Structure-activity relationships of 2, 4-disubstituted pyrimidines as dual ERα/VEGFR-2 ligands with anti-breast cancer activity

Article abstract of DOI:10.1016/j.ejmech.2018.03.018

Both ERα and VEGFR-2 are important targets for cancer therapies. Here a series of 2, 4-disubstituted pyrimidine derivatives were designed, synthesized and evaluated as dual ERα/VEGFR-2 ligands. Most of the derivatives exhibited potent activities in both e

Full text of DOI:10.1016/j.ejmech.2018.03.018

Accepted Manuscript
Structure-activity relationships of 2, 4-disubstituted pyrimidines as dual ERα/
VEGFR-2 ligands with anti-breast cancer activity
Guoshun Luo, Zhichao Tang, Kejing Lao, Xinyu Li, Qidong You, Hua Xiang
PII:
S0223-5234(18)30253-8
10.1016/j.ejmech.2018.03.018
EJMECH 10283
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2017
Revised Date: 26 February 2018
Accepted Date: 5 March 2018
Please cite this article as: G. Luo, Z. Tang, K. Lao, X. Li, Q. You, H. Xiang, Structure-activity
relationships of 2, 4-disubstituted pyrimidines as dual ERα/VEGFR-2 ligands with anti-breast cancer
activity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.018.
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Graphical abstract

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Structure-Activity Relationships of 2, 4-disubstituted Pyrimidines as
Dual ERα/VEGFR-2 Ligands with Anti-Breast Cancer Activity
Guoshun Luoa,b, Zhichao Tanga,b, Kejing Laoc, Xinyu Lia,b, Qidong Youa,b, Hua
Xianga,b*
aState Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009,China
bDepartment of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
Nanjing 210009, China
cInstitute of Basic and Translational Medicine, and School of Basic Medical Science, Xi'an
Medical University, No.1 Xinwang Road, Xi'an, 710021, China
* 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
Both ERα and VEGFR-2 are important targets for cancer therapies. Here a series of 2,
4-disubstituted pyrimidine derivatives were designed, synthesized and evaluated as dual
ERα/VEGFR-2 ligands. Most of the derivatives exhibited potent activities in both enzymatic
and cellular assays. Structure-activity relationship studies showed that a hydrogen-bonding
interaction in the head section is important factors for the enhancement of ERα-binding
affinity. The most potent compound II-9OH, an analog of 2-(4-hydroxylphenyl)pyrimidine,
was 19-fold more efficacious than tamoxifen in MCF-7 cancer cells and exhibited the best
ERα binding affinity (IC50=1.64 µM) as well as excellent VEGFR-2 inhibition (IC50=0.085
µM). Furthermore, this dual targeted compound II-9OH exerted significantly antiestrogenic
property via suppressing the expression of progesterone receptor (PgR) mRNA in MCF-7
cells and also showed obvious in vivo angiogenesis inhibitory effects in CAM assay. An
induction of apoptosis and a decrease in cell migration, accompanied by transduction
inhibition of Raf-1/MAPK/ERK pathway, were observed in MCF-7 cells after treatment with
II-9OH, suggesting that II-9OH is a promising candidate for the development of
multifunctional agents targeting ERα and VEGFR-2 in the therapy of some breast cancers.
Keywords: 2, 4-disubstituted Pyrimidines; ERα; VEGFR-2; antiangiogenesis; anti-breast
cancer.
1. Introduction
Breast cancer (BC) is one of the most common types of cancer and the most frequent
cause of cancer death among females worldwide. Estrogen receptor alpha (ERα) positive
breast cancer accounts for approximately 70% of these cases [1]. Selective estrogen receptor
modulators (SERMs) which act as antagonists in breast tissue but agonists in other tissues
such as cardiovascular system and bone represent first-line treatment for ER+ patients [2, 3].
Tamoxifen, the first generation of SERM, is the most commonly used drug for the treatment
of estrogen receptor positive breast cancer and has been in clinical use for four decades [4, 5].
Since then, multiple additional SERMs with various scaffolds mimicking Tamoxifen have
been developed for the treatment or prevention of breast cancer [5-8]. Many alternative
scaffolds for ER modulators have also been reported, e.g., the heterocyclic
propylpyrazoletriol (PPT) and m-carborane-containing compound (Fig. 1A) [9-11].
However, up to 50% of patients either do not respond or acquire resistance within 5

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years of SERM treatment [12]. Multiple mechanisms contribute to the development of an
ER+ and treatment resistant (TR) phenotype, in which growth is endocrine independent,
including alterations in crosstalk between ER and growth factor-mediated signaling pathways
[13, 14] Increasing studies have revealed that aberrant activation of Raf-1/MAPK/ERK
pathway interplays with ER in regulating estrogen-dependent gene expression and is widely
observed in the endocrine resistance [15, 16]. Vascular endothelial growth factor receptor-2
(VEGFR-2) is a member of the receptor tyrosine kinase (RTK) family and the upstream
effector of Raf-1/MAPK/ERK signaling in promoting cell proliferation, migration and
angiogenesis in cancer [17]. Recently, the combination of Tamoxifen with Brivanib, a
VEGFR-2 inhibitor, was reported not only to maximize therapeutic efficacy but also to retard
SERM resistant tumor growth [18]. Therefore, development of a multiple ligand of ER and
VEGFR-2, exerting enhanced activity against BC with fewer disadvantages is highly needed.
In the course of identifying various chemical fragments that could serve as a scaffold for
new antitumor agents, the literature revealed that pyrimidine is an essential druglike nucleus
bearing enormous biological applicability and has the ability to interact with diverse target
sites i.e., enzymes, receptors, DNA, protein targets, etc [19, 20]. Particularly, the pyrimidine
ring was widely used as a common skeleton in various kinases including VEGFR-2 and
proved to exhibit potent anticancer activities (Fig. 1B) [21-24]. As our ongoing interest to
explore different scaffold structures as multiple ligands of ER and VEGFR-2 [25, 26], we
designed and synthesized a series of 2, 4-disubstituted pyrimidine derivatives with basic side
chain to inhibit the proliferation of breast cancer cells (Fig. 1C). Firstly, the pyrimidine
moiety was utilized as a core scaffold to initiate the interactions with ERα ligand-binding
domain (LBD) as m-carborane and pyrazole did as well as the interactions with VEGFR-2
kinase hinge as adenine did [11, 22]. Then a substituted aromatic ring was introduced to the 2-
or 5-position to occupy the hydrophobic pocket of ER receptor and VEGFR-2 kinase. Next,
various N, N-dialkylamino containing side chains were introduced at para-position of
4-phenyl substituent, which is the most noteworthy feature of SERMs and serves to inhibit the
binding of co-activators by moving helix-12 of the receptor to an unfavorable position [27].
Finally, our exploration focused on the biological effect of the bridging atoms between the
phenyl and pyrimidine moieties, in addition to the established NH linker, bioisosteric group O
atom was also employed. Herein, we disclosed the synthesis of 24 novel pyrimidine-based
analogs and preliminary pharmacological data from receptor binding, enzyme inhibition as
well as antiproliferation assays. A lead compound was identified and its antiangiogenic
activity, anti-estrogenic property together with the effect on VEGFR-2/Raf-1/MAPK/ERK

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signaling pathway were further evaluated. Molecule docking was also carried out for ERα and
VEGFR-2 in order to explore the potential binding mode of the novel dual-action inhibitor.
Fig. 1. Structures of selective estrogen receptor modulators (A), pyrimidine-based VEGFR-2
inhibitors (B) and proposed pyrimidine-based ERα/VEGFR-2 Ligands (C).
2. Results and discussion
2.1 Chemistry
The synthesis of compounds I-1~I-9 and II-1~II-9 is described in Schemes 1. The key
intermediates 6a-6b were prepared from commercially available 4-methoxybenzaldehyde
according to literature procedure in high yields [28]. Briefly, 4-methoxybenzaldehyde reacted
with hydroxylamine under reflux to yield benzonitrile 2, which was then converted to the
corresponding 4-methoxybenzimidamide 3. Further treatment of 3 with diethyl malonates 4a
or 5a in reflux MeOH gave cyclized compound 5a or 5b, respectively. Next, 4,
6-dihydroxypyrimidine 5a and 5b were then subjected to a selective chlorination in the
presence of POCl3 to provide the key intermediates 6a and 6b. Finally, combination of
different side chains with compound 6a and 6b afforded the target compounds I-1~I-9 and

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II-1~II-9, respectively. Compounds II-4OH, II-5OH and II-9OH were prepared from their
corresponding methoxy compounds II-4, II-5 and II-9 by demethylation with BBr3 (Scheme
2).
Scheme 1. Synthetic routes of I-1~I-9 and II-1~II-9. Reagents and conditions: (i) NH2OH·HCl,
DMSO, 100 °C, 0.5 h; (ii) dry methanol, CH3ONa, rt, 48 h; NH4Cl, rt, 24 h; (iii) dry methanol,
CH3ONa, reflux, 4 h; (iv) POCl3, 110 °C, 5 h; (v) dry dioxane, Pd(PPh3)2Cl2, K2CO3, 100 °C, 8 h;
(vi) dry DMF, K2CO3, 80 °C, 4 h.

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Scheme 2. Synthetic routes of II-4OH, II-5OH and II-9OH. Reagents and conditions: (i) dry
dichloromethane, BBr3, 0 °C-rt, 4 h.
2.2. Biological evaluation.
2.2.1. ERα binding affinity assay and antiproliferative activity.
The ERα binding affinities of title molecules were initially evaluated using ER-α
competitor assay kit (Polar Screen ER-α Competitor Assay Kit, Green, Life Technology).
Additionally, in order to verify the probability of synthesized compounds as novel ERα
antagonists, the potential cytotoxic effects were also investigated in MCF-7 cell (ER+) using
MTT assay. It is known that long term treatment of tamoxifen will stimulate endometrial cell
growth and induce endometrial cancer; antiproliferative activity of new compounds on
endometrial cells was also measured. For comparison, clinically approved SERMs tamoxifen
and raloxifene was used as the positive control in these two assays.
These result data can be analyzed in two groups, 5-hydrogen analogues (I-1~I-9) and 5-
phenyl substituted analogues (II-1~II-9), and are depicted in Table 1. As a global observation,
most of the 2, 4-disubstituted pyrimidines showed promising binding affinities with more than
50% inhibition at 10 µM indicating that pyrimidine scaffold could favorably bind to ERα
ligand-binding domain. Most 5-hydrogen prototypes were more active in ERα binding assay
than their phenyl-substituted counterpart compounds except II-9 which exhibited the best
ERα binding affinity (IC50= 4.57 µM). Compounds I-4, II-4, II-5, and II-6 showed significant
in vitro cytotoxicity against both MCF-7 and Ishikawa cells that surpassed those of clinical
SERM tamoxifen and raloxifene. It was interesting to find that compounds (I-4, II-4, II-5 and
II-6) which were less potent in ERα binding assay manifested promising anti-proliferative
activity against MCF-7 cells with IC50 values of 3.54, 7.56, 8.59 and 7.84 µM respectively,
which confirmed that the obtained antiproliferative activity was not only through ERα but a
multi-target effect. Investigation on the side chain revealed an unfavorite in acetamide side
chain in MCF-7 compared with the ethoxy chain and a decrease in activity was observed (I-1
vs I-4, I-2 vs I-5, II-1 vs II-4, II-2 vs II-5 and II-3 vs II-6). And similar trend was also seen
in Ishikawa cells (I-1 vs I-4, I-2 vs I-5, I-3 vs I-6, II-1 vs II-4, II-2 vs II-5 and II-3 vs II-6).

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Table 1
ERα binding affinity and antiproliferation activity of compounds -1~I-9, II-1~II-9, II-4OH,
II-5OH and II-9OH
Antiproliferation (IC50,
ERα binding affinitya
µM)c
Cmpd
R
R1
Inh% at 10
IC50 (µM)
NDb
ND
MCF-7
>40
Ishikawa
>40
µM
I-1
I-2
H
H
64.32
66.64
80.47
65.35
77.22
65.97
74.53
77.69
72.74
55.39
65.76
60.59
69.47
42.33
51.68
>40
5.81
I-3
H
2.86
ND
23.35
3.54
36.82
43.7
>40
18.35
5.67
I-4
H
I-5
H
4.74
ND
11.67
17.28
>40
I-6
H
I-7
H
5.96
5.13
6.22
ND
I-8
H
49.61
21.32
17.66
>40
24.86
25.51
17.14
>40
I-9
H
II-1
II-2
II-3
II-4
II-5
II-6
Ph
Ph
Ph
Ph
Ph
Ph
ND
ND
>40
>40
ND
7.56
8.59
7.84
6.34
ND
9.98
ND
3.81

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II-7
II-8
II-9
Ph
Ph
Ph
58.17
58.79
84.87
ND
ND
21.75
>40
17.72
23.46
14.12
2.17
32.43
II-4OH
II-5OH
II-9OH
80.44
54.23
90.15
3.42
ND
33.1
35.6
0.81
37.13
12.02
5.93
1.64
Tamoxifen
Raloxifene
-
-
-
-
92.36
-
1.42
-
15.46
13.18
26.52
24.37
aPercent inhibition of each compound was calculated from the polarization values. The IC50 values
are calculated when the Inh% at 10 µM is >70%.
bND stands for not determined
cIC50: concentration that inhibits 50% of cell growth
It is known that the hydrogen bonds formed between the two hydroxyl group of estradiol
(endogenous ERα ligand) and Glu353, Arg394 and His524 are important and required to keep
the conformation stable when estradiol binds to ERα [29]. The ability to form hydrogen bonds
with these three key amino acids is widely accepted as required to identify ERα ligands. For
the purpose of exploring the SAR of substituent group in 2-phenyl ring, we next studied the
hydrogen donor group on activity by designing compounds II-4OH, II-5OH and II-9OH
(Table 1). It was supposed that hydroxyl group at C-4’ position of 2-phenyl substituent would
form more hydrogen bonds with essential amino acids than methoxyl group in the ligand
binding domain of ERα. Expectedly, it is obvious to note that hydroxyl containing derivatives
II-4OH (IC50=3.42 µM) and II-9OH (IC50=1.64 µM) possessed apparently better affinities to
ERα than methoxyl substituted lead compound II-4 and II-9 (IC50=2.17 µM). However, to our
disappointment, their hydroxy analogs II-4 (clogP=6.59) and II-5 (clogP=7.67) displayed
poor cytotoxicity in the same assay system which might be due to their lower cellular uptake.
The highest binding affinity was observed with compound II-9OH, exhibiting binding
affinity value of 1.64 µM very close to that of tamoxifen (1.42 µM). Among all the active
compounds, II-9OH also showed maximum inhibition of cell growth against MCF-7 cells
with an IC50 of 0.81 µM, almost 20 and 16 fold more potent than tamoxifen (15.46 µM) and

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raloxifene (13.18 µM), respectively.
2.2.2. Antiangiogenic activity on VEGFR-2 enzyme and HUVEC cells
All the synthesized 2, 4-disubstituted pyrimidine derivatives I-1~I-9, II-1~II-9, II-4OH,
II-5OH and II-9OH were next evaluated for their enzymatic activities against VEGFR-2 and
anti-proliferative activity towards VEGFR-2 overexpressed human umbilical vein endothelial
cells (HUVEC). The approved VEGFR-2 inhibitor drug sunitinib was used as a positive
control. As shown in Table 2, most of the tested compounds exhibited moderate VEGFR-2
inhibitory activities with more than 50% inhibition at 10 µM. Among these compounds,
compound II-5 which displayed the most potent inhibitory activity against VEGFR-2 with
IC50 of 0.067 µM was also the most potent one against HUVEC with the IC50 of 4.34 µM.
While compound II-9OH which showed the best binding affinity in previous ERα assay
retained promising antiangiogenic activity on both VEGFR-2 enzyme (IC50= 0.085 µM) and
HUVEC cells (IC50= 4.57 µM) compared with sunitinib (IC50= 0.054 µM and 4.57 µM,
respectively) indicating that II-9OH is a potent dual ERα/VEGFR-2 inhibitor.
Most of these compounds demonstrated moderate to good anti-proliferative activities.
Compounds I-3, I-4, II-5, II-6 and II-9OH showed better activities against HUVEC cells
than the reference drug sunitinib. A close observation of the data showed that the 4-N-linked
compounds were more active than their 4-O-linked counterpart (I-4~I-6 vs I-7~I-9 and
II-5~II-6 vs II-8~II-9). It is worthy to note that compounds I-3, I-4 and II-6 which were less
active in VEGFR-2 assay also showed noteworthy inhibition on HUVEC proliferation
indicating another mechanism for their acquired activity. On the contrary, II-3 showing
promising activity against VEGFR-2 from the cell-free enzyme assay (IC50= 0.203 µM) was
not active in HUVEC, which might due to its poor membrane permeability (ClogP = 6.9).
Such discrepancies between the inhibitory activity toward HUVEC proliferation and
VEGFR-2 inhibition concluded in this manuscript implied that other modes of action or other
parameters such as: physicochemical property, cell membrane permeability or target activity
could have a significant role in the antiproliferative activity of our novel compounds.
Table 2
VEGFR-2 inhibition and antiproliferation activity against HUVEC of all the designed compounds
VEGFR-2 inhibition
Antiproliferation (IC50, µM)b
Cmpd
Inh% at 10 µM
IC50 (µM)
HUVEC
>40
I-1
I-2
I-3
66.24±3.32
44.05±4.78
61.29±5.79
>1
>1
>1
15.04
4.82

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I-4
I-5
40.00±2.88
>1
0.174
>1
6.31
9.24
89.70±2.50
51.88±1.65
61.25±1.84
79.99±2.53
39.84±3.12
50.77±1.85
40.22±5.46
87.35±2.06
50.94±0.57
90.27±2.22
49.38±3.03
90.86±2.47
60.19±1.47
75.32±2.15
68.45±2.03
76.92±1.89
91.07±1.45
92.74±1.96
I-6
11.32
>40
I-7
>1
I-8
>1
23.27
15.84
10.35
>40
I-9
>1
II-1
>1
II-2
>1
II-3
0.203
>1
>40
II-4
14.17
4.34
II-5
0.067
>1
II-6
5.02
II-7
0.101
>1
11.72
15.09
10.62
13.83
8.81
II-8
II-9
>1
II-4OH
II-5OH
II-9OH
Sunitinib
>1
>1
0.085
4.57
0.054
6.36
aPercentage inhibition of each compound on VEGFR-2 at 10 µM was measured using
homogeneous time resolved fluorescence (HTRF) assay, shown as mean ± SD of three
experiments. The IC50 values are calculated when Inh% at 1µM is >50%.
bIC50: concentration that inhibits 50% of cell growth
According to above bioassay results, some of the 2, 4-disubstituted pyrimidines analogs
especially I-5, II-5 and II-9OH were potent dual-profile ligands. We found that compound
II-9OH exhibited the strongest ERα binding affinity (IC50= 1.64 µM) as well as the best
cytotoxicity against MCF-7 cells (IC50 = 0.81 µM), also was a promising anti-angiogenesis
agent (IC50= 0.085 µM for VEGFR-2 inhibition; IC50= 4.57 µM against HUVEC cells). This
synergetic inhibitory effect of II-9OH on ERα and VEGFR-2 suggested it as a promising
dual-target agent against breast cancer. Thus, II-9OH was selected for our further
investigation of its multifunctional effects.
2.2.3. Cytostatic activity and antiestrogenic activity of II-9OH
To assess the potential of compound II-9OH towards breast cancer, their cytostatic
potency was additionally tested against human breast cancer cells T47D (ERα+) and
MDA-MB-231 (ERα-). Results presented in Fig. 2A indicated that II-9OH strongly inhibited
proliferation of both estrogen-responsive and -nonresponsive cells in a dose-dependent
manner. Compound II-9OH exhibited promising antiproliferative activities against T47D and
MDA-MB-231 cells with IC50 values at 2.73 and 3.81 µM, respectively. As for ER+ and ER-

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breast cancer cells, compound II-9OH could inhibit their growth on the same degree which
also indicated that the antiproliferative activity was acquired not only through ERα but a
multi-target effect.
We further adapted quantitative real-time polymerase chain reaction (RT-PCR) in the ER
positive MCF-7 cells to investigate antiestrogenic activity of II-9OH. It is known that the
expression of ERα-regulated target gene progesterone receptor (PgR) is commonly associated
with estrogenic or antiestrogenic activity. As shown in Fig. 2B, compound II-9OH at the
concentration of 0.4 µM in combination with 10 nM E2 dramatically reduced the expression
of PgR mRNA induced by E2 (*** P < 0.001 vs. E2 group), indicating that II-9OH presented
significantly antiestrogenic property.
Figure 2. (A) In vitro anti-proliferative properties of II-9OH against T47D and MDA-MB-231
cell lines. (B)The increased mRNA expression of PR induced by E2 was reversed by II-9OH 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,
0.001
P<
.
2.2.4. II-9OH exhibits anti-angiogenic activity in vivo and downregulates the protein
expression of VEGFR-2 in HUVEC cells
It is well known that vascular endothelial growth factor (VEGF)/VEGFR-2 signaling has
a predominant role in angiogenesis, the process of new blood vessel growth from the
quiescent pre-existing vessels [30]. Compound II-9OH which exhibited potent VEGFR-2
inhibition in enzymatic assay was then selected to evaluate the in vivo angiogenetic activity
by chick chorioallantoic membrane (CAM) Assay [31]. Compound II-9OH dissolved in
DMSO were placed on sterile methyl cellulose filter papers at 1 µM, 10 µM and 20 µM with
phosphate buffered saline (PBS) as the blank control and sunitinib (1µM) as the positive
control. Results are shown in Fig. 3A. Compared with blank control group, compound

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II-9OH could significantly inhibit angiogenesis in a dose-dependent manner. And the
inhibitory activity of compound II-9OH was more potent than sunitinib in CAM assay at the
same dose (1 µM). Overall, compounds showed potential anti-angiogenesis activities in vivo.
The inhibitory effect of compound II-9OH on VEGFR-2 was further determined by
assessing the phosphorylation level of VEGFR-2 in VEGFR-2 overexpressed HUVEC cells
using western blot. As shown in Fig. 3B, II-9OH was able to block the phosphorylation of
VEGFR-2 significantly compared with control group (*P < 0.05). However, this inhibition
effect of II-9OH was weaker than that of positive control sunitinib.
Figure 3. (A) In vivo Inhibitory effects of compound II-9OH on angiogenesis of CAM (B)
II-9OH inhibits the phosphorylation of VEGFR-2 in HUVEC cells. Expression of p-VEGFR-2
and VEGFR-2 in HUVEC cells were examined by western blots. 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.
2.2.5. II-9OH induces apoptosis and inhibits cancer cell migration in MCF-7 cells through
Raf-1/MAPK/ERK signaling inhibition
Because compound II-9OH caused a significant reduction in the growth of both ER+
and ER- breast cancer cells, the underlying mechanisms were investigated. First, we measured
the induction of apoptosis by this compound using Annexin-V/PI staining and flow cytometry.

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Briefly, MCF-7 cells were cultured with 0.5 µM, 1µM of II-9OH or 1% DMSO. After
incubation for 24 h, cells were subjected to flow cytometric analysis (Fig. 4A). Compound
II-9OH caused significant induction of apoptosis in a concentration-dependent manner. When
treated with 0.5 and 1 µM compound II-9OH for 24 h; the percentages of apoptotic cells were
23.39% and 33.49% (Q2 + Q4), respectively, compared with 10.47% of vehicle control.
Metastasis is the major cause of death of the advanced breast cancer patients [32].
Migration of cancer cells is an essential phenomenon exhibited by many types of cancer cells
during metastasis. For this assay, a transwell assay was performed to evaluate the ability of
MCF-7 cells to pass through the transwell membrane barrier in the presence of II-9OH. As
shown in Fig. 4B, MCF-7 cells treated with 0.5 µM and 1µM of II-9OH showed reduced cell
migration as compared to cells treated with vehicle control (DMSO). These results suggested
that II-9OH played a role in the inhibition of cell migration in breast cancer.
Several cellular signaling pathways are involved in the evolution, aggressiveness and
metastatic potential of breast tumors. Currently, studies have revealed that over-expression
and aberrant activation of Raf-1/MAPK/ERK pathway is associated with the resistance of
endocrine therapy [65]. Crosstalk of estrogen receptor pathway with Raf-1/MAPK/ERK
pathway is also relative to breast cancer cell migration, invasion, and angiogenesis [66].
Therefore, we made further investigation of II-9OH on inhibiting 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 II-9OH affected the
Raf-1/MAPK/ERK pathway. As expected, the results indicated that 0.5 µM of II-9OH
significantly inhibited p-Raf-1 and p-ERK1/2 activation, while the total protein level of Raf-1
was not altered (Fig. 4C). To further confirm that the observed transduction inhibition of
Raf-1/MAPK/ERK pathway was acquired through VEGFR-2 blockade, a full kinase
screening panel is required in further study on mechanism of action.

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Figure 4. (A) Flow cytometry analysis of II-9OH on MCF-7 cell apoptosis using Annexin
V-FITC/PI. (B) The inhibition of II-9OH on MCF-7 cell migration using transwell cell migration
assay. (C) Expression of p-Raf-1, Raf-1, p-ERK1/2, and ERK1/2 by 0.5 µM II-9OH were
examined by western blots in MCF-7 cells. Densitometric analysis was performed to determine
the phosphorylation rate of Raf-1 and ERK1/2. Values are mean ± SM (n = 3)
2.3. Molecular docking studies
In order to investigate the possible binding mode of synthesized compounds in
ERα/VEGFR-2, molecular docking studies on the potent compound II-9OH were performed
using the Discovery Studio 3.0/CDOCKER protocol. As shown in Fig. 5A, in the binding
pocket of ERα, the pyrimidine scaffold of II-9OH is favorably located into the ligand binding
domain and the side chain stretches toward the edge of the pocket. Furthermore, an excellent
superimposition of II-9OH over the structure of 4-hydroxytamoxifen (OHT, co-crystallized
ligand) was observed indicating that the designed compounds exhibit the same binding
patterns as that of OHT. Detailed docking analysis revealed that that hydroxyl group at C-4’
position of 2-phenyl substituent formed hydrogen bonding interactions with Glu353 and
Arg394, which is basic requirements for high-affinity ligand binding to ERα (Fig. 5B) [33].
Further docking study of II-9OH with VEGFR-2 kinase (PDB ID: 3CJF) confirmed its

ACCEPTED MANUSCRIPT
predicted mode of binding within the ATP site of VEGFR-2 LBD, with the pyrimidine N-1
making hydrogen acceptor bond with essential amino acid Asn921 (Fig. 6) [22]. Moreover,
hydroxyl group at C-4’ position of 2-phenyl substituent formed another hydrogen bonding
interaction with Asn1031, providing further stabilization for the ligand-receptor complex.
These docking results provide additional insights into the protein-ligand interactions and
potential structural modifications for further activity improvement.
Figure 5. (A) Superimposed poses of OHT (orange) with designed compound II-9OH (white),
binding to ERα (PDB ID: 3ERT), the protein was shown in surface. (B) Docking interactions of
II-9OH within ERα active site. Dotted lines (red) represent the hydrogen bonding interaction.
Figure 6. Docking poses of II-9OH (white) within ATP site of VEGFR-2 (PDB ID: 3CJF). Dotted
lines (red) represent the hydrogen bonding interaction.
3. Conclusion
To explore different scaffold structures as dual ligands of ERα and VEGFR-2, we have
designed and synthesized a series of 2, 4-disubstituted pyrimidines derivatives with basic side
chain. Most of them not only exhibited potent ERα binding affinities and VEGFR-2 inhibition,
but also showed promising anti-proliferative activities against both cancer cells and
angiogenesis-related cells in vitro. Among them, Compound II-9OH was found to be the
most potential inhibitor on the both targets. Further mechanism investigation revealed that

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II-9OH was able to induce apoptosis and suppress migration in MCF-7 cells by inhibiting
signaling transduction of Raf-1/MAPK/ERK pathway. Moreover, II-9OH significantly
inhibited blood vessel growth in CAM and inhibited the protein expression of VEGFR-2.
Collectively, our results demonstrated that the newly developed pyrimidine analogue II-9OH
showed significant anti-breast cancer efficacy as well as antiangiogenesis activity via a
multiple inhibition on ERα/VEGFR-2 and has the potential to be further developed into a
promising anti-breast cancer agent.
4. Experimental section
4.1. Chemistry
4.1.1. General.
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. Infrared (IR) spectra (KBr) were recorded on a Nicolet Impact 410
instrument (KBr pellet). 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 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.
4.1.2. Synthesis of 4-methoxybenzonitrile 2
To a solution of hydroxylammonium chloride (2.57 g, 37 mmol) in 25 ml DMSO was
added 4-anisaldehyde (2.27 mL, 20 mmol). The reaction mixture was stirred under reflux for
0.5 h. The resulting mixture was cooled, and the precipitate was collected by filtration,
washed with diethyl ether, and then dried in vacuum to obtain 2 as a yellow solid (2.1 g,
78.9%), MS (ESI) m/z: 134.1 ([M+H]+).
4.1.3. Synthesis of 4-methoxy-benzamidine hydrochloride 3
A solution of 4-methoxybenzonitrile 2 (2.1 g, 15.7 mmol), sodium methylate (86 mg,
1.57 mmol) in 20 mL of anhydrous methanol was stirred at room temperature for 48 h. Then
ammonium chloride (0.84 g, 15.7mmol) was added, and the reaction group was stirred for

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another 24 h. the precipitate was collected by filtration, washed with diethyl ether, and then
dried in vacuum to obtain 3 as a white solid (1.3 g, 44.5%), MS (ESI) m/z: 151.1 ([M+H]+).
4.1.4. Synthesis of 2-(4-methoxyphenyl)pyrimidine-4,6-diol 5a
To a solution of 4-methoxy-benzamidine hydrochloride (0.46 g, 2.47 mmol) in 10 ml
anhydrous methanol was added malonate (0.28 mL, 2.47 mmol). A solution of sodium
methylate dissolved in 5 ml anhydrous methanol was added to the previous reaction mixture
dropwise, and then the reaction mixture was stirred under reflux for 4 h. After the reaction
was completed, the resulting mixture was cooled, poured to 50 ml ice water and was dropped
by 1 M HCl to pH=4. The precipitate was collected by filtration as a white solid (0.24 g, 45%),
MS (ESI) m/z: 217.1 ([M-H]-).
4.1.5. Synthesis of 2-(4-methoxyphenyl)-5-phenylpyrimidine-4,6-diol 5b
To a solution of 4-methoxy-benzamidine hydrochloride (0.46 g, 2.47 mmol) in 10 ml
anhydrous methanol was added malonate (0.53 mL, 2.47 mmol). A solution of sodium
methylate dissolved in 5 ml anhydrous methanol was added to the previous reaction mixture
dropwise, and then the reaction mixture was stirred under reflux for 4 h. After the reaction
was completed, the resulting mixture was cooled, poured to 50 ml ice water and was dropped
by 1 M HCl to pH=4. The precipitate was collected by filtration as a white solid (0.3 g, 41%),
MS (ESI) m/z: 295.1 ([M+H]+).
4.1.6. Synthesis of 4,6-dichloro-2-(4-methoxyphenyl)pyrimidine 6a
A solution of 2-(4-methoxyphenyl)pyrimidine-4,6-diol 5a (1.8 g, 8.24 mmol) in 4 ml
phosphorus oxychloride was stirred at 110°C for 5 h. When the reaction was completed, the
resulting mixture was cooled, poured to 50 ml ice water and then extracted with ethyl acetate.
The organic phase was collected, dried with Na2SO4 and evaporated to get crude product
which was then purified by column chromatograph to give 6a as a white solid (1.5 g, 71%),
MS (ESI) m/z: 256.1 ([M+H]+).
4.1.7. Synthesis of 4,6-dichloro-2-(4-methoxyphenyl)-5-phenylpyrimidine 6b
Compound 6b was synthesized from 2-(4-methoxyphenyl)-5-phenylpyrimidine-4,6-diol
5b (1.7 g, 5.78 mmol) according to the synthetic procedure of 6a in a yield of 68%, 1.3g. MS
(ESI) m/z: 331.1 ([M+H]+).

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4.1.8. General synthesis of final compounds I-1~I-6 and II-1~II-6
A mixture of intermediate 6a or 6b (1 equiv), various 4-acetamidoanilines or
4-thoxyanilines (1.5 equiv.), K2CO3 (2.0 equiv.) and Pd(PPh3)2Cl2 in anhydrous dioxane (5.0
mL) was stirred at 100 °C for 8 h. After completion of the reaction as indicated by TLC, the
mixture was cooled to room temperature and then extracted, evaporated and purified by flash
column chromatography (CH2Cl2/CH3OH) on silica gel to afford the corresponding pure
products I-1~I-6 and II-1~II-6.
4.1.9.
N-(4-((6-chloro-2-(4-methoxyphenyl)pyrimidin-4-yl)amino)phenyl)-2-(dimethylamino)acetam
ide I-1
Yellow solid (0.12g, 34.0%). Mp 118-120 oC; IR (KBr): 3295, 3173, 3077, 2931, 2809, 1663,
1
1562, 1508, 1372, 1253, 1095, 985, 833 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 9.37 (s,
1H, CONH), 8.34-8.37 (d, 2H, 2-Ar-2’-H, J=8.97Hz), 7.65-7.68 (d, 2H, 4-Ar-3’-H,
J=8.73Hz), 7.36-7.39 (d, 2H, 4-Ar-2’-H, J=8.82Hz), 6.97-7.00 (d, 2H, 2-Ar-3’-H, J=8.94Hz),
6.86 (s, 1H, 5-Ar-H), 6.48 (s, 1H, NH), 3.89 (s, 3H, OCH3), 3.12 (s, 2H, COCH2), 2.35 (S,
6H). 13C NMR (75 MHz, CDCl3): δ 170.3, 164.7, 162.1, 160.6, 134.5, 134.1, 130.4, 129.3,
123.6, 120.5, 113.8, 100.1, 57.5, 53.6, 44.5. MS (ESI, m/z): 412.2[M+H]+.
4.1.10.
N-(4-((6-chloro-2-(4-methoxyphenyl)pyrimidin-4-yl)amino)phenyl)-2-(diethylamino)acetamid
e I-2
Yellow solid (0.13 g, 37.9%). Mp 127-128oC; IR (KBr): 3316, 3153, 3076, 2964, 2818, 1656,
1
1561, 1508, 1373, 1253, 1096, 985, 835 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 9.51 (s,
1H, CONH), 8.34-8.36 (d, 2H, 2-Ar-2’-H, J=8.79Hz), 7.65-7.71 (d, 2H, 4-Ar-3’-H,
J=8.67Hz), 7.35-7.38 (d, 2H, 4-Ar-2’-H, J=8.58Hz), 6.96-6.99 (d, 2H, 2-Ar-3’-H, J=8.88Hz),
6.90 (s, 1H, 5-Ar-H), 6.48 (s, 1H, NH), 3.89 (s, 3H, OCH3), 3.18 (s, 2H, COCH2 ), 2.64-2.72
(q, 4H, NCH2, J=7.05Hz), 1.10-1.15 (t, 6H, NCH2CH3, J=7.11Hz). 13C NMR (75 MHz,
CDCl3): δ 170.4, 164.6, 162.1, 160.5, 134.8, 133.9, 130.1, 129.3, 123.5, 120.5, 113.7, 100.1,
58.0, 55.4, 48.9, 12.5. MS (ESI) m/z: 440.3 [M+H]+, 462.3[M+Na]+.
4.1.11.
N-(4-((6-chloro-2-(4-methoxyphenyl)pyrimidin-4-yl)amino)phenyl)-2-(pyrrolidin-1-yl)acetami
de I-3

ACCEPTED MANUSCRIPT
Yellow solid (98 mg, 28.6%). Mp 208-210oC; IR (KBr): 3447, 3304, 3211, 2953, 2812, 1676,
1
1560, 1511, 1370, 1244, 1096, 984, 825 cm-1; H-NMR (300MHz, DMSO): δ (ppm) 9.81 (s,
1H, CONH), 9.72 (s, 1H, 5-Ar-H), 8.22-8.24 (d, 2H, 2-Ar-2’-H, J=8.76Hz), 7.63-7.69 (m, 4H,
4-Ar-H), 7.06-7.09 (d, 2H, 2-Ar-3’-H, J=8.79Hz), 6.60 (s, 1H, NH), 3.83 (s, 3H, OCH3), 3.23
(s, 2H, COCH2), 2.58 (brs, 4H, 2, 5-pyrrolidyl-H), 1.74 (brs, 4H, 3, 4-pyrrolidyl-H). 13C NMR
(75 MHz, CDCl3): δ 170.5, 164.7, 162.1, 160.5, 134.9, 133.9, 130.1, 129.4, 123.6, 120.5,
113.8, 100.0, 58.1, 55.5, 49.9, 23.8. MS (ESI, m/z): 438.3[M+H]+.
4.1.12.
6-chloro-N-(4-(2-(dimethylamino)ethoxy)phenyl)-2-(4-methoxyphenyl)pyrimidin-4-amine I-4
Yellow solid (98 mg, 31.5%). Mp 113-116oC; IR (KBr): 3447, 2933, 2822, 2777, 1593, 1569,
1
1507, 1397, 1247, 1166, 1208, 844 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.32-8.35 (d,
2H, 2-Ar-2’-H, J=8.82Hz), 7.23-7.26 (d, 2H, 4-Ar-2’-H, J=8.58Hz), 6.93-6.98 (m, 5H,
4-Ar-3’-H, 2-Ar-3’-H, 5-Ar-H), 6.37 (s, 1H, NH), 4.08-4.12 (t, 2H, OCH2CH2, J=5.52Hz),
3.88 (s, 3H, OCH3), 2.75-2.79 (t, 2H, OCH2CH2, J=5.46Hz), 2.37 (s, 6H, N(CH3)2). 13C NMR
(75 MHz, CDCl3): δ 163.6, 162.8, 162.3, 162.1, 161.2, 155.8, 132.4, 131.4, 130.1, 129.5,
125.6, 124.9, 115.5, 115.2, 113.7, 113.4, 66.2, 58.3, 55.3, 45.9, 37.1. MS (ESI, m/z):
397.2[M-H]-.
4.1.13.
6-chloro-N-(4-(2-(diethylamino)ethoxy)phenyl)-2-(4-methoxyphenyl)pyrimidin-4-amine I-5
Yellow solid (0.12 g, 36.0%). Mp 122-124oC; IR (KBr): 2975, 2930, 2811, 1585, 1565, 1507,
1
1389, 1304, 1235, 1168, 1028, 820 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.32-8.35 (d,
2H, 2-Ar-2’-H, J=7.59Hz), 7.22-7.26 (d, 2H, 4-Ar-2’-H), 6.95-6.97 (m, 4H, 4-Ar-3’-H,
2-Ar-3’-H), 6.79 (s, 1H, 5-Ar-H), 6.36 (s, 1H, NH), 4.07 (t, 2H, OCH2CH2), 3.88 (s, 3H,
OCH3), 2.90 (t, 2H, OCH2CH2), 2.63-2.67 (q, 4H, N(CH2CH3)2, J=6.66Hz), 1.07-1.11 (t, 6H,
N(CH2CH3)2, J=6.39Hz ). 13C NMR (75 MHz, CDCl3): δ 164.6, 162.9, 162.1, 160.6, 157.0,
130.1, 130.1, 129.3, 125.7, 115.4, 113.7, 99.2, 66.9, 55.4, 51.7, 47.8, 11.8. MS (ESI, m/z):
427.2[M+H]+.
4.1.14.
6-chloro-2-(4-methoxyphenyl)-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pyrimidin-4-amine I-6
Yellow solid (0.13 g, 39.2%). Mp 140-142oC; IR (KBr): 3421, 2970, 2933, 2804, 1592, 1568,
1
1508, 1395, 1246, 1161, 1035, 840 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.34-8.36 (d,

ACCEPTED MANUSCRIPT
2H, 2-Ar-2’-H, J=7.26Hz), 7.23-7.26 (d, 2H, 4-Ar-2’-H), 6.92-6.96 (m, 4H, 4-Ar-3’-H,
2-Ar-3’-H), 6.77 (s, 1H, 5-Ar-H), 6.36 (s, 1H, NH), 4.12 (brs, 2H, OCH2CH2), 3.87 (s, 3H,
OCH3), 2.92 (brs, 2H, OCH2CH2), 2.64 (brs, 4H, 2, 5-pyrrolidyl-H), 1.60 (brs, 4H, 3,
4-pyrrolidyl-H). 13C NMR (75 MHz, CDCl3): δ 164.7, 162.9, 162.3, 160.7, 157.0, 130.1,
130.0, 129.0, 125.8, 115.4, 113.7, 99.1, 67.0, 55.4, 51.8, 48.9, 24.1. MS (ESI, m/z):
423.1[M-H]-.
4.1.15.
N-(4-((6-chloro-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-yl)amino)phenyl)-2-(dimethylamin
o)acetamide II-1
Yellow solid (145 mg, 50%). Mp 171-173oC; IR (KBr): 3410, 3297, 2942, 2835, 2787, 1675,
1
1550, 1513, 1404, 1246, 1173, 844, 789 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 9.15 (s,
1H, CONH), 8.37-8.39 (d, 2H, 2-Ar-2’-H, J=6.99Hz), 7.55-7.60 (m, 7H, 5-Ar-H, 4-Ar-3’-H),
7.44 (d, 2H, 4-Ar-2’-H), 6.98-7.00 (d, 2H, 2-Ar-3’-H, J=6.24Hz), 6.45 (s, 1H, NH), 3.89 (s,
3H, OCH3), 3.09 (s, 2H, CH2), 2.39 (s, 6H, N(CH3)2). 13C NMR (75 MHz, DMSO): δ 170.0,
162.1, 161.8, 160.0, 158.0, 134.8, 134.8, 132.7, 130.8, 129.9, 129.7, 129.2, 129.1, 123.7,
119.5, 115.4, 114.5, 57.9, 55.6, 45.3. MS (ESI, m/z): 488.3 [M+H]+.
4.1.16.
N-(4-((6-chloro-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-yl)amino)phenyl)-2-(diethylamino)
acetamide II-2
Yellow solid (150 mg, 49.7%). Mp 131-132oC; IR (KBr): 3409, 3299, 3970, 2934, 2824, 1682,
1
1545, 1514, 1405, 1250, 1173, 842, 791 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 9.43 (s,
1H, CONH), 8.37-8.39 (d, 2H, 2-Ar-2’-H, J=7.11Hz), 7.52-7.61 (m, 7H, 5-Ar-H, 4-Ar-3’-H),
7.42-7.44 (d, 2H, 4-Ar-2’-H, J=6.78Hz), 6.98-7.00 (d, 2H, 2-Ar-3’-H, J=7.02Hz), 6.45 (s, 1H,
NH), 3.89 (s, 3H, OCH3), 3.15 (s, 2H, COCH2), 2.62-2.69 (q, 4H, NCH2, J=6.84Hz),
1.08-1.12(t, 6H, CH2CH3, J=7.05Hz). 13C NMR (75 MHz, DMSO): δ 170.1, 162.2, 161.8,
159.9, 157.9, 134.8, 134.8, 132.8, 130.8, 129.9, 129.7, 129.3, 129.2, 123.7, 119.5, 115.3,
114.5, 57.8, 55.8, 48.3, 12.4. MS (ESI, m/z): 516.2[M+H]+.
4.1.17.
N-(4-((6-chloro-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-yl)amino)phenyl)-2-(pyrrolidin-1-
yl)acetamide II-3
Yellow solid (110 mg, 35.7%). Mp 162-165oC; IR (KBr): 3408, 3290, 2939, 2813, 1678, 1547,

ACCEPTED MANUSCRIPT
1
1516, 1405, 1250, 1170, 842, 790 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 9.30 (s, 1H,
CONH), 8.37-8.39 (d, 2H, 2-Ar-2’-H, J=6.93Hz), 7.52-7.61 (m, 7H, 5-Ar-H, 4-Ar-3’-H),
7.42-7.44 (d, 2H, 4-Ar-2’-H, J=6.6Hz), 6.98-7.00 (d, 2H, 2-Ar-3’-H, J=7.05Hz), 6.45 (s, 1H,
NH), 3.89 (s, 3H, OCH3), 3.08 (s, 2H, COCH2), 2.56 (brs, 4H, 2,5-pyrrolidyl-H), 1.66 (brs,
4H, 3, 4-pyrrolidyl-H). 13C NMR (75 MHz, DMSO): δ 168.8, 162.1, 161.8, 159.9, 157.99,
134.9, 134.7, 132.8, 130.8, 129.9, 129.7, 129.3, 129.2, 123.7, 119.6, 115.3, 114.4, 63.1, 55.8,
54.6, 25.9, 23.9. MS (ESI, m/z): 514.3[M+H]+.
4.1.18.
6-chloro-N-(4-(2-(dimethylamino)ethoxy)phenyl)-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-a
mine II-4
Yellow solid (93 mg, 32.6%). Mp 68-70°C; IR (KBr): 3621, 3417, 3321, 2940, 2831, 1601,
1
1573, 1540, 1408, 1254, 1168, 1029, 795 cm-1; H-NMR (300MHz, CDCl3): δ (ppm)
8.34-8.36 (d, 2H, 2-Ar-2’-H, J=6.81Hz), 7.42-7.57 (m, 7H, 5-Ar-H, 4-Ar-2’-H), 6.91-6.98 (m,
4H, 2-Ar-3’-H, 4-Ar-3’-H), 6.36 (s, 1H, NH), 4.07 (brs, 2H, OCH2), 3.88 (s, 3H, OCH3), 2.73
(brs, 2H, OCH2CH2N), 2.35(s, 6H, N(CH3)2). 13C NMR (75 MHz, CDCl3): δ 162.8, 162.0,
159.3, 157.8, 155.5, 132.6, 131.6, 130.2, 130.1, 129.7, 129.6, 129.2, 122.9, 114.7, 114.5,
113.7, 66.3, 58.3, 55.4, 45.9. MS (ESI, m/z): 475.2[M+H]+.
4.1.19.
6-chloro-N-(4-(2-(diethylamino)ethoxy)phenyl)-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-am
ine II-5
Yellow solid (120 mg, 39.8%). Mp 47-49°C; IR (KBr): 3620, 3413, 3287, 2977, 2838, 1601,
1
1571, 1540, 1407, 1259, 1166, 1029, 979, 795 cm-1; H-NMR (300MHz, CDCl3) : δ (ppm)
8.34-8.36 (d, 2H, 2-Ar-2’-H, J=7.17Hz), 7.41-7.58 (m, 7H, 5-Ar-H, 4-Ar-2’-H), 6.96-6.98 (d,
2H, 2-Ar-3’-H, J=7.53), 6.89-6.92 (d, 2H, 4-Ar-3’-H, J=7.95), 6.38 (s, 1H, NH), 4.06 (t, 2H,
OCH2, J=5.27Hz), 3.88 (s, 3H, OCH3), 2.89 (t, 2H, OCH2CH2N, J=5.37Hz), 2.63-2.67 (m,
4H, NCH2CH3), 1.06-1.10 (t, 6H, NCH2CH3, J=6.57Hz). 13C NMR (75 MHz, CDCl3): δ 162.8,
162.0, 159.3, 157.8, 155.5, 132.6, 131.5, 130.2, 130.1, 129.7, 129.6, 129.2, 122.9, 114.6,
114.5, 113.7, 66.8, 55.4, 51.7, 47.8, 11.8. MS (ESI, m/z): 503.2[M+H]+.
4.1.20.
6-chloro-2-(4-methoxyphenyl)-5-phenyl-N-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)pyrimidin-4-a
mine II-6

ACCEPTED MANUSCRIPT
Yellow solid (98 mg, 32.7%) Mp 87-89°C; IR (KBr): 3611, 3418, 3343, 2965, 2826, 1600,
1
1573, 1539, 1407, 1256, 1167, 1026, 981, 795 cm-1; H-NMR (300MHz, CDCl3): δ (ppm)
8.34-8.36 (d, 2H, 2-Ar-2’-H, J=7.08Hz), 7.42-7.57 (m, 7H, 5-Ar-H, 4-Ar-2’-H), 6.90-6.98 (m,
4H, 2-Ar-3’-H, 4-Ar-3’-H), 6.36 (s, 1H, NH), 4.11 (brs, 2H, OCH2), 3.88 (s, 3H, OCH3), 2.91
(brs, 2H, OCH2CH2N), 2.63 (brs, 4H, 2, 5-pyrrolidyl-H), 1.82 (brs, 4H, 3, 4-pyrrolidyl-H).
13C NMR (75 MHz, CDCl3): δ 162.8, 162.0, 159.3, 157.8, 155.6, 132.6, 131.4, 130.2, 130.0,
129.7, 129.6, 129.2, 122.8, 114.6, 114.5, 113.7, 66.9, 55.4, 51.7, 47.8, 23.5.MS (ESI, m/z):
501.2[M+H]+.
4.1.21. General synthesis of final compounds I-7~I-9 and II-7~II-9
A mixture of intermediate 6a or 6b (1 equiv), various 4-acetamidophenoxy or
4-thoxyphenoxy (1.0 equiv) and K2CO3 (1.0 equiv.) in anhydrous DMF (8.0 mL) was stirred
at 80 °C for 4 h. After completion of the reaction as indicated by TLC, the mixture was cooled
to room temperature and then extracted, evaporated and purified by flash column
chromatography (CH2Cl2/CH3OH) on silica gel to afford the corresponding pure products
I-7~I-9 and II-7~II-9.
4.2.22.
2-(4-((6-chloro-2-(4-methoxyphenyl)pyrimidin-4-yl)oxy)phenoxy)-N,N-dimethylethanamine
I-7
1
Yellow solid (83 mg, 26.6%). H-NMR (300MHz, CDCl3): δ (ppm) 8.24-8.27 (dd, 2H,
2-Ar-2’-H, J=6.99Hz, J=1.95Hz), 7.11-7.14 (dd, 2H, 4-Ar-2’-H, J=6.81Hz, J=2.28Hz),
6.98-7.01 (dd, 2H, 4-Ar-3’-H, J=6.84Hz, J=2.28Hz), 6.91-6.94 (dd, 2H, 2-Ar-3’-H, J=7.08Hz,
J=1.92Hz), 6.61 (s, 1H, 5-Ar-H), 4.09-4.13 (t, 2H, OCH2CH2N, J=5.67Hz), 3.87 (s, 3H,
OCH3), 2.76-2.80 (t, 2H, OCH2CH2N, J=5.64Hz), 2.38 (s, 6H, N(CH3)2). δ 170.8, 164.9,
162.4, 161.9, 156.7, 145.7, 130.4, 128.2, 122.6, 115.4, 113.7, 103.6, 66.5, 57.9, 55.3, 55.1.
MS (ESI, m/z): 400.1[M+H]+.
4.2.23.
2-(4-((6-chloro-2-(4-methoxyphenyl)pyrimidin-4-yl)oxy)phenoxy)-N,N-diethylethanamine I-8
Yellow solid (0.187 g, 56.0%). Mp 59-61°C; IR (KBr): 3448, 3113, 2965, 2934, 2785, 1664,
1
1607, 1541, 1503, 1253, 1198, 1036, 962 cm-1; H-NMR (300MHz, CDCl3): δ (ppm)
8.24-8.27 (d, 2H, 2-Ar-2’-H, J=8.73Hz), 7.10-7.13 (d, 2H, 4-Ar-2’-H, J=8.88Hz), 6.91-6.99
(m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 6.60 (s, 1H, 5-Ar-H), 4.07-4.11 (t, 2H, OCH2CH2N,

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J=6.15Hz), 3.86 (s, 3H, OCH3), 2.89-2.93 (t, 2H, OCH2CH2N, J=5.64Hz), 2.64-2.71 (q, 4H,
N(CH2CH3)2, J=7.11Hz), 1.08-1.13 (t, 6H, N(CH2CH3)2, J=7.11Hz). 13C NMR (75 MHz,
CDCl3): δ 170.8, 164.8, 162.5, 162.0, 156.6, 145.7, 130.4, 128.4, 122.4, 115.4, 113.8, 103.6,
66.4, 57.9, 55.4, 55.1, 25.9. MS (ESI, m/z): 428.2[M+H]+.
4.2.24. 4-chloro-2-(4-methoxyphenyl)-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)pyrimidine I-9
Yellow solid (0.167 g, 50.3%). Mp 82-84°C; IR (KBr): 3422, 3073, 2922, 2818, 2786, 1609,
1
1541, 1502, 1253, 1192, 1087, 964 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.24-8.27 (d,
2H, 2-Ar-2’-H, J=8.91Hz), 7.10-7.13 (d, 2H, 4-Ar-2’-H, J=9.03Hz), 6.91-7.01 (m, 4H,
4-Ar-3’-H, 2-Ar-3’-H), 6.61 (s, 1H, 5-Ar-H), 4.13-4.17 (t, 2H, OCH2CH2N, J=5.91Hz), 3.86
(s, 3H, OCH3), 2.93-2.97 (t, 2H, OCH2CH2N, J=5.88Hz), 2.66 (brs, 4H, 2, 5-pyrrolidyl-H),
13
1.84 (brs, 4H, 3, 4-pyrrolidyl-H). C NMR (75 MHz, CDCl3): δ 170.9, 164.8, 162.4, 162.1,
156.5, 145.8, 130.4, 128.3, 122.4, 115.4, 113.8, 103.7, 66.3, 57.9, 55.5, 46.3, 23.8. MS (ESI,
m/z): 426.1[M+H]+.
4.2.25.
2-(4-((6-chloro-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-yl)oxy)phenoxy)-N,N-dimethyletha
namine II-7
Yellow solid (0.273 g, 63.0%). Mp 97-100°C; IR (KBr): 3448, 2932, 2818, 2770, 1601, 1570,
1
1500, 1406, 1253, 1189, 972, 845 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.17-8.20 (d,
2H, 2-Ar-2’-H, J=8.37Hz), 7.48-7.52 (m, 5H, 5-Ar-H), 7.07-7.10 (d, 2H, 4-Ar-2’-H),
6.90-6.98 (m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 4.09-4.12 (t, 2H, OCH2CH2N, J=5.07Hz), 3.86 (s,
3H, OCH3), 2.76-2.79 (t, 2H, OCH2CH2N, J=4.80Hz), 2.39 (s, 6H, N(CH3)2). 13C NMR (75
MHz, CDCl3): δ 167.6, 162.40, 162.3, 160.2, 156.2, 146.3, 131.8, 130.3, 130.2, 128.5, 128.3,
122.6, 117.8, 114.9, 113.8, 66.4, 58.4, 55.4, 46.0. MS (ESI, m/z): 476.3[M+H]+.
4.2.26.
2-(4-((6-chloro-2-(4-methoxyphenyl)-5-phenylpyrimidin-4-yl)oxy)phenoxy)-N,N-diethylethana
mine II-8
Yellow solid (0.262 g, 57.1%). Mp 93-96°C; IR (KBr): 3447, 2969, 2917, 2848, 1602, 1569,
1
1498, 1405, 1251, 1188, 1168, 971 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 8.17-8.20 (d,
2H, 2-Ar-2’-H, J=8.82Hz), 7.45-7.52 (m, 5H, 5-Ar-H), 7.07-7.10 (d, 2H, 4-Ar-2’-H,
J=8.97Hz), 6.90-6.96 (m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 4.07-4.11 (t, 2H, OCH2CH2N,
J=6.15Hz), 3.86 (s, 3H, OCH3), 2.89-2.94 (t, 2H, OCH2CH2N, J=6.18 Hz), 2.65-2.72 (q, 4H,

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N(CH2CH3)2, J=7.11Hz), 1.09-1.13 (t, 6H, N(CH2CH3)2, J=7.11Hz). 13C NMR (75 MHz,
CDCl3): δ 167.6, 162.4, 162.3, 160.2, 156.2, 146.3, 131.8, 130.3, 130.2, 128.5, 128.3, 122.6,
117.8, 114.9, 113.8, 67.0, 55.4, 51.8, 47.9, 11.9. MS (ESI, m/z): 504.4[M+H]+.
4.2.27.
4-chloro-2-(4-methoxyphenyl)-5-phenyl-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)pyrimidine
II-9
Yellow solid (0.281 g, 61.5%). Mp 121-123°C; IR (KBr): 3448, 2956, 2931, 2812, 1602,
1
1571, 1500, 1405, 1250, 1192, 1028, 973 cm-1; H-NMR (300MHz, CDCl3): δ (ppm)
8.17-8.20 (d, 2H, 2-Ar-2’-H, J=8.76Hz), 7.46-7.52 (m, 5H, 5-Ar-H), 7.06-7.09 (d, 2H,
4-Ar-2’-H, J=8.91Hz), 6.90-6.97 (m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 4.13-4.17 (t, 2H,
OCH2CH2N, J=5.82Hz), 3.86 (s, 3H, OCH3), 2.93-2.97 (t, 2H, OCH2CH2N, J=5.79Hz), 2.68
(brs, 4H, 2, 5-pyrrolidyl-H), 1.85 (brs, 4H, 3, 4-pyrrolidyl-H). 13C NMR (75 MHz, CDCl3): δ
167.7, 162.4, 162.3, 160.1, 156.2, 146.4, 131.9, 130.2, 130.2, 128.5, 128.3, 122.7, 117.9,
115.0, 113.9, 67.0, 55.4, 51.8, 48.5, 12.1.MS (ESI, m/z): 502.4[M+H]+.
4.2.28. General synthesis of final compounds II-4OH, II-5OH and II-9
A solution of II-4 or II-5 or II-9 (0.23 mmol) in dry CH2Cl2 (5mL) was cooled to -30°C
by cryotrap. A solution of BBr3 (2.4mmol) in dry CH2Cl2 (2.4mL) was added dropwise under
nitrogen. The mixture was gently warmed to room temperature and stirred for 4 h. The
reaction mixture was quenched with ice water, then saturated sodium bicarbonate solution
was adjusted to pH=8 and further purified by silica gel column chromatography to afford the
demethylation products II-4OH, II-5OH and II-9.
4.2.29.
4-(4-chloro-6-((4-(2-(dimethylamino)ethoxy)phenyl)amino)-5-phenylpyrimidin-2-yl)phenol
II-4OH
Yellow solid (62.3%). 1H-NMR (300MHz, CDCl3) : δ (ppm) 10.09 (s, 1H, OH), 8.17-8.20 (dd,
2H, 2-Ar-2’-H, J=7.05Hz, J=1.98Hz), 7.46-7.52 (m, 5H, 5-Ar-H), 7.07-7.10 (dd, 2H,
4-Ar-2’-H, J=6.81Hz, J=2.19Hz), 6.90-6.96 (m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 4.13-4.17 (t, 2H,
OCH2CH2N, J=5.70Hz), 3.86 (s, 3H, OCH3), 2.73 (brs, 2H, OCH2CH2N), 2.35(s, 6H,
N(CH3)2).MS (ESI, m/z) : 461.2[M+H]+.
4.2.30.

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4-(4-chloro-6-((4-(2-(diethylamino)ethoxy)phenyl)amino)-5-phenylpyrimidin-2-yl)phenol
II-5OH
Yellow solid (54.1%); IR (KBr) : 3178, 2937, 2852, 2792, 1561, 1506, 1380, 1251, 1169,
1
1035, 823 cm-1; H-NMR (300MHz, CDCl3): δ (ppm) 10.15 (s, 1H, OH), 8.16-8.19 (dd, 2H,
2-Ar-2’-H, J=6.96Hz, J=2.04Hz), 7.46-7.52 (m, 5H, 5-Ar-H), 7.06-7.09 (dd, 2H, 4-Ar-2’-H,
J=6.81Hz, J=2.25Hz), 6.89-6.95 (m, 4H, 4-Ar-3’-H, 2-Ar-3’-H), 4.12-4.16 (t, 2H,
OCH2CH2N, J=5.82Hz), 3.85 (s, 3H, OCH3), 2.89 (t, 2H, OCH2CH2N, J=5.37Hz), 2.63-2.67
(m, 4H, NCH2CH3), 1.06-1.10 (t, 6H, NCH2CH3, J=6.57Hz). MS (ESI, m/z): 490.2[M+H]+.
4.2.31. 4-(4-chloro-5-phenyl-6-(4-(2-(pyrrolidin-1-yl)ethoxy)phenoxy)pyrimidin-2-yl)phenol
II-9OH
Yellow solid (57 mg, 58.5%). Mp 203-205°C; IR (KBr): 3431, 2919, 2481, 1569, 1499, 1408,
1
1379, 1236, 1188, 1165, 972 cm-1; H-NMR (300MHz, DMSO): δ (ppm) 10.17 (s, 1H, OH),
7.90-7.93 (d, 2H, 2-Ar-2’-H, J=8.82Hz), 7.42-7.59 (m, 5H, 5-Ar-H), 7.16-7.19 (d, 2H,
4-Ar-2’-H, J=9.06Hz), 6.99-7.02 (d, 2H, 4-Ar-3’-H, J=9.09Hz), 6.80-6.83 (d, 2H, 2-Ar-3’-H,
J=8.79Hz), 4.10-4.14 (t, 2H, OCH2CH2N, J=5.79Hz), 2.91 (brs, 2H, OCH2CH2N), 2.65 (brs,
4H, 2, 5-pyrrolidyl-H), 1.72 (brs, 4H, 3, 4-pyrrolidyl-H). 13C NMR (75 MHz, DMSO): δ
164.6, 162.9, 162.1, 160.7, 157.0, 130.2, 130.1, 129.3, 125.7, 115.5, 113.7, 99.2, 66.2, 57.9,
55.4, 55.1, 25.9. MS (ESI, m/z): 488.2[M+H]+.
4.2 Biological evaluation
4.2.1. ERα Binding Affinity Assay.
The recombinant ERα (Thermo Fisher Scientific Inc., Invitrogen, USA) and the
fluorescent estrogen ligands (self-made) were removed from the -80oC freezer and thawed on
ice for 1 h prior to use. The fluorescent estrogen ligand was added to the ERα and screening
buffer (ES2 Screening Buffer, Invitrogen, USA) was added to make the final concentration 9
nM for fluorescent estrogen and 30 nM for ERα. Test compounds were accurately weighed
and dissolved in DMSO, screening buffer was added to dilute to required concentration. Test
compound (1 µL) was added to 49 µL screening buffer in each well (384-well microplate,
Corning, USA). To this 50 µL of the fluorescent estrogen/ER complex was added to make up
a final volume of 100 µL. A positive control contained 50 µL estradiol buffer (1nM) and 50
µL fluorescent estrogen/ER complexes. A negative control contained 50 µL screening buffer
and 50 µL fluorescent estrogen/ER complexes. The negative control was used to determine
the polarization value when no competitor was present (theoretical maximum polarization).

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The microplate was incubated in the dark at room temperature for 2 h and shaken on a plate
shaker. The polarization values were read on a Safire microplate reader and used to calculate
the IC50 values.
4.2.2. VEGFR-2 kinase inhibitory assay
VEGFR-2 kinase assay was conducted using homogeneous time resolved fluorescence
(HTRF) assay (HTRF ® KinEASE™ TK, Cisbio Bioassays, Codolet, France). The general
procedures were as following: VEGFR-2 kinase, substrates, ATP and test compound were
mixed and incubated in a final buffer with the total volume of 10 µL in 384-well microplate.
Wells containing the substrate and the kinase without compound were used as total reaction
control. The assay plate was incubated at 30oC in the dark for 1 h. Detection was performed
with HTRF module of Beckman Coulter detection platform. Inhibition %= [1-(Sample
Ratio-Negative Ratio) / (Positive Ratio-Negative Ratio)] ×100%.
4.2.3. MTT assay for anti-proliferative activities
Cells were cultured in RPMI1640 medium (containing 10% (v/v) FBS, 100 U/mL
Penicillin and 100 mg/mL Streptomycin) in a 5% CO2-humidified atmosphere at 37oC. Cells
were trypsinized and seeded at a density of 1 x 105/mL into a 96-well plate (100 mL/well) and
incubated at 37oC, 5% CO2 atmosphere for 24 h. After this time they were treated with 100
mL/well medium containing test compounds which had been pre-prepared to provide the
concentration range of 4 x 10-5 mol/L, 2 x 10-5 mol/L, 1 x 10-5 mol/L, 1 x 10-6 mol/L, 1 x
10-6 mol/L and 1 x 10-7 mol/L, and re-incubated for a further 48 h. Control wells were added
the equivalent volume of medium containing 1% (v/v) DMSO. 20 µL MTT (5 mg/mL) was
added and cells continued to incubate in darkness at 37oC for 4 h. The culture medium was
then removed carefully and 150 mL DMSO was added. The cells were maintained at room
temperature in darkness for 20 min to ensure thorough color diffusion before reading the
absorbance. The absorbance values were read at 490 nm for determination of IC50 values.
4.2.4. Real-Time Polymerase Chain Reaction (RT-PCR).
RNA samples were reverse transcribed to cDNA and the PCR reactions were performed
using TaKaRa SYBR Green Master Mix (Code. no. 638320) carried out in StepOnePlusTM
Real-Time PCR instrument (4376600, Life Technologies). The program for amplification was
1 cycle of 95°C for 2 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 95°C for
10 s. The PCR results were normalized to GAPDH expression and were quantified by the

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∆∆CT method.
4.2.5. Chicken chorioallantoic membrane (CAM) assay
Fertilized eggs were incubated for 7 days in a humidified environment at 37oC with 5%
CO2 in air and saturated humidity. Then, a window of approximately 1 cm2 was opened on the
egg shell to expose the CAM. Test compounds and positive control Sunitinib dissolved in
DMSO were placed on sterile methyl cellulose filter papers with phosphate buffered saline as
the blank control. The papers were then placed on the CAM. The window was sealed with
sterile cellophane tape. The eggs were further incubated at 37oC under a constant relative
humidity of 60% for 72 h. After fixed with acetone and ethanol for 10min, the CAM was cut
and papers removed to observe angiogenesis. Images of the control and sample-treated areas
were captured.
4.2.6. Cellular apoptosis study
Annexin V/PI staining assay was used to assess the mechanism of cell death. MCF-7
cells were treated with II-9OH at concentration of 10 µM for 48 h. Then, cells were collected,
washed with PBS, and stained with annexin V fluorescein isothiocyanate (FITC) and PI for
15 min at RT. After that, the samples were analyzed by flow cytometry using FACScalibur
(Becton Dickinson). The cell distributions were calculated using Cell Quest software (Becton
Dickinson).
4.2.7. Transwell invasion assay
Invasion assays were performed following the manufacturer’s instructions (BD
Biosciences). Cells were treated at time of plating. After 24 h (as noted in the legend),
invasion cells were fixed to the Matrigel (BD Biosciences)-coated transwell membranes with
a seeding density of 5×104 cells per well for 48 h and stained with crystal violet (0.1 % in 20 %
methanol), and invaded cells visualized and quantified by microscopy.
4.2.8. Western Blots.
Cells with different treatments for 24 h were washed twice with PBS, then collected and
lysed in lysis buffer (100 mM of Tris-Cl, pH 6.8, 4% (m/v) SDS, 20% (v/v) glycerol, 200 mM
of β-mercaptoethanol, 1mM of PMSF, 0.1 mM NaF and DTT) for 1 h on the ice. The lysates
were then subjected to centrifugation (13,000 rpm) at 4 °C for 20 min. Protein concentration
in the supernatants was detected by BCA protein assay (Thermo, Waltham, MA). Then equal

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amount of protein was separated with 12% SDS-PAGE and transferred to polyvinylidene
difluoride (PVDF) membranes (Millipore, Bedford, MA) using a semi-dry transfer system
(Bio-rad, Hercules, CA). Proteins were detected using specific antibodies overnight at 4°C
followed by HRP-conjugated secondary antibodies for 1 h at 37 °C. All of the antibodies were
diluted in PBST containing 1% BSA. Enhanced chemiluminescent reagents (Beyotime,
Jiangsu, China) were used to detect the HRP on the immunoblots, and the visualized bands
were captured by film. The bands were quantified by Quantity One software (Vision 4.62,
Bio-rad, Hercules, CA), and the relative protein level were normalized to β-actin.
4.2.9. Molecular modeling
The molecular modeling was performed with Discovery Studio.3.0/CDOCK protocol
(Accelrys Software Inc.). The crystal structures of ERα complexed with 4-hydroxytamoxifen
(PDB code: 3ERT) and VEGFR-2 (PDB ID code: 3CJF) were downloaded from Protein Data
Bank. Compound II-9OH was drowned and optimized using Hyperchem v7.0. The protein
and ligand were optimized and charged with CHARMm force field to perform docking. Up to
10 conformations were retained, and binding modes presented graphically are representative
of the highest-scored conformations.
Supplementary data
Supplementary data (the 1H NMR and 13C NMR spectra of target compounds associated with
this article can be found, in the online version, at.
Acknowledgements
This work was supported by grants from the Natural Science Foundation of China (NSFC,
81373279).
Abbreviations
ER, estrogen receptor; SERMs, selective estrogen receptor modulators; VEGF, Vascular
endothelial growth factor; VEGFR-2, Vascular endothelial growth factor receptor-2; RTK,
receptor tyrosine kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular
regulated kinases; SAR, structure-activity relationship; HUVEC, human umbilical vein
endothelial cell; RT-PCR, real-time polymerase chain reaction; PgR, progesterone receptor;
E2, estradiol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.