
adequate threeꢀdimensional topology for binding the receptor. Finally, for comparison,
we also synthesized some conjugates that have methyl ether groups on the resveratrol
unit, or replacement of the resveratrol moiety with transꢀstilbene or
3ꢀhydroxypinosylvin, and we evaluated a range of biological activities in vitro and in
vivo.
Figure 2. The design of bifunctional OBHSꢀRES conjugates.
RESULTS AND DISCUSSION
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Chemistry.
OBHSꢀRES conjugates were obtained via the DielsꢀAlder reaction of furan
derivative 17a or 23 with different dienophiles (Scheme 3), and the synthetic route of
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compound 23 has been reported in our previous work . Regarding the important
intermediate (E)ꢀ5ꢀ(4ꢀ(4ꢀ(4ꢀHydroxyphenyl)furanꢀ3ꢀyl)styryl)benzeneꢀ1,3ꢀdiol 17a in
the series I products, the synthetic pathway is presented in Scheme 1 and the
Supporting Information section. We used Nꢀbromosuccinimide as the bromide reagent
and pꢀtoluenesulfonic acid as an acid catalyst to react with 4ꢀmethoxyacetophenone 7
to provide αꢀbromoꢀ4ꢀmethoxyacetophenone 8, which was treated with
4ꢀbromophenylacetic acid 9 and triethylamine in acetonitrile solution to generate
compound 10. Subsequently, an intramolecular aldol reaction of compound 10 under
the basic condition provided compound 11. Demethylation of 11 with three
equivalents of boron tribromide afforded butenolide 12. Then, compound 12 was
reduced at ꢀ78 °C in the presence of diisobutylaluminum hydride, after careful
acidification at a low temperature, to yield the furan 13. The Heck reaction of 13 and
15aꢀb, using bisꢀ(triphenylphosphine) palladium(II) dichloride as the catalyst,
provided the furan derivatives 16aꢀb. Among them, compound 15a was obtained by a
+
ꢀ
3 3
Wittig reaction with commercially available compound 14a and Ph P CH Br .
Compound 15b was commercially available. Finally, by employing pyridine
hydrochloride to demethylate compound 16a, we could acquire the important
intermediate 17a.
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a
Scheme 1. The synthetic route of compound 17a .
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Reaction conditions: (a) NBS, pꢀtoluenesulfonic acid monohydrate, CHCl , rt, 12 h; (b) TEA,
3 3
CH CN, rt, 12 h; (c) NaH, DMSO, rt, 2 h; (d) BBr , DCM, 0 °C, 10 h; (e) diisobutylaluminum
+
ꢀ
3 3 3 2 2 3
hydride, THF, ꢀ78 °C, 12 h. (f) Ph P CH Br , NaH, THF, reflux, 2 h; (g) Pd(PPh ) Cl , Et N,
DMF, 120 °C, 12 h; (h) Py·HCl, 200 °C, 2 h.
The synthetic route of target dienophiles 20aꢀe and 22aꢀr is described in Scheme
. The synthesis of vinyl sulfonate 20e started with commercially available
2
pterostilbene through a twoꢀstep reaction. First, pterostilbene 18a was allowed to react
with 2ꢀchloroethanesulfonyl chloride under basic conditions in an ice bath to generate
compound 20a, which was subsequently demethylated with boron tribromide at 0 °C
to generate the vinyl sulfonate 20e (Scheme 2A). Vinyl sulfonates 20bꢀd can be
obtained under similar conditions. The synthesis of compounds 18b and 18c is
described in the supporting information.
The synthesis of vinyl sulfonates 22aꢀr is shown in Scheme 2B. In the presence
of base, introduction of the vinyl sulfonate functionality was achieved by treatment of
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different phenols with 2ꢀchloroethanesulfonyl chloride. The target OBHSꢀRES
conjugates were then obtained via the reaction of 17a with the appropriate 22aꢀr as
well as 23 with 20a or 20e (Scheme 3). The DielsꢀAlder reaction between furans (17a
or 23) and vinyl sulfonates went smoothly, and the product yields were generally good.
Moreover, for additional SAR investigations, we expanded the diversity of conjugates
by appending 16a onto vinyl sulfonates 22aꢀc, q (Scheme 3A, series I) or appending
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23 onto vinyl sulfonates 20bꢀd (Scheme 3A, series II). Additionally, starting from 16b,
we synthesized a conjugate 25r, in which transꢀstilbene replaced the 6ꢀphenol group
of OBHS, to investigate the importance of the 3,5ꢀdihydroxyl group.
a
Scheme 2. The synthetic route of analogues 20aꢀe and 22aꢀr .
R2
R2
A)
O
S
Cl
a
+
Cl
O
R1
R1
19
20a-e
1
8a-c
2
2
0a: R1 = 4-ethene sulfonate, R2 = 3',5'-dimethoxy
0b: R1 = 4-ethene sulfonate, R2 = H
1
1
1
8a: R1 = 4-OH, R2 = 3',5'-dimethoxy
8b: R1 = 4-OH, R2 = H
8c: R1 = 3-OH, R2 = 3',5'-dimethoxy
20c: R1 = 3-ethene sulfonate, R2 = 3',5'-dimethoxy
2
2
b
b
0d: R1 = 3-ethene sulfonate, R2 = 3',5'-dihydroxyl
0e: R1 = 4-ethene sulfonate, R2 = 3',5'-dihydroxyl
O
R
O
S O
R
B)
a
Cl
S
+
HO
Cl
O
O
19
21
22a-r
22a: R = H, 93%
22g: R = 2-CH3, 84%
22h: R = 2-CF3, 79%
22m: R = 3-CH , 81%
22n: R = 4-OH, 86%
22o: R = 3-Br, 83%
3
2
2
2
2b: R = 4-Br, 82%
2
2r:
84%
2c: R = 3-OCH , 84%
22i: R = 3-CF , 77%
3
3
2d: R = 4-F, 84%
22j: R = 4-Ph, 78%
22k: R = 3-Cl, 84%
22l: R = 3-OH, 88%
22p: R = 4-CH , 87%
3
22e: R = 4-Cl, 82%
2f: R = 2-Cl, 88%
22q: R = 2-CH CH , 88%
2
3
2
a
3
Reaction conditions: (a) TEA, DCM, 0 °C, 12 h; (b) BBr , DCM, 0 °C, 10 h.
Scheme 3. The synthetic route of target compounds 24aꢀd, 25aꢀr and 26aꢀe.
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It is worth noting that there was a high stereoselectivity in the cycloaddition
4
1
reaction, as we have observed previously. The thermodynamically favorable exo
products were more easily generated through this DielsꢀAlder reaction, which was
presumed to be due to the high rate and ready reversibility. Because compound 23 is
symmetrical, conjugates 26aꢀe were studied as single isomers. Because 26a showed
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the highest cell antiꢀproliferative activity and antiꢀinflammatory activity, we separated
their enantiomers to further investigate their activity (see below, Scheme 4 and Table
5). Synthesis of the target compounds 25aꢀq was accomplished after 17a reacted with
0
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vinyl sulfonates, which interestingly were formed as mainly the one regioisomer; the
other regioisomers were hardly found (see supporting information for more details of
NOESYꢀNMR of 25b). The major regioisomers were studied as racemates.
OBHSꢀRES Conjugates Exhibit Binding Affinity. We used a competitive
4
8, 49
fluorometric receptorꢀbinding assay
to examine whether the OBHSꢀRES
conjugates could bind to ER, and the results are reported in Table 1.
a
Table 1. Relative binding affinity (RBA) of OBHSꢀRES conjugates to ERα and ERβ .
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a
estradiol
compound
The RBA values were calculated according to the formula: IC50
/IC50
× 100 ± the
range. We set the RBA value of estradiol as 100%.
b
i d d
K = (100/RBA) × K . For estradiol, the K value was 3.49 nM and 4.12 nM for ERα and ERβ,
respectively.
Overall, the position of the resveratrol group in the OBHS scaffold could
significantly influence the binding affinity. According to previous work, we realized
that the phenolic group could directly affect whether the compounds could effectively
50
bind to the ER ligand. One phenolic ring in OBHS mimics the "A ring" of estrogen,
which plays a key role in the binding affinity, while the other can tolerate large
substituents and its deletion would reduce the binding affinity to ERα to some
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extent. In further studies, it has been found that replacement of phenolic ring with
44
active groups also sometimes produces very good biological activity. In fact, RBA
values are lower for ERα in the majority of series I compounds, excluding compound
25p, which possesses a methyl group on the paraꢀposition of phenyl sulfonate unit.
0
1
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0
2
5p possesses the highest ERα binding affinity as 33.21 and has the highest α/β
subtype selectivity up to 23 among all the conjugates (Table 1, entry 20). The good
binding affinity of 25p may derive from its ability to form four hydrogen bonds with
ERα (computer model of the complex shown in Supporting Information). The RBA
values of OBHS were 8.11 and 3.39 for ERα and ERβ, respectively. However, some
of our final products presented even better ERα binding affinity and selectivity than
OBHS. These results may be understood based on the observation that resveratrol is
capable of modulating the inflammatory response as the pathwayꢀselective ligand for
3
6
ERα, OBHSꢀRES conjugates could bind to ER by using resveratrol moiety to
increase the binding affinity. From the computer model of the ERαꢀcompound 25p
complex, we observed that OBHSꢀRES conjugates could fill the cavity very well, thus
possibly increasing the binding affinity. It is evident that the position and size of the
substituents on the phenyl sulfonate could largely influence the RBA values.
Additionally, compounds 24aꢀc, which have a pterostilbene unit, showed lower ERα
binding affinity as well as decreased α/β selectivity, compared to 25aꢀc, which have a
resveratrol unit (Table 1, entries 1ꢀ3 vs 5ꢀ7). In addition, compound 25r, with
transꢀstilbene replacing the 6ꢀphenol group of OBHS, exhibited lower ERα binding
affinity and reduced subtype selectivity compared with compound 25a (Table 1,
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entries 22 vs 5). Thus, we conclude that the resveratrol unit makes a positive
contribution to the binding affinity of ERα in series I compounds.
Taking 25a as an example, in which the phenyl sulfonate moiety lacks the
substituent, only moderate ERα binding affinity was found. However, the introduction
of an electronꢀwithdrawing group, such as a fluorine, chlorine and trifluoromethyl
group, at the orthoꢀposition or paraꢀposition significantly decreased the ERα binding
affinity (analogues 25dꢀf and 25h Table 1, entries 8ꢀ10 and 12). Additionally, the
ERα binding affinity would increase when we introduced an electronꢀdonating group
on the phenyl sulfonate (analogues 25c, 25g and 25p, Table 1, entries 7, 11 and 20).
Substituents at the orthoꢀposition of the phenyl sulfonate unit could hardly affect the
RBA value (analogues 25f and 25g, Table 1, entries 10 and 11), except 25h with a
trifluoromethyl group, a strong electronꢀwithdrawing group, displayed almost 8ꢀfold
and 3ꢀfold reduced affinity compared with 25a for ERα and ERβ, respectively (entries
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
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4
4
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5
5
5
5
5
5
5
6
0
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2
3
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5
6
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0
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3
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5
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9
0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12 vs 5). Finally, conjugates 26a and 26e in series II possessing the pterostilbene or
resveratrol unit in the sulfonate part also demonstrated good RBA values for both ERs
and a preferential selectivity for ERα (entries 23 and 27). Compared with conjugates
2
6c and 26d, conjugates 26a, 26b and 26e (entries 25 and 26 vs entries 23, 24 and 27)
could better bind to ERα, suggesting that the resveratrol unit was preferred to the
ꢀhydroxypinosylvin unit.
3
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A
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0
Figure 3. (A) The RBA values of compounds with substituents at the metaꢀposition of
the phenyl sulfonate. (B) The RBA values of compounds with substituents at the
paraꢀposition of phenyl sulfonate.
Figure 3A shows the effect of the substituents at the metaꢀposition of the phenyl
sulfonate on binding affinity. It illustrates that halogen and trifluoromethyl
substituents had good RBA values and tended to bind to ERβ, especially compound
25i and 25k, showing 5ꢀfold and 7ꢀfold higher affinity for ERβ than ERα. In addition,
compound 25o displayed higher ERα selectivity, which might be attributed to
bromine possessing a larger atomic radius. Figure 3B shows the binding affinities of
compounds with substituents at the paraꢀposition of phenyl sulfonate. The RBA value
results for 25b, 25d, 25e, 25n and 25p (Table 1, entries 6, 8, 9, 18 and 20) indicated
that a lipophilic character and suitable size were necessary substituents. In addition,
compound 25p showed the best binding to ERα, indicating that the paraꢀmethyl group
on the phenyl sulfonate moiety provided a useful contribution to the binding affinity.
Based on other changes in the substitutions, we observed that compounds 25j and 25q
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had a bulkier substituent compared with 25a (Table 1, entries 14 and 21 vs 5),
resulting in a decrease in RBA values for ERα and further suggesting the importance
of the appropriate size of the substituents.
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4
Transcription Activation Assays. We used luciferase reporter gene assays to test
the ER transcriptional activities of various OBHSꢀRES conjugates, and the results are
described in Table 2.
a
Table 2. The transcriptional activity of OBHSꢀRES conjugates for ERα and ERβ .
a
In HEK 293T cells, a luciferase reporter assay was used to test the luciferase activity and
2
normalized to the control as 0% and 10 nM E as 100%. Data show the mean ± SD from an
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average of three experiments. Omitted parts could not be accurately determined. For details, see
the Experiment Section.
ER agonists are typically classified as normal agonists, partial agonists or
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51
superagonists. Overall, most compounds have high efficacy as ERα antagonists and
ERβ agonists and show higher efficacy in comparison to OBHS. Compared with
compound 24a, compound 25a (Table 2, entries 1 vs 5) with an improved RBA value
of ERβ demonstrates an approximately 9ꢀfold higher efficacy but decreased potency
as an ERβ agonist; moreover, it displays an antagonistic effect on ERα. From
conjugate 25r, which has a transꢀstilbene replacing the 6ꢀphenol group of OBHS, we
found that in comparison to 25a, the antagonistic effect on ERα and the agonistic
effect on ERβ decreased 2.4ꢀfold and 4.2ꢀfold, respectively, suggesting the
importance of the 3,5ꢀdihydroxyl groups for the transcriptional activity of OBHSꢀRES
conjugates (Table 2, entries 22 vs 5). Surprisingly, compared with compounds 24a,
24b and 24c, which act as partial ERβ agonists or ERα antagonists, compound 24d
exhibited good ERβ agonistic and ERα antagonistic effects (Table 2, entries 1ꢀ3 vs 4).
Compound 25a, an analogue of OBHS with replacement of resveratrol by a 6ꢀphenol
group, acted as an ERα antagonist and ERβ agonist. In addition, there was a large
change in activity on ERα or ERβ after modifying the phenyl sulfonate unit
(compounds 25bꢀ25q, Table 2, entries 6ꢀ21). The potency of these conjugates as ERα
antagonists or ERβ agonists was completely altered after a very slight change in the
phenyl sulfonate unit. For example, the introduction of halogens into phenyl sulfonate
significantly influenced transcriptional activity. The fluoroꢀsubstituted compound 25d,
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trifluoromethylꢀsubstituted compounds 25h and 25i, displayed agonist activity at ERβ;
moreover, 25i was a superantagonist on ERα (Table 2, entries 8, 12 and 13). However,
the chloro analogues 25e, 25f and bromo analogue 25b (Table 2, entries 9, 10 and 6)
were profiled as ERβ antagonists. In fact, these compounds all showed an antagonistic
effect on ERα. Indeed, by comparing the ERα antagonistic activities of 25b, 25d, 25e,
1
1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
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3
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3
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3
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0
25j, 25n and 25p (Table 2, entries 6, 8, 9, 14, 18 and 20), we observed that
substitution in the paraꢀposition of the phenyl sulfonate unit had remarkable effects,
with the preferred substituent exhibiting lipophilic properties and a suitable size. In
addition, the results for 25c, 25i, 25k, 25l and 25m (Table 2, entries 13, 15 vs 7, 16
3
and 17) suggested that the use of electronꢀwithdrawing substituents (CF or Cl) at the
metaꢀposition of the phenyl sulfonate unit might convey higher ERα antagonist
efficacy than electronꢀdonating (Me, OMe or OH) groups. Of note, the use of a larger
group such as αꢀnaphthyl (25q) to replace the phenyl resulted in stronger ERα
antagonistic activity. Compounds in series II also showed impressive activities.
Compounds possessing a pterostilbene or resveratrol unit in the sulfonate part were
capable of antagonizing ERα and agonizing ERβ efficiently, with compound 26a
having EC50 or IC50 values up to 4 nM and 7 nM, respectively (Table 2, entries 23).
Conjugates 26b, 26c and 26d possessing transꢀstilbene or 3ꢀhydroxypinosylvin in
place of the resveratrol moiety were also moderate ERα antagonists and ERβ agonists
(Table 2, entries 24ꢀ26).
Antiꢀinflammatory Assay for the Inhibition of Nitric Oxide Production in
Macrophage RAW 264.7 Cell Lines. NFꢀκB promotes gene expression for a number
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of cytokines and enzymes in inflammatory process, including inducible nitric oxide
24
synthase (iNOS), the expression of which is increased in macrophage of patients.
iNOS, an enzyme catalyzing nitric oxide (NO) production, is an effector located
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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19
downstream of cytokines and NFꢀκB, which could link inflammation to cancer. NO
20
is a key signaling molecule and a critical component of inflammatory responses;
furthermore, continuous and excessive production of NO by iNOS causes
pathophysiological problems, such as chronic inflammatory diseases and cancer
39, 52, 53
development.
Therefore, we chose to inhibit the generation of NO in
macrophage RAW 264.7 cell lines as an index to examine the antiꢀinflammatory
activity of OBHSꢀRES conjugates.
a
Table 3. Antiꢀinflammatory activity of OBHSꢀRES conjugates .
NO inhibition
IC50 (µM)a
NO production
% inhibitionb
NO inhibition
IC50 (µM)
NO production
% inhibition
Entry Compound
Entry Compound
1
2
3
4
5
6
7
8
9
24a
24b
24c
24d
25a
25b
25c
25d
25e
25f
25g
25h
25i
25j
25k
25l
>
>
100
100
20.44 ± 0.30
30.75 ± 2.01
60.07 ± 1.79
65.39 ± 1.59
70.87 ± 0.55
22.03 ± 1.15
42.29 ± 0.41
71.87 ± 0.21
62.07 ± 2.65
60.86 ± 0.13
39.07 ± 0.64
38.46 ± 1.38
41.65 ± 2.21
43.04 ± 0.37
62.08 ± 2.67
12.29 ± 1.14
17
18
19
20
21
22
23
24
25
26
27
28
29
30
25m
25n
25o
25p
25q
25r
26a
26b
26c
26d
26e
RES
OBHS
4OHT
2.41 ± 0.33
15.5 ± 1.94
2.53 ± 0.33
12.5 ± 0.16
3.29 ± 0.68
9.7 ± 0.20
0.44 ± 0.23
2.23 ± 0.39
6.37 ± 1.18
16.7 ± 0.63
0.93 ± 0.07
13.1 ± 1.11
18.4 ± 2.33
>100
68.42 ± 0.57
41.03 ± 0.04
60.86 ± 0.55
42.29 ± 0.72
44.38 ± 0.41
40.41 ± 1.01
95.58 ± 0.21
72.44 ± 0.72
55.07 ± 2.73
24.75 ± 2.01
80.97 ± 2.65
48.03 ± 0.64
41.48 ± 1.38
21.51 ± 0.39
80.42 ± 1.93
1
0
6
1
.26 ± 0.57
.79 ± 0.04
.02 ± 0.55
6.5 ± 1.15
1
1.1 ± 0.41
8
6
5
2
1
.88 ± 0.21
.50 ± 2.65
.00 ± 0.13
8.38 ± 0.64
7.22 ± 1.38
14.6 ± 2.21
8.7 ± 0.37
.14 ± 1.02
10
1
1
12
13
14
15
16
1
2
31 RES+OBHS
11.1 ± 0.66
>
100
a
Experimental values represent the average of three independent experiments ± standard deviation
b
(mean ± SD). Determined at a compound concentration of 10 µM.
First, to promote the protein expression of iNOS, we used lipopolysaccharide
(LPS) as a stimulant to activate macrophages (the RAW 264.7 cell line). Following
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the over expression of iNOS, NO production increased, which can be assayed using
an NO assay kit. The results of our compounds when coꢀtreated with LPS for RAW
264.7 cell lines are displayed in Table 3. We also tested the parent compounds OBHS,
1
1
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1
1
1
1
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1
1
2
2
2
2
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resveratrol and the physical mixture of OBHS and resveratrol for comparison.
Interestingly, most OBHSꢀRES conjugates, such as 24c, 24d, 25a, 25e, 25f, 25k, 25m,
25o, 25q, 26a, 26b, 26c and 26e, displayed excellent NO inhibition, while 4OHT
(4ꢀhydroxytamoxifen), a classic SERM for the treatment of breast cancer, did not
show any NO inhibition activity. Compared with their parent compounds and physical
mixture group, compound 26a, with a pterostilbene group in the sulfonate moiety,
was the best one, with an IC50 up to 0.44 µM and a percent inhibition rate determined
at a concentration of 10 µM reaching 95% (Figure 4). In general, the conjugates with
the resveratrol unit in the phenyl sulfonate part demonstrated excellent NO inhibition
(26a and 26e, Table 3, entries 23 and 27). Although 24c and 24d had moderate
binding affinity, their NO inhibition was good, suggesting the absence of a direct
correspondence between ER binding affinity and antiꢀinflammatory activity.
Interestingly, we found that the substituents at the orthoꢀ or paraꢀposition of the
phenyl sulfonate part had to be as small as possible, such as compounds 25b, 25dꢀh
and 25p (Table 3, entries 6, 8ꢀ12 and 20). Unexpectedly, compound 25l exerted poor
NO inhibition, with an IC of more than 100 µM, and compound 25n exhibited only
50
moderate NO inhibition, indicating that the hydrophilic groups did not augment the
antiꢀinflammatory activity. Although compound 25r showed a moderate IC50 for
antiꢀinflammatory activity, the inhibition rate was not appreciable, demonstrating the
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importance of the resveratrol unit for activity.
In addition, compared with compounds 26c and 26d, compounds 26a and 26e
(Table 3, entries 25 and 26 vs 23 and 27) displayed much better antiꢀinflammatory
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activity, again suggesting the need for the resveratrol unit.
Figure 4. Doseꢀresponse curves of the NO inhibitory activities for 26a, RES, OBHS,
and RES+OBHS.
Antiꢀproliferative Activity toward Breast Cancer Cells. To test the
antiꢀproliferative activity, MCFꢀ7 cells were treated with the target compounds, and
the results are shown in Table 4. Overall, compared with OBHS, the antiꢀproliferative
activity against MCFꢀ7 cell lines of most OBHSꢀRES conjugates was much stronger,
exceeding even their physical mixture group. Comparison to the approved drug
4ꢀhydroxytamoxifen revealed that compounds 25j, 25p, 26a and 26b showed higher
antiꢀproliferative activity in MCFꢀ7 cell lines (Table 4, entries 14, 20, 23 and 24). In
addition, compounds 25c, 25i, 25q, 26c and 26e were equipotent to 4OHT against
MCFꢀ7 cells. Furthermore, the compounds possessing pterostilbene and transꢀstilbene
substituents instead of the 6ꢀphenol group of OBHS (compounds 24aꢀd and 25r,
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Table 4, entries 1ꢀ4 and 22) displayed lower antiꢀproliferative activity. These results
indicated that the antiꢀproliferative activity of these compounds might mainly derive
from the antagonism toward ER, and the antiꢀinflammatory activity could also
improve their potency against MCFꢀ7 cell lines.
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Table 4. Cell antiꢀproliferative activity of OBHSꢀRES conjugates (IC50, ꢁM) .
a
Experimental values represent the mean ± SD from an average of three experiments.
The Antiꢀinflammatory Activity of 26a Contributes to Its Increased
Antiꢀproliferative Activity. Based on the aboveꢀdescribed experimental results, we
identified the best compound 26a, which displayed excellent antiꢀproliferative activity
and antiꢀinflammatory activity. According to our knowledge, antiꢀproliferative activity
may originate from excellent antagonistic activity on ERα. To determine the
contribution of the antiꢀinflammatory to the antiꢀproliferative activity, we selected
compound 26a for further study. We used JSHꢀ23, which can abrogate NFꢀκB nuclear
translocation, with 26a to coꢀtreat MCFꢀ7 cells for 48 h. Based on the results, we
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found that cell inhibition was significantly decreased in the coꢀtreatment group
compared with the 26a only treatment group (Figure 5). These findings suggested that
the antiꢀproliferative activity exhibited by 26a was partly due to its antiꢀinflammatory
activity, although its antiꢀproliferative activity was likely mainly derived from its
excellent ERα antagonistic activity.
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Figure 5. A comparison of the antiꢀproliferative activity of 26a and JSHꢀ23
cotreatment with 26a in MCFꢀ7 cells. JSHꢀ23 (10 ꢁM) and compound 26a (5 ꢁM, 10
ꢁM and 20 ꢁM) coꢀtreatment of MCFꢀ7 cells for 48 h. White columns show the
results for only treatment with 26a, and black columns show the results for the
coꢀtreatment group.
In Vivo MCFꢀ7 Breast Cancer Model in Balb/c Nude Mice. Considering the
excellent activity of compound 26a, we evaluated the antitumor potency of this
compound in vivo in murine MCFꢀ7 breast cancer tumor models in Balb/c nude mice
3
(
shown in Figure 6). After the tumor size reached ~80 mm , the nude mice were
randomly divided into four groups consisting of 5 animals each and systemically
administered vehicle control (consisting of cremophorꢀEL/DMSO/PBS 1:1:8, total
100 ꢁl), tamoxifen (25 mg/kg), or compound 26a (17.5 mg/kg or 25 mg/kg) once
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every two days. We observed that the group treated with compound 26a at a dose of
17.5 mg/kg had a reduced tumor volume and burden (Figure 6A and 6C). In addition,
the group treated with compound 26a at a dose of 25 mg/kg, a dose equivalent to
tamoxifen, had a reduced tumor burden by ~55% on day 12, whereas tamoxifen was
somewhat less efficacious, reducing the tumor burden by ~48%. Moreover, on day 27,
the group treated with compound 26a at 25 mg/kg inhibited tumor growth by up to
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~
69%, whereas the group treated with tamoxifen at 25 mg/kg showed only moderate
inhibition by ~33%.
It is noteworthy that compound 26a did not significantly reduce body weight
(Figure 6B), indicating its low toxicity to normal body tissues. These studies
demonstrated that compound 26a was more potent than tamoxifen in a murine tumor
model of human breast cancer.
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Figure 6. Antitumor potency of compound 26a in vivo in a murine MCFꢀ7 breast
cancer tumor model in Balb/c nude mice. (A) The changes in tumor volume in
different treatment groups measured every 3 days. (B) The changes in body weight in
different treatment groups measured every 3 days. (C) Average weight of the tumors
excised at the end of treatment. (D) Representative photos of the tumors at the end of
treatment.
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OBHSꢀRES Conjugates Have Antiꢀinflammatory Effects during Antiꢀtumor
Progression in Balb/c Nude Mice. Many inflammatory cells and signaling molecules
are present in the inflammatory microenvironment, which is beneficial for the
malignant progression of transformed cells. The inflammatory microenvironment can
facilitate the invasion and migration of tumor cells via breakage of the basement
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membrane.
In many studies
, inhibition of inflammatory factor expression,
such as NFꢀκB, interleukinꢀ6 (ILꢀ6) and tumor necrosis factor α (TNFꢀα), has been
shown to improve the outcome of tumor treatment.
Consequently, we further evaluated the antiꢀinflammatory activity of compound
26a in vivo in murine MCFꢀ7 breast cancer tumor models in Balb/c nude mice. After
3
the tumor size reached ~80 mm , the nude mice were randomly divided into four
groups consisting of 4 animals each and systemically administered the vehicle control
(consisting of cremophorꢀEL/DMSO/PBS 1:1:8, total 100 ꢁl), tamoxifen (25 mg/kg),
or compound 26a (17.5 mg/kg or 25 mg/kg) once every two days. After two weeks,
immunohistochemistry was used to evaluate the level of inflammatory parameters in
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the tissue around the tumor. Based on the result, we found that compound 26a
exhibited good antiꢀinflammatory activity in the tissue around the tumor, as assessed
by the inhibition of expression of NFꢀκB, ILꢀ6 and TNFꢀα (Figure 7). We believe that
this is an indication that the antiꢀtumor activity we observed in the therapeutic model
was accompanied by a reduction of inflammation. Based on this evidence, we could
conclude that the antiꢀinflammatory activity of 26a might contribute to its antiꢀtumor
activity.
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A
ILꢀ6 (2 weeks)
B
TNFꢀα (2 weeks)
C
p65 (2 weeks)
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Figure 7. Antiꢀinflammatory effect of compound 26a during antiꢀtumor progression
in a MCFꢀ7 breast cancer model in Balb/c nude mice. (A) ILꢀ6 immunohistochemistry
in the tissue around the tumor after 2 weeks of treatment. Scale bar, 50 ꢁm.
Quantification of the ILꢀ6 staining signal in the tissue around the tumor (n = 4) is
displayed in the column chart. (B) TNFꢀα immunohistochemistry in the tissue around
the tumor after 2 weeks of treatment. Scale bar, 50 ꢁm. Quantification of the TNFꢀα
staining signal in the tissue around the tumor (n = 4) is displayed in the column chart.
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(C) NFꢀκB activity monitored by immunohistochemistry of p65 in the tissue around
the tumor after 2 weeks of treatment. Scale bar, 50 ꢁm. Quantification of the p65
staining signal in the tissue around the tumor (n = 4) is displayed in the column chart.
Synthesis, Separation and Biological Evaluation of the Enantiomers of
Compound 26a. As we know, chirality of compounds is very common in medicinal
chemistry, and usually the precise structure of a ligand can help to determine whether
5
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a receptor for hormones and drugs is activated. The relatively rigid endogenous
steroid ligands normally bind to steroid hormone receptors, while conformationally
flexible lipids have the ability to bind to other nuclear receptor superfamily members
57
and exhibit the plasticity in the ligandꢀbinding pocket to some extent. However, ER
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can bind and respond to various steroidal or nonsteroidal ligands. Given the
aforementioned experimental results, we wondered whether one of the two isomers of
the best compound 26a, which has a chiral center, might play a main role in this
excellent activity.
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Initially, we attempted to use normal phase Chiralpak columns to separate this
compound, but this attempt failed. Fortunately, considering work conducted by
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Katzenellenbogen and his colleagues, we used orthogonal protecting groups and
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chiral HPLC and successfully separated the two isomers. First, the two phenols of
compound 23 were protected as a benzyl ether and a sterically bulky
tertꢀbutyldiphenylsilyl (TBDPS) ether. We use the DielsꢀAlder reaction of this
diprotected furan 28 and vinyl sulfonate 20a to obtain a mixture of regioisomers,
which could be isolated by normal phase silica chromatography. The structures of
these regioisomers were verified by twoꢀdimensional NOESYꢀNMR (see Supporting
Information for details). The next challenge was to explore the appropriate HPLC
conditions to separate the enantiomers from one or both regioisomers. After using
different chiral columns, we successfully achieved the separation of the two
enantiomers 29a and 29b using a normal phase Chiralpak IB column with
hexane/isopropanol as the eluent. Then, to complete the synthesis, the orthogonal
protecting groups had to be removed. TBDPS could be cleaved by
tetrabutylammonium fluoride (TBAF)ꢀpromoted deprotection to provide 31 with good
yield. However, the final step involving cleavage of the benzyl protecting group
seemed to be more challenging than we anticipated. Our first attempt utilized
TMSIꢀmediated debenzylation, but it was unsuccessful. Next, we used the Pd/C
hydrogenolysis conditions, which led to debenzylation but a further reduction of
olefin. Surprisingly, it also included a reduction of the double bond in the oxabicyclic
3
ring. Another debenzylation condition using AlCl allowed us to obtain the desired
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