Design, synthesis and biological evaluation of novel indone derivatives as selective ERβ modulators

Article abstract of DOI:10.1007/s00044-019-02355-z

To reduce the endometrial toxicity and improve the efficacy of current selective estrogen receptor modulators used in breast cancer treatment by enhancing ERβ selectivity, inspired by active resveratrol oligomer, a series of analogs (5a–f, 6a–b, 7a–d, 8a–

Full text of DOI:10.1007/s00044-019-02355-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02355-z
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of novel indone
derivatives as selective ERβ modulators
Xi-Xi Liu1 Mei-Lin Tang1,4 Chen Zhong1 Yun Tang3 Jian-Ming Yu1 Xun Sun1,2
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Received: 26 November 2018 / Accepted: 30 April 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
To reduce the endometrial toxicity and improve the efficacy of current selective estrogen receptor modulators used in breast
cancer treatment by enhancing ERβ selectivity, inspired by active resveratrol oligomer, a series of analogs (5a–f, 6a–b,
7a–d, 8a–d) were designed, synthesized and biologically evaluated. Among them, the chiral indone analog (2R,3R)-8a
exhibited best antiproliferative activity against both breast cancer cell lines (MDA-MB-231 and MCF-7) and better safety
profile on uterus than tamoxifen. Analog (2R,3R)-8a demonstrated good binding affinity and selectivity toward ERβ, which
was further proved by both molecular docking and radiometric competitive binding assay. Other studies for (2R,3R)-8a also
have been explored including cell cycle and apoptosis evaluation and in vitro metabolic stability studies. These results
demonstrated that (2R,3R)-8a could be a promising lead compound for future exploration of selective ERβ anti-breast cancer
agents.
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Keywords Resveratrol oligomer ERβ Breast cancer SERM
Introduction
along with various drawbacks, such as high rates of recur-
rence, metastasis, severe toxicity, and drug resistance.
Selective estrogen receptor modulators (SERMs) (Fig. 1)
are widely used in preventing or treating estrogen-related
diseases including breast cancer and osteoporosis (Howell
and Evans 2013). However, despite their promising effec-
tiveness and widely usage, SERMs still suffer from
unsolved safety issues. For example, tamoxifen enhances
the risk of endometrial cancer and liver damage, whereas
raloxifene may increase the chance of stroke (Jordan 2004;
Ellis et al. 2015). Since the discovery of the two subtypes,
ERα and ERβ (Mosselman et al. 1996; Paterni et al. 2014),
the lack of ER subtype and tissue selectivity of these
SERMs has been considered to be responsible for most of
the side effects. So far, most preliminary SERM studies
were focused on the investigation of ERα-selective agents.
However, disadvantages have been observed for ERα-
selective SERMs such as causing severe side effects in ERα
highly expressed tissues including liver, uterus, and cardi-
ovascular system (Skliris et al. 2008). Meanwhile, the tissue
distribution of ERβ is quite different from ERα. ERβ
expresses very few in liver and uterus, which may reduce
the influence on these tissues by selective ERβ modulators
(Skliris et al. 2008; Dotzlaq 1997). Thus, ERβ-selective
ligands could be utilized as novel anti-breast cancer agents
Breast cancer has become a great threat to females,
accounting for 24.2% of all cancer cases in women and
causing 626,679 deaths worldwide in 2018, according to the
global cancer statistics 2018 (Bray et al. 2018). Surgery,
chemotherapy, and newly antibody therapies like HER2-
targeted therapy have been used to cure different types of
breast cancer (Figueroa-Magalhães et al. 2014), but still
Supplementary Information The online version of this article
(https://doi.org/10.1007/s00044-019-02355-z) contains supplementary
material, which is available to authorized users.
* Xun Sun
sunxunf@shmu.edu.cn
1
Department of Natural Products Chemistry, School of Pharmacy,
Fudan University, Shanghai, China
2
The Institutes of Integrative Medicine of Fudan University,
Shanghai, China
3
Shanghai Key Laboratory of New Drug Design, School of
Pharmacy, East China University of Science and Technology,
Shanghai, China
4
State Key Laboratory of Molecular Engineering and Institutes of
Biomedical Sciences, Fudan University, 220 Handan Road,
Shanghai 200433, China

Medicinal Chemistry Research
which is more consistent with ERβ ligands model. Herein,
two strategies were applied to design novel analogs by
maintaining or reducing hydroxyl group(s) on phenyl
rings (analogs 5 and 7), which may strengthen its good
binding affinity to ERβ or methylating hydroxyl group(s)
on phenyl rings (analogs 6 and 8), which may increase the
metabolic stability. Thus, total 16 novel analogs (5a–f,
6a–b, 7a–d, and 8a–d) were designed and synthesized
and their biological activities were evaluated.
N
N
O
O
N
O
O
O
O
OH
S
HO
S
HO
Tamoxifen (1)
Arzoxifene (3)
Raloxifene (2)
Fig. 1 Selective estrogen receptor modulators (SERMs)
Chemistry
with potentially less side effects (Fox et al. 2008; Hartman
et al. 2009; Ström et al. 2004).
The target compounds 5–8 were synthesized by following
the procedure described in Scheme 1. Commercially avail-
able 1-chloromethyl-4-methoxybenzene and magnesium
were used to produce a Grignard reagent that reacted with
chlorodiphenyl phosphine and then was oxidized to give (4-
methoxybenzyl) diphenylphosphine oxide (Dotzlaq 1997).
After de-methylation, the obtained intermediate 10 reacted
with 2-(benzyloxy)ethyl-4-methylbenzenesulfonate to give
11 in 78% yield. Compound 12 was achieved by deben-
zylation with AlCl3 condition in almost quantative yield.
Direct replacement of tosyl group with various amines
provided 14a–f in 52–81% yields. Subjected 14a–f to
substituted benzils 15 or 16 gave corresponding α,
β-unsaturated ketone intermediates 17a–f or 18a–f as a
mixture of E- and Z-isomer via Wittig–Horner reaction.
Compounds (5a–f, 6a–b, 7a–d, and 8a–d) were synthesized
via Nazarov cyclization using conditions either BF3·Et2O or
BBr3. In order to study the effect of stereochemistry,
enantiomerically enriched compounds (2R,3R)-8a and
(2S,3S)-8a were also separated from its racemic form by
Daicel Chiral Technologies (China) Co., Ltd. using high-
performance liquid chromatography (HPLC) and chiral
column. The absolute configurations were determined by
the circular dichroism (CD) spectra method. The same CD
spectral patterns were observed among previous chiral
synthesized compounds and (2R,3R)-8a, indicating that
they have identical stereochemistry that confirmed the
absolute configurations of two enantiomerically enriched
compounds (Tang et al. 2016).
Previously, Paul Mak et al. (2006) reported that the
natural compound Apigenin can suppress breast cancer
cell growth and induce apoptosis through ERβ. Interest-
ingly, natural compound resveratrol is a polyphenol,
which widely exists in several kinds of plants, and its
anticancer activity has been confirmed by many studies
(Bishayee 2009). Many efforts have been done in our
group in the past years and lots of oligomers have been
synthesized including oligomer derivatives from Car-
agana sinica acting as phytoestrogens (Luo et al. 2001;
Ma et al. 2010; Sun et al. 2006, 2007; Zhu et al. 2008;
Zhong et al. 2011, 2013; Wang et al. 2015; Tang et al.
2016). After biological evaluation of these derivatives, it
was found that isopaucifloral F (Jordan 2004) (Fig. 2)
showed good in vivo anti-osteoporosis activity in rat,
preferring ERβ activity in yeast two-hybrid assay and low
toxicity (Hao et al. 2015). However, isopaucifloral F
(Jordan 2004) did not show anticancer activity in MTT
assay on human breast cancer cell lines MCF-7 and MDA-
MB-231. Based on the general structural formula brought
up by Grese et al. (1997), it was speculated that both trans
1,2-stilbene-like core structure and a dialkylamine side
chain could be pivotal in SERMs for their anticancer
activity. Isopaucifloral F (Jordan 2004) possesses a trans
1,2-stilbene-like core structure but lacks the key dialky-
lamine side chain. Thus, incorporated a dialkylamino-
ethoxyphenyl substituent into isopaucifloral F (Jordan
2004) may enhance its anticancer activity. According to
previous studies, it was found that piperidine or pyrroli-
dine may contribute to achieve better antagonist activity
(Grese et al. 1997). Therefore, when designing the dif-
ferent dialky-substituents on the basic amine of the side
chain, we chose piperidine and its derivatives as the
source for basic amine.
Results and discussion
Cytotoxicity against human breast cancer cells
To further enhance ERβ selectivity, we compared iso-
paucifloral F (Jordan 2004) structure with ERα and ERβ
ligand model developed by Katzenellenboen’s group
(Meri De Angelis et al. 2005). Isopaucifloral F (Jordan
2004) has compact core structure and less substituents,
To evaluate the anticancer activity of designed compounds,
two cancer cell lines MCF-7 (expressing both in ERα and
ERβ) and MDA-MB-231 (expressing only in ERβ) were
chosen to study their in vitro cytotoxicity using standard MTT
assay (Dotzlaq 1997; Mak et al. 2006). The ER expression

Medicinal Chemistry Research
Fig. 2 Strategy for designing new analogs 5–8
model of the two cell lines was confirmed by western blot
analysis (Fig. 3). The activity is expressed as the concentra-
tion of drug inhibition at 50% cell growth (IC50) and the data
are presented in Table 1. Tamoxifen, raloxifene and iso-
paucifloral F were used as controls. Initially, analogs 5, 6, and
7 were taken into examination. The results showed similar
trends in all two cell lines. Isopaucifloral F (Jordan 2004) and
analogs 5, which have four hydroxyls did not show any
anticancer activity for both MDA-MB-231 and MCF-7 cell
lines. However, 7a–b (with two hydroxyls) and 6a–b (with
four methoxys) showed slightly improved activity, whereas
7c–d were inactive for both cell lines even after 72 h treat-
ment. The results indicated that either methylation of hydro-
xyl groups or reduction of hydroxyl groups on the A and C
rings may have positive influence on the anticancer activity.
Based on these results, further structure–activity relationship
study was carried out to design new analogs 8 with methoxyl
substituted on R2. As speculated, analogs 8 did show the best
inhibitory activity in four series.
As rationalized, the side chains of alkylamino groups do
affect the biological activities. Overall, compounds with
piperidine ring (6a and 8a) displayed better activities
against two cell lines, whereas compounds with morpholine
ring (7c and 8d) did not show any or weaker activity.
Compared with control drugs, tamoxifen and raloxifene,
compounds 8a, 8b, and 8c showed similar or improved
activity. Especially, analog 8a exhibited 2.2- and 1.4-fold
more potent than tamoxifen and 2.8- and 1.8-fold more

Medicinal Chemistry Research
Scheme 1 The syntheses of new analogs of 5a–f, 6a–b, 7a–d, and 8a–d
difference on activities. Analog (2R,3R)-8a was more potent
than (2S,3S)-8a against both MDA-MB-231 (3.94 vs.
7.45 µM) and MCF-7 (7.16 vs. 11.64 µM) cell line after 72 h
treatment. This indicated that stereochemistry of com-
pounds does influence the antiproliferative effect on ERβ
expressed cell lines.
Docking analysis
To explore the ER subtype selectivity of active com-
pounds and elucidate the binding model of active com-
pounds with ERβ receptor, molecule docking analysis was
undertaken using Schrodinger software (Schrödinger
version 9.3, Schrödinger, LLC, New York, NY). The
crystal structure of human ER5β ligand-binding domain
(LBD) in complex with 4-hydroxytamoxifen (PDB ID:
2FSZ) and estrogen receptor α LBD in complex with
raloxifene (PDB ID: 2JFA) were used for the docking.
The protein was prepared using Maestro module of
Schrodinger software. During protein preparation, correct
bond orders were assigned and hydrogen along with other
missing atoms were added. Residues and charges were
assigned to atoms using OPLS-AA force field and the co-
crystallized waters were retained for docking. The
Fig. 3 The expression of ERα and ERβ in MCF-7 and MDA-MB-231
was shown by western blot analysis. No ERα was detected in MDA-
MB-231, whereas both ERα and ERβ were detected in MCF-7
potent than raloxifene against MDA-MB-231 and MCF-7
cell line, respectively, after 72 h treatment.
As 8a had optical enantiomers, to further study whether
the chiral structure affected the anticancer activity of the
compounds, we separated 8a with HPLC and chiral column
to give (2S,3S)-8a (½αŠ20 = +151.5 (c = 0.4 in MeOH)) and
D
(2R,3R)-8a (½αŠ20 = −151.5 (c = 0.4 in MeOH). Interest-
D
ingly, the separated chiral compounds showed the

Medicinal Chemistry Research
Table 1 New compound in vitro cytotoxicity on human breast cancer cell lines MDA-MB-231 and MCF-7
NA not active, means IC50 cannot be calculated from MTT assay data; ND not determined
aResults were showed as means SD (n = 3) of at least three independent experiments

Medicinal Chemistry Research
docking was done using Glide module of the Schrodinger
software. The docking grid was generated using co-
crystallized ligand (4-hydroxytamoxifen) as grid center
with default settings. The ligands were sketched in
Maestro module of Schrodinger software and were pre-
pared using Ligprep module of Schrodinger software. The
docking was performed with standard precision mode of
Glide.
Compounds with strong activity against breast cancer
cell lines were examined. Compound (2R,3R)-8a had better
selectivity (ERβ/α = 1.93) than tamoxifen (ERβ/α = 0.89)
(Table 2). Also, (2R,3R)-8a achieved good glide score with
ERβ (glide score = −9.203) (Table 2). The docking results
for (2R,3R)-8a showed a similar binding mode as 4-
hydroxytamoxifen in ERβ-binding site, and the best pose
was selected on the basis of the best docking score. The
indone scaffold fitted into the hydrophobic pocket consists
of Ile373 and Met336 residues, which distinguish ERβ from
ERα (Gim et al. 2014). The methoxyl groups of the mole-
cule formed hydrogen-bonding interactions with His475
and Arg346 and the ππ stacking interaction was also
observed between 4-methoxylphenyl and Phe356. Further,
the nitrogen atom of the piperidine group on (2R,3R)-8a had
an electrostatic interaction with Asp303 (Fig. 4).
Oil/water partition coefficient assessment
The ability of organic chemicals to elicit certain therapeutic
effect is strongly influenced by the partitioning tendency of
these chemicals between the organism and water. The oil/
water partition coefficient (log P) could be expressed as a ratio
of n-octanol solubility to aqueous solubility. We determined
the log P value of 2 in n-octanol/water at 37 °C. The result
showed that (2R,3R)-8a has an appropriate oil/water partition
coefficient (log P = 2.3365, R2 = 0.9999), According to the
Lipinski’s rule of five (Lipinski et al. 2001), (2R,3R)-8a
showed its potential as an orally active drug.
Uterine wet weight assay and morphometric
analysis
The uterotrophic assay is a well-established method to
measure the effect of a compound on uterine. As the
induction of uterine cancer is a main side effect of tamox-
ifen, its estrogenicity of increasing uterine wet weight and
luminal epithelial cell height (LECH) was detected by this
method reported in previous studies. Fong et al. (2007) To
investigate the stimulatory effect on uterine of (2R,3R)-8a,
the uterotrophic assay was taken and uterine wet weight,
luminal epithelial cell height, and luminal circumference
were measured. An ovariectomized (OVX) mice model and
30 healthy female kunming mouse were randomly assigned
to five groups: a sham operated group in which only a piece
of fat around the ovary was ablated (SHAM) and four
groups of OVX mouse, including the placebo (OVX+NS),
the tamoxifen positive control (OVX+TAM), low dosage
and high dosage (OVX+(2R,3R)-8a) group. Compared with
the vehicle OVX+NS group, OVX+TAM group and
SHAM group showed significant increase in all three indi-
cators, while OVX+(2R,3R)-8a groups exhibited no stimu-
lating effects on any aspects measured (Table 4 and Fig. 5),
which indicated that (2R,3R)-8a may have beneficial com-
pared with tamoxifen in overcoming the endometrial side
effect.
ER-binding affinity
Based on the result of the MTT assay and molecule dock-
ing, the most-effective compounds were chosen to investi-
gate their binding affinity and selectivity to ERα and ERβ
by a radiometric competitive binding assay (Table 3). All
compounds showed better selectivity for ERβ than ERα. In
particular, (2R,3R)-8a exhibited 6.98 times higher
ERβ-binding affinity than ERα. The ERβ-binding results
exhibited good consistency with the cytotoxicity results.
Compound (2R,3R)-8a displayed better binding affinity
than its counterpart enantiomer, which indicates that ste-
reochemistry configurations have significant effects on
receptor–ligand interactions. Compound (2R,3R)-8a, with
the highest binding affinity among tested compounds, the
IC50 value for ERβ was determined to be 0.513 μM.
Cell circle analysis and evaluation of apoptosis with
flow cytometry
Further investigation of effects of (2R,3R)-8a on MCF-7
cell apoptosis and cell cycle suggested that it might induce
MCF-7 cell apoptosis dose dependently and affect cell
circle by arresting cells at G0/G1 phase at a concentration
of 4.50 μM (Table 5). G0/G1 arrest caused by tamoxifen
was also observed by other studies (Khamis et al. 2018).
In Annexin V-FITC/PI dual staining assay, MCF-7 cells
were treated with (2R,3R)-8a for 72 h at three different
concentrations, respectively, to examine the apoptotic
effect (Table 6). The results demonstrated that (2R,3R)-8a
Table 2 Docking results of active compounds (8a and 8c) and ERs
Compound
Glide score
β/α
ERα
ERβ
−9.20
(2R, 3R)-8a
(2S, 3S)-8a
(2R, 3R)-8c
(2S, 3S)-8c
Tamoxifen
−4.76
−3.87
–
1.93
1.54
–
−5.98
−3.610
−3.49
−3.44
−12.96
1.01
0.89
−11.54

Medicinal Chemistry Research
Fig. 4 Interactions between
ERβ-binding pocket and
(2R,3R)-8a;
Table 3 Results of radiometric competitive binding assay
commonly used as candidate screening tool in the early
stage of drug development. Analog (2R,3R)-8a was
incubated with rat liver microsome at 37 °C for 1 h. The
results showed that T1/2 and CLint of (2R,3R)-8a were
63 min and 0.022 mL/min/mg, whereas T1/2 and CLint of
isopaucifloral F were > 145 min and < 0.001 mL/min/mg.
The results indicated the metabolic stability of (2R,3R)-8a
and contributed to its druggability. Shorter T1/2 might be
the influenced by the oxidation of its side chain substitute.
Further in vivo study and analysis will be undertook to
confirm the prediction and explore the metabolic
pathways.
Compound
ER-binding affinity %
max (20 μM)
β/α ratio
ERα
ERβ
8a
42.48
36.74
30.10
49.28
170.97
67.52
4.02
1.84
6.98
2.74
8c
(2R,3R)-8a
(2S,3S)-8a
210.33
135.30
showed a trend to induce apoptosis against MCF-7 cells.
It induced 44.7%, 59.0%, and 88.6% early and late
apoptosis at 4.50, 6.00 and 7.50 μM concentrations,
respectively, for 72 h. Although the cell cytostatic and
cytotoxic capability of (2R,3R)-8a demonstrated similar
patterns with tamoxifen, further studies on gene expres-
sion level are still needed to learn more about the detailed
mechanism.
Materials and methods
Materials and physical measurements
Reagents were purchased from Aldrich, TCI, Shaoyuan and
Energy Chemical companies. [2,4,6,7-3H]-E2 (81.0 ci/
mmol) was purchased from PerkinElmer Company. All
solvents were purified and dried in accordance with stan-
dard procedures unless otherwise indicated. Oxygen-free
and water-free operations were carried out under N2 in dried
glassware unless otherwise noted. Reactions were
In vitro metabolic stability studies
As analog (2R,3R)-8a has performed noticeably in former
studies, it was decided to conduct in vitro metabolic sta-
bility experiment with rat liver microsome, which is

Medicinal Chemistry Research
Table 4 Effect of (2R,3R)-8a on uterine wet weight, luminal epithelial cell height (LECH), and luminal circumference
Uterine wet weight (g)a
Luminal epithelial cell height (μm)b
Luminal circumference (mm)b
OVX+NS
0.06 0.03
0.05 0.01
0.06 0.01
0.16 0.04*
0.17 0.05*
48.83 5.95
42.20 11.00
–
0.69 0.28
1.09 0.32
–
OVX+(2R,3R)-8a-low
OVX+(2R,3R)-8a-high
OVX+TAM
114.57 2.65*
77.07 7.29*
1.79 0.57*
2.85 2.03*
SHAM
OVX+NS ovariectomized mice with sesame oi, OVX+TAM ovariectomized mice with 0.1 mg/kg tamoxifen, OVX+(2R,3R)-8a-low ovariectomized
mice with 0.1 mg/kg (2R,3R)-8a, OVX+(2R,3R)-8a-high ovariectomized mice with 1 mg/kg (2R,3R)-8a, SHAM sham-operated group
*P < 0.05 vs OVX+NS
an = 6
bn = 3
Fig. 5 Hematoxylin and eosin-
stained sections of uterine tissue
at × 100 magnification; after
three daily doses of a OVX with
sesame oil, b SHAM with
sesame oil, c OVX with 100 µg/
kg TAM, and d OVX with
100 µg/kg (2R,3R)-8a. TAM
treatment and endogenous
estrogen exposure (SHAM)
induced increases in luminal
epithelial cell height. Luminal
circumference is increased to a
greater degree by endogenous
estrogen exposure than TAM
Table 5 Effect of different concentration of (2R,3R)-8a on cell circle
of MCF-7 cell line after 24 h treatment
recorded on Varian Mercury 300 (300 MHz) or Bruker
DRX-400 (400 MHz) spectrometers and 13C-NMR spectral
Concentration (μM)
apoptosis
G0/G1 phase
S phase
data were recorded on a Bruker DPX-400 (100 MHz)
spectrometer. Low-resolution mass spectra (ESI) were
obtained on a Shimadzu LCMS-2010EV and higher reso-
lution mass spectra were obtained on an IonSpec 4.7 Tesla
FTMS (MALDI) or a Bruker Daltonics, Inc. APEXIII7.0
TESLA FMS (ESI). Melting points (m.p.) were determined
using an X-4 microscope melting point apparatus; Infrared
radiation (IR) spectra were recorded on a Thermo Scientific
Nicolet 380 FT-IR spectrometer. Optical rotations were
measured on an Auto-pol V polarimeter.
0
4.50%
27.81%
40.44%
53.65%
45.70%
31.65%
41.87%
26.48%
27.90%
4.50
6.00
monitored by thin-layer chromatography using Yantai
(China) GF254 silica gel plates (5 × 10 cm). Silica gel col-
umn chromatography was performed on silica gel (300–400
mesh) from Yantai (China). All 1H-NMR spectra were

Medicinal Chemistry Research
Table 6 Effect of different
concentration of (2R,3R)-8a on
apoptosis of MCF-7 cell line
after 72 h treatment
Concentration (μM)
Living cells
Early apoptotic cells
Late apoptotic cells
Apoptosis rate
0
59.2%
54.3%
40.5%
7.1%
5.2%
10.5%
9.7%
35.4%
34.2%
49.3%
78.2%
40.6%
44.7%
59.0%
88.6%
4.50
6.00
7.50
10.4%
1
Chemistry
1056, 753 cm−1; H-NMR (400 MHz, CD3OD) δ 3.73 (d,
J = 13.1 Hz, 2H), 6.58 (d, J = 8.3 Hz, 2H), 6.82–6.99 (m,
(4-Methoxybenzyl)diphenylphosphine oxide (9)
2H), 7.41–7.83 (m, 10H); ESIMS m/z: 309.2 (M+H)+
.
In a 250 mL round-bottom flask, magnesium (3.00 g,
0.13 mol), trace of iodine, and anhydrous tetrahydrofuran
(THF) (190 mL) were charged under N2 at room temperature.
A solution of p-MeO-PhCH2Cl (19.58 g, 0.13 mol) in anhy-
drous THF (10 mL) was added dropwise to the reaction. Then
the reaction was heated and refluxed for 4.5 h until magne-
sium was completely consumed. After the reaction mixture
was cooled down in an ice bath, a mixture of Ph2PCl
(21.24 mL, 0.12 mmol) and anhydrous THF (10 mL) was
added dropwise. Then the reaction mixture was heated to
reflux overnight. The reaction mixture was cooled down and
30% H2O2 (20 mL) was added. After evaporated most of
THF, the residue was extracted with CH2Cl2 (100 mL × 3).
The combined organic layer was washed with saturated
NaHCO3, brine, dried with anhydrous Na2SO4 and con-
centrated. The obtained light yellow solid was washed sub-
sequently with ethyl acetate and methanol to give 9 (28.07 g,
67%) as a white solid, mp: 233–235 °C; IR (film) 2927, 2849,
1666, 1540, 1426, 1310, 1311, 1120, 1046, 1015, 810,
503 cm−1; 1H-NMR (CDCl3, 400 MHz) δ: 3.59 (d, J =
13.2 Hz, 2H), 3.74 (s, 3H), 6.73 (d, J = 8.8 Hz, 2H), 7.02 (d,
J = 7.2 Hz, 2H), 7.43–7.71 (m, 10H); 13C-NMR (CDCl3,
75 MHz) δ: 36.7, 37.3, 55.2, 113.7, 113.9, 113.9, 122.8,
122.9, 128.4, 128.5, 131.1, 131.1, 131.1, 131.2, 131.8, 131.8,
132.8, 158.5, 158.5; MS m/z: 322 (M)+
(4-(2-(Benzyloxy)ethoxy)benzyl)diphenylphosphine oxide (11)
To a solution of (4-hydroxybenzyl)diphenylphosphine
oxide (Fox et al. 2008) (3.08 g, 10 mmol) and 2-(benzy-
loxy)ethyl-4-methylbenzenesulfonate (3.06 g, 10 mmol)
in anhydrous dimethylformamide (DMF), anhydrous
cesium carbonate (13.03 g, 40 mmol), and tetrabutyl
ammonium iodide (TBAI) (154 mg) were added. The
reaction was stirred at 80 °C for 2 h and then quenched
with water. The mixture was extracted with ethyl acetate
(100 mL × 3) and the combined organic layer was washed
with water, brine and dried with anhydrous Na2SO4. After
evaporated the solvent, the obtained solid was washed
with ethyl ether (30 mL) to give 11 (3.45 g, 78%) as white
powder. mp: 221–222 °C; IR (film) 2917, 2822, 1709,
1623, 1514, 1477, 1354, 1347, 1266, 1078, 754 cm−1; 1H-
NMR (400 MHz, CDCl3) δ 3.61 (d, J = 13.5 Hz, 2H),
3.75–3.82 (m, 2H), 4.04–4.10 (m, 2H), 6.75 (d, J =
8.5 Hz, 2H), 7.00 (dd, J = 2.0, 8.6 Hz, 2H), 7.26–7.68 (m,
15H); 13C-NMR (100 MHz, CDCl3) δ 67.3, 68.4, 73.3,
114.6, 127.7, 127.8, 128.4, 128.5, 131.0, 131.1, 131.2,
131.7; ESIMS m/z: 443.0 (M+H)+
.
(4-(2-Hydroxyethoxy)benzyl)diphenylphosphine oxide (12)
To a solution of {4-[2-(benzyloxy)ethoxy]benzyl}diphe-
nylphosphine oxide (Hartman et al. 2009) (4.42 g, 10 mmol)
in anhydrous CH2Cl2 (100 mL), aluminum trichloride
(2.00 g, 15 mmol) was added under ice bath. The reaction
mixture was stirred at room temperature for 2 h, cooled to
0 °C, and quenched with water. After evaporated most of
CH2Cl2, the solution was extracted with ethyl acetate
(100 mL × 3). The combined organic layer was washed with
water, brine and dried with anhydrous Na2SO4. After eva-
porated the solvent, the obtained solid was further washed
with ethyl ether to give 12 (3.45 g, 98%) as white powder.
mp: 137–138 °C; IR (film) 3324, 3010, 2925, 2873, 1737,
(4-Hydroxybenzyl)diphenylphosphine oxide (10)
To a solution of (4-methoxybenzyl)diphenylphosphine
oxide (Dotzlaq 1997) (3.22 g, 10 mmol) in anhydrous
CH2Cl2 (100 mL), BBr3 (2.4 mL, 25 mmol) was added
under N2 at 0 °C. The reaction was stirred at room tem-
perature for 8 h and then quenched with water. The mix-
ture was extracted with ethyl acetate (100 mL) and the
organic layer was washed with water, brine and dried with
anhydrous Na2SO4. After evaporated the solvent, the
obtained solid was washed with CH2Cl2 to give pure 10
(2.03 g, 66%) as white powder. mp: 177–178 °C; IR (film)
3354, 3005, 2934, 1716, 1688, 1433, 1423, 1334, 1266,
1639, 1482, 1354, 1337, 1206, 1162, 1067, 1023, 853 cm−1
1H-NMR (400 MHz, CDCl3) δ 3.59 (d, J = 13.3 Hz, 2H),
;

Medicinal Chemistry Research
3.90–3.92 (m, 2H), 3.99–4.01 (m, 2H), 6.73 (d, J = 8.4 Hz,
2H), 7.01 (d, J = 8.3 Hz, 2H), 7.41–7.70 (m, 10H);
δ 1.82 (s, 4H), 2.64 (s, 4H), 2.89 (t, J = 5.8 Hz, 2H), 3.61
(d, J = 13.3 Hz, 2H), 4.06 (t, J = 5.9 Hz, 2H), 6.76 (d, J =
8.2 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 7.44–7.73 (m, 10H);
13C-NMR (100 MHz, CDCl3) δ 23.4, 36.8, 37.4, 54.7, 55.0,
66.8, 114.5, 123.0, 128.4, 128.5, 131.0, 131.1, 131.2,
ESIMS m/z: 353.2 (M+H)+
.
2-(4-((Diphenylphosphoryl)methyl)phenoxy)ethyl-4-
methylbenzenesulfonate (13)
131.7, 131.8, 132.9, 157.7; ESIMS m/z: 406.2 (M+H)+
.
To a solution of [4-(2-hydroxyethoxy)benzyl]diphenylpho-
sphine oxide (Ström et al. 2004) (3.52 g, 10 mmol) in
anhydrous CH2Cl2 (100 mL), triethylamine (50 mL) and
paratoluensulfonyl chloride (1.99 g, 10.5 mmol) were
added. The resulting mixture was heated to 50 °C for 20 h
and then quenched with water. The mixture was extracted
with CH2Cl2 (100 mL × 3) and the combined organic layer
was washed with water, brine and dried with anhydrous
Na2SO4. After evaporated the solvent, the obtained solid
was further washed with ethyl ether to give 13 (3.09 g,
61%) as white powder. mp: 245–246 °C; IR (film) 2919,
1742, 1634, 1489, 1356, 1321, 1211, 1150, 1121, 1029,
Diphenyl(4-(2-(2-methylpiperidin-1-yl)ethoxy)benzyl)phos-
phine oxide (14c) White solid, yield 71%. mp: 168–169 °C;
IR (film) 2930, 1724, 1620, 1499, 1357, 1314, 1206, 1142,
1
1133 1009 cm−1; H-NMR (400 MHz, CDCl3) δ 1.12 (d, J
= 5.4 Hz, 3H), 1.31 (s, 2H), 1.60 (s, 4H), 2.35 (s, 2H), 2.80
(s, 1H), 2.94 (d, J = 9.9 Hz, 1H), 3.07 (d, J = 6.5 Hz, 1H),
3.59 (d, J = 13.3 Hz, 2H), 4.02 (s, 2H), 6.72 (d, J = 8.4 Hz,
2H), 7.00 (d, J = 8.3 Hz, 2H), 7.43–7.71 (m, 10H); 13C-
NMR (100 MHz, CDCl3) δ 23.8, 25.8, 34.3, 36.7, 37.4,
52.5, 53.3, 56.4, 65.3, 114.4, 114.4, 122.9, 123.0, 128.4,
128.5, 131.0, 131.1, 131.2, 131.7, 131.7, 131.8, 132.8,
157.6; ESIMS m/z: 434.2 (M+H)+
.
1
1011, 850, 520 cm−1; H-NMR (400 MHz, CDCl3) δ 2.44
(s, 3H), 3.58 (d, J = 13.3 Hz, 2H), 4.03–4.11 (m, 2H),
4.26–4.36 (m, 2H), 6.60 (d, J = 8.4 Hz, 2H), 6.99 (d, J =
6.8 Hz, 2H), 7.26–7.81 (m, 14H).
Diphenyl(4-(2-(4-methylpiperidin-1-yl)ethoxy)benzyl) phos-
phine oxide (14d) White solid, yield 81%. mp: 178–179 °C;
IR (film) 2929, 1734, 1656, 1483, 1337, 1332, 1223, 1145,
1057 cm−1; 1H-NMR (400 MHz, CDCl3)
δ 0.92 (d,
General procedure for the syntheses of (4-(2-(naphthenic
amino)ethoxy)- benzyl)diphenylphosphine oxide (14a–g)
J = 6.1 Hz, 3H), 1.17–1.41 (m, 3H), 1.62 (d, J = 11.8 Hz,
2H), 1.98–2.14 (m, 2H), 2.75 (s, 2H), 2.94 (d, J = 10.3 Hz,
2H), 3.59 (d, J = 13.3 Hz, 2H), 4.04 (t, J = 5.8 Hz, 2H),
6.73 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 6.8 Hz, 2H),
7.29–7.80 (m, 10H); 13C-NMR (100 MHz, CDCl3) δ 21.8,
30.5, 34.1, 36.8, 37.4, 54.5, 57.5, 65.9, 114.5, 123.0, 128.4,
128.5, 131.0, 131.1, 131.1, 131.2, 131.7, 131.9, 132.8,
To a solution of 2-{4-[(diphenylphosphoryl)methyl]phenoxy}
ethyl-4-methylbenzenesulfonate (Mak et al. 2006) (1.00 mmol)
and naphthenic amino (1.00 mmol) in anhydrous DMF
(anhydrous cesium carbonate (4.00 mmol) and TBAI (25 mg)
were added. The reaction was stirred at 80 °C for 2 h and
quenched with water. The mixture was extracted with ethyl
acetate (100 mL × 3) and the combined organic layer was
washed with water, brine and dried with anhydrous Na2SO4.
After evaporated the solvent, the obtained solid was washed
with ethyl ether (20 mL) to give 14a–g as white powder.
157.7; ESIMS m/z: 434.2 (M+H)+
.
Diphenyl(4-(2-(4-cyclohexylpiperazin-1-yl)ethoxy)benzyl)
phosphine oxide (14e) White solid, yield 67%. mp:
199–200 °C; IR (film) 2933, 1754, 1657, 1466, 1369, 1329,
1310, 1140, 1029 cm−1 1H-NMR (400 MHz, CDCl3) δ
;
0.99–1.30 (m, 6H), 1.61 (d, J = 11.5 Hz, 1H), 1.77 (s, 2H),
1.88 (d, J = 8.3 Hz, 2H), 2.23 (s, 1H), 2.30 (s, 1H), 2.61 (s,
6H), 2.76 (t, J = 5.7 Hz, 2H), 3.57 (d, J = 13.3 Hz, 2H),
4.01 (dd, J = 8.2, 13.9 Hz, 2H), 6.71 (d, J = 7.6 Hz, 2H),
6.99 (d, J = 8.4 Hz, 2H), 7.33–7.80 (m, 10H); 13C-NMR
(100 MHz, CDCl3) δ 25.8, 26.2, 28.9, 36.7, 37.4, 45.9,
48.8, 54.0, 57.2, 63.5, 65.7, 76.7, 77.0, 77.3, 114.5, 114.5,
122.9, 123.0, 128.4, 128.5, 131.0, 131.1, 131.2, 131.7,
Diphenyl(4-(2-(piperidin-1-yl)ethoxy)benzyl)phosphine
oxide (14a) White solid, yield 79%. mp: 199–200 °C; IR
(film) 2920, 2850, 1699, 1620, 1413, 1401, 1358, 1308,
1271, 1201, 1147, 1020 cm−1; 1H-NMR (400 MHz, CDCl3)
δ 1.46 (s, 2H), 1.63 (s, 4H), 2.53 (s, 4H), 2.78 (s, 2H), 3.58
(d, J = 13.3 Hz, 2H), 4.07 (s, 2H), 6.72 (d, J = 8.4 Hz, 2H),
7.00 (d, J = 7.3 Hz, 2H), 7.26–7.71 (m, 10H); 13C-NMR
(100 MHz, CDCl3) δ 23.9, 25.6, 36.7, 37.4, 54.9, 57.7,
114.5, 128.4, 128.5, 131.0, 131.2, 131.7, 131.7, 131.8,
131.7, 131.8, 132.8, 157.7; ESIMS m/z: 503.2 (M+H)+
.
132.8; ESIMS m/z: 420.2 (M+H)+
(4-(2-Morpholinoethoxy)benzyl)diphenylphosphine oxide
(14f) White solid, yield 52%. mp: 179–180 °C; IR (film)
2936, 1778, 1623, 1456, 1479, 1338, 1320, 1231, 1204,
.
Diphenyl(4-(2-(pyrrolidin-1-yl)ethoxy)benzyl)phosphine
oxide (14b) White solid, yield 73%. mp: 189–190 °C; IR
(film) 2923, 2855, 1734, 1619, 1517, 1428, 1300, 1222,
1
1153, 1119, 1045, 1067, 836 cm−1; H-NMR (400 MHz,
CDCl3) δ 2.50 (t, J = 4.3 Hz, 4H), 2.69–2.72 (m, 2H), 3.54
(d, J = 13.3 Hz, 2H), 3.67 (t, J = 4.7 Hz, 4H), 3.97–4.00
1
1143, 1110, 1037, 826 cm−1; H-NMR (400 MHz, CDCl3)

Medicinal Chemistry Research
(m, 2H), 6.68 (d, J = 8.6 Hz, 2H), 6.96–6.98 (m, 2H),
yield 70%. IR (film) 2946, 2845, 1701, 1633, 1576, 1473,
1
7.35–7.67 (m, 10H); ESIMS m/z: 422.0 (M+H)+
1446, 1348, 1288, 1214, 1154, 1140, 1028, 732 cm−1; H-
.
NMR (400 MHz, CDCl3) δ 1.11 (t, J = 8.7 Hz, 3H),
1.25–1.35 (m, 2H), 1.61 (ddd, J = 7.1, 16.0, 33.7 Hz, 4H),
2.27–2.41 (m, 2H), 2.70–2.80 (m, 1H), 2.92 (d, J =
11.4 Hz, 1H), 3.00–3.13 (m, 1H), 3.57–3.90 (m, 12H),
3.95–4.06 (m, 2H), 6.40–6.75 (m, 6H), 7.10–7.22 (5H, m);
(4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy)benzyl)
diphenylphosphine oxide (14g) White solid, yield 52%.
mp: 177–178 °C; IR (film) 2945, 2811, 1725, 1613, 1517,
1
1456, 1348, 1210, 1214, 1130, 1028, 811 cm−1; H-NMR
(400 MHz, CDCl3) δ2.87−2.97 (m, 6H), 3.59 (d, J =
13.2 Hz, 2H), 3.77 (s, 2H), 4.14 (t, J = 5.6 Hz, 2H), 6.76 (d,
J = 8.4 Hz, 2H), 7.01 (dd, J = 8.4 Hz, J = 2 Hz, 3H),
7.08–7.13 (m, 3H), 7.41–7.52 (m, 6H), 7.66–7.71 (m, 4H);
ESIMS m/z: 546.2 (M+H)+
.
1,2-Bis(3,5-dimethoxyphenyl)-3-(4-(2-(4-methylpiperidin-1-
yl)ethoxy)-phenyl)prop-2-en-1-one (17d) Yellowish oil,
yield 72%. IR (film) 2940, 1715, 1624, 1496, 1427, 1367,
1306, 1211, 1148, 1021 cm−1; 1H-NMR (400 MHz, CDCl3)
δ 0.92 (d, J = 6.1 Hz, 3H), 1.24–1.31 (m, 3H), 1.62 (d, J =
12.3 Hz, 2H), 2.07 (t, J = 10.8 Hz, 2H), 2.74 (t, J = 6.0 Hz,
2H), 2.93 (d, J = 11.5 Hz, 2H), 3.76 (s, 6H), 3.77 (s, 6H),
4.04 (t, J = 6.0 Hz, 2H), 6.40–6.75 (m, 6H), 7.10–7.22 (m,
ESIMS m/z: 468.0 (M+H)+
.
General procedure for the synthesis of 1,2-bis(3,5-
dimethoxyphenyl)-3-(4- (2-(aminoalkyl-1-yl)ethoxy) phenyl)
prop-2-en-1-ones (17a–f) and 1,2-bis(4-methoxy-phenyl)-3-
(4-(2-(aminoalkyl-1-yl)ethoxy) phenyl)prop-2-en-1-ones
(18a–f)
5H); ESIMS m/z: 546.2 (M+H)+
.
To a solution of oxybenzoic acid substituted by methyl (15
or 16) (3.02 mmol) and diphenyl{4-[2-(aminoalkyl-1-yl)
ethoxy]benzyl}phosphine oxide (3.02 mmol) in anhydrous
toluene (100 mL), NaHMDS (2 M, 6.05 mmol) in THF was
added under N2 at room temperature. The reaction mixture
was quenched with water after 0.5 h stirring. The reaction
mixture was concentrated and then diluted with ethyl
acetate. The organic layer was washed with water (30 mL)
and brine (30 mL), dried over Na2SO4, filtered, and eva-
porated. The obtained residue was further purified by silica
gel column chromatography (0–10% ethyl acetate/hexanes)
to give the desired product as yellowish oil.
3-(4-(2-(4-Cyclohexylpiperazin-1-yl)ethoxy)phenyl)-1,2-bis
(3,5-dimethoxy-phenyl)prop-2-en-1-one (17e) Yellowish
oil, yield 52%. IR (film) 2935, 1734, 1611, 1426, 1445,
1356, 1326, 1211, 1037, 555, 460 cm−1
;
1H-NMR
(400 MHz, CDCl3) δ 1.07–1.30 (m, 6H), 1.58–2.21 (m,
7H), 2.58 (s, 6H), 2.73 (s, 2H), 3.73 (s, 6H), 4.00 (s, 2H),
6.36–6.71 (m, 6H), 6.92–7.22 (m, 5H); ESIMS m/z: 515.0
(M+H)+
.
1,2-Bis(3,5-dimethoxyphenyl)-3-(4-(2-morpholinoethoxy)
phenyl)prop-2-en-1-one (17f) Yellowish oil, yield 57%. IR
(film) 2923, 2850, 1708, 1627, 1532, 1486, 1456, 1420,
1
1322, 1310, 1226, 1205, 1150, 1018, 670, 452 cm−1; H-
1,2-Bis(3,5-dimethoxyphenyl)-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)prop-2-en-1-one (17a) Yellowish oil, yield
67%. IR (film) 2932, 1722, 1637, 1449, 1433, 1418, 1314,
NMR (400 MHz, CDCl3) δ 2.55 (s, 4H), 2.74–2.77 (m, 2H),
3.70–3.73 (m, 4H), 3.75 (s, 6H), 3.76 (s, 6H), 4.03–4.06 (m,
2H), 6.39–6.74 (m, 6H), 7.08–7.21 (m, 5H); ESIMS m/z:
1204, 1130, 1026 cm−1
;
1H-NMR (400 MHz, CDCl3) δ
534.0 (M+H)+
.
1.43 (d, J = 4.7 Hz, 2H), 1.59 (dt, J = 5.4, 10.8 Hz, 4H),
2.48 (s, 4H), 2.72 (t, J = 5.9 Hz, 2H), 3.75 (s, 6H), 3.76 (s,
6H), 4.04 (t, J = 6.0 Hz, 2H), 6.36–6.75 (m, 6H), 7.06–7.28
1,2-Bis(4-methoxyphenyl)-3-(4-(2-(piperidin-1-yl)ethoxy)
phenyl)prop-2-en-1-one (18a) Yellowish oil, yield 57%.
IR (film) 2921, 2828, 1711, 1632, 1590, 1411, 1425, 1312,
(m, 5H); ESIMS m/z: 532.2 (M+H)+
.
1
1316, 1218, 1205, 1137, 1056, 1047, 788, 641 cm−1; H-
1,2-Bis(3,5-dimethoxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)
ethoxy)phenyl)prop-2-en-1-one (17b) Yellowish oil, yield
65%. IR (film) 2917, 1707, 1602, 1477, 1439, 1355, 1306,
NMR (400 MHz, CDCl3) δ 1.43 (d, J = 5.1 Hz, 2H),
1.55–1.61 (m, 4H), 2.47 (s, 4H), 2.72 (t, J = 6.1 Hz, 2H),
3.78 (s, 3H), 3.80 (s, 3H), 4.02 (t, J = 6.0 Hz, 2H), 6.70 (d,
J = 8.6 Hz, 2H), 6.82–6.86 (m, 4H), 7.00 (s, 1H), 7.20 (d,
J = 8.6 Hz, 2H), 7.36 (d, J = 9.0 Hz, 2H), 7.97 (d, J =
1
1223, 1203, 1141, 1027, 900 cm−1; H-NMR (400 MHz,
CDCl3) δ 1.82 (d, J = 2.9 Hz, 4H), 2.62 (s, 4H), 2.88 (t, J =
6.0 Hz, 2H), 3.66–3.89 (m, 12H), 4.06 (t, J = 5.9 Hz, 2H),
6.41–6.78 (m, 6H), 7.11–7.28 (m, 5H); ESIMS m/z: 518.2
8.6 Hz, 2H); ESIMS m/z: 472.0 (M+H)+
.
(M+H)+
1,2-Bis(4-methoxyphenyl)-3-(4-(2-(4-methylpiperidin-1-yl)
ethoxy)phenyl)-prop-2-en-1-one (18b) Yellowish oil, yield
56%. IR (film) 2942, 2834, 1633, 1622, 1567, 1456, 1422,
.
1,2-Bis(3,5-dimethoxyphenyl)-3-(4-(2-(2-methylpiperidin-1-
yl)ethoxy)-phenyl)prop-2-en-1-one (17c) Yellowish oil,
1
1297, 1250, 1233, 1071, 1038, 763, 554 cm−1; H-NMR

Medicinal Chemistry Research
(400 MHz, (CDCl3) δ 1.25–1.34 (m, 6H), 1.60 (d, J =
11.2 Hz, 2H), 2.08 (t, J = 11.6 Hz, 2H), 2.75 (t, J =
11.6 Hz, 2H), 2.94 (d, J = 11.6 Hz, 2H), 3.76 (s, 3H), 3.77
(s, 3H), 4.03 (t, J = 6.0 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H),
6.82 (t, J = 8.8 Hz, 4H), 6.99 (s, 1H), 7.19 (d, J = 8.8 Hz,
2H), 7.35 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H);
hydroxy-2-(4-hydroxyphenyl)-3-(4-(2-(aminoalkyl -1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-ones (7a–d)
To an ice-cold solution of 17a–f or 18a or 18c or 18e–f
(0.1 mmol) in CH2Cl2 (12 mL), BBr3·OEt2 (1.5 mmol) was
dropwise added via syringe. After stirred at room tem-
perature for 8 h under N2, the reaction was quenched with
water (1–2 mL). After CH2Cl2 was evaporated, the saturated
sodium bicarbonate solution was added to the mixture. The
resulting solution was extracted with ethyl acetate (50 mL ×
2) and the combined organic phase was washed with brine,
dried over Na2SO4 and evaporated. The residue was further
purified by silica gel column chromatography eluting with
CH3CN/H2O/TFA (1: 2: 0.1%) to give the desired product
as brown solid.
ESIMS m/z: 486.0 (M+H)+
.
1,2-Bis(4-methoxyphenyl)-3-(4-(2-morpholinoethoxy)phe-
nyl)prop-2-en-1-one (18c) Yellowish oil, yield 52%. IR
(film) 2934, 2847, 1612, 1500, 1433, 1456, 1245, 1240,
1075, 1026, 860, 542 cm−1; 1H-NMR (400 MHz, CDCl3) δ
2.56 (m, 4H), 2.76 (t, J = 5.6 Hz, 2H), 3.72 (t, J = 4.8 Hz,
4H), 3.78 (s, 3H), 3.80 (s, 3H), 4.04 (t, J = 5.6 Hz, 2H),
6.70 (d, J = 8.8 Hz, 2H), 6.84 (t, J = 8.8 Hz, 4H), 7.00 (s,
1H), 7.21 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.97
(d, J = 8.8 Hz, 2H); ESIMS m/z: 474.0 (M+H)+
2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-(piperidin-1-
.
yl)ethoxy)-phenyl)-2,3-dihydro-1H-inden-1-one
(5a)
1,2-Bis(4-methoxyphenyl)-3-(4-(2-(4-cyclohexylpiperazin-1-
yl)ethoxy)-phenyl)prop-2-en-1-one (18d) Yellowish oil,
yield 52%. IR (film) 2926, 1693, 1576, 1478, 1447, 1411,
Brown solid, yield 43%. mp: 233–234 °C; IR (film) 3345,
2923, 1760, 1633, 1450, 1432, 1417, 1355, 1325, 1234,
1154, 1076, 1022, 752, 521 cm−1;1H-NMR (400 MHz,
CD3OD) δ 1.45–1.57 (m, 1H), 1.80 (t, J = 11.3 Hz, 3H),
1.93 (d, J = 11.9 Hz, 2H), 3.02 (t, J = 11.6 Hz, 2H), 3.36 (d,
J = 2.5 Hz, 1H), 3.48–3.54 (m, 2H), 3.59 (d, J = 11.9 Hz,
2H), 4.25–4.36 (m, 2H), 4.43 (d, J = 2.2 Hz, 1H), 5.98 (d,
J = 1.7 Hz, 2H), 6.16 (s, 1H), 6.64 (dd, J = 1.7, 30.1 Hz,
2H), 6.93 (dd, J = 8.5, 30.6 Hz, 4H); 13C-NMR (100 MHz,
CD3OD) δ 22.5, 24.0, 52.3, 54.8, 57.1, 63.0, 67.0, 100.6,
102.3, 107.2, 110.9, 115.7, 129.3, 136.2, 138.5, 140.0,
143.1, 157.2, 157.7, 159.9, 160.9, 208.4; ESIMS m/z: 476.2
(M+H)+; C28H30NO6: HRMS calcd. 476.2068 (M+H)+,
found 476.2064.
1
1354, 1301, 1242, 1211, 1104, 1029, 668, 560 cm−1; H-
NMR (400 MHz, CDCl3) δ 2.56 (m, 4H), 2.76 (t, J =
5.6 Hz, 2H), 3.72 (t, J = 4.8 Hz, 4H), 3.78 (s, 3H), 3.80 (s,
3H), 4.04 (t, J = 5.6 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H), 6.84
(t, J = 8.8 Hz, 4H), 7.00 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H),
7.36 (d, J = 8.8 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H); ESIMS
m/z: 555.1 (M+H)+
.
3-(4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy)phenyl)-
1,2-bis(4-methoxyphenyl)prop-2-en-1-one (18e) Yellow-
ish oil, yield 43%. IR (film) 3000, 2912, 1686, 1581, 1489,
1478, 1429, 1310, 1244, 1211, 1037, 1026, 876, 563 cm−1
;
1H-NMR (400 MHz, CDCl3) δ 3.85–3.96 (m, 6H), 4.76 (s,
2H), 4.86 (s, 3H), 4.89 (s, 3H), 5.22 (t, J = 6.0 Hz, 2H),
7.89 (d, J = 8.8 Hz, 2H), 7.99–8.03 (m, 4H), 8.12–8.17 (m,
4H), 8.19 (s, 1H), 8.37 (d, J = 8.4 Hz, 2H), 8.49 (d, J =
8.8 Hz, 2H), 9.03 (d, J = 8.8 Hz, 2H); ESIMS m/z: 520.0
2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-(pyrrolidin-
1-yl)ethoxy)-phenyl)-2,3-dihydro-1H-inden-1-one
(5b)
Brown solid, yield 47%. mp: 205–206 °C; IR (film) 3356,
2941, 2824, 1711, 1632, 1486, 1442, 1400, 1324, 1300,
1221, 1151, 1023, 1050, 1012, 840, 811, 741, 502 cm−1
;
(M+H)+
1H-NMR (400 MHz, CD3OD) δ 1.98–2.02 (m, 4H),
3,11–3.16 (m, 2H), 3.34 (d, J = 2.35 Hz, 1H), 3.56–3.59
(m, 2H), 3.67 (s, 2H), 4.24 (t, J = 4.7 Hz, 2H), 4.41 (d, J =
2.7 Hz, 1H), 5.97 (d, J = 2.0 Hz, 2H), 6.14 (t, J = 2.0 Hz,
1H), 6.59 (d, J = 2.3 Hz, 1H), 6.66 (d, J = 2.3 Hz, 1H), 6.88
(d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H); 13C-NMR
(100 MHz, CD3OD) δ 21.0, 23.9, 52.3, 55.2, 55.7, 64.3,
67.0, 100.6, 102.4, 107.3, 111.0, 115.8, 129.4, 136.3,
138.6, 140.1, 143.1, 157.3, 157.8, 159.9, 160.0, 161.0,
208.5; ESIMS m/z: 462.2 (M+H)+; C27H28NO6: HRMS
calcd. 462.1911 (M+H)+, found 462.1911.
.
1,2-Bis(4-methoxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)ethoxy)
phenyl)prop-2-en-1-one (18f) Yellowish oil, yield 49%. IR
(film) 2923, 1755, 1724, 1627, 1517, 1480, 1454, 1427,
1
1329, 1216, 1228, 1148, 1023, 668, 573 cm−1; H-NMR
(400 MHz, CDCl3) δ 1.79 (m, 4H), 2.59 (m, 4H), 2.85 (t, J =
6 Hz, 2H), 3.79 (s, 3H), 3.81 (s, 3H), 4.03 (t, J = 6.0 Hz, 2H),
6.72 (d, J = 8.4 Hz, 2H), 6.84 (t, J = 8.4 Hz, 4H), 7.00 (s,
1H), 7.20 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 7.97
(d, J = 8.4 Hz, 2H); ESIMS m/z: 458.0 (M+H)+
.
General procedure for the synthesis of 2-(3,5-
dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-(aminoalkyl-1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-ones (5a–f) and 5-
2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-morpholi-
noethoxy)phenyl)-2,3-dihydro-1H-inden-1-one (5c) Brown
solid, yield 42%. mp: 217–219 °C; IR (film) 3356, 2916,

Medicinal Chemistry Research
1734, 1622, 1478, 1429, 1433, 1353, 1300, 1214, 1167,
(400 MHz, CD3OD) δ 1.17 (m, 6H), 1.69 (d, J = 12.9 Hz,
1H), 1.92 (d, J = 12.5 Hz, 2H), 2.10 (d, J = 10.6 Hz, 2H),
3.36 (d, J = 2.7 Hz, 1H), 3.48–3.57 (m, 10H), 4.27–4.29
(m, 2H), 4.42 (d, J = 2.4 Hz, 1H), 5.99 (d, J = 2.0 Hz, 2H),
6.16–6.17 (m, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.69 (d, J =
2.0 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 9.0 Hz,
2H); 13C-NMR (100 MHz, CD3OD) δ 26.0, 28.0, 48.3,
49.9, 51.2, 52.3, 57.1, 65.1, 67.0, 67.0, 100.7, 102.4, 107.3,
111.1, 115.7, 129.3, 136.4, 138.1, 140.1, 143.2, 157.3,
158.2, 159.9, 160.0, 160.9, 208.6; ESIMS m/z: 559.3 (M
+H)+; C30H41NO9: HRMS calcd. 559.2776 (M+H)+,
found 559.2789.
1
1123, 1036, 666, 453 cm−1; H-NMR (400 MHz, CD3OD)
δ 3.26 (m, 2H), 3.35 (d, J = 2.7 Hz, 1H), 3.55 (m, 2H), 3.60
(t, J = 4.7 Hz, 2H), 3.80 (m, 2H), 4.03 (m, 2H), 4.33–4.35
(m, 2H), 4.43 (d, J = 2.4 Hz, 1H), 5.98 (d, J = 2.0 Hz, 2H),
6.15 (t, J = 2.0 Hz, 1H), 6.60 (d, J = 1.6 Hz, 1H), 6.68 (d,
J = 2.0 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.98 (d, J =
8.6 Hz, 2H); 13C-NMR (100 MHz, CD3OD) δ 52.3, 53.6,
57.5, 62.8, 64.9, 67.1, 100.6, 102.4, 107.3, 111.0, 115.8,
129.4, 136.3, 138.7, 140.1, 143.1, 157.3, 157.7, 160.0,
161.0, 208.5; ESIMS m/z: 478.0 (M+H)+; C27H28NO7:
HRMS calcd. 478.1860 (M+H)+, found 478.1862.
2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-(4-methylpi-
peridin-1-yl)-ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one
(5d) Brown solid, yield 60%. mp: 221–222 °C; IR (film)
3337, 2918, 2833, 1754, 1629, 1487, 1434, 1352, 1323,
5-Hydroxy-2-(4-hydroxyphenyl)-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one (7a) Brown
solid, yield 67%. mp: 234–235 °C; IR (film) 3321, 2932,
1745, 1633, 1521, 1499, 1451, 1354, 1320, 1212, 1147,
1215, 1142, 1032, 754, 632 cm−1
;
1H-NMR (400 MHz,
1110, 1027 cm−1
;
1H-NMR (400 MHz, CD3OD)
δ
CD3OD) δ 0.99 (d, J = 6.7 Hz, 3H), 1.41–1.91 (m, 5H),
2.98–3.04 (m, 2H), 3.36 (d, J = 2.7 Hz, 1H), 3.49 (t, J =
4.7 Hz, 2H), 3.60 (d, J = 12.5 Hz, 2H), 4.29 (t, J = 4.7 Hz,
2H), 4.42 (d, J = 2.7 Hz, 1H), 5.98 (d, J = 2.3 Hz, 2H),
6.15–6.16 (m, 1H), 6.61 (d, J = 2.3 Hz, 1H), 6.68 (d, J =
2.0 Hz, 1H), 6.88 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz,
2H); 13C-NMR (100 MHz, CD3OD) δ 21.4, 29.7, 32.4,
52.3, 54.7, 57.3, 63.0, 67.0, 100.6, 102.3, 107.2, 111.0,
115.7, 129.3, 136.3, 138.5, 140.0, 143.1, 143.2, 157.3,
157.7, 159.9, 159.9, 160.9, 208.5; ESIMS m/z: 490.2 (M
+H)+; C29H32NO6: HRMS calcd. 490.2224 (M+H)+,
found 490.2223.
1.52–1.57 (m, 1H), 1.75–1.85 (m, 3H), 1.95 (d, J = 14.8 Hz,
2H), 3.02–3.08 (m, 2H), 3.54 (t, J = 4.7 Hz, 2H), 3.59 (s,
1H), 3.63 (d, J = 4.4 Hz, 2H), 4.32–4.35 (m, 2H), 4.39 (d,
J = 4.5 Hz, 1H), 6.54 (d, J = 1.1 Hz, 1H), 6.71 (d, J =
8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.90–6.92 (m, 1H),
6.96 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 2H), 7.67 (d, J
= 8.2 Hz, 1H); 13C-NMR (150 Hz, CDCl3) δ 197.5, 161.5,
157.0, 141.3, 139.9, 137.9, 137.7, 136.9, 136.6, 129.3,
123.9, 123.5, 123.3, 122.5, 122.1, 121.0, 106.5, 95.8, 55.2,
54.9, 34.3, 20.8. HRMS calcd. 444.2169 (M+H)+, found
444.2165.
3-(4-(2-(3,4-Dihydroisoquinolin-2(1H)-yl)ethoxy)phenyl)-5-
hydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-1H-inden-1-one
(7b) Brown solid, yield 67%. mp: 215–216 °C; IR (film)
3356, 2937, 1806, 1623, 1511, 1476, 1344, 1320, 1276,
2-(3,5-Dihydroxyphenyl)-4,6-dihydroxy-3-(4-(2-(2-methylpi-
peridin-1-yl)-ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one
(5e) Brown solid, yield 51%. mp: 251–252 °C; IR (film)
3356, 2924, 1736, 1622, 1478, 1436, 1329, 1320, 1210,
1142, 789, 531 cm−1; 1H-NMR (400 MHz, CD3OD) δ 1.44
(d, J = 5.9 Hz, 3H), 1.54–2.00 (m, 6H), 3.03–3.09 (m, 1H),
3.36 (d, J = 2.7 Hz, 1H), 3.38–4.34 (m, 6H), 4.42 (d, J =
2.7 Hz, 1H), 5.99 (d, J = 2.0 Hz, 2H), 6.15–6.16 (m, 1H),
6.61 (d, J = 2.3 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 6.89 (d,
J = 8.6 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H); 13C-NMR
(100 MHz, CD3OD) δ 18.3, 22.9, 24.3, 30.7, 32.8, 52.3,
53.7, 54.2, 62.2, 63.0, 67.0, 100.6, 102.3, 107.2, 111.0,
115.7, 129.3, 136.3, 138.5, 140.0, 143.2, 157.3, 157.7,
159.9, 160.9, 208.5; ESIMS m/z: 490.0 (M+H)+;
C29H32NO6: HRMS calcd. 490.2224 (M+H)+, found
490.2221.
1
1201, 1157, 1133, 1021, 1026, 786, 577 cm−1; H-NMR
(400 MHz, CD3OD) δ 3.23 (m, 2H), 3.30 (m, 2H), 3.63 (d,
J = 5.2 Hz, 1H), 3.74 (t, J = 4.4 Hz, 2H), 4.39 (d, J =
4.8 Hz, 1H), 4.44 (t, J = 4.4 Hz, 2H), 6.55 (s, 1H), 6.71(d,
J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.91 (dd, J =
8.4 Hz, J = 2 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 7.20 (d,
J = 7.6 Hz, 1H), 7.26–7.34 (m, 3H), 7.67 (d, J = 8.4 Hz,
1H); 13C-NMR (100 MHz, CD3OD) δ 26.0, 48.4, 49.6,
51.4, 54.7, 55.5, 55.6, 56.1, 63.0, 63.2, 65.7, 112.9, 116.1,
116.5, 118.0, 127.9, 128.3, 129.6, 129.9, 130.3, 130.9,
137.4, 157.7, 158.2, 161.6, 166.5, 206.5; HRMS calcd.
492.2169 (M+H)+, found 492.2165.
5-Hydroxy-2-(4-hydroxyphenyl)-3-(4-(2-morpholinoethoxy)
phenyl)-2,3-dihydro-1H-inden-1-one (7c) Brown solid,
yield 45%. mp: 211–212 °C; IR (film) 3345, 2930, 1710,
1623, 1450, 1332, 1323, 1162, 1132, 1078, 1076, 690,
3-(4-(2-(4-Cyclohexylpiperazin-1-yl)ethoxy)phenyl)-2-(3,5-
dihydroxy-phenyl)-4,6-dihydroxy-2,3-dihydro-1H-inden-1-
one (5f) Brown solid, yield 42%. mp: 233–234 °C; IR
(film) 3334, 2959, 1711, 1622, 1437, 1422, 1351, 1319,
1
532 cm−1; H-NMR (400 MHz, CD3OD) δ 2.02 (m, 2H),
1205, 1148, 1050, 1022, 757, 632 cm−1
;
1H-NMR
2.15 (m, 2H), 3.19 (m, 2H), 3.61 (t, J = 4.8 Hz, 3H), 4.28 (t,

Medicinal Chemistry Research
J = 4.8 Hz, 2H), 4.37 (d, J = 4.8 Hz, 1H), 6.52 (s, 1H), 6.69
(d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.89 (d, J =
8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz,
2H), 7.65 (d, J = 8.4 Hz, 1H); 13C-NMR (100 MHz,
CD3OD) δ 21.9, 28.7, 46.6, 47.4, 53.2, 53.6, 53.7, 62.3,
63.7, 110.9, 114.1, 114.5, 116.0, 124.8, 127.0, 128.2,
128.5, 129.0, 135.3, 155.6, 156.3, 159.6, 164.4, 204.5;
HRMS calcd. 446.1962 (M+H)+, found 446.1965.
6.90 (d, J = 8.6 Hz, 2H); 13C-NMR (150 Hz, CDCl3) δ
196.4, 156.2, 152.3, 149.6, 140.8, 138.2, 137.9, 135.8,
129.2, 128.8, 128.5, 126.8, 123.2, 106.8, 103.4, 78.7, 55.7,
55.5, 40.6, 20.7, 20.3.HRMS calcd. 532.2694 (M+H)+,
found 532.2694.
2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-(2-(4-methyl-
piperidin-1-yl)-ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one
(6b) Brown solid, yield 62%. mp: 201–202 °C; IR (film)
3337, 3011, 2935, 1716, 1622, 1473, 1454, 1347, 1328,
5-Hydroxy-2-(4-hydroxyphenyl)-3-(4-(2-(pyrrolidin-1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one (7d) Brown
solid, yield 52%. mp: 214–215 °C; IR (film) 3347, 3001,
2946, 2852, 1711, 1627, 1490, 1411, 1352, 1320, 1210,
1233, 1136, 1029, 730, 569 cm−1 1H-NMR (400 MHz,
;
CD3OD) δ 1.25–1.42 (m, 8H), 2.13 (t, J = 6 Hz, 2H), 2.81
(t, J = 6 Hz, 2H), 3.01 (d, J = 11.6 Hz, 2H), 3.60 (d, J =
2.8 Hz, 1H), 3.65 (s, 3H), 3.73 (s, 6H), 3.87 (s, 3H), 4.09 (t,
J = 5.6 Hz, 2H), 4.49 (d, J = 2.8 Hz, 1H), 6.22 (d, J =
2.4 Hz, 2H), 6.35 (t, J = 2 Hz, 1H), 6.68 (d, J = 2 Hz, 1H),
6.77 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 2 Hz, 1H), 6.91 (d, J
= 8.8 Hz, 2H); 13C-NMR (100 MHz, CDCl3) δ 14.0, 21.5,
22.6, 29.3, 29.6, 30.1, 31.8, 50.8, 54.1, 55.1, 55.2, 55.5, 55.7,
57.1, 65.1, 65.2, 76.7, 77.0, 77.3, 96.3, 98.8, 105.9, 106.5,
114.4, 127.9, 135.6, 138.3, 138.4, 141.4, 157.0, 157.6,
160.9, 161.8, 205.3; HRMS calcd. 546.2850 (M+H)+, found
546.2854.
1142, 1078, 805, 773 cm−1 1H-NMR (400 MHz, CD3OD)
;
δ 3.49 (m, 2H), 3.62 (m, 3H), 3.85–3.99 (m, 6H), 4.37 (m,
3H), 6.54 (s, 1H), 6.70 (d, J = 8.4 Hz, 2H), 6.85 (d, J =
8.4 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 8.4 Hz,
2H), 7.06 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 1H);
13C-NMR (100 MHz, CD3OD) δ 28.7, 47.9, 51.8, 53.6,
55.5, 60.9, 62.9, 63.7, 110.9, 114.1, 114.6, 116.0, 124.8,
127.0, 128.2, 128.5, 129.0, 135.3, 155.6, 156.1, 159.6,
164.5, 204.5; HRMS calcd. 430.2013 (M+H)+, found
430.2010.
General procedure for the synthesis of 2-(3,5-
5-Methoxy-2-(4-methoxyphenyl)-3-(4-(2-(piperidin-1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-one (8a) Yellow-
ish solid, yield 82%. mp: 236–237 °C; IR (film) 3338, 3001,
1942, 1716, 1646, 1556, 1487, 1439, 1354, 1236, 1200,
1147, 1036, 1029, 850, 468 cm−1; 1H-NMR (400 MHz,
CD3OD) δ 1.37–1.77 (m, 6H), 2.96 (s, 2H), 3.42–3.61 (m,
5H), 3.74 (s, 3H), 3.77 (s, 3H), 4.27 (s, 2H), 4.34 (d, J =
4.7 Hz, 1H), 6.63–7.31 (m, 9H), 7.74–7.82 (m, 2H); 13C-
NMR (100 MHz, CDCl3) δ 23.4, 25.0, 53.8, 54.3, 54.6, 55.1,
57.1, 63.7, 65.0, 108.9, 113.6, 114.3, 115.8, 125.1, 125.2,
128.3, 128.7, 128.9, 130.4, 134.1, 157.2, 158.0, 158.7, 165.2,
203.3; HRMS calcd. 472.2482 (M+H)+, found 472.2487.
We separated 8a with HPLC and chiral column to give
dimethoxyphenyl)-4,6-dimethoxy-3-(4-(2-(aminoalkyl -1-yl)
ethoxy)phenyl)-2,3-dihydro-1H-inden-1-ones (6a–b) and 5-
methoxy-2-(4-methoxyphenyl)-3-(4-(2- (aminoalkyl-1-yl)
ethoxy)-phenyl)-2,3-dihydro-1H-inden-1-ones (8a–d)
To an ice-cold solution of 18a–d or 17a or 17d (1 mmol)
in CH2Cl2 (20 mL), BF3·OEt2 (0.2 mL) was dropwise
added via syringe. After stirred at room temperature for
72–120 h under N2 protection, the reaction was quenched
with water (1–2 mL) and CH2Cl2 was evaporated. Then
saturated sodium bicarbonate solution was added to the
mixture. The resulting solution was extracted with ethyl
acetate and organic phase was washed with brine and
dried over Na2SO4. After evaporated the solvent, the
residue was further purified by reversed phase silica gel
column chromatography (CH3CN/H2O/TFA = 1: 2: 0.1%)
to give the desired product as yellowish or brown
solid.
(2S,3S)-8a (½αŠ20 = +151.5 (c = 0.4 in MeOH)) and (2R,3R)-
D
8a (½αŠ20 = −151.5 (c = 0.4 in MeOH).
D
5-Methoxy-2-(4-methoxyphenyl)-3-(4-(2-(4-methylpiperi-
din-1-yl)ethoxy)-phenyl)-2,3-dihydro-1H-inden-1-one (8b)
Yellowish solid, yield 82%. mp: 200–201 °C; IR (film)
3356, 2913, 1716, 1638, 1487, 1446, 1352, 1334, 1200,
2-(3,5-Dimethoxyphenyl)-4,6-dimethoxy-3-(4-(2-(piperidin-
1142, 1056, 1027, 668, 542 cm−1 1H-NMR (400 MHz,
;
1-yl)ethoxy)-phenyl)-2,3-dihydro-1H-inden-1-one
Yellowish solid, yield 75%. mp: 212–213 °C; IR (film)
(6a)
CD3OD) δ 0.94 (d, J = 4.4 Hz, 3H), 1.45 (s, 2/H), 1.67 (s,
2/H), 2.32 (t, J = 10.8 Hz, 2H), 2.97 (t, J = 5.2 Hz, 2H),
3.16 (d, J = 10.8 Hz, 2H), 3.67 (d, J = 4.8 Hz, 1H), 3.76 (s,
3H), 3.80 (s, 3H), 4.21 (t, J = 5.2 Hz, 2H), 4.37 (d, J =
4.4 Hz, 1H), 6.66 (s, 1H), 6.81–6.84 (m, 4H), 6.97–6.99 (m,
5H), 7.79 (d, J = 8.8 Hz, 1H); 13C-NMR (100 MHz, CDCl3)
δ 20.4, 29.1, 53.8, 54.6, 55.1, 63.6, 64.3, 85.9, 108.8, 112.3,
113.7, 114.3, 115.3, 115.8, 125.1, 127.2, 128.5, 128.7,
3369, 2919, 1724, 1621, 1493, 1352, 1311, 1202, 1130,
1
670, 498 cm−1; H-NMR (400 MHz, CD3OD) δ 1.40–2.10
(m, 6H), 2.82–2.88 (m, 2H), 3.42 (s, 2H), 3.54 (s, 1H), 3.62
(s, 3H), 3.70 (s, 6H), 3.74 (s, 1H), 3.79 (s, 1H), 3.84 (s, 3H),
4.42 (s, 2H), 4.47 (d, J = 1.9 Hz, 1H), 6.19 (s, 2H), 6.33 (s,
1H), 6.66 (s, 1H), 6.75 (d, J = 8.2 Hz, 2H), 6.86 (s, 1H),

Medicinal Chemistry Research
130.3, 130.4, 131.5, 158.1, 158.5, 165.3, 203.0; HRMS
FBS and antibiotics. Cells were collected and washed twice
with phosphate-buffered saline (PBS). After trypsin diges-
tion, the centrifuged supernatant was discarded. Cell pre-
cipitations were lysed with 1 × LDS Sample buffer (Life
Technologies, USA). For analysis, 20 μg of protein were
resolved on 10% sodium dodecyl sulphate-polyacrylamide
gel. After electrophoresis, the gel was transferred into
“sandwich”, and applied 180 mA for 120 min. After transfer
membrane, polyvinylidene difluoride membrane was rinsed
in TBST three times every 10 min. The membrane was
placed in TBS/T blocking buffer containing 5% (w/v)
skimmed milk powder and shaken for 1 h at room tem-
perature. And the membrane was followed by overnight
incubation with appropriate dilution of primary antibody:
ERα (Cell Signaling Technology, USA), ERβ (Proteintech,
USA) and β-actin (Bioworld Technology, USA). Protein
bands were detected using Immobilon Western Chemilu-
minescent HRP Substrate (MILLIPORE, USA) and visua-
lized using an image analyzer (BIO-RAD, USA).
calcd. 486.2639 (M+H)+, found 486.2643.
3-(4-(2-(4-Cyclohexylpiperazin-1-yl)ethoxy)phenyl)-5-meth-
oxy-2-(4-methoxyphenyl)-2,3-dihydro-1H-inden-1-one
(8c) Yellowish solid, yield 82%. mp: 207–208 °C; IR
(film) 3368, 2914, 1716, 1637, 1455, 1429, 1346, 1300,
1
1217, 1210, 1147, 1033, 754 cm−1; H-NMR (400 MHz,
CD3OD) δ 1.11–1.39 (m, 8H), 1.47 (d, J = 10.4 Hz, 2H),
1.69 (d, J = 15.6 Hz, 1H), 1.91 (d, J = 12.0 Hz, 2H), 2.25
(s, 2H), 2.90 (s, 2H), 3.08 (s, 4H), 3.60 (d, J = 4.8 Hz, 1H),
3.77 (s, 3H), 3.81 (s, 3H), 4.08 (s, 2H), 4.36 (d, J = 4.8 Hz,
1H), 6.66 (s, 1H), 6.81–6.84 (m, 4H), 6.98–7.03 (m, 5H),
7.79 (d, J = 8.8 Hz, 1H); 13C-NMR (100 MHz, CDCl3) δ
24.4, 26.1, 29.1, 47.6, 49.2, 52.8, 53.7, 54.7, 55.1, 55.7,
63.7, 65.1, 108.9, 113.7, 114.3, 115.7, 125.1, 128.4, 128.7,
128,9, 130.4, 158.1, 158.6, 165.2, 203.1; HRMS calcd.
555.3217 (M+H)+, found 555.3220.
5-Methoxy-2-(4-methoxyphenyl)-3-(4-(2-morpholinoethoxy)
phenyl)-2,3-dihydro-1H-inden-1-one (8d) Yellowish solid,
yield 76%. mp: 217–218 °C; IR (film) 3357, 3003, 2937,
1715, 1622, 1583, 1479, 1446, 1335, 1227, 1216, 1178,
Cell cytotoxicity assay
Cytotoxicity was measured using a MTT assay. MCF-7 and
MDA-MB-231 cells were seeded into flat-bottomed 96-well
microplates for 24 h and followed by the treatment of
tamoxifen, raloxifene, isopaucifloral F and synthesized
isopaucifloral F derivatives dissolved in dimethyl sulfoxide
(DMSO) (100, 50, 25, 12.5, 6.25, 3.62, 1.81 μM as con-
centration gradient). After treatment, cell viability was
assessed using the MTT method. The cytotoxicity assays
were performed in triplicate.
1146, 1008, 1026, 751, 512 cm−1 1H-NMR (400 MHz,
;
CD3OD) δ 3.07 (s, 2H), 3.52 (s, 2H), 3.65 (d, J = 4.8 Hz,
1H), 3.70 (s, 2H), 3.77 (s, 3H), 3.81 (s, 3H), 3.98 (s, 4H),
4.36–4.38 (m, 3H), 6.65 (s, 1H), 6.79–6.84 (m, 4H),
6.91–7.01 (m, 5H), 7.79 (d, J = 8.8 Hz, 1H); 13C-NMR
(100 MHz, CDCl3) δ 28.5, 52.0, 54.4, 55.2, 55.5, 62.4,
63.3, 70.4, 73.7, 81.7, 93.6, 100.5, 108.0, 112.3, 113.1,
113.6, 113.7, 131.4, 131.6, 132.0, 132.0, 187.6, 211.3;
HRMS calcd. 474.2275 (M+H)+, found 474.2278.
Radiometric competitive receptor–ligand binding
Cell culture
assay
MCF-7 and MDA-MB-231 cell lines were obtained from
Chinese Academy of Sciences. MCF-7 cell lines were cul-
tured in RPMI1640 (HyClone Life technologies), supple-
mented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS) as well as 1% (v/v) penicillin and streptomycin (P/S).
MDA-MB-231 cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (HyClone Life tech-
nologies) cell culture medium supplemented with 10% FBS
and 1% penicillin and streptomycin. All cell lines were
maintained in a humidified atmosphere of 5% CO2 at 37 °C.
The competitive binding assay method was performed as
previously study described with slightly modification (Gim
et al. 2014). The recombinant human ER (1.28 nM) was
incubated with test compounds for 20 min at 4 °C in the
presence of 5 nM of [2,4,6,7-3H]-E2 (81.0 ci/mmol). Then
50% (v/v) hydroxyapatite PBS slurry (100 μL) was added to
the reaction mixture. After sufficiently mixing, the mixture
was kept for 15 min at 4 °C. Bound and free radioligand
were separated by centrifugation at 12,000 × g for 2 min,
and the radioactivity of the supernatant was determined
using a liquid scintillation counter (LS-6500, Beckman
counter, CA, USA). The amount of the receptor-bound
[3H]-E2 in the presence of the test compounds was pre-
sented relative to maximum receptor-bound ligand (2 × 10
Western blot analysis
The expression pattern of ER was confirmed by western blot
analysis. MCF-7, MDA231 were purchased from American
Type Culture Collection (ATCC) and cultured according to
the supplier’s recommendations, supplemented with 10%
−8
M E2). The data are expressed as the ratio of the receptor-
bound [3H]-E2 in the presence of the compounds to the
0.1% DMSO used as a control.

Medicinal Chemistry Research
Cell circle analysis
uterine was placed in 10% neutral buffered formalin to fix
for histological preparation. After fixed for over 48 h, par-
affin embedding, 5 µm sectioning, mounting, and hema-
toxylin and eosin staining were completed by Shanghai Liqi
Biological according to standard techniques. Morphometric
analysis was performed using Scion Image analysis software
(Scioncorp, Frederick, MD). Histological markers of utero-
trophy, including LECH, luminal circumference and number
of endometrial glands were quantified for each slide.
MCF-7 cells were plated in six-well plate and allowed to
grow overnight. Then the cells were incubated with com-
pound (2R, 3R)-8a at concentrations of 6, 12, and 18 μM for
48 h. Then untreated and treated cells were harvested,
washed with PBS, fixed in ice-cold 70% ethanol, and
stained with propidium iodide (Beyotime). Cell cycle ana-
lysis was performed by flow cytometry (FACScan, BD
Biosciences, USA).
In vitro metabolic stability studies
Evaluation of apoptosis with flow cytometry
Compound (2R,3R)-8a was incubated at a concentration of
20 μM with rat liver microsomes and NADPH (10 mM) at
37 °C for 60 min. The reaction was quenched by addition of
four volume of acetonitrile at 0 and 60 min. After quenching
by acetonitrile, samples were vortexed for 60 s and cen-
trifuged at 12000 rpm for 5 min. The supernatant was
injected into HPLC. Peak areas of test compounds at 0 and
60 min incubation were used to calculate the % remaining
MCF-7 cells were plated in six-well plate and allowed to
grow overnight. The medium was then replaced and cells
were treated with desired concentrations of lead compounds
for 72 h. Cells treated with vehicle (0.001% DMSO in cul-
ture medium) were included as controls for all experiments.
After 24 h of treatment, cells from supernatant and adherent
monolayer cells were harvested by trypsinization and
washed with PBS at 300 g. After that, the cells were stained
with Annexin V-FITC and propidium iodide using the
Annexin V-PI apoptosis detection kit (Beyotime). Flow
cytometry was performed using a FACScan (Becton Dick-
inson) equipped with a single 488-nm argon laser. Annexin
V-FITC was analyzed using excitation and emission settings
of 488 and 535 nm (FL-1channel) whereas PI was recorded
with 488 and 610 nm (FL-2 channel). Debris and clumps
were gated out using forward and orthogonal light scatter.
values with the formula: %Remaining = Peak Area60min
/
Peak Area0min. T 1/2 and CLint was calculated by the standard
formula (Li et al. 2007).
Ln2 0:693
T1=2
¼
¼
ke
ke
0:693
1
CLint
¼
Á mg
In vitro T1=2 mL microsomal protein in reaction system
Uterine wet weight assay and morphometric
analysis
Conclusions
The protocol employed here was approved by the Local
Ethics Committee for Animal Experiments at Fudan Uni-
versity. An OVX mice model was used. Thirty healthy
female kunming mouse (Sippr/BK, Shanghai, China)
weighing 30–40 g were housing in the specific pathogen
free animal facilities under controlled conditions (tempera-
ture 21 1 °C; relative humidity 50 10%; 12:12 h
light–dark cycle). After acclimatization to laboratory con-
ditions for 10 days, the mouse were randomly assigned to
five groups of six mouse: a sham-operated group (in which
only a piece of fat around the ovary was ablated) and four
groups of OVX mouse, including the placebo (OVX + NS),
the positive control (OVX + tamoxifen, 0.1 mg/kg), low
dosage group (OVX + (2R,3R)-8a, 0.1 mg/kg) and high
dosage group (OVX + (2R,3R)-8a, 1 mg/kg). Oral admin-
istration of samples was performed once daily for 3 days.
Animals were weighted and killed 24 h after the final
treatment. The uterus was transected at the border of the
cervix and stripped of extraneous connective tissue and fat.
Whole-uterine weights were recorded (wet weight) and the
In conclusion, we have designed and synthesized 16 iso-
paucifloral F (Jordan 2004) derivatives, and their potencies of
breast cancer inhibition were evaluated. The ERα/β LBD-
binding affinity of these compounds were also examined.
Among them, (2R,3R)-8a demonstrated significant binding
affinity and high ERα/β LBD selectivity. Through chiral
resolution, it was found that (2R,3R)-8a displayed good
antiproliferative propriety against human breast cancer cell
lines MCF-7 (ERα+/β+) and MDA-MB-231 (ERα−/β+)
than both positive controls tamoxifen and raloxifene.
Besides, it also showed the trend to induce apoptosis in
human breast cancer cell line MCF-7. Moreover, (2R,3R)-8a
was demonstrated both significant binding affinity and good
ERα/β LBD selectivity. Molecular docking analysis further
confirmed specific interactions with ERβ-binding pocket. In
addition, (2R,3R)-8a exhibited no uterine stimulation in the
uterotrophic assay. As a result, (2R,3R)-8a could be a pro-
mising lead compound for future exploration of selective
ERβ anti-breast cancer agents. Further evaluation of (2R,3R)-

Medicinal Chemistry Research
8a in vivo anticancer experiments and development of its
chiral synthetic method are ongoing in our laboratory.
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Acknowledgements This work was supported by the National Natural
Science Foundation of China (No: 81673297), Shanghai Municipal
Committee of Science and Technology (No: 17JC1400200, 17431902500),
and China Postdoctoral Science Foundation (BX20180065).
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Funding The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.
Author contributions Conceptualization and methodology, X.S., C.Z.,
and X.-X.L.; software, X.-X.L. and Y.T.; validation, X.-X.L.; formal
analysis, X.-X.L.; investigation, X.-X.L.; resources, X.S.; data cura-
tion, X.-X.L.; writing—original draft preparation, X.-X.L.; writing—
review and editing, X.S., J.-M.Y. and M.-L.T.; supervision, X.S.;
project administration, X.-X.L.; funding acquisition, X.S.
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
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