Novel SERMs based on 3-aryl-4-aryloxy-2H-chromen-2-one skeleton - A possible way to dual ERα/VEGFR-2 ligands for treatment of breast cancer

Article abstract of DOI:10.1016/j.ejmech.2017.09.015

There is considerable interest in developing new SERMs as multifunctional agents in women's health. Development of dual selective estrogen receptor modulators/VEGFR-2 inhibitors (SERMs/V-2I) has been an attractive strategy for the discovery of new breast cancer therapeutic agents. Our previous efforts led to the preparation of a series of 3-aryl-4-anilino-2H-chromen-2-ones endowed with potent estrogen receptor binding affinity and anti-proliferative efficacy. In this study, various structurally related 3-aryl-4-anilino/aryloxy-2H-chromen-2-one analogues were rationally designed, synthesized and evaluated as a new chemo-type of dual ERα and VEGFR-2 inhibitors. Most of the derivatives exhibited potent activities in both enzymatic and cellular assays. SAR investigation revealed that introducing of bioisosteric O atom at the C-4 position of coumarin scaffold is beneficial to improve the inhibitory potency, especially in ERα binding affinity assay. Furthermore, most of the piperidyl substituted compounds showed better inhibitory activity against MCF-7 and Ishikawa cells than lead compounds BL-18d, tamoxifen and Vandetanib. Optimization of the hit compound, identified in an ERα binding affinity assay, led to compound 42d, exhibiting an IC₅₀ for ERα binding affinity of 2.19 μM while retaining an excellent inhibition on VGFR-2 as well as a potent suppression on the growth of angiogenesis-related cells. In RT-PCR assay, 42d exerted significantly antiestrogenic property via suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7 cells, which was consistent with the ERα antagonistic property of a selective estrogen receptor modulator. Further mechanism investigation demonstrated that compound 42d could inhibit the activation of VEGFR-2 and subsequent signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells. All these results together with molecular modeling studies open a new avenue for the development of multifunctional agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.

Full text of DOI:10.1016/j.ejmech.2017.09.015

Accepted Manuscript
Novel SERMs based on 3-aryl-4-aryloxy-2H-chromen-2-one skeleton - A possible
way to dual ERα/VEGFR-2 ligands for treatment of breast cancer
Guoshun Luo, Xinyu Li, Guoqing Zhang, Chengzhe Wu, Zhengpu Tang, Linyi Liu,
Qidong You, Hua Xiang
PII:
S0223-5234(17)30697-9
10.1016/j.ejmech.2017.09.015
EJMECH 9730
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 July 2017
Revised Date: 24 August 2017
Accepted Date: 10 September 2017
Please cite this article as: G. Luo, X. Li, G. Zhang, C. Wu, Z. Tang, L. Liu, Q. You, H. Xiang, Novel
SERMs based on 3-aryl-4-aryloxy-2H-chromen-2-one skeleton - A possible way to dual ERα/VEGFR-2
ligands for treatment of breast cancer, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.09.015.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Novel SERMs based on 3-aryl-4-aryloxy-2H-chromen-2-one Skeleton - A Possible
Way to Dual ERα/VEGFR-2 Ligands for treatment of Breast cancer
Guoshun Luoa, Xinyu Lia, Guoqing Zhanga, Chengzhe Wua, Zhengpu Tanga, Linyi Liua,
Qidong Youa, Hua Xianga*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang,
Nanjing 210009, PR China
* Corresponding author. Tel.: +86 025 83271096; Fax: +86 025 83271096 (H. Xiang).
E-mail addresses: xianghua@cpu.edu.cn (H. Xiang)
Abstract
There is considerable interest in developing new SERMs as multifunctional agents in women's
health. Development of dual selective estrogen receptor modulators/VEGFR-2 inhibitors (SERMs/V-2I)
has been an attractive strategy for the discovery of new breast cancer therapeutic agents. Our previous
efforts led to the preparation of a series of 3-aryl-4-anilino-2H-chromen-2-ones endowed with potent
estrogen receptor binding affinity and anti-proliferative efficacy. In this study, various structurally
related 3-aryl-4-anilino/aryloxy-2H-chromen-2-one analogues were rationally designed, synthesized
and evaluated as a new chemo-type of dual ERα and VEGFR-2 inhibitors. Most of the derivatives
exhibited potent activities in both enzymatic and cellular assays. SAR investigation revealed that
introducing of bioisosteric O atom at the C-4 position of coumarin scaffold is beneficial to improve the
inhibitory potency, especially in ERα binding affinity assay. Furthermore, most of the piperidyl
substituted compounds showed better inhibitory activity against MCF-7 and Ishikawa cells than lead
compounds BL-18d, tamoxifen and Vandetanib. Optimization of the hit compound, identified in an
ERα binding affinity assay, led to compound 42d, exhibiting an IC50 for ERα binding affinity of 2.19
µM while retaining an excellent inhibition on VGFR-2 as well as a potent suppression on the growth of
angiogenesis-related cells. In RT-PCR assay, 42d exerted significantly antiestrogenic property via
suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7 cells, which was
consistent with the ERα antagonistic property of a selective estrogen receptor modulator. Further
mechanism investigation demonstrated that compound 42d could inhibit the activation of VEGFR-2
and subsequent signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells. All these results
together with molecular modeling studies open a new avenue for the development of multifunctional
agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.
Keywords: Estrogen receptor; VEGFR-2; Coumarin; Anti-breast cancer; Docking studies.

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1. Introduction
Breast cancer is the most prevalent form of cancer diagnosed in women worldwide, with an
incidence that rises dramatically with age. An estimated 1.7 million women will be diagnosed with
breast cancer in 2020 - a 26% increase from current levels [1]. Estrogens are a group of steroid
hormones and key regulators of growth, differentiation and function of the female reproductive organs
including the breast, uterus and ovaries [2]. The biological actions of estrogens are mediated through
intracellular transcription factors called estrogen receptors (ER) which belong to the nuclear receptor
superfamily. Estrogen receptors (ER) mediate estrogen activity in many important physiological
processes and play vital roles in the development of therapeutic agents for breast cancer treatment [3,
4]. Despite a remarkable increase in the depth of our understanding and management of breast cancer
in the past 50 years, the disease remains a significant public health problem worldwide and poses
significant challenges [5].
It is well known that approximately 70% of human breast cancers are hormone-dependent and
ERα-positive [6]. Endocrine therapy which aims to block the ER transcription effect is regarded as an
effective treatment. Thus ERα has provided an ideal pharmaceutical target and a lot of ERα ligands
have been developed as antagonists against ERα positive breast cancer [7]. Selective estrogen receptor
modulators (SERMs) are a special group of ligands which act as antagonists in breast tissue but
agonists in other tissues such as cardiovascular system and bone [8, 9]. Due to their special action
mode, SERMs remain as important anti-breast cancer agents with benefits in cardiovascular system and
maintaining bone density compared with aromatase inhibitors and pure anti-estrogens [10, 11].
Tamoxifen (a SERM, 1a), containing a triphenylethylene scaffold and a basic side chain, was the first
SERM approved for the treatment of breast cancer and have provided invaluable treatments for a
number of patients [12-14]. Its active metabolite, 4-hydroxy [4-OH] tamoxifen 1b, exhibits a high
binding affinity for the ER. Since then, many SERMs with various scaffolds mimicking Tamoxifen
have been developed for the treatment or prevention of breast cancer [15, 16]. Many alternative
scaffolds for ER modulators have also been reported, e.g., the naphthalene ring system (3),
benzopyran-based analogue (ormeloxifene, 4), dihydrobenzoxathiin platform (5) and coumarin-based
benzopyranone analogues (6, BL-16d and BL-18d) (Figure 1) [17-21].

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Figure 1. Previously reported selective estrogen receptor modulators (SERMs)
Although SERMs have improved the outcome for ERα positive breast cancer patients,
unfortunately, undesired side effects severely limit their therapeutic use. For instance, long-term
treatment of Tamoxifen increases the occurrence of endometrial cancer due to their partial estrogenic
activity on the endometrium [22]. Intrinsic and acquired drug resistance is another common deficiency
that limits the use of SERMs, in which breast tumors become refractory to endocrine therapies and
relapse [23, 24]. Therefore, there is still a need for developing new ER ligands with better therapeutic
effect.
Recently, intensive interest in discovery of novel anti-breast cancer drugs based on different
mechanisms of action is highlighted by the pursuit of multiple ligands acting at multiple biomolecular
targets. These multifunctional conjugates may exert favorable advantages of improved efficacy and
lower incidence of side effects [25]. Several attempts have been made to improve efficacy through
coupling a potent ER ligand to a second component, such as an antimetabolite agent (E2-nucleoside
conjugate 7) [26], alkylating agent (E2-chlorambucil conjugate 8) [27], tubulin agent (dual ER-tubulin
agent 9) [28], histone deacetylase (HDAC) inhibitor (dual ER-HDAC agent 10) [29], and aromatase
agent (dual ER-aromatase agent 11) [30] (Fig. 2A).
However, with the development of acquired resistance in breast and endometrial tumors it is
axiomatic that selective estrogen receptor modulators (SERMs, tamoxifen and raloxifene) stimulated
tumors must induce angiogenesis to grow [31]. Tumor angiogenesis is vital to cancer growth,
metastasis and invasion, and thus represents an attractive therapeutic target [32]. Vascular endothelial
growth factor receptor-2 (VEGFR-2) is a member of the receptor tyrosine kinase (RTK) family and the
predominant effector of VEGF/VEGFR signaling in promoting angiogenesis in cancer [33]. The
phosphorylation of VEGFR-2 will activate the Raf-1/MAPK/ERK signaling which is very important

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for cell proliferation, migration, invasion, and angiogenesis [34]. Moreover, studies have revealed that
the Raf-1/MAPK/ERK interplays with ER in regulating estrogen-dependent gene expression and is
widely observed in the endocrine resistance [35, 36]. Monotherapy of VEGFR-2 inhibitors were
reported in the treatment of breast cancer but are not sufficient as single therapy [37]. We thus
hypothesize that building VEGFR-2 inhibitory activity into SERMs could be an effective way to
improve the efficacy and decrease the side effects associated with them. Indeed, a combination of
Tamoxifen with Brivanib, a VEGFR-2 inhibitor, was recently reported not only to maximize
therapeutic efficacy but also to retard SERM resistant tumor growth [31]. Moreover, 2, 3-diaryl
isoquinolinone (12) and 6-aryl-indenoisoquinolone (13) were reported by our group as multiple ligands
of ERα and VEGFR-2, which showed inhibition on both targets and potent anti-cancer activity [38, 39]
(Fig. 2B). These inspiring evidences suggest that simultaneous ER and VEGFR-2 inhibition could be a
promising approach in breast cancer therapy.
Figure 2. (A) Previously referenced dual-acting ER ligand conjugates (B) multiple ligands of ERα and VEGFR-2
together with outstanding biological results reported by our group previously
Coumarins constitute an important class of pharmacologically active scaffolds known to exert
versatile biological activities including anticancer [40-43], anti-HIV [44], anti-microbial [45], and
anti-inflammatory activity [46]. The therapeutic applications of coumarins depend upon the pattern of
substitution and have attracted intense interest in recent years due to their diverse pharmacological

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properties [47]. Among these properties, their anticancer effects were the most extensively examined
[48, 49]. Studies have revealed the mechanism behind the anticancer effect of coumarin analogs
which include anti-angiogenesis and induction of apoptosis independently [50-53]. As the current
strategies in cancer drug development shifted toward the multiple mechanistic approach [54], such
validation of structure-activity relationship of coumarins with respect to angiogenesis and
anti-proliferation activity leads to dual-action anti-tumor activities deserve to be investigated.
Previously,
we
have
prepared
a
series
of
novel
SERMs
based
on
3-aryl-4-anilino-2H-chromen-2-one core, of which the piperidyl substituted analogues BL-16d (with
OMe at C-7 position of the coumarin nucleus) and BL-18d (with OH at C-7 position of the coumarin
nucleus) demonstrated strong ERα binding affinities and excellent anti-proliferative activities against
MCF-7 and Ishikawa cells [21]. As an extension of our study in the development of novel SERMs with
high therapeutic index, here we adopted 3, 4-disubstituted-2H-chromen-2-ones (BL-18d and BL-16d)
as promising lead compounds in this study for further optimized as novel anti-breast cancer agents with
multiple mechanistic profiles.
2. Structure-Based Drug Design
Encouraged by the promising research results mentioned above, our further efforts focused on
discovery of novel coumarin-based anti-breast cancer agents dual targeting ERα and VEGFR-2. To
rationalize the modification of 3, 4-disubstituted-2H-chromen-2-one, docking study of BL-18d with
ERα active site was carried out by CDOCKER protocol in Discovery studio 3.0 (PDB: 3ERT). As
shown in Figure 3A, the hydrogen bond interactions of lead compound’s 7-OH with Glu353 and
Arg394 and 4'-OMe with His524 play important roles in stabilizing the binding mode of BL-18d to
ERα. Furthermore, It has been indicated that that an oxygen linker confers a substantial increase in
estrogen antagonist potency [55]. Our first exploration focused on the biological effect of the bridging
atoms between the phenyl and coumarin moieties, in addition to the established NH linker, bioisosteric
group O atom was also employed. Subsequently, two more groups (F and OH) were further attached to
C-4' position of 3-phenyl substituent. Studies showed that incorporation of fluorine to a bioactive
molecule causes minimal steric alterations and, hence, can facilitate interactions of that biomolecule
with enzyme active sites, receptor recognition sites and other biological systems [56, 57]. Additionally,
these hydroxyl containing derivatives bearing more hydrogen bond donors could retain essential
hydrogen bonds with essential amino acid residues of targeted ERα receptor and VEGFR-2 enzyme.
Finally, various medium size tertiary amines including dimethyl amine, diethyl amine, pyrrolidinyl and
piperidyl were incorporated to terminal basic side chain to obtain diverse derivatives.
In the past decades, a number of potent VEGFR inhibitors including Vandetanib, Vatalanib
(PTK787) and Cabotantinib have been identified and brought to clinical trials in the treatment of

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angiogenesis-related diseases [58, 59]. The essential pharmacophoric features in the representative
VEGFR-2 inhibitors (Figure 3B) include fused aromatic system and substituted aniline or aryloxy
moiety which target the kinase domain of VEGFR. Recently, several coumarin-type compounds were
reported to block angiogenesis by inhibiting endothelial cell growth and have attracted considerable
attention as antitumor agents [52, 53]. In this context, 3, 4-disubstituted-2H-chromen-2-one containing
fused aromatic ring and solvent side chain region in particular emerged as an interesting scaffold for
designing VEGFR inhibitors. On the other hand, the structural optimization was also guided and
corroborated by molecular docking of a putative compound 17d (R1=R2=OH) into the VEGFR-2 ligand
binding domain (PDB: 1YWN; Figure 3C). Analysis of the molecular docking results revealed both
hydrogen bond interactions and Pi-Pi interactions of the coumarin moiety in the bind site formed by
critical amino acid residues phe916 (6.30 Å), Cys917 (2.62 and 2.96 Å) and Lys866 (2.78 Å), which
we believe will contribute, at least in part, to VEGFR-2 inhibition. Overall, the design strategy and
structures of the title compounds were represented in Fig. 3D.

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s
o
OH
4'
p
'
NH
O
4
-
C
t
O
a
R
H
O
R2
NH
O
HO
O
O
Further exploring
substitutions
at C-4' position
17a-d
O
O
R1
O
F
a
t
N
H
C
O
-
R
4
'
p
O
o
O
N
O
F
s
Asp351
i
t
i
o
Antiestrogenic
side chain
NH
O
n
N
4'
bridging N atom
R2
H
His524
O
O
H
N
R1: H, OMe, OH
R
O
R1
O
NH
4
N
substituted Coumarin skeleton
X
4'
Optimizing
strategy
4'
4
23a-d, 29a-d, 30a-d
O
O
3'
H
6
O
O
O
1'
2'
7
O
O
R1
O
O
O
O
n
BL-18d
HN
o
i
t
R
R
7
HN
Glu353
i
s
o
H
HN
p
O
OH
'
4
-
H
O
O
Demethylation
C
t
O
N
4'
a
e
bridging O atom
M
O
NH
O
r
o
H
Arg394
O
O
O
O
O
HO
O
O
R
R2
39a-d
41a-d
Further exploring
substitutions
at C-4' position
O
O
ER (PDB: 3ERT)
α
O
N
R1
O
O
F
O
a
R
NH
t
C
-
4
F
'
p
o
O
s
i
t
i
o
4'
n
R1
O
O
R1: OMe, OH
substituted Coumarin skeleton
R1
O
O
R1= OMe: BL-16d
R1= OH: BL-18d
40a-d, 42a-d
Coumarin-based SERMs
previously reported by our group
Figure 3. The rationally design of targeted compounds based on 3-aryl-4-anilino/aryloxy-2H-chromen-2-one scaffold. (A) Schematic representation of the catalytic
pocket of ERα and the chemical modulation strategy. (B) Previously reported VEGFR-2 inhibitor and the design of novel inhibitor for VEGFR-2 kinase (C) Docking
analysis of a putative compound 17d within active site of VEGFR-2 (for clarity, only interacting residues are labeled. Hydrogen-bonding and hydropholic
interactions are shown by red dashes). (D) Schematic representation showing the overall designing strategy of dual ERα and VEGFR-2 ligands.
Herein, we describe the synthesis of 32 novel coumarin-based analogs, preliminary pharmacological data from receptor binding and cellular
assays, and mechanism investigation on Raf-1/MAPK/ERK pathway. This allowed a straightforward evaluation of ERα binding affinity,
anti-proliferative potency, antiangiogenic activity, anti-estrogenic property as well as the expression of essential proteins from
VEGFR-2/Raf-1/MAPK/ERK signaling pathway. Molecule docking was further carried out for ERα and VEGFR-2 in order to explore the potential
binding mode of the novel dual-action inhibitors.

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3. Results and discussion
3.1 Chemistry.
The synthetic route of two hydroxyl containing 3-aryl-4-anilino-2H-chromen-2-one derivatives
17a-d was shown in Scheme 1. This synthetic strategy utilized a key starting material,
7-methoxy-3-(4-methoxyphenyl)-2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate (14), which was
synthesized through a facile route previously reported by our group [43]. Nucleophilic substitution of
14 with previously synthesized 4-[(2-aminoethoxy)]-substituted anilines 15a-d [21] afforded
compounds 16a-d, which were then converted to the final compounds 17a-d through demethylation .
Scheme 1. Synthetic routes of 17a-d. Reagents and conditions: (a) K2CO3, EtOH, 75 oC, 3 h; (b) BBr3, CH2Cl2, rt,
12 h.
The design and synthesis of 4’-fluorine substituted 3-aryl-4-anilino-2H-chromen-2-one analogues
23a-d, 32a-d and 34a-d were successfully processed following the previously reported procedures [43].
The synthetic sequence of 23a-d began with the commercially available methyl salicylate 18, which
were easily converted to the esterified product 20 (Scheme 2). The following cyclized compound 21
was prepared from corresponding intermediate 20 via intramolecular Claisen condensation. Further
treatment of 21 with paratoluensulfonyl chloride provided sulfonate precursor which were then coupled
with various 4-[(2-aminoethoxy)]-substituted anilines 15a-d via the nucleophilic substitution to yield
the target compounds 23a-d in good to moderate yields.

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Scheme 2. Synthetic routes of 23a-d. Reagents and conditions: (a) POCl3, pyridine, 0-10 °C, 2 h; (b) KOH,
pyridine, rt, 4 h; (c) TsCl, Et3N, dry DCM, rt, 0.5 h; (d) arylamines, K2CO3, EtOH, 75 oC, 2-3 h.
The synthesis of compounds 29a-d and 30a-d is shown in Scheme 3. Briefly, resorcinol reacted
with 4-hydroxybenzoic acid to yield benzophenone 25 under Friedel-Crafts reaction conditions, which
was then converted to the corresponding 4-methoxy deoxybenzoin 26 through alkylation of 4-hydroxyl
with methanol via Mitsunobu reaction. Further treatment of 26 with diethyl carbonate (DEC)/NaH in
reflux toluene gave cyclized compound 27, which were then subjected to an esterification reaction in
the presence of TsCl and TEA in DCM to provide the key intermediates 28. Subsequently, the
introduction of different substituents 15a-d on the C-4 position of 28 provided targeted compounds
29a-d. The removal of the methyl group of compounds 29a-d with BBr3 in DCM produced desired
analogues 30a-d.
o
Scheme 3. Synthetic routes of 29a-d and 30a-d. Reagents and conditions: (a) BF3-OEt2, 85 C, 2.5 h; (b) MeOH,
o
o
PPh3, DIAD, THF, 0 C-rt, 0.5 h; (c) NaH, DEC, 120 C, 2 h; (d) TsCl, Et3N, dry DCM, 0.5 h; (e) arylamines,
K2CO3, EtOH, 75 oC, 2-3 h; (f) BBr3, CH2Cl2, rt, 12 h.
Subsequently, we designed and synthesized the 4-O-linked 3-aryl-4- aryloxy-2H-chromen-2-one
derivatives to further investigate the effects of the bridging O atom between the phenyl and coumarin
moieties on biological activity. The Synthetic routes of the 4-[(2-aminoethoxy)]-substituted phenols
35a-d and the desired 4-anilino-3-aryl-2H-chromen-2-ones 39a-d, 40a-d, 41a-d and 42a-d are outlined
in Scheme 4. The 4-[(2-aminoethoxy)]-substituted phenols 35a-d were obtained in four steps involving:
i) selective etherification of hydroquinone 31 with methoxymethyl chloride; ii) reaction of the resulting
mon-etherified product 32 with 1, 2-dichloroethane; iii) treatment of the 2-chloroethyl derivative 33
with selected amines; and, finally iv) removal of the methoxymethyl group in the aminated derivatives
34a-d.
The bromination of preexisting 4-hydroxy coumarins 36 [43] and 27 with excess phosphorus
oxybromide was carried out to give the key 4-bromocoumarins 37, 38 [60]. The corresponding

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O-linked compounds 39a-d and 40a-d were prepared by coupling of intermediates 37 and 38 with
various 4-[(2-aminoethoxy)]-substituted phenols 35a-d. Demethylation was thus carried out on the
compounds 39a-d and 40a-d to afford the required derivatives 41a-d and 42a-d.
Scheme 4. Synthetic routes of 39a-d, 40a-d, 41a-d and 42a-d. Reagents and conditions: (a) K2CO3, dry acetone,
o
MOMCl, rt, 2 h; (b) 5N NaOH solution, ClCH2CH2Cl, TBAB, 75 C, 4 h; (c) RCl or RCl·HCl, K2CO3, KI, DMF,
100 °C, 4 h; (d) concentrated HCl, MeOH, rt, 4 h; (e) POBr3 (1.1 equiv), 1,2-dichlorobenzene, 170 °C, 4 h; (f)
arylamines, Cs2CO3 (2 equiv), MeCN, 75 °C, 2-3 h; (g) BBr3, CH2Cl2, rt, 12 h.
3.2. Biological evaluation.
3.2.1. ERα binding affinity assay and cell proliferation assay.
The ERα binding affinities of title molecules were initially measured by following a fluorescence
polarization procedure [21, 38 and 39]. Their potency to inhibit the proliferation of human breast
cancer cell line MCF-7 (ER+) and human endometrial cancer cell line Ishikawa was also performed
using MTT assay. Clearly, the fact that long term treatment of tamoxifen will stimulate endometrial cell
growth and increase the incidence of endometrial cancer is a significant concern for its clinical
application. Thus, it is essential to evaluate the anti-proliferative activity of new compounds on
endometrial cells. For comparison, lead compound BL-18d, Tamoxifen and Vandetanib were also used
as the positive control in these two assays. These result data can be analyzed in two groups, 4-N-linked
analogues and 4-O-linked analogues, and are depicted in Table 1 and Table 2 respectively.
As is shown in Table 1, most of the N-linked compounds (piperidyl substituted as terminal basic
group) showed promising binding affinities with IC50 values less than 10 µM indicating that coumarin
scaffold could favorably mimic that of extradiol. It was supposed that hydroxyl group at C-4’ position
had formed more hydrogen bonds with essential amino acid His524 than methoxyl group in the ligand
binding domain of ERα. Expectedly, it is obvious to note that derivatives 17d with hydroxyl substituted
at C-4’ position (IC50 = 3.82 µM) possessed apparently better affinities to ERα than methoxyl

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substituted lead compound BL-18d (IC50=6.64 µM). Overall, the more H donor groups (hydroxyl) were
incorporated to coumarin skeleton the better binding affinities were observed in the binding assay (17d
vs. BL-18d). Furthermore, replacement of methoxy with F at p-position of 3-phenyl substituent
resulted in slightly increased binding affinity (30d vs. BL-18d), which was consistent with our idea of
title compound design. The F containing compounds 30a-d all held the inhibition rate of more than 90%
which were very close to that of Tamoxifen. The highest affinity was observed with compound 17d,
exhibiting binding affinity value of 3.82 µM, which is almost twice higher than BL-18d (IC50=6.64
µM).
As a global observation, most of N-linked compounds presented significant anti-proliferative
activity on these two cancer cell lines compared to positive control. Moreover, all the piperidine
substituted compounds 17d, 23d, 29d and 30d which showed the best binding affinity in ERα assay
within their respective group also demonstrated the best anti-proliferative activity against MCF-7 cells
(IC50 = 18.74, 11.32, 12.58 and 7.06 µM, respectively), which indicated an ERα pathway on the
anti-proliferative activity (Table 1). A close observation of the data showed that fluorine containing
prototypes (30a-d) were more active than their hydroxyl counterpart compounds 17a-d. Contrary to the
trend observed in ERα binding assay, it was interesting to find that an increase in the number of
hydroxy groups decreased their anti-proliferative activities, which might be due to their lower cellular
uptake. Of all the active compounds, 30d showed maximum inhibition of cell growth against MCF-7
cells with an IC50 of 7.06 µM, about 2 fold more potent than both Tamoxifen (14.35 µM) and
Vandetanib (16.52 µM), whereas, compound 29d showed maximum inhibitory effect against Ishikawa
cells with an IC50 of 8.71 µM.
Table 1
SAR of C-4 substituted N-aryl analogs 17a-d, 23a-d, 29a-d and 30a-d.
ERα binding affinitya
Cytotoxicity (IC50, µM)b
Compound
R1
R2
OH
OH
OH
OH
F
R
Inh% at 10 µM
IC50 (µM)
MCF-7
Ishikawa
OH
OH
OH
OH
H
68.46 ± 5.17
86.26 ± 6.07
83.13 ± 4.06
91.61 ± 7.48
34.21 ± 2.09
31.76 ± 2.27
36.92 ± 2.27
8.21
28.35 ± 4.35
30.21 ± 4.24
17a
17b
17c
17d
23a
23b
23c
5.97
6.43
3.82
NDc
ND
27.52 ± 5.17
20.65 ± 3.12
18.74 ± 2.84
32.46 ± 6.43
26.38 ± 3.27
26.11 ± 4.63
29.07 ± 5.94
26.69 ± 4.36
24.59 ± 5.27
28.49 ± 4.48
24.65 ± 2.94
25.73 ± 3.67
H
F
H
F
ND

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H
F
F
F
F
F
F
F
F
F
76.48 ± 5.15
34.97 ± 3.83
37.34 ± 6.77
43.29 ± 4.58
79.52 ± 5.15
90.38 ± 4.97
90.88 ± 7.39
93.67 ± 4.82
94.94 ± 8.75
7.79
ND
11.32 ± 2.25
31.57 ± 4.74
26.31 ± 2.66
21.53 ± 3.15
12.58 ± 1.89
19.29 ± 3.32
17.46 ± 1.29
13.94 ± 2.08
7.06 ± 1.41
10.26 ± 1.34
13.54 ± 1.49
17.89 ± 2.05
12.48 ± 2.12
8.71 ± 1.53
24.84 ± 3.17
17.18 ± 2.78
21.47 ± 3.09
10.44 ± 2.62
23d
29a
29b
29c
29d
30a
30b
30c
30d
OMe
OMe
OMe
OMe
OH
ND
ND
7.96
6.62
4.37
4.53
3.86
OH
OH
OH
-
-
-
86.47 ± 5.49
94.79 ± 9.87
6.64
2.67
-
11.39 ± 3.63
14.35 ± 3.47
16.52 ± 2.12
14.38 ± 2.82
20.65 ± 4.52
BL-18d
Tamoxifen
Vandetanib
-
-
-
-
-
-
-
-
aPercent inhibition of each compound was calculated from the polarization values, shown as mean ± SD of three
experiments. The IC50 values are calculated when the Inh% at 10 µM is more than 50%.
bThe concentration of compound that inhibit 50% of the cell growth after 48 h of drug exposure measured by MTT
assay. Each value was shown as mean ± SD of three experiments.
cND stands for not determined (IC50 >10 µM for ERα binding affinity).
On the other hand, overall, the series of O-linked analogues showed a similar trend in both ERα
binding affinity assay and cell proliferation assay (Table 2): i) more donor group (hydroxyl) led to
higher affinities while their dihydroxy analogs 41a-d displayed poor cytotoxicity against these two
cancer cell lines; ii) among these four basic side substituents, piperidyl (39d, 40d, 41d and 42d) was
the most optimized group which made great contribution to their ERα binding affinity and
anti-proliferative activity. Expectedly, it seemed that introducing of bioisosteric O atom at the C-4
position of coumarin scaffold is beneficial to improve the inhibitory potency, especially in ERα binding
affinity assay (30a-d vs. 42a-d). Interestingly, the dimethoxyl compounds 40a-d which were less
potent in ERα binding assay manifested slightly greater potency than the corresponding dihydroxyl
compounds 41a-d against both MCF-7 and Ishikawa cells which also confirmed that the obtained
antiproliferative activity might not be only through ERα. What’s more, among these O-linked
analogues, 40d was found to exhibit the strongest inhibitory effect against MCF-7 cells (IC50 = 9.32 ±
1.29 µM) and Ishikawa cells (IC50 = 8.41 ± 1.12 µM). Finally, among all the compounds of two series,
compound 42d (R1=OH, R2=F) were found to be the most potent ERα ligand embodying the highest
affinity with IC50 value of 2.19 µM.
Table 2
SAR of C-4 substituted O-aryl analogs 39a-d, 40a-d, 41a-d and 42a-d.

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ERα binding affinitya
Cytotoxicity (IC50, µM)b
MCF-7 Ishikawa
Compound
R1
R2
R
Inh% at 10 µM
IC50(µM)
OMe OMe
OMe OMe
OMe OMe
OMe OMe
34.37 ± 5.64
NDc
17.84 ± 2.23 11.44 ± 2.55
15.35 ± 3.69 9.32 ± 1.14
14.38 ± 2.43 8.53 ± 1.08
39a
39b
39c
39d
40a
40b
40c
40d
41a
41b
41c
41d
42a
42b
42c
42d
38.93 ± 6.89
34.28 ± 4.58
62.48 ± 4.42
72.86 ± 8.52
46.83 ± 6.89
38.32 ± 8.42
85.79 ± 10.23
68.76 ± 3.57
46.36 ± 4.84
86.52 ± 7.13
92.46 ± 8.12
84.32 ± 7.47
93.67 ± 9.38
91.25 ± 8.53
95.74 ± 8.45
ND
ND
9.42
8.96
ND
9.62 ± 1.59
7.47 ± 1.57
OMe
OMe
OMe
OMe
OH
F
15.26 ± 2.38 17.34 ± 2.25
16.64 ± 2.35 15.72 ± 1,63
14.56 ± 3.64 15.14± 1.76
F
F
ND
F
6.87
8.43
ND
9.32 ± 1.29
41.38 ± 6.32 36.97 ± 6.79
>50 >50
8.41 ± 1.12
OH
OH
OH
OH
F
OH
OH
6.84
2.62
6.43
3.55
4.27
2.19
23.88 ± 3.64 24.53 ± 4.06
19.68 ± 2.55 28.37 ± 3.94
16.61 ± 2.94 18.7 ± 2.37
21.33 ± 3.62 17.4 ± 1.96
16.78 ± 1.59 22.61 ± 4.18
OH
OH
OH
F
OH
F
OH
F
9.54 ± 2.65
11.12 ± 2.34
86.47 ± 5.49
94.79 ± 9.87
-
6.64
2.67
-
11.39 ± 3.63 14.38 ± 2.82
14.35 ± 3.47 20.65 ± 4.52
BL-18d
Tamoxifen
Vandetanib
-
-
-
-
-
-
-
-
-
16.52 ± 2.12
-
aPercent inhibition of each compound was calculated from the polarization values, shown as mean ± SD of three
experiments. The IC50 values are calculated when the Inh% at 10 µM is >50%.
bThe concentration of compound that inhibit 50% of the cell growth after 48 h of drug exposure measured by MTT
assay. Each value was shown as mean ± SD of three experiments.
cND stands for not determined (IC50 >10 µM for ERα binding affinity).
Based on the above results, we found that all the piperidine substituted compounds 17d, 23d, 29d,
30d, 39d, 40d, 41d and 42d which exhibited the best binding affinity in ERα assay within their
respective group also demonstrated potent anti-breast cancer activity against MCF-7, as well as good
anti-endometrial hyperplasia activity against Ishikawa cells with IC50 value less than 25 µM. Results
presented in Figure 4A indicate that all the tested compounds inhibited cell proliferation in a
dose-dependent manner. From Figure 4B it is evident that most of the piperidine containing compounds
displayed comparable to higher potency than BL-18d and tamoxifen against MCF-7 and Ishikawa

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cells.
Figure 4. In vitro anti-proliferative properties of selected compounds against MCF-7 and Ishikawa cell lines. (A)
Growth inhibition assay performed on MCF-7 and Ishikawa cell cells using MTT after 48 h incubation with a
range of drug concentrations. Points: % of cell proliferation as compared to untreated control cells, means of at
least three individual experiments. (B) Histogram representation of the micromolar concentration of the
compounds required to inhibit 50% of MCF-7 and Ishikawa cell proliferation after 48 h drug incubation (IC50).
Columns: means of at least three individual experiments; bars: SD.
3.2.2. Antiangiogenic activity evaluation.
On the basis of preliminary ERα binding affinity and cancer cell proliferation activity, several
potent compounds were screened in vitro for their antiproliferative potency against VEGFR-2
overexpressed human umbilical vein endothelial cells (HUVEC) and VEGFR-2 inhibitory activity with
Vandetanib as positive control. As shown in Table 4, most of the tested compounds manifested
considerable inhibitory activity on VEGFR-2 compared with Vandetanib. Three compounds 17d, 30d
and 42d showed 82.91-88.42% inhibition against VEGFR-2 in contrast to Vandetanib which showed
inhibition percentage of 95.73%. Results of HUVEC cell proliferation assay indicated that five of
selected compounds (29d, 30d, 39d, 40d and 42d) displayed potent antiproliferative activities with IC50
values ranging from 7.64 to 15.73 µM. Their potency were comparable with the reference drug
Vandetanib but better than BL-18d (IC50=16.19 µM). Particularly, 39d showed the highest
anti-proliferative activity with IC50 values of 7.64 µM. It is worthy to note that the dihydroxyl

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containing compounds 17d and 41d only showed fair cell-growth inhibitory effect against HUVEC,
which was in accordance with their low potency against MCF-7 and Ishikawa cells.
Table 3
Antiangiogenic activity of representative compounds 17d, 23d, 29d, 30d, 39d, 40d, 41d and 42d.
VEGFR-2
compound
HUVEC (IC50, µM)b
Inhibitiona (%)
95.73 ± 2.15
59.34 ± 4.64
83.25 ± 8.83
41.84 ± 3.26
66.01 ± 6.71
88.42 ± 8.12
67.63 ± 2.05
55.13 ± 3.68
66.08 ± 6.32
82.91 ± 6.43
6.76 ± 0.68
16.19 ± 2.07
30.79 ± 4.52
17.15 ± 1.63
9.05 ± 1.38
9.92 ± .084
7.64 ± 0.97
13.57 ± 1.01
23.94 ± 3.62
15.73 ± 1.42
Vandetanib
BL-18d
17d
23d
29d
30d
39d
40d
41d
42d
aPercentage inhibition of each compound on VEGFR-2 at 40 µM was measured using homogeneous time resolved
fluorescence (HTRF) assay, shown as mean ± SD of three experiments.
bThe concentration of compound that inhibit 50% of the cell growth after 48 h of drug exposure measured by MTT
assay. Each value was shown as mean ± SD of three experiments.
In summary, we found that compound 42d exhibited the strongest ERα binding affinity (IC50=
2.19 µM) and excellent cytotoxicity for MCF-7 and Ishikawa cells (IC50 = 9.54 ± 2.65 and 11.12 ± 2.34
µM, respectively), also was a promising anti-angiogenesis agent (82.91% of VEGFR-2 inhibition;
IC50= 15.73 µM against HUVEC cells). Thus, 42d was selected for our further investigation of its
multifunctional effects.
3.2.3. Effect of 42d on migration of MCF-7 cells
Metastasis is the major cause of death of the advanced breast cancer patients. Although many
breast cancers respond well to chemotherapy initially, they become aggressive, recur and metastasize
once they develop resistance [61]. Cell migration is usually associated with a metastatic phenotype;
herein we adopted a wound-healing migration assay to determine the ability of 42d to inhibit the
migration of MCF-7 cells. The results showed that in the absence of 42d, the cells migrated within 48 h
to fill most of scratched area, but the noncytotoxic treatment of 42d significantly prevented this
migration of MCF-7cells in a dose- and time-dependent manner (Fig. 5).

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Figure 5. Effects on the migration of MCF-7. Representative images of MCF-7 cells treated with serum-free 1640
medium containing 42d (2 and 4 µM) for 24 and 48 h were photographed under phase contrast microscopy
(magnification, 100×). Control was treated with serum-free 1640 medium.
3.2.4. Effect of 42d on Cell Cycle distribution in human breast cancer MCF-7 cells
In MCF-7 human breast cancer cells, tamoxifen exerts anti-estrogenic effects, marked by slowing
of cellular proliferation, with the cells arrested in the G1 phase of the cell cycle [62]. Thus, we further
evaluated the effect of 42d on the distribution of the cells in cell cycle with Tamoxifen as positive
control. Representative cell-cycle changes were detected in MCF-7 cells after exposure to DMSO
(control), Tamoxifen (2 µM) and 42d (2 µM, 4 µM and 6 µM) for 24 h (Fig. 6). As is seen from the
results, compound 42d concentration-dependently arrested cell cycle at G0/G1 phase, accompanied
with decrease of cells at G2/M and S phase for MCF-7 cells. This indicates that the G0/G1 arrest in
MCF-7 cells by compounds 42d also made contribution to its potent anti-proliferative activity.
Figure 6. (A) Cell cycle analysis of MCF-7 cells treatment of compound 42d (2, 4 and 6 µM) and Tamoxifen (2
µM) as positive control for 24 h.
3.2.5. The inhibition activity of compound 42d against the expression of progestrone receptor
microRNA in MCF-7 cells.
To evaluate the transcriptional level of ERα-regulated target genes, we further adapted quantitative
real-time polymerase chain reaction (RT-PCR) in the ER positive MCF-7 cells to investigate the
expression of progesterone receptor (PgR). The progesterone receptor expression is commonly
associated with estrogenic or antiestrogenic activity. As shown in Fig. 7, presence of 10 nM estradiol
(E2) was able to remarkably elevate the mRNA expression of PgR gene compared to the vehicle
control. Observably, compound 42d at the concentration of 1µM in combination with 10 nM E2
dramatically reduced the expression of PgR mRNA induced by E2 (*** P < 0.001 vs. E2 group).

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Furthermore, 42d exhibited stronger antagonism against the expression of PgR mRNA than positive
control Tamoxifen at the concentration of 1 µM in the presence of 10 nM E2, indicating that 42d
presented significantly antiestrogenic property.
Figure 7. The increased mRNA expression of PR induced by E2 was reversed by 42d in MCF-7 cells. The mRNA
expression of PR was examined by Real-time PCR. Values are mean ±SD (n=3). * P < 0.05, ** P < 0.01, *** P <
0.001 vs. E2 group. # P< 0.05, ## P< 0.01, ### P< 0.001.
3.2.6. Effect of 42d on VEGFR-2 phosphorylation
VEGF signalling pathway through the VEGFR-2 tyrosine kinase phosphorylation displays a
crucial role in angiogenesis. What’s more, VEGFR-2 is expressed at high level in many kinds of
cancers [63]. Disruption of VEGF signaling pathway by either specific binding of circulating VEGF or
inhibiting receptor tyrosine kinases with small molecules has been found to inhibit angiogenesis, tumor
progression and dissemination. To confirm our hypothesis, a Western blotting assay for total and
phosphorylated (active) VEGFR-2 was performed. As shown in Fig. 8, 42d was able to block the
phosphorylation of VEGFR-2 in a dose dependent manner. Sunitinib was used as the positive control.
At the concentration of 4 µM, 42d significantly decreased the active form of VEGFR-2 (Fig. 8B) (***
p < 0.001 vs. control). Moreover, at concentrations of 2 µM, compounds 42d presented a slightly better
effect than Sunitinib.

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Figure 8. 42d inhibits the phosphorylation of VEGFR-2 in MCF-7 cells. (A) Expression of p-VEGFR-2 and
VEGFR-2 in MCF-7 cells were examined by western blots. (B) Densitometric analysis was performed to
determine the phosphorylation rate of VEGFR-2. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P <
0.001 vs. Control group.
3.2.7. 42d inhibits the MAPK signaling cascade as the result of VEGFR-2 phosphorylation
blockade in MCF-7 cells.
Multifactorial pathways have been shown to develop tamoxifen resistance in both preclinical and
clinical studies, including the crosstalk between ER and growth factor pathways [64, 65]. The
Raf-1/MAPK/ERK pathway is an important cell signaling pathways and controls multiple cellular
processes, such as cell proliferation and differentiation [66]. Currently, studies have revealed that
over-expression and aberrant activation of Raf-1/MAPK/ERK pathway is associated with the resistance
of endocrine therapy [67]. Crosstalk of estrogen receptor pathway with Raf-1/MAPK/ERK pathway is
also relative to breast cancer cell migration, invasion, and angiogenesis [68]. Therefore, the blockage of
Raf-1/MAPK/ERK pathway is supposed to enhance the effect of anti-hormonal therapy and retard
endocrine resistance. Compound 42d has shown its anti-angiogenesis effect through inhibition on
VEGFR-2 activation; we made further investigation of 42d on inhibiting the downstream
Raf-1/MAPK/ERK pathway transduction in MCF-7 cells to explore its underlying mechanism of action.
Two effectors, Raf-1 and ERK were examined to study how 42d affected the Raf-1/MAPK/ERK
pathway. As expected, the results indicated that 42d significantly inhibited p-Raf-1 and p-ERK1/2
activation in a dose-dependent manner, while the total protein level of Raf-1 and ERK1/2 were not
altered (Fig. 9).

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Figure 9. 42d inhibits the activity of the Raf-1/ERK pathway in MCF-7 cells. (A) Expression of p-Raf-1, Raf-1,
p-ERK1/2, and ERK1/2 were examined by western blots in MCF-7 cells. (B) Densitometric analysis was
performed to determine the phosphorylation rate of Raf-1 and ERK1/2. Values are mean ± SD (n = 3). *P < 0.05,
**P < 0.01, ***P < 0.001 vs. Control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Control group.
3.2.8. Potential mechanisms of designed compound 42d on ERα- and VEGFR-2-meidated
signaling pathways
Estrogen receptor-α (ERα), a member of the nuclear receptor family, is activated by the
endogenous estrogen 17β-estradiol (E2) to regulate proliferation-dependent gene expression. Our
compound 42d was able to compete with circulating estrogen for binding (ERα), consequently leading
to proliferation inhibition and PgR gene down-regulation (Fig. 10). One the other hand, cancer cell
proliferation and migration as well as angiogenesis promoted by the crosstalk of estrogen receptor
pathway with Raf-1/MAPK/ERK pathway were also restrained by 42d via the phosphorylation
blockade of VEGFR-2, Raf-1 and ERK1/2. This synergetic inhibitory effect of 42d on ERα and
VEGFR-2/Raf-1/MAPK/ERK pathway suggested it a promising multifunctional agent against breast
cancer. And this work opens a novel way to the design of multi-targeted agents targeting ERα and
VEGFR-2 in the therapy of some breast cancers.

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Figure 10. Schematic representation elucidating the role of 42d in interfering with signaling pathways initiated by
ERα and VEGFR-2
3.3. Molecular modeling studies.
The designed inhibitors were subsequently subjected to docking analysis in ERα (PDB ID: 3ERT)
and VEGFR-2 (PDB ID: 1YWN) proteins. The docking interaction energies obtained for the designed
candidates docked within the active site of both the proteins showed favorable binding modes for all
docked compounds, where all ligands had good scores between -49.59 and -69.26 kcal/mol (Table 4).
Compound 41d endowed with two H-bond donor groups (hydroxyl) achieved the lowest docking score
amongst all synthesized compounds within both proteins, which was in line with the our idea of drug
design. Furthermore, the O linked analogues 40d, 41d and 42d exhibited better scores within ERα
protein than their N linked counterparts 29d, 17d and 30d, which was in line with the experimental
ERα binding affinities indicating a good correlation between predicted binding energies and the
corresponding experimental affinities scoring functions. The docking energy scores observed within
both ERα and VEGFR-2 proteins are mentioned in the supplementary data.
To further support our structural modifications, a deeper binding mode analysis was performed on
the promising compounds 42d. As depicted in Fig. 11A, the docked pose of 42d in ERα active site
revealed that the core skeleton of 3-aryl-4-aryloxy-2H-chromen-2-one is favorably positioned into the
binding site. Furthermore, an excellent superimposition of 42d over the structure of
4-hydroxytamoxifen (OHT, co-crystallized ligand) was observed. And its basic side chain pointed
toward Asp351 to generate an antagonistic conformation as that of 4-OHT. Detailed docking results
from Fig. 11B showed that hydroxy group of the coumarin moiety formed hydrogen bonding
interaction with Glu353 (2.86 Å) and Arg394 (2.80 Å), while the p-methoxy of C-3 substituted benzyl
ring interacted with His524 amino acid residue (3.09 Å) and formed an essential hydrogen bond. This
indispensable ERα binding mode of 42d which is the typical character of selective estrogen receptor

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modulator contributed greatly to its antiestrogenic property.
Figure 11. (A) Superimposed poses of OHT (blue) with representative derivatives 42d (yellow), binding to ERα,
the protein was shown in surface. (B) Docking interactions of 42d within ERα active site (surrounding residues
shown as green sticks). Dotted lines (red) represent the hydrogen bonding interaction.
The binding orientation of 42d within the ligand binding domain of VEGFR-2 showed a binding
mode which was different with Sunitinib but similar to that of diaryl urea VEGFR-2 inhibitors [69].
The docking analysis of compound 42d within VEGFR-2 protein showed hydrogen bonding interaction
of hydroxy group of coumarin with an essential amino acid Cys917 (2.68 and 2.92 Å) in the ATP hinge
region (Fig. 12). Moreover, the coumarin ring was docked into a hydrophobic pocket, creating a π-π
stacking interaction with the aromatic ring of Phe916 (6.12 Å), providing further stabilization for the
ligand-receptor complex. The side chain of 42d pointed toward a hydrophobic region which was
commonly occupied by the diaryl urea fragment. These docking results may provide additional insights
into the protein-ligand interactions and potential structural modifications for further activity
improvement.
Figure 12. The binding mode of compound 42d (ball and stick in yellow) docked into the VEGFR-2 binding site
(surrounding residues shown as gray sticks). Hydrogen bonding and hydropholic interactions are shown as red

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dotted lines with distances in angstrom.
4. Conclusion
In summary, what described here is the rationally guided optimization of a series of
3-aryl-4-aniline/aryloxy-2H-chromen-2-one derivatives through structure-based modification of lead
compound LE-18d in order to obtain multifunctional effect towards ERα and VEGFR-2. By means of
bioisosteric replacement, O atom was attached to the 4-position of coumarin moiety, while F atom was
incorporated to C-4' position of 3-phenyl substituent with the hope of retaining necessary interactions
with essential amino acid residues of targeted ERα receptor and VEGFR-2 enzyme. Among them, O
linked compounds not only exhibited potent ERα binding affinities, but also showed promising
anti-proliferative activities against both cancer cells and angiogenesis-related cells in vitro. Further
mechanism investigation revealed that 42d was able to inhibit MCF-7 cell migration and arrest cell
cycle at G0/G1 phase in MCF-7 cells in a concentration-dependent manner. In addition to its significant
antiestrogenic property observed in RT-PCR, 42d was also able to inhibit the activation of VEGFR-2
and the signaling transduction of Raf-1/MAPK/ERK pathway in MCF-7 cells. Molecular docking
analysis of 42d indicated this molecule assumed a well-known ERα binding mode embodying three
essential hydrogen bonding interactions, which is the typical character of selective estrogen receptor
modulator. Additionally, docking interaction within VEGFR-2 revealed that 42d has the ability to
occupy the ATP region of VEGFR-2 enzyme via forming a stronger π-π stacking interaction and two
hydrogen bonding interactions with key amino acid residues.
Finally, all these results together open a new avenue for the development of multifunctional agents
targeting ERα and VEGFR-2 in the therapy of some breast cancers. Further in vivo anticancer effects of
promising compound 42d on 4T1 induced BALB/c mice breast cancer models are in progress and will
be reported in due course.
5. Experimental section
5.1 Chemistry
5.1.1. General.
Most chemicals and solvents were of analytical grade and, when necessary, were purified and
dried by standard methods. Reactions were monitored by thin-layer chromatography (TLC) using
precoated silica gel plates (silica gel GF/UV 254), and spots were visualized under UV light (254 nm).
Melting points (uncorrected) were determined on a Mel-TEMP II melting point apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were recorded with a Bruker Avance 300 MHz
spectrometer at 300 K, using TMS as an internal standard. MS spectra were recorded on a Shimadzu

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GC-MS 2050 (ESI) or an Agilent 1946A-MSD (ESI) Mass Spectrum. Column chromatography was
performed with silica gel (200-300 mesh). Chemical shifts (d) are expressed in parts per million
relative to tetramethylsilane, which was used as an internal standard, coupling constants (J) are in hertz
(Hz), and the signals are designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet.
Analytical HPLC for assessing purity was performed on Agilent 1260 Infinity System equipped
with a UV detector at 254 nm. The column employed is an Agilent Zorbax AB-C18 chromatography
column (5 µm, 4.6 × 250 mm). Unless otherwise indicated, the purity of the final products was >95%
(Supplementary data).
5.1.2. General method I: sulfonic esterification
Compounds 21 or 27 (2 mmol) was dissolved in 10 mL of dry dichloromethane and to it was
added dry TEA (0.56 g, 5.60 mmol) and stirred at room temperature until the reaction mixture became
homogeneous. The total reaction mixture was then put on an ice bath, 4-toluenesulfonylchloride (0.42 g,
2.2 mmol) was added dropwise stirred for 10 more minutes; the ice bath was removed and the reaction
mixture continued to be stirred at room temperature for another 30 min. The solvent was evaporated
and the solid purified by column chromatograph to give a white solid 22 or 28.
5.1.3. General method II: SN2 nucleophilic substitution.
A mixture of compound 14 or 22 or 28 (0.47 mmol), four kind of anilines (1.0 equiv.), and K2CO3
(2.0 equiv.) in ethanol (6.0 mL) was stirred at 75 °C for 2-3 h. After completion of the reaction as
indicated by TLC, the mixture was cooled to room temperature and then extracted, evaporated and
purified directly by flash column chromatography on silica gel to afford the corresponding pure
products 16a-d, 23a-d and 29a-d.
5.1.4. General method III: demethylation.
A solution of 16a-d or 29a-d or 39a-d or 40a-d (0.4 mmol) in dry CH2Cl2 (10 mL) was cooled to
o
-30 C by cryotrap. A solution of BBr3 (2.4 mmol) in dry CH2Cl2 (2.4 mL) was added dropwise under
nitrogen. The mixture was gently warmed to room temperature and stirred for 12 h. The reaction
mixture was quenched with H2O (50 mL), then saturated sodium bicarbonate solution was adjusted to
pH=8. The precipitate was collected by filtration, and further purified by silica gel column
chromatography to afford the demethylation product 17a-d, 30a-d, 41a-d and 42a-d.
5.1.5. Synthesis of intermediates 16a-d
Intermediates 16a-d were prepared by our group previously and associated NMR and HR-MS
characterization were reported in previous published paper [21].
5.1.6. General synthesis of final compounds 17a-d
Final compounds 17a-d were prepared according to General method III: demethylation, yield
44.5-61.6%.

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5.1.7.
4-((4-(2-(dimethylamino)ethoxy)phenyl)amino)-7-hydroxy-3-(4-hydroxyphenyl)-2H-ch
romen-2-one (17a)
1
Light yellow solid, yield 44.5%, mp 156-158oC; H NMR (300 MHz, DMSO) δ (ppm): 9.33 (s,
1H), 8.12 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.74 – 6.63 (m, 4H), 6.63 (d, J =
8.8 Hz, 2H), 6.52 (d, J = 8.4 Hz, 2H), 3.92 (t, J = 5.9 Hz, 2H), 3.68 (s, 3H), 2.59 (t, J = 5.5 Hz, 2H),
2.23 (s, 6H). 13C NMR (75 MHz, DMSO) δ: 162.3, 161.1, 156.4, 154.5, 154.5, 148.2, 134.8, 132.2,
126.4, 125.0, 123.5, 114.8, 114.7, 112.5, 108.9, 103.6, 102.4, 66.4, 57.9, 55.4, 45.8. MS (ESI) m/z:
455.1 ([M+Na] +). HRMS (ESI) for C25H24N2O5+H calcd 432.1720, found 432.1742.
5.1.8.
4-((4-(2-(diethylamino)ethoxy)phenyl)amino)-7-hydroxy-3-(4-hydroxyphenyl)-2H-chr
omen-2-one (17b)
1
Light yellow solid, yield 48.9%, mp 168-171oC; H NMR (300 MHz, DMSO) δ (ppm): 9.32 (s,
1H), 8.11 (s, 1H), 7.69 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.78 – 6.67 (m, 4H), 6.62 (d, J =
8.9 Hz, 2H), 6.53 (d, J = 8.5 Hz, 2H), 3.94 (t, J = 5.9 Hz, 2H), 2.82 (s, 2H), 2.64 (d, J = 6.7 Hz, 4H),
1.01 (t, J = 7.1 Hz, 6H). 13C NMR (75 MHz, DMSO) δ: 162.4, 160.8, 156.3, 154.5, 154.5, 148.2, 134.9,
132.2, 126.3, 125.0, 123.5, 114.8, 114.7, 112.5, 109.1, 103.7, 102.5, 66.4, 51.5, 47.5, 11.8. MS (ESI)
m/z: 483.1 ([M+Na] +). HRMS (ESI) for C27H28N2O5+H calcd 461.2071, found 461.2071.
5.1.9.
7-hydroxy-3-(4-hydroxyphenyl)-4-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)-2H-chro
men-2-one (17c)
1
Light yellow solid, yield 53.2%, mp 134-138 oC; H (300 MHz, DMSO) δ (ppm): 9.34 (s, 1H),
8.20 (s, 1H), 7.72 (d, J = 9.3 Hz, 1H), 6.93 (d, J = 8.5 Hz, 2H), 6.65 (t, J = 6.3 Hz, 4H), 6.65 (d, J = 8.5
Hz, 2H), 6.56 (d, J = 8.9 Hz, 2H), 3.92 (t, J = 5.6 Hz, 2H), 2.75 (t, J = 5.7 Hz, 2H), 2.53 (d, J = 1.8 Hz,
4H), 1.67 (s, 4H). 13C NMR (75 MHz, DMSO) δ: 162.2, 160.7, 157.8, 154.5, 154.3, 147.6, 134.0, 131.5,
126.7, 125.6, 123.6, 113.9, 112.7, 112.0, 108.5, 102.1, 101.7, 66.6, 54.0, 53.8, 23.0. MS (ESI) m/z:
481.1 ([M+Na] +). HRMS (ESI) for C27H26N2O5+H calcd 459.1854, found 4593.1862.
5.1.10.
7-hydroxy-3-(4-hydroxyphenyl)-4-((4-(2-(piperidin-1-yl)ethoxy)phenyl)amino)-2H-chr
omen-2-one (17d)
1
Light yellow solid, yield 61.6%, mp 241-243oC; H NMR (300 MHz, DMSO) δ (ppm): 9.47 (s,
1H), 8.04 (s, 1H), 7.66 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 8.5 Hz, 2H), 6.76 – 6.64 (m, 4H), 6.60 (d, J =
8.9 Hz, 2H), 6.52 (d, J = 8.5 Hz, 2H), 3.92 (t, J = 6.0 Hz, 2H), 2.58 (t, J = 5.9 Hz, 2H), 2.47 – 2.35 (m,
4H), 1.57 – 1.45 (m, 4H), 1.37 (d, J = 4.9 Hz, 2H). 13C NMR (75 MHz, DMSO) δ: 162.4, 160.8, 156.3,
154.5, 154.5, 148.2, 134.93, 132.2, 126.3, 125.0, 123.5, 114.8, 114.7, 112.5, 109.1, 103.6, 102.5, 66.4,
+
51.5, 47.5, 11.8. MS (ESI) m/z: 495.1 ([M+Na] ). HRMS (ESI) for C28H28N2O5+H calcd 473.2071,
found 473.2075.
5.1.11. 4-flourinephenylacetic acid, 2-(methoxycarbonyl)phenyl ester (20)

ACCEPTED MANUSCRIPT
To a mixture of methyl salicylate (1.52 g, 10 mmol) and 4-flourinephenylacetic acid (11 mmol) in
10 mL of pyridine at 0-10 °C was added dropwise POCl3 (1.1 mL, 12 mmol) within 0.5 h under stirring.
After the above mixture was stirred for 2 h at 0-10 °C, the concentrated HCl was added to adjust pH
5-6 at the same temperature. The mixture was filtered and washed with water to obtain the crude
product with a yield >80%, mp 73-77oC. MS (ESI) m/z: 289.1 ([M+H] +).
5.1.12. 3-(4-flourinephenyl)-4-hydroxy-2H-chromen-2-one (21)
The 4-flourinephenylacetic ester of methyl salicylate (5 mmol) was mixed with anhydrous
pyridine (10 mL), followed by the addition of KOH powder (0.7 g, 12.5 mmol) at room temperature.
After the mixture was stirred for 4 h in a nitrogen atmosphere, 1N HCl was added to adjust pH 3-4. The
mixture was filtered and washed with water to obtain the crude product with a yield >45%, mp
234-237oC. MS (ESI) m/z: 255.1 ([M-H] -).
5.1.13. 2-Oxo-3-(4-methoxyphenyl)-2H-chromen-4-yl-4-flourinebenzenesulfonate (22)
Intermediate 22 was prepared according to General method I: sulfonic esterification as a white
solid, yield 77.8%, mp 176-179oC. 1H (300 MHz, CDCl3) δ (ppm): 8.23 (d, J = 15.5 Hz, 1H), 7.68 (m,
1H), 7.41 (m, 2H), 7.42 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.2 Hz, 2H), 6.68 (d,
J = 8.2 Hz, 2H), 2.41(s, 3H). MS (ESI) m/z: 433.1 ([M+Na] +).
5.1.14. General synthesis of final compounds 23a-d
Final compounds 23a-d were prepared according to General method II: SN2 nucleophilic
substitution, yield 49.6-64.3%.
5.1.15. 4-((4-(2-(dimethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-2H-chromen-2-one
(23a)
1
Yellow solid, yield 64.3%, mp 155-156oC; H NMR (300 MHz, DMSO) δ (ppm): 8.60 (s, 1H),
8.06 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.31 (dd, J = 13.3, 8.0 Hz, 2H), 7.01 (dd, J = 8.5, 5.7
Hz, 2H), 6.77 (t, J = 8.9 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 8.8 Hz, 2H), 3.84 (t, J = 5.7 Hz,
2H), 2.52 – 2.46 (m, 2H), 2.13 (s, 6H). 13C NMR (75 MHz, DMSO) δ: 162.8, 162.11, 159.5, 155.0,
152.7, 148.0, 133.6, 133.4, 133.3, 132.2, 131.1, 124.8, 124.4, 124.0, 117.2, 114.6, 114.4, 114.1, 102.7,
+
66.5, 57.9, 45.9. MS (ESI) m/z: 419.1 ([M+H] ). HRMS (ESI) for C25H23FN2O3+H calcd 419.1765,
found 419.1766.
5.1.16. 4-((4-(2-(diethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-2H-chromen-2-one
(23b)
1
Yellow solid, yield 62.4%, mp 138-141oC; H NMR (300 MHz, DMSO) δ (ppm): 8.58 (s, 1H),
8.04 (d, J = 7.9 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.36 – 7.20 (m, 2H), 7.00 (dd, J = 8.5, 5.8 Hz, 2H),
6.75 (t, J = 8.9 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 6.46 (d, J = 8.8 Hz, 2H), 3.80 (t, J = 6.0 Hz, 2H), 2.63
(t, J = 6.0 Hz, 2H), 2.46 – 2.40 (m, 4H), 0.89 (t, J = 7.1 Hz, 7H). 13C NMR (75 MHz, DMSO) δ: 162.8,

ACCEPTED MANUSCRIPT
162.1, 159.5, 155.1, 152.7, 148.0, 133.6, 133.4, 133.3, 132.1, 131.2, 124.8, 124.4, 123.9, 117.14, 114.6,
+
114.4, 114.1, 102.7, 66.9, 51.7, 47.5, 12.2. MS (ESI) m/z: 469.1 ([M+H] ). HRMS (ESI) for
C27H27FN2O3+H calcd 447.2078, found 447.2085.
5.1.17. 3-(4-fluorophenyl)-4-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)-2H-chromen-2-one
(23c)
1
Yellow solid, yield 58.7%, mp 155-157oC; H NMR (300 MHz, CDCl3) δ 7.42 – 7.33 (m, 1H),
7.32 – 7.17 (m, 4H), 6.97 (ddd, J = 15.3, 12.8, 5.0 Hz, 3H), 6.80 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.9 Hz,
2H), 3.98 (t, J = 5.9 Hz, 2H), 2.81 (t, J = 5.9 Hz, 2H), 2.54 (s, 4H), 1.72 (d, J = 3.4 Hz, 4H). 13 C NMR
(75 MHz, DMSO) δ 162.3, 161.6, 159.0, 154.6, 152.2, 147.5, 133.0, 132.9, 132.8, 131.7, 130.6, 124.3,
123.9, 123.5, 116.7, 114.1, 113.9, 113.6, 102.2, 66.9, 54.1, 53.9, 23.1. MS (ESI) m/z: 467.1 ([M+Na] +).
HRMS (ESI) for C27H25FN2O3+H calcd 445.1922, found 445.1921.
5.1.18. 3-(4-fluorophenyl)-4-((4-(2-(piperidin-1-yl)ethoxy)phenyl)amino)-2H-chromen-2-one
(23d)
1
Yellow solid, yield 49.6%, mp 157-159oC; H NMR (300 MHz, DMSO) δ (ppm): 8.70 (s, 1H),
8.14 (d, J = 8.0 Hz, 1H), 7.67 – 7.60 (m, 1H), 7.41 (dd, J = 8.3, 1.0 Hz, 1H), 7.38 – 7.32 (m, 1H), 7.12
– 7.06 (m, 2H), 6.87 – 6.80 (m, 2H), 6.73 – 6.68 (m, 2H), 6.59 – 6.52 (m, 2H), 3.92 (t, J = 5.9 Hz, 2H),
2.56 (t, J = 5.9 Hz, 2H), 2.45 – 2.35 (m, 4H), 1.49 (dd, J = 10.9, 5.5 Hz, 4H), 1.41 – 1.35 (m, 2H). 13C
NMR (75 MHz, DMSO) δ: 162.4, 162.1, 159.9, 155.0, 152.7, 148.0, 133.5, 133.4, 133.3, 132.1, 131.1,
124.7, 124.4, 124.0, 117.2, 114.6, 114.4, 114.2, 102.7, 66.4, 57.6, 54.8, 26.1, 24.4. MS (ESI) m/z: 481.1
([M+Na] +). HRMS (ESI) for C28H27FN2O3+H calcd 459.2078, found 459.2086.
5.1.19. 1-(2,4-dihydroxyphenyl)-2-(4-fluorophenyl)ethanone (25)
A mixture of resorcinol (6.67 g, 60.3 mmol) and an appropriate 4-flourinephenylacetic (60.3 mmol)
in BF3-OEt2 (150 mL) was heated at 85°C for 2.5 hours. The reaction mixture was poured into ice-cold
water (300 mL) and extracted with EtOAc twice. The combined organic layer was washed with
saturated sodium bicarbonate solution (NaHCO3) and then brine, dried over magnesium sulfate
(Na2SO4), and concentrated under reduced pressure. The resulting compound was further purified by
recrystallization (EtOH and PE). Yellow crystals; yield: 79.8%, mp 143-146oC; 1H (300 MHz, DMSO)
δ (ppm): 11.64 (s, 1H), 9.26 (s, 1H), 6.18-7.84 (m, 7H), 4.33 (s, 2H). MS (ESI) m/z: 245.1 ([M-H] -).
5.1.20. 2-(4-fluorophenyl)-1-(2-hydroxy-4-methoxyphenyl)ethanone (26)
At 0 °C, to a solution of compound 25 (15.5 mmol) and MeOH (15.5 mmol) in dry THF (60 mL)
were added PPh3 (15.5 mmol) and DIAD (15.5 mmol) and the mixture warmed to room temperature
over 0.5 h. The solution was diluted with EtOAc (150 mL), washed twice with water and brine dried
over Na2SO4 and concentrated under reduced pressure. Finally, the crude product was purified by
column chromatography. White solid; yield: 84.6%, MS (ESI) m/z: 249.1 ([M-H] -).

ACCEPTED MANUSCRIPT
5.1.21. 3-(4-fluorophenyl)-4-hydroxy-7-methoxy-2H-chromen-2-one (27)
Intermediate 26 (10.29 mmol) was dissolved in 40 ml of diethyl carbonate and was added 2.4 g
NaH (102.9 mmol) under an ice bath with vigorous stirring. The temperature was slowly lifted and then
stirred at 120 °C for 2 hours under nitrogen atmosphere. After the completion of the reaction, the
solvent was evaporated at reduced pressure and the remaining solid was poured on crushed ice. The pH
was adjusted to 3 by adding 6 N of hydrochloric acid. The precipitate was collected and purified by
column chromatography. White solid; 69.7%, MS (ESI) m/z: 285.1 ([M-H] -).
5.1.22. 3-(4-fluorophenyl)-7-methoxy-2-oxo-2H-chromen-4-yl 4-methylbenzenesulfonate (28)
Intermediate 28 was prepared according to General method I: sulfonic esterification as a yellow
solid, yield 86.4%, mp 212-215oC. 1H (300 MHz, CDCl3) δ (ppm): 7.86 (d, J = 9.0 Hz, 1H), 7.26-7.18
(m, 2H), 7.08 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 8.2 Hz, 2H), 6.90 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J =
+
2.4 Hz, 1H), 6.57 (d, J = 8.8 Hz, 2H), 3.83 (s, 3H), 2.42 (s, 3H). MS (ESI) m/z: 441.1 ([M+H] ). MS
(ESI) m/z: 463.1 ([M+Na] +).
5.1.23. General synthesis of final compounds 29a-d
Final compounds 29a-d were prepared according to General method II: SN2 nucleophilic
substitution, yield 48.9-63.2%.
5.1.24. 4-((4-(2-(dimethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-7-methoxy-2H-chrom
en-2-one (29a)
Yellow solid, yield 63.2%, mp 146-148 oC; 1H (300 MHz, DMSO) δ (ppm): 8.48 (s, 1H), 7.91 (d,
J = 8.9 Hz, 1H), 7.04 – 6.94 (m, 2H), 6.91 – 6.82 (m, 2H), 6.74 (t, J = 8.8 Hz, 2H), 6.61 (d, J = 8.7 Hz,
2H), 6.47 (d, J = 8.7 Hz, 2H), 3.84 – 3.75 (m, 5H), 2.45 (t, J = 5.7 Hz, 2H), 2.11 (s, 6H). 13C NMR (75
MHz, DMSO) δ: 162.7, 162.5, 162.4, 159.5, 155.0, 154.5, 148.5, 133.7, 133.4, 133.3, 131.3, 125.7,
124.7, 114.6, 114.4, 114.1, 111.8, 110.2, 101.1, 100.7, 66.5, 57.9, 56.3, 45.9. MS (ESI) m/z: 471.1
([M+Na] +). HRMS (ESI) for C26H25FN2O4+H calcd 449.1871, found 449.1880.
5.1.25. 4-((4-(2-(diethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-7-methoxy-2H-chrome
n-2-one (29b)
Yellow solid, yield 58.7%, mp 145-147oC; 1H (300 MHz, DMSO) δ (ppm): 8.47 (s, 1H), 7.91 (d, J
= 8.9 Hz, 1H), 7.03 – 6.95 (m, 2H), 6.85 (dd, J = 12.5, 3.3 Hz, 2H), 6.74 (t, J = 8.7 Hz, 2H), 6.61 (d, J
= 8.5 Hz, 2H), 6.47 (d, J = 8.6 Hz, 2H), 3.80 (d, J = 6.2 Hz, 5H), 2.61 (t, J = 5.9 Hz, 2H), 2.49 – 2.42
(m, 4H), 0.88 (t, J = 7.0 Hz, 6H). 13C NMR (75 MHz, DMSO) δ: 162.7, 162.5, 162.3, 159.5, 155.1,
154.5, 148.5, 133.7, 133.4, 133.3, 131.3, 125.7, 124.7, 114.6, 114.4, 114.1, 111.8, 110.2, 101.1, 100.7,
+
67.1, 56.3, 51.7, 47.5, 12.3. MS (ESI) m/z: 499.1 ([M+Na] ). HRMS (ESI) for C28H29FN2O4+H calcd
477.2193, found 477.2184.
5.1.26. 3-(4-fluorophenyl)-7-methoxy-4-((4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)amino)-2H-chrom

ACCEPTED MANUSCRIPT
en-2-one (29c)
Yellow solid, yield 48.9%, mp 129-131oC; 1H (300 MHz, DMSO) δ (ppm): 8.50 (s, 1H), 7.95 (d, J
= 8.9 Hz, 1H), 7.08 – 7.01 (m, 2H), 6.97 – 6.88 (m, 2H), 6.81 (t, J = 8.8 Hz, 2H), 6.67 (d, J = 8.7 Hz,
2H), 6.53 (d, J = 8.9 Hz, 2H), 3.88 (dd, J = 13.2, 7.4 Hz, 5H), 2.83 (t, J = 5.7 Hz, 2H), 2.54 (s, 4H),
1.74 (d, J = 3.4 Hz, 4H). 13C NMR (75 MHz, DMSO) δ: 162.5, 162.3, 159.5, 154.9, 154.5, 148.5, 133.8,
133.4, 133.3, 131.3, 125.7, 124.7, 114.7, 114.4, 114.1, 111.8, 110.2, 101.1, 100.8, 66.4, 55.8, 54.3, 23.1.
MS (ESI) m/z: 497.1 ([M+Na] +). HRMS (ESI) for C28H27FN2O4+H calcd 475.2033, found 475.2028.
5.1.27. 3-(4-fluorophenyl)-7-methoxy-4-((4-(2-(piperidin-1-yl)ethoxy)phenyl)amino)-2H-chrome
n-2-one (29d)
Yellow solid, yield 52.7%, mp 133-135oC; 1H (300 MHz, DMSO) δ (ppm): 8.46 (s, 1H), 7.90 (d, J
= 8.9 Hz, 1H), 6.99 (dd, J = 8.3, 5.8 Hz, 2H), 6.89 – 6.81 (m, 2H), 6.74 (t, J = 8.8 Hz, 2H), 6.61 (d, J =
8.7 Hz, 2H), 6.47 (d, J = 8.8 Hz, 2H), 3.81 (dd, J = 12.5, 6.6 Hz, 5H), 2.47 (t, J = 5.8 Hz, 2H), 2.30 (s,
4H), 1.40 (s, 4H), 1.29 (d, J = 4.9 Hz, 2H). 13C NMR (75 MHz, DMSO) δ: 162.7, 162.5, 162.35, 159.5,
155.0, 154.5, 148.5, 133.7, 133.4, 133.3, 131.3, 125.7, 124.7, 114.7, 114.4, 114.1, 111.8, 110.2, 101.1,
+
100.8, 66.4, 57.6, 56.3, 54.80, 26.1, 24.4. MS (ESI) m/z: 511.1 ([M+Na] ). HRMS (ESI) for
C29H29FN2O4+H calcd 489.2184, found 489.2188.
5.1.28. General synthesis of final compounds 30a-d
Final compounds 30a-d were prepared according to General method III: demethylation, yield
48.6-60.5%.
5.1.29. 4-((4-(2-(dimethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-7-hydroxy-2H-chrom
en-2-one (30a)
White solid, yield 48.6%, mp 189-191oC; 1H (300 MHz, DMSO) δ (ppm): 8.45 (s, 1H), 7.87 (d, J
= 8.8 Hz, 1H), 7.07 – 6.99 (m, 2H), 6.80 (t, J = 8.9 Hz, 2H), 6.74 – 6.63 (m, 4H), 6.52 (d, J = 8.8 Hz,
2H), 3.88 (t, J = 5.8 Hz, 2H), 2.52 (d, J = 5.8 Hz, 2H), 2.18 (s, 6H). 13C NMR (75 MHz, DMSO) δ:
162.5, 162.2, 161.2, 159.8, 154.86, 154.5, 148.7, 133.9, 133.4, 133.3, 131.4, 125.9, 124.6, 114.7, 114.4,
+
114.1, 112.7, 108.9, 102.6, 100.3, 66.4, 57.9, 45.8. MS (ESI) m/z: 457.2 ([M+Na] ). HRMS (ESI) for
C25H23FN2O4+H calcd 435.1715, found 435.1719.
5.1.30. 4-((4-(2-(diethylamino)ethoxy)phenyl)amino)-3-(4-fluorophenyl)-7-hydroxy-2H-chrome
n-2-one (30b)
Yellow solid, yield 52.8%, mp 168-170oC; 1H (300 MHz, DMSO) δ (ppm): 8.43 (s, 1H), 7.84 (d, J
= 8.8 Hz, 1H), 7.05 – 6.95 (m, 2H), 6.75 (t, J = 8.9 Hz, 2H), 6.69 – 6.59 (m, 4H), 6.48 (d, J = 8.6 Hz,
2H), 3.82 (d, J = 5.3 Hz, 2H), 2.68 (s, 2H), 2.51 (d, J = 7.0 Hz, 4H), 0.91 (t, J = 7.0 Hz, 6H). 13C NMR
(75 MHz, DMSO) δ: 162.4, 161.2, 159.4, 154.9, 154.5, 148.7, 133.9, 133.4, 133.3, 131.4, 125.9, 124.5,
114.6, 114.4, 114.1, 112.7, 108.9, 102.6, 100.3, 66.7, 51.6, 47.5, 12.1. MS (ESI) m/z: 485.1 ([M+Na] +).