3, 9-di-O-substituted coumestrols incorporating basic amine side chains act as novel apoptosis inducers with improved pharmacological selectivity

Article abstract of DOI:10.1016/j.bioorg.2018.12.024

There is much interest in the use of phytoestrogens such as coumestrol in breast cancer intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However, the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of coumestrol led to the most promising compound 7e with improved antiproliferative activity (1.7-fold increase) against MCF-7 cells, decreased estrogen activity (>20 times weaker ERα binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with distinct pharmacological properties, providing an excellent starting point to future development of novel tumor-vascular disrupting agents targeting tubulin.

Full text of DOI:10.1016/j.bioorg.2018.12.024

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Accepted Manuscript
3, 9-Di-O-substituted coumestrols incorporating basic amine side chains Act as
novel apoptosis inducers with improved pharmacological selectivity
Guoshun Luo, Zhengpu Tang, Xinyu Li, Qiangqiang Hou, Yu Chen, Kejing
Lao, Hua Xiang
PII:
S0045-2068(18)30920-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.024
YBIOO 2688
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 August 2018
7 November 2018
18 December 2018
Please cite this article as: G. Luo, Z. Tang, X. Li, Q. Hou, Y. Chen, K. Lao, H. Xiang, 3, 9-Di-O-substituted
coumestrols incorporating basic amine side chains Act as novel apoptosis inducers with improved pharmacological
selectivity, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.024
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3, 9-Di-O-substituted coumestrols incorporating basic amine side
chains Act as novel apoptosis inducers with improved
pharmacological selectivity
Guoshun Luoa,b,#, Zhengpu Tanga,b,#, Xinyu Lia,b, Qiangqiang Houa,b, Yu Chenb,c,
Kejing Laod, Hua Xianga,b*
a
State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing
210009, China
c
Key Laboratory of Smart Drug Delivery, Ministry of Education School of Pharmacy, Fudan
University, Lane 826, Zhangheng Road, Shanghai 201203, China
d Shaanxi Key Laboratory of Brain Disorders and Institute of Basic and Translational Medicine, Xi'an
Medical University, Xi'an, Shaanxi 710021, China
# Same contribution to this paper
*Corresponding author. Tel.: +86 025 83271096; Fax: +86 025 83271096 (H. Xiang).
E-mail addresses: xianghua@cpu.edu.cn (H. Xiang)

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Abstract
There is much interest in the use of phytoestrogens such as coumestrol in breast cancer
intervention due to their antiestrogenic activity and multiple modes of tumor cell death. However,
the clear beneficial effects of naturally occurring estrogen mimetic coumestrol remain
controversial due to experimental evidence that it has been shown to stimulate MCF-7 cell
proliferation via agonist effect on estrogen receptor at low concentration. Herein, to disconnect the
ER interaction and apoptosis-specific mechanism of coumestrol, various 3, 9-di-O-substituted
coumestrols (7a-7e) and their furan ring-opened analogs (5a-5e) were synthesized and assessed
for antiproliferative properties. Attachment of a dimethylamine-containing side chain to 3-O of
coumestrol led to the most promising compound 7e with improved antiproliferative activity
(1.7-fold increase) against MCF-7 cells, decreased estrogen activity (> 20 times weaker ERα
binder) and a novel action to induce apoptosis. Mechanistic studies revealed that 7e is a tubulin
polymerization inhibitor, which could arrest cell cycle at G2/M phase and induce apoptosis along
with the decrease of mitochondrial membrane potential. In summary, such subtle modifications to
the 3, 9-di-hydroxyl groups of coumestrol allow the generation of a novel apoptosis inducer with
distinct pharmacological properties, providing an excellent starting point to future development of
novel tumor-vascular disrupting agents targeting tubulin.
Keywords: coumestrol; estrogenic activity; apoptosis; cell cycle; tubulin polymerization inhibitor.

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1. Introduction
With their diverse structures and bioactivity, natural products historically have been a rich
source of lead compounds for drug development. Of the drugs approved by the US Food and Drug
Administration (FDA), more than 40% of them developed over the past 30 years are from NP or
their structural scaffolds [1, 2]. For anticancer drugs, over 60% of these drugs approved since
1940 can be traced to a natural product [3]. Discovery and development of anticancer agents from
plants have shown promise for cancer prevention and therapeutics. As part of our recent research
to develop new anti-cancer agents, we were attracted to the coumarin-based derivatives [4, 5],
which are common components of biologically active natural products [6].
Coumestan (6H-[1]benzofuro[3,2-c]chromen-6-one) is an important class of phytochemicals
containing a planar and oxygenated tetracyclic scaffold in which one coumarin ring is fused with a
benzofuran ring (Figure 1). Natural coumestans are widely distributed in a variety of plant species
belonging to botanical families such as Fabaceae, Leguminosae and Asteraceae [7] and exhibites
various biological activities such as antioxidative [8], antitubercular [9], and antineoplastic effects
[10], depending on their diverse structures modified by substituents on the ring system.
Coumestrol (Figure 1) is a representative member in the coumestan family and was first identified
as a phytoestrogen with estrogenic properties by E. M. Bickoff in ladino clover and alfalfa in 1957
[11]. It has garnered great research interest stemming from its antiestrogenic activity and
prevalence in many human diets, including soybeans, brussels sprouts, spinach and a variety of
legumes [12]. These two hydroxy groups of coumestrol orients in the same position as those in the
endogenous estradiol (E2) (Figure 1), allowing it to disrupt the activity of estrogen receptor (ER),
which highlighted the potential of coumestrol as a therapeutic approach targeting
hormone-dependent breast cancer. However, the role of phytoestrogens like coumestrol in treating
breast cancer remains contradictory and elusive. Several analyses that assessed soy exposure and
breast cancer risk revealed that the chemoprotective effects of phytoestrogens are dependent on
early exposure and high soy consumption [13-16]. There is evidence that at low pharmacologic
concentrations, coumestrol produces negative effects, such as inducing further growth of existing
breast cancer tumors by activating ERα receptors [17-19].
Recently, prof. Jordan VC and colleagues have investigated this matter and concluded that
phytoestrogens may be used as an effective treatment for breast cancer due to their apoptotic
properties [20]. When it comes to its apoptotic activity, coumestrol was also reported to exhibit
broad-spectrum anti-proliferative effects [21-23] through a variety of mechanisms which involve
increasing ROS generation and modulation of cell cycle progression in breast and colon cancer
[24-26], stimulating mitochondria-mediated intrinsic apoptosis in breast and prostate cancer cells
[27, 28] and regulating PI3K and MAPK signal transduction cascades in prostate and ovarian
cancer cells [29].
Concerns regarding the effects of coumestrol on promoting cell growth and proliferation in
ER positive breast tumors have been linked to its agonistic activity on ERα leading to upregulation