Design, synthesis and biological evaluation of chalcones as reversers of P-glycoprotein-mediated multidrug resistance

Article abstract of DOI:10.1016/j.ejmech.2019.05.053

Overexpression of P-glycoprotein (P-gp) is one of the major causes for multidrug resistance (MDR), which has become a major obstacle in cancer therapy. One hopeful approach to reverse the MDR is to develop inhibitors of P-gp in expression and/or function. Here, we designed and synthesized a series of chalcone derivatives as P-gp inhibitors and evaluated their potential reversal activities against MDR. Among them, the most active compound MY3 had little intrinsic cytotoxicity and showed the highest activity (RF = 50.19) in reversing DOX resistance in MCF-7/DOX cells. Further studies demonstrated that MY3 could increase intracellular accumulation of DOX and inhibit expression of P-gp at mRNA and protein levels. More importantly, MY3 significantly enhanced the efficacy of DOX against the tumor xenografts bearing MCF-7/DOX cells with the precondition of unchanged body weight. Therefore, MY3 might represent a promising lead to develop MDR reversal agents for cancer chemotherapy.

Full text of DOI:10.1016/j.ejmech.2019.05.053

Accepted Manuscript
Design, synthesis and biological evaluation of chalcones as reversers of P-
glycoprotein-mediated multidrug resistance
Huanhuan Yin, Jingjing Dong, Yingchun Cai, Ximeng Shi, Hao Wang, Guixia Liu, Yun
Tang, Jianwen Liu, Lei Ma
PII:
S0223-5234(19)30463-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.053
EJMECH 11363
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2019
Revised Date: 24 April 2019
Accepted Date: 19 May 2019
Please cite this article as: H. Yin, J. Dong, Y. Cai, X. Shi, H. Wang, G. Liu, Y. Tang, J. Liu, L. Ma,
Design, synthesis and biological evaluation of chalcones as reversers of P-glycoprotein-mediated
multidrug resistance, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.05.053.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Chalcones as
Reversers of P-Glycoprotein-Mediated Multidrug Resistance
Huanhuan Yin§, Jingjing Dong§, Yingchun Cai§, Ximeng Shi, Hao Wang, Guixia Liu, Yun Tang*,
Jianwen Liu*, Lei Ma*
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237, China
§These authors contributed equally to the work.
Corresponding Authors
*Y.T.: phone, +86-21-64251052; e-mail, ytang234@ecust.edu.cn; *J.L.: phone, +86-21-64252044;
e-mail, liujian@ecust.edu.cn; *L.M.: phone, +86-21-64253691; e-mail, malei@ecust.edu.cn.

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Graphical Abstract

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Abstract: Overexpression of P-glycoprotein (P-gp) is one of the major causes for multidrug
resistance (MDR), which has become a major obstacle in cancer therapy. One hopeful approach to
reverse the MDR is to develop inhibitors of P-gp in expression and/or function. Here, we designed
and synthesized a series of chalcone derivatives as P-gp inhibitors and evaluated their potential
reversal activities against MDR. Among them, the most active compound MY3 had little intrinsic
cytotoxicity and showed the highest activity (RF = 50.19) in reversing DOX resistance in
MCF-7/DOX cells. Further studies demonstrated that MY3 could increase intracellular
accumulation of DOX and inhibit expression of P-gp at mRNA and protein levels. More
importantly, MY3 significantly enhanced the efficacy of DOX against the tumor xenografts
bearing MCF-7/DOX cells with the precondition of unchanged body weight. Therefore, MY3
might represent a promising lead to develop MDR reversal agents for cancer chemotherapy.
Keywords: Chalcones; P-glycoprotein inhibitors; Structure-activity relationship; Multidrug
resistance reversers; Inhibitors docking

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1. Introduction
Cancer is the second leading cause of death after cardiovascular disease worldwide, mainly
because of development of multidrug resistance [1]. It designates a phenomenon where resistance
to a wide variety of drugs with unrelated chemical structures and different mechanisms of action
[2-3]. The mechanisms underlying multidrug resistance (MDR) are complicated, one of them is
the overexpression of P-glycoprotein (P-gp, MDR1/ABCB1), belongs to the large superfamily of
ABC transporters [4-6]. P-gp which features ATPase activity decreases the concentrations of
intracellular anticancer drugs by pumping drugs out of the cells, and consequently leading to drug
insensitivity [4,7]. Down-regulation and/or inhibition of P-gp are two of the most effective
approaches to reverse the MDR phenotype without directly killing MDR cells [8].
Within the last decade, several inhibitors of P-gp-medicated drug efflux have been identified.
But their clinical efficacy is disappointing for many reasons, such as poor potency, inherent
toxicity and adverse interaction with anticancer drugs [9]. One obvious challenge of the first
generation of P-gp inhibitors is inherent toxicity. For instance, the use of verapamil to inhibit P-gp
leads to cardiotoxicity, while the use of cyclosporine A induces and increases of hepatic, renal,
myeloid, and neurological toxicities [10]. Third-generation MDR modulators, such as tariquidar
(XR9576) and zosuquidar (LY335979) inhibit P-gp at the nanomolar level but still suffer the
unexpected toxicity in clinical trials [4]. There are currently no approved drugs available for clinic
use in cancer chemotherapies to reverse MDR by inhibiting P-glycoprotein.
In order to find more potent reversal agents with minimal adverse effects, researchers have
aimed at natural products. As a promising example, chalcones are widely occurring in plants, as
precursors of flavonols and other flavonoids synthesis. Due to their simple chemistry, ease of
synthesis, several replaceable hydrogen to yield a large number of derivatives [11], with a variety
of promising biological activities such as anti-cancer [12], anti-retroviral [13], anti-diabetic [14],
anti-inflammatory [15], anti-hypertensive [16], anti-histaminic [17], anti-malarial [18], and
anti-oxidant [19], they have attracted growing attentions. Many chalcones have been approved for
clinic use, including the choleretic drug metochalcone and the gastrointestinal medication
sofalcone (Figure 1) [20].
Chalcones have been reported to bind to P-gp [21-22] and inhibit its transport activity [6]. For
example, bifendateechalcone hybrids were synthesized and evaluated by Gu et al. The best

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compound not only showed little intrinsic cytotoxicity, but also increased accumulation of
Rhodamine 123 in K562/A02 cells, compared to bifendate and verapamil for inhibition of P-gp
efflux [4]. Liu et al. synthesized a series of basic chalcone analogs to inhibit P-gp by the
calcein-AM accumulation assay on MDCKII/MDR1 cells. The most potential compound
increased calcein-AM accumulation to a greater extent than verapamil, which enhanced the uptake
of doxorubicin (DOX) by MCF-7 cells with over-expressed P-gp [6]. These results strengthen the
evidence that chalcones are potential candidates in reversing P-gp-mediated MDR.
As a member of this huge family, 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC,
Figure 1), is accumulated in the buds of Cleistocalyx operculatus, which has been used for
preparing tonic drinks in southern China for centuries [23]. The MDR reversal effect of DMC has
been extensively studied by Qian et al and co-workers. They discovered that DMC enabled us to
potentiate the cytotoxicity of DOX against drug resistant MCF-7/DOX cells by increasing the
intracellular accumulation of DOX and modulating the expression of MDR1 gene both in vitro
and in vivo [24]. However, the poor accessibility of 3,5-dimethyl-substituted ring A severely
limited further biological screening. Therefore, in this work, we designed a series of chalcone
derivatives without 3,5-dimethyl on ring A to optimize structure and develop more effective
compounds to combat drug resistance.
To better explore the template of DMC, our drug design strategies were guided by
ligand−receptor interaction. Starting with the scaffold of DMC, molecular docking analysis and
binding free energy score indicated that lipophilicity groups at C-3 position of ring A could stretch
into the hydrophobic pockets of P-gp and increase the reversal activity. Hence, first of all, series I
derivatives with isopentenyl group at C-3 position were synthesized to explore the effect of
lipophilicity on reversal activity. In addition, a survey on MDR modulators concluded that a
common feature shared by many MDR modulators is the presence of at least one basic nitrogen,
frequently a piperazine group. So we evaluated the reversal activity of series II derivatives with
heterocyclic nitrogen group on ring B. After preliminary evaluation of reversal effect in vitro,
compound MY3 showed the greatest inhibitory potency and the lowest intrinsic cytotoxicity.
Therefore, we further investigated the mechanism of MY3 on reversing P-gp-mediated MDR.
MY3 not only showed inhibitory activity on P-gp by accumulation assay, but also decreased
expression of P-gp at mRNA and protein levels, respectively, by RT-PCR and western blot. More

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importantly, MY3 significantly enhanced the efficacy of DOX against the tumor xenografts
bearing MCF-7/DOX cells in nude mice with the precondition of unchanged body weight, which
indicated that MY3 did not increase the adverse side effects of the chemotherapeutic agents.
Figure 1. Some typical structures of chalcones.
2. Results and Discussion
2.1. Chemistry
The investigated chalcones were prepared via the classical method of Claisen–Schmidt
reaction. Acetophenone 3 reacted with corresponding benzaldehydes in the presence of 40%
KOH in a mixture of water and ethanol to obtain chalcones MC1-13 in Scheme 1. To get the
target 3, firstly, 2-hydroxy-4,6-dimethoxyacetophenone was treated with prenyl bromide in the
presence of DBU as base, in anhydrous THF, at room temperature under nitrogen atmosphere.
However, we were not able to gain that result. But when we changed
2-hydroxy-4,6-dimethoxyacetophenone
to
1,
the
desired
C-prenylation
of
2,4,6-trihydroxyacetophenone was obtained in 40% yield in the same conditions. Then,
compound 3 was synthesized by methylation of hydroxylated acetophenone 2 using methyl
p-toluenesulfonate in the presence of K2CO3.
Compounds 5a-d were prepared via palladium-catalyzed arylation of compounds 4a-b with
aryl halides and corresponding secondary amines (1-methyl-piperazine, morpholine,
1-boc-piperazine). The subsequent tandem reactions yielded the corresponding benzaldehydes

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6a-d, which reacted with 3, 7 and 8 via Claisen–Schmidt reaction to obtain MY1-2, MY4,
MY6-8, MY10, MY12 and 9 in Scheme 2. TFA is commonly used for deprotection of Boc. But
under acidic condition of TFA, the ortho-allylchalcones MY2 and MY4 led to intramolecular
allylation–cyclisation product. So we replaced TFA to HCl for deprotection of N-boc-piperazinyl
to get MY2 and MY4. Chalcones MY9, MY11 and MY14 were prepared by deprotection of
Boc in MY8, MY10 and 9, under acidic condition of TFA in dry dichloromethane.
1-Boc-piperazine was treated with carboxylate 10 to comprise N-substituted piperazine
derivative 11 catalyzed by weak base (DMAP) and conducted at room temperature. Subsequently,
Compounds 12, 13, 14 and MY13 were synthesized using the same procedure as MY9, 5a-d, 6a-d,
MC1 respectively in scheme 3.
The synthesis of starting materials of MY15-19 (Scheme 4) has been already reported.
Therefore, it won’t be discussed here [25].
Scheme 1. Synthesis of chalcones MC1-13a
aReagents and conditions: (a) BrCH2CH=C(CH3)2, DBU, THF, 30 oC, 24 h; (b) TsOCH3, K2CO3,
EtOH, 80 oC, 4 h; (c) 40% KOH, EtOH, r.t. overnight.

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Scheme 2. Synthesis of chalcones MY1-MY12, MY14a
.
aReagents and conditions: (a) Pd(OAC)2, BINAP, NaO-t-Bu, toluene,100 oC, 24 h; (b) FeCl3 6H2O,
DCM, r.t. 12 h; (c) 40% KOH, EtOH, r.t. overnight; (d) HCl, DCM, 0 oC, 0.5 h; (e) TFA, DCM, 0
oC, 0.5 h;

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Scheme 3. Synthesis of chalcones MY13a
aReagents and conditions: (a) EDCI, DMAP, DCM, r.t. N2, 3 h; (b) TFA, DCM, 0 oC, 0.5 h; (c)
.
Pd(OAC)2, BINAP, NaO-t-Bu, toluene, 100 oC, 24 h; (d) FeCl3 6H2O, DCM, r.t. 12 h; (e) 40%
KOH, EtOH, r.t. overnight.

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Scheme 4. Synthesis of chalcones MY15-19a
.
o
aReagents and conditions: (a) TFA, Et3SiH, r.t., 12 h; (b) AC2O, BF3 Et2O, EA, 40 C, 10 h; (c)
.
(CH3(CH2)nO)2O, n = 1-4, BF3 Et2O, EA, 40 oC, 10 h; (d) TsOCH3, K2CO3, EtOH, 80 oC, 4 h; (e)
40% KOH, EtOH, r.t. overnight; (f) TFA, DCM, 0 oC, 0.5 h.
2.2. Biological assays
2.2.1 Effect of target compounds to reverse DOX resistance in MCF-7/DOX cells
Since an ideal P-gp inhibitor should reverse MDR at non-toxic concentrations to exclude the
possible false positive results caused by the potential toxic compounds themselves [26]. Therefore,
we first evaluated the potential cytotoxicity of all tested compounds against MCF-7/DOX cells

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with overexpressed P-gp (induced by DOX) [27] by MTT assay, to determine the concentrations
of all chalcone derivatives.
As shown in Tables S1-2, the survival rates of most compounds were above 90% when their
concentrations were 12.5 µM. Consequently, in the following MDR reversal experiments, we
chose 12.5 µM as the suitable incubation concentration.
Firstly, we investigated the chemo-sensitizing effect of the target compounds in MCF-7/DOX
cells by MTT assay. We used the reversal-fold (RF) as the potency of MDR reversers, via the ratio
of DOX IC50 values on MCF-7/DOX cells with and without MDR inhibitors, and 0.1% DMSO
was used as solvent control. It can be seen from Tables 1-2 that single treatment of the anticancer
drug DOX has little toxicity on MCF-7/DOX cells (IC50 = 51.19 ± 0.95 µM). However, the
combination of compounds or verapamil (VRP) with DOX showed improved toxic effects on
MCF-7/DOX cells to different extents, suggesting that most of the examined compounds could
reverse DOX-resistance.
We evaluated the reversal activity of MC1-13 to DOX resistance, and from the
structure-activity relationship (SAR) studies, we found that the lipophilicity and position of
substitutions were important for the reverse activity. As depicted in Table 1, the reversal activity
of MC1-11 increased due to introducing the isopentenyl group on ring A compared with DMC,
suggesting the importance of hydrophobicity to the potency of 3′-prenylated chalcones. Moreover,
once substitutions were introduced at C-3 on ring B, whether they were electron-withdrawing or
donating groups, MC2, MC4, MC7, MC9 and MC11 exhibited potent chemo-sensitizing effect
(RF = 4.05-8.18), while the substitutions at C-4 on ring B showed no positive effect on reversing
DOX-resistance and MC3, MC5, MC8 and MC10 exhibited slightly higher RF values than DMC.
Furthermore, the effect of multiple substitutions with methoxy groups at the phenyl moiety was
investigated. Nevertheless, MC12 and MC13 with three methoxy groups didn’t show positive
effect, perhaps due to steric hindrance.
On the basis of above survival results, we synthesized compounds MY1-6 with nitrogen
heterocyclic groups on ring B to check the role of the substitutions with nitrogen atom. First of all,
as expected, a similar trend was observed on the position of substitution in comparison of MY2
and MY3 with MY4 and MY5, respectively (Table 2). In addition, compound MY3 with free N
terminal piperazine group at C-3 on ring B showed a significant increase of RF value compared

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with MY1-2, MY6 with other nitrogen heterocyclic substituents. The results indicated that the
high reversal activity may be closely concerned to piperazinyl without substitutions at the terminal
nitrogen atom, which may be related to the stronger hydrogen bond interaction with P-gp.
Lipophilicity of chalcones is commonly cited as an important factor for P-gp inhibitory activity
[6]. To verify the importance of hydrophobicity at C-3 of ring A for the reversal activity, we
synthesized compounds MY7-13, which appeared different reversal effects. Expectedly, MY9
kept potent chemo-sensitizing effect. Based on MY3, the activity of MY9 was in a sharp decline.
The reason would be explained in the following molecular docking section in detail. On the basis
of these data from above analysis, to improve lipophilicity, MY14 without 2-hydroxy and
MY15-19 with hydrocarbyl groups at C-3 of ring A were synthesized. Unexpectedly, compounds
MY14 and MY15-19 were less potent than the corresponding compounds MY9 and MY3. It is
possible that the hydrogen-bond interaction of hydroxy group contributed to the potency of MY9.
Moreover, carbon chain length is critical for MDR reversal activity, and compounds MY15-18
with hydrocarbyl group (n = 0-3) displayed better reversal activity than MY19 (n = 4).
After the completion of evaluation for reversal activity of all compounds, SAR was summarized
in Figure 2. In general, above findings manifested that the high reversal activity may be closely
related to lipophilicity, position of substitutions and hydrogen-bonding interaction with P-gp.
Specifically, on ring A, the 2-hydroxy group of chalcones is favorable for the formation of
hydrogen-bonding interaction. Besides, suitably extending carbon chain length of alkyl at C-3 of
ring A can increase the reversal activity due to the increase of lipophilicity. On ring B, position of
substitutions and hydrogen-bonding interactions were important for the reverse activity. In other
words, substitutions that can form strong hydrogen bonds with P-gp at C-3 of ring B were the
contributor to the substantially improved potency of reversal activity. So MY3 presented more
remarkable MDR reversal activity than DMC owing to isopentene group stretching into the
hydrophobic pockets and 3-piperazinyl which engaged in hydrogen bond with P-gp.

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Table 1. DOX-Resistance Reversal Activity of MC1-13 at 12.5 µM in MCF-7/DOX Cells
Compd.
DMC
R1
IC50 DOX (µM)a
24.73 ± 1.19
20.64 ± 1.07
7.86 ± 0.70
RFb
2.07
MC1c
H
2.48
6.51
2.47
5.45
2.35
2.94
4.05
2.85
8.18
2.17
5.80
1.86
1.93
5.07
MC2
3-OCH3
4-OCH3
3-Cl
MC3c
MC4
20.72 ± 1.32
9.39 ± 0.59
MC5
4-Cl
21.78 ± 2.02
17.41 ± 1.53
12.64 ± 1.13
17.96 ± 1.49
6.26 ± 0.61
MC6
4-N(CH3)2
3-F
MC7
MC8
4-F
MC9
3-CF3
MC10
MC11
MC12
MC13c
VRPd
4-CF3
23.59 ± 2.05
8.83 ± 0.37
3-NO2
2,3,4-OCH3
3,4,5-OCH3
27.52 ± 2.34
26.56 ± 1.14
10.10 ± 1.24
51.19 ± 0.95
Control (DOX)
aIC50 values were obtained from three independent repeats and represented as the mean ± SD.
bThe reversal fold is calculated as a ratio of IC50 (DOX) to IC50 (P-gp inhibitor + DOX).
c Known compound
dVerapamil.
Table 2. DOX-Resistance Reversal Activity of MY1-MY19 at 12.5 µM in MCF-7/DOX
Compd. R1
R2
R3
IC50 DOX
RFb
2.17
(µM)a
MY1
MY2
MY3
MY4
MY5
MY6
3-(1-methyl-piperazinyl)
CH2CHC(CH3)2
CH2CHC(CH3)2
CH2CHC(CH3)2
CH2CHC(CH3)2
CH2CHC(CH3)2
CH2CHC(CH3)2
OH
OH
OH
OH
OH
OH
23.59 ± 1.70
20.07 ± 1.21
1.02 ± 0.04
52.21 ± 3.01
14.84 ± 1.21
31.60 ± 1.14
3-(1-boc-piperazinyl)
3-piperazinyl
2.55
50.19
0.98
4-(1-boc-piperazinyl)
4-piperazinyl
3.45
3-morpholinyl
1.62

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MY7
3-(1-methyl-piperazinyl)
3-(1-boc-piperazinyl)
3-piperazinyl
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
17.96 ± 0.52
39.95 ± 2.05
2.63 ± 0.04
59.23 ± 2.01
16.62 ± 1.18
18.75 ± 1.68
42.31 ± 1.51
2.85
1.28
19.46
0.86
3.08
2.73
1.21
MY8
MY9
MY10
MY11
MY12
MY13
4-(1-boc-piperazinyl)
4-piperazinyl
3-morpholinyl
3-(1-(3,4-dimethoxybenzoyl)
piperazinyl)
MY14
MY15
MY16
MY17
MY18
MY19
VRPc
3-piperazinyl
H
H
4.90 ± 0.17
4.00 ± 0.12
4.69 ± 0.09
5.12 ± 0.43
3.57 ± 0.16
23.59 ± 1.52
10.10 ± 1.24
51.19 ± 0.95
10.45
12.78
10.92
10.00
14.33
2.17
3-piperazinyl
CH3
OH
OH
OH
OH
OH
3-piperazinyl
CH2CH3
(CH2)2CH3
(CH2)3CH3
(CH2)4CH3
3-piperazinyl
3-piperazinyl
3-piperazinyl
5.07
Control
(DOX)
aIC50 values were obtained from three independent repeats and represented as the mean ± SD.
bThe reversal fold is calculated as a ratio of IC50 (DOX) to IC50 (P-gp inhibitor + DOX).
cVerapamil.
Figure 2. SAR for MDR reversal activity of chalcone derivatives of DMC
2.2.2 Molecular docking analysis
The top-scoring poses of two molecules MY3 and MY9 were docked into the binding pocket of
P-gp (exhibited in Figure 3). Their Glide scores were −8.995 kcal/mol and −8.14 kcal/mol, while
their MM-GBSA ∆Gbinding values were −83.792 kcal/mol and −74.41 kcal/mol, respectively. Both
Glide score and MM-GBSA ∆Gbinding indicated that MY3 had better binding affinity than MY9.
Then we analyzed their binding poses and compared the binding differences between them.
As shown in Figure 3, both compounds were docked into the binding site located in the top
cavity of P-gp (Figure 3A). The core structures of both compounds were mainly stabilized in a

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large hydrophobic hole which was surrounded by amounts of aromatic and hydrophobic residues,
including PHE 332, ILE 863, ILE 864, ILE 860, MET 944, MET 945, TYR 949, CYS 952, PHE
974, and ILE 977. Their piperazinyl rings were located in the top of the pocket composed by TYR
949, CYS 952, PHE 974, and ILE 977, while their tails of benzenes were far away from the top
and deep into an area formed by ILE 860, MET 944, ILE 864, and ILE 863.
From the molecular docking results, we could see that two pi-pi stacking interactions were
formed between the benzene ring connecting with piperazinyl of MY3 and the benzene ring in
side chain of PHE 332 as well as TYR 949 (Figure 3D). However, as to MY9, there was only one
pi-pi stacking interaction formed between the benzene ring connecting with piperazinyl and the
benzene ring in side chain of PHE 974 (Figure 3C). Then from the difference of binding poses
between MY3 and MY9 (Figure 3B), we found that the part of piperazinyl in MY9 standed up
and formed a spatial resistance to prevent TYR 949 from forming pi-pi stacking interaction with
benzene ring. But this situation was not observed for MY3. Moreover, the isopentenyl group in
MY3 contributes to the hydrophobic force formed by the pocket of P-gp. Docking pose analysis
led to relatively high binding affinity for MY3 compared to MY9, which supports the
experimental observation and analysis above. Based on these results, we could conclude that MY3
exhibited better binding affinity than that of MY9.

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Figure 3. Binding modes of compounds MY3 and MY9 to P-gp predicted by molecular docking.
(A) Global ribbon diagram of 3D structural in-ward facing conformation of P-gp. The two
compounds were docked into the pocket. (B) The alignment of docking poses of MY3 and MY9
to compare the binding difference. (C) The docking pose of MY9 (ball and stick model in orange).
(D) The docking pose of MY3 (ball and stick model in grey) Key residues were depicted in stick
model, and the green dotted line represents pi-pi stacking interaction.
Considering the flexibility of the P-gp binding pocket, we performed induced-fit docking for
MY3 to re-evaluate its binding pose and above docking results. The best pose of MY3 in the P-gp
binding pocket was exhibited in Figure 4A. Its induced-fit docking score was -9.55 kcal/mol. As
shown in this figure, its piperazidine ring demonstrated similar pose to that of previous Glide
docking. Besides, its tail of benzenes formed a pi-pi stacking interaction with TYR 949. The long
isopentene group of MY3 was also bridged with TYR 949 by a hydrogen-bonding interaction to
further stabilize its binding conformation in the flexible and large pocket of P-gp. Then, we
aligned our docking pose to that of QZ59, a perfect superposition was found between them. This

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alignment provided an evidence to show that MY3 would have better binding affinity.
Figure 4. (A) The binding mode of compound MY3 to P-gp predicted by induced-fit docking. The
amino acid residues were shown in stick only, while MY3 was shown in ball-and-stick. The green
dotted line represents pi-pi stacking aromatic interaction, and the yellow dotted line represents
H-bond interaction. (B) The alignment of docking poses of QZ59 and MY3 to compare the
difference between them. The two compounds were shown in ball-and-stick (QZ59 in green and
MY3 in grey).
2.2.3 Determination of the intrinsic cytotoxicity
The most potent compound MY3 was selected for further intrinsic cytotoxicity assay using four
pairs of drug-susceptible cell lines and their MDR-sublines including H69 (small cell lung cancer),
parental MDCK II (Madin-Darby canine kidney), MCF-7 (breast cancer), H69AR, MDCK II
BCRP, MCF-7/DOX. Cytotoxic activities are described as cell growth inhibition resulting in a cell
survival of 50% of the control (GI50) in the MTT assay. One aim of this investigation was to
evaluate a possible benefit for in vivo applications. As shown in Table 3, MY3 demonstrated
GI50 values of more than 100 µM in all tested cell lines, except H69AR cell line. So we could
conclude that MY3 is a nontoxic and potent MDR modulator.

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Table 3. Intrinsic Toxicity of MY3 on Sensitive and Drug resistant cell lines
GI50 (µM)a on drug-sensitive and drug-resistant cancer cell lines
Compd.
MDCK
II-mock
>100
MCF- MCF-7/DO
H69
H69AR
MDCK II BCRP
>100
7
X
MY3
>100
91.74 ± 2.35
>100
>100
aHalf-maximal growth inhibition values (GI50) of MY3. The data were obtained from at least three
independent experiments as mean values.
2.2.4 Screening for MRP1 (ABCC1) and BCRP (ABCG2) inhibition
The selectivity toward P-gp/ABCB1 was investigated for MY3 by MTT assay using H69AR
(MRP1/ABCC1) and MDCK II BCRP (BCRP/ABCG2) cell lines overexpressing the
corresponding transport protein. The screening was carried out at MY3 concentration of 12.5 µM
and 25 µM, and the ability to reverse the MDR toward the corresponding transporter was
determined by using the corresponding cytostatic drug DOX and mitoxantrone. Inhibition of the
transport protein led to a decreased cell viability compared to the treatment of single cytostatic
drug. The results of the screening illustrated only low reversal activity toward both cell lines at 25
µM (Figure 5), which confirmed the high selectivity of MY3.
Figure 5. Inhibitory effect of MY3 toward ABCC1 overexpressing cell line H69AR and ABCG2
overexpressing cell line MDCK II BCRP.
2.2.5 Effect of MY3 on DOX accumulation
To verify whether MY3 could induce the intracellular accumulation of DOX in MCF-7/DOX
cells, we used fluorescence microscopy to detect the intracellular DOX concentration in the
drug-resistant MCF-7/DOX cells in which P-gp was overexpressed. As shown in Figure 6,
MCF-7/DOX cells had very little intracellular DOX accumulation when cells were exposed to
single DOX (8 µM) treatment (Figure 6). However, when MCF-7/DOX cells were treated with the
combination of MY3 and DOX, an increase of intracellular DOX-fluorescence (red) was showed

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(Figure 6). These results demonstrated that the combination of MY3 and DOX could inhibit the
drug-efflux function, and promoted the accumulation of DOX in cells.
Figure 6. Effect of the combination of MY3 (12.5 µM) and DOX on the accumulation of DOX in
MCF-7/DOX cells. Cells were stained with DAPI (nuclear stain). As shown, DAPI alone had blue
fluorescence, DOX had red fluorescence.
2.2.6 Effect of MY3 on P-gp expression
Inhibiting the transport function and/or down-regulating the expression level of P-gp may also
cause the reversal of P-gp-mediated MDR [28]. In the following assays, we used both RT-PCR
and western blot to separately evaluate whether compound MY3 and DOX could effectively
decrease the expression level of P-gp at mRNA and protein levels. Western blotting assay showed
that there was no detectable P-gp expression in MCF-7 cells but high P-gp expression (Figure 7A)
in MCF-7/DOX cells. Further western blotting assay demonstrated that MY3 (12.5 µM) combined
with DOX (8 µM) could inhibit P-gp expression in a time-dependent manner (Figure 7A). To
illustrate the effect of the combination on the expression of P-gp in MCF-7/DOX cells, RT-PCR

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assay was also performed. The results demonstrated that the combination of MY3 and DOX could
reverse P-gp-mediated MDR by inhibiting P-gp expression, the same as those from western
blotting assay (Figure 7B).
Figure 7. Effect of the combination of MY3 (12.5 µM) and DOX on P-gp expression. (A) P-gp
protein levels in MCF-7/DOX cells which were exposed to MY3 (12.5 µM), DOX (8 µM) or the
combined treatment for 24 h and 48 h. Combined treatments were able to suppress P-gp
expression in time-dependent manner compared with the single drug treatments. (B) P-gp gene
levels in MCF-7/DOX cells which were exposed to MY3 (12.5 µM), DOX (8 µM) and the
combined treatment for 48 h. Combined treatments were able to suppress P-gp gene expression.
Significant differences were indicated by *p < 0.05, **p < 0.01 and ***p < 0.001vs control; #p <
0.05, ##p < 0.01 and ###p < 0.001 vs DOX treated group.
2.2.7 Effect of MY3 on reversal of P-gp-mediated MDR in mice
To further evaluate the reversal effect of MY3, we examined the growth of tumor xenografts
bearing MCF7/DOX cells in nude mice. Mice were randomly divided into 6 groups (i) vehicle, (ii)
MY3 (5 mg/kg), (iii) MY3 (15 mg/kg), (iv) DOX (2 mg/kg), (v) MY3 (5 mg/kg) + DOX (2 mg/kg)
or (vi) MY3 (15 mg/kg) + DOX (2 mg/kg). Mice were treated for 30 days, and we measured and
recorded the weights and the tumor volumes of the mice every 2 days (Figure 8). As shown in
Figure 8A, single MY3 treatment had no cytotoxicity in xenograft model and the mice had stable
increasing weight, which demonstrated that MY3 could not inhibit the growth of mouse tumors
neither in tumor volume nor weight compared with the control group (Figure 8B and 8C). When
mice were treated with DOX, it failed to inhibit tumor growth (Figure 8B and 8C). When mice
were treated with the combination of MY3 and DOX, the tumor growth was apparently inhibited

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with the precondition of unchanged body weight compared with the single treatment of DOX.
These results displayed that as a potent P-gp inhibitor MY3 could efficiently restore the sensitivity
of MDR human tumors to chemotherapeutic DOX at well-tolerated doses.
Figure 8. MY3 combined with DOX showed effective antitumor activities in mice. (A) During
the treatments, the weight of the mice was weighed every two days. (B) On the 30th day, the
tumors were taken out from the body. (C) Mean tumor weight in six groups after 30 days of
treatment. Mean ± SEM was used to represent error bars. Significant differences were indicated by
*p < 0.05, **p < 0.01 and ***p < 0.001vs control; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs
DOX treated group.
2.2.8 Effect of MY3 on the migratory ability of MCF-7 cells and MCF-7/DOX cells
Many studies indicated that the breast cancer cells displayed great capability of migration.
Therefore, wound-healing assays were performed to determine whether MY3 could inhibit MCF-7
cells and MCF-7/DOX cells migration by observing the wounded confluent monolayers of each
group of cells. The wound of MY3 treated cells was obviously broader than the control cells

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(Figure 9A). So MY3 markedly inhibited the migration of MCF-7 cells and MCF-7/DOX cells
compared with the behavior observed in the control cells. Besides, transwell assays showed that
MY3 inhibited the migratory ability of MCF-7 cells and MCF-7/DOX cells (Figure 9B).
Therefore, the above results suggested that MY3 can inhibit breast cancer and resistant breast
cancer migration.
Figure 9. MY3 inhibited the migratory ability of MCF-7 cells and MCF-7/DOX cells. (A)
Wound-healing assays showed the effects of MY3 on migration of the MCF-7 cells and
MCF-7/DOX cells in each group, cells were treated with MY3 for 24 h. The right was the
quantitative analysis of the migration. Each point represent means ± SD, n = 3. *** p < 0.001 for
MY3 treated cells versus control cells. Scale bar, 50 µM; (B) Transwell assays showed the effects

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of MY3 on migration of the MCF-7 cells and MCF-7/DOX cells in each group, cells were treated
with MY3 for 36 h. The right was the quantitative analysis of the migration. Each point represent
means ± SD, n = 3. *** p < 0.001 for MY3 treated cells versus control cells. Scale bar, 50 µM.
3. Conclusions
In this study, the reversal activities of all the compounds against P-gp-mediated MDR were
evaluated, and most of the target compounds exhibited stronger P-gp inhibitory effect than the
lead compound DMC. One of these compounds, MY3, showed the most potency. Then, it
displayed other potential functions including increasing accumulation of DOX and inhibiting
expression of P-gp at mRNA and protein levels. Additionally, the growth of tumor xenografts
bearing MCF7/DOX cells in nude mice was significantly inhibited by DOX combined with MY3
compared to single drug treatment of DOX or MY3. Besides, the cytotoxicity and the screening
assays of MY3 showed its nontoxicity and high selectivity toward ABCB1. MY3 also showed
inhibition of migration of MCF-7cells and MCF-7/DOX cells. So our findings indicate that MY3
might represent a promising lead to develop P-gp-mediated MDR reversal agent in cancer
chemotherapy. To our knowledge, this is the first report to illuminate the combined functions of
3-piperazinylchalcone and DOX against MCF-7/DOX cells.
4. Experimental Section
4.1 General chemistry
All commercial chemicals and solvents are reagent grade and were used without further
purification unless otherwise noted. All reactions are stirred magnetically.1H (400 MHz) NMR
spectra are recorded on a Bruker AV-400 instrument in CDCl3, (CD3)2CO-d6, DMSO-d6.
Chemical shifts are given in parts per million (ppm) down field from internal reference Me4Si in δ
units, and coupling constants (J) are given in hertz (Hz). Selected data are reported in the
following manner: chemical shift, multiplicity, coupling constant. High resolution mass (HRMS,
ESI) spectral data are acquired by use of a Bruker Daltonics 7T mass spectrometer in positive ion
mode. All final products are purified to > 95% purity by HPLC analysis, using an Essential LC
solution 15C with a Diamonsil C18 column at 35 °C. The gradient solvent system is from 80% A

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and 20% B to 100% A and 0% B (20 min) or 20% A and 80% B to 80% A and 20% B (15 min) to
100% A and 0% B (10 min) (A = CH3CN, B = H2O) with a flow rate of 0.5 mL/min. Melting
point was tested using a WRS-1B digital melting point apparatus. Flash chromatographic
separations are performed using silica gel (particle size 0.040−0.045mm). Solvents are removed
by rotary evaporation under vacuum using a standard rotovap equipped with a cold alcohol
condenser. All filtrations are performed with a vacuum unless otherwise noted.
4.2 2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone (DMC)
DMC was isolated from C. operculatus in our lab as described by Salem et al [29]. DMC purity
was above 97% and the structure of the compound is shown in Figure 1 and analyzed by 1H NMR
and 13C NMR.
Yellow solid, 1H NMR (400 MHz, CDCl3) δ 13.61 (s, 1H, 2′-OH), 7.99 (d, J = 15.7 Hz, 1H, α-H),
7.84 (d, J = 15.7 Hz, 1H, β-H), 7.65 (dd, J = 7.3, 2.1 Hz, 2H, 2,6-ArH), 7.43-7.39 (m, 3H,
3,4,5-ArH), 3.66 (s, 3H, 6′-OCH3), 2.16 (s, 3H, 5′-CH3), 2.13 (s, 3H, 3′-CH3); 13C NMR (100
MHz, CDCl3) δ 193.5 (CO), 162.1 (2′-C), 159.4 (4′-C), 158.9 (6′-C), 143.0 (α-C), 135.4 (1-C),
130.3 (4-C), 129.0 (3,5-C), 128.5 (2,6-C), 126.7 (β-C), 109.1 (3′-C), 109.0 (1′-C), 106.7 (5′-C),
62.4 (6′-OCH3), 8.3 (5′-CH3), 7.7 (3′-CH3). HRMS (ESI): Calcd for C18H18O4 [M+H]+ 299.1283;
Found: 299.1281.
4.3 General procedure for chalcones MC1-13
4.3.1 2,4,6-trihydroxy-3-(3-methylbut-2-en-1-yl) acetophenone (2)
To a stirred solution of 2,4,6-trihydroxyacetophenone 1 (1.68 g, 0.01 mol) in dry methylbenzene
(150 mL) was added prenyl bromide (1.17 mL, 0.01 mol) in the presence of DBU (1.49 mL, 0.1
mol) as base, at room temperature under nitrogen atmosphere, for 48 h. After completion of the
reaction as indicated by TLC, the mixture was diluted with water and extracted with ethyl acetate
(3 × 150 mL). The combined organic layer was washed with brine, dried over Na2SO4, and
concentrated under vacuum, the residue was chromatographed on silica gel to afford 2 [30].
4.3.2 2-hydroxy-3-(3-methylbut-2-en-1-yl)-4,6-dimethoxyacetophenone (3)
To a mixture of compound 2 (1.18 g, 5 mmol) and K2CO3 (1.4 g, 10 mmol) in ethanol (150 mL)
was added TsOCH3 (1.9 mL, 12.5 mmol) dropwise at room temperature, then refluxed for 3 h.
The resulting solution was cooled and was added H2O (2 mL), then extracted with CH2Cl2 (3 ×
150 mL). The organic layer was concentrated under vacuum. Then the residue was subjected to

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column chromatography to afford the desired product 3, 92%.
MC1−13 were synthesized using Claisen–Schmidt reaction as previously described in published
articles [31], in which a mixture of 3 (1.0 mmol), corresponding benzaldehyde (1.2 mmol), 40%
aqueous solution of potassium hydroxide (5 mL) and ethanol (15 mL) was used.
4.3.3
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(phenyl)prop-2-en-1-one
(MC1)
Yellow solid, yield 62%; 1H NMR (600 MHz, DMSO-d6) δ 14.01 (s, 1H, 2′-OH), 7.90 (d, J = 15.6
Hz, 1H, α-H), 7.82-7.61 (m, 3H, β-H, 2,6-ArH), 7.46 (d, J = 7.0 Hz, 3H, 3,4,5-ArH), 6.28 (s, 1H,
5′-ArH), 5.11 (t, J = 7.1 Hz, 1H, -CH=), 3.99 (s, 3H, -OCH3), 3.92 (s, 3H,-OCH3), 3.18 (d, J = 7.1
Hz, 2H, -Ar-CH2), 1.71 (s, 3H, -CH3), 1.61 (s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ
193.0, 164.0, 163.3, 161.8, 142.4, 135.4, 130.9, 130.8, 129.5, 128.9, 128.0, 123.1, 108.9, 106.1,
88.2, 56.7, 56.4, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for C22H24O4 [M+H]+ 353.1753; Found:
353.1754.
4.3.4
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3-methoxyphenyl)prop-2-e
n-1-one (MC2)
Yellow solid, yield 61%; 1H NMR (400 MHz, DMSO-d6) δ 13.95 (s, 1H, 2′-OH), 7.88 (d, J = 15.6
Hz, 1H, α-H), 7.68 (d, J = 15.6 Hz, 1H, β-H), 7.38 (t, J = 7.8 Hz, 1H,-ArH), 7.32 (d, J = 7.6 Hz,
1H, 6-ArH), 7.27 (s, 1H, 2-ArH), 7.03 (dd, J = 7.9, 1.9 Hz, 1H, -ArH), 6.29 (s, 1H, 5′-ArH), 5.10
(t, J = 6.9 Hz, 1H, -CH=), 3.99 (s, 3H, -OCH3), 3.92 (s, 3H, -OCH3), 3.82 (s, 3H, -OCH3), 3.17 (d,
J = 6.9 Hz, 2H, -Ar-CH2), 1.70 (s, 3H, -CH3), 1.61 (s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6)
δ 193.0, 164.0, 163.2, 161.8, 160.1, 142.3, 136.8, 130.8, 130.6, 128.3, 123.1, 121.1, 116.7, 114.0,
108.9, 106.1, 88.3, 56.8, 56.5, 55.6, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for C23H26O5 [M+H]+
383.1858; Found: 383.1857.
4.3.5
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-methoxyphenyl)prop-2-e
n-1-one (MC3)
Yellow solid, yield 63%; 1H NMR (400 MHz, CDCl3) δ 14.13 (s, 1H, 2′-OH), 7.78 (d, J = 10.8 Hz,
2H, -CH=CH-), 7.56 (d, J = 8.7 Hz, 2H, 2,6-ArH), 6.92 (d, J = 8.7 Hz, 2H, 3,5-ArH), 6.00 (s, 1H,

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5′-ArH), 5.21 (t, J = 7.0 Hz, 1H, -CH=), 3.94 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 3.85 (s, 3H,
-OCH3), 3.30 (d, J = 7.0 Hz, 2H, -Ar-CH2), 1.78 (s, 3H, -CH3), 1.67 (s, 3H, -CH3); 13C NMR (150
MHz, DMSO-d6) δ 192.8, 163.7, 163.3, 161.7, 142.8, 130.8, 130.7, 128.0, 125.3, 123.2, 115.0,
108.9, 106.0, 88.1, 56.7, 56.4, 55.8, 25.9, 21.5, 18.1. HRMS (ESI): Calcd for C23H26O5 [M+H]+
383.1858; Found: 383.1854.
4.3.6
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3-chlorophenyl)prop-2-en-
1-one (MC4)
Yellow solid, yield 65%; 1H NMR (400 MHz, CDCl3) δ 13.92 (s, 1H, 2′-OH), 7.84 (d, J = 15.6 Hz,
1H, α-H), 7.66 (d, J = 15.6 Hz, 1H, β-H), 7.57 (s, 1H, 2-ArH), 7.49-7.42 (m, 1H, 4-ArH), 7.34 (dd,
J = 4.8, 1.6 Hz, 2H, 5,6-ArH), 6.01 (s, 1H, 5′-ArH), 5.28-5.15 (m, 1H, -CH=), 3.96 (s, 3H, -OCH3),
3.92 (s, 3H, -OCH3), 3.30 (d, J = 7.0 Hz, 2H, -Ar-CH2), 1.78 (s, 3H, -CH3), 1.68 (s, 3H, -CH3);
13C NMR (150 MHz, DMSO-d6) δ 192.9, 164.2, 163.1, 161.9, 140.4, 137.7, 134.3, 131.3, 130.8,
130.4, 129.7, 128.7, 127.1, 123.1, 108.9, 106.1, 88.3, 56.8, 56.5, 25.9, 21.4, 18.1. HRMS (ESI):
Calcd for C22H23ClO4 [M+H]+ 387.1363; Found: 387.1364.
4.3.7
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-chlorophenyl)prop-2-en-
1-one (MC5)
1
Yellow solid, yield 67%; H NMR (400 MHz, CDCl3) δ 13.98 (s, 1H, 2′-OH), 7.82 (d, J = 15.6 Hz,
1H, α-H), 7.68 (d, J = 15.6 Hz, 1H, β-H), 7.51 (d, J = 8.5 Hz, 2H, 3,5-ArH), 7.36 (d, J = 8.5 Hz,
2H, 2,6-ArH), 5.99 (s, 1H, 5′-ArH), 5.20 (t, J = 7.1 Hz, 1H, -CH=), 3.94 (s, 3H, -OCH3), 3.90 (s,
3H, -OCH3), 3.29 (d, J = 7.0 Hz, 2H, -Ar-CH2), 1.78 (s, 3H, -CH3), 1.67 (s, 3H, -CH3); 13C NMR
(150 MHz, DMSO-d6) δ 192.8, 164.1, 163.3, 161.8, 140.8, 135.3, 134.4, 130.8, 130.5, 129.5,
128.7, 123.1, 108.9, 106.0, 88.2, 56.7, 56.4, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for C22H23ClO4
[M+H]+ 387.1363; Found: 387.1367.
4.3.8
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-dimethylaminophenyl)pr
op-2-en-1-one (MC6)
Orange solid, yield 61%; 1H NMR (600 MHz, DMSO-d6) δ 14.44 (s, 1H, 2′-OH), 7.74 (d, J = 15.4
Hz, 1H, α-H), 7.70 (d, J = 15.4 Hz, 1H, β-H), 7.57 (d, J = 8.7 Hz, 2H, 2,6-ArH), 6.75 (d, J = 8.7
Hz, 2H, 3,5-ArH), 6.26 (s, 1H, 5′-ArH), 5.10 (t, J = 6.7 Hz, 1H, -CH=), 3.98 (s, 3H, -OCH3), 3.91

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(s, 3H, -OCH3), 3.16 (d, J = 6.9 Hz, 2H, -Ar-CH2), 3.01 (s, 6H, -N(CH3)2), 1.70 (s, 3H, -CH3),
1.61 (s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 192.5, 163.4, 163.3, 161.4, 152.4, 144.5,
130.9, 130.6, 123.3, 122.6, 121.7, 112.4, 108.8, 106.1, 88.0, 56.6, 56.3, 26.0, 21.5, 18.1. HRMS
(ESI): Calcd for C24H29NO4 [M+H]+ 395.2097; Found: 395.2100.
4.3.9
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3-fluorophenyl)prop-2-en-
1-one (MC7)
Yellow solid, yield 56%; 1H NMR (400 MHz, CDCl3) δ 13.94 (s, 1H, 2′-OH), 7.84 (d, J = 15.6 Hz,
1H, α-H), 7.69 (d, J = 15.6 Hz, 1H, β-H), 7.38-7.33 (m, 2H, -ArH), 7.29 (dd, J = 11.0, 1.8 Hz,
1H,-ArH), 7.11-7.04 (m, 1H, -ArH), 6.00 (s, 1H, 5′-ArH), 5.20 (t, J = 7.1 Hz, 1H, -CH=), 3.96 (s,
3H, -OCH3), 3.91 (s, 3H, -OCH3), 3.30 (d, J = 7.0 Hz, 2H, -Ar-CH2), 1.78 (s, 3H, -CH3), 1.67 (s,
3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 192.9, 164.2, 163.2, 161.9, 140.7, 138.0, 131.5,
130.8, 129.5, 125.1, 123.1, 117.4, 115.1, 108.9, 106.1, 88.2, 56.8, 56.5, 25.9, 21.4, 18.1. HRMS
(ESI): Calcd for C22H23FO4 [M+H]+ 371.1658; Found: 371.1660.
4.3.10
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-fluorophenyl)prop-2-en-
1-one (MC8)
Yellow solid, yield 59%; 1H NMR (600 MHz, DMSO-d6) δ 13.98 (s, 1H, 2′-OH), 7.84 (d, J = 15.7
Hz, 1H, α-H), 7.81 (dd, J = 8.6, 5.6 Hz, 2H, 3,5-ArH), 7.71 (d, J = 15.6 Hz, 1H, β-H), 7.29 (t, J =
8.8 Hz, 2H, 2,6-ArH), 6.28 (s, 1H, 5′-ArH), 5.11 (t, J = 7.2 Hz, 1H, -CH=), 3.99 (s, 3H, -OCH3),
3.92 (s, 3H, -OCH3), 3.17 (d, J = 7.1 Hz, 2H, -Ar-CH2), 1.70 (s, 3H, -CH3), 1.61 (s, 3H, -CH3);13C
NMR (150 MHz, DMSO-d6) δ 192.9, 164.3 (d, J = 83.1 Hz), 163.1 (d, J = 51.9 Hz), 161.8, 141.2,
132.0 (d, J = 2.7 Hz), 131.2, 131.2, 130.8, 127.8, 123.1, 116.6, 116.5, 108.9, 106.0, 88.2, 56.7,
56.4, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for C22H23FO4 [M+H]+ 371.1658; Found: 371.1656.
4.3.11
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3-trifluoromethylphenyl)pr
op-2-en-1-one (MC9)
1
Yellow solid, yield 56%; H NMR (400 MHz, DMSO-d6) δ 13.84 (s,1H, 2′-OH), 8.07 (d, J = 8.7
Hz, 2H, 2,4-ArH), 7.96 (d, J = 15.8 Hz, 1H, α-H), 7.79 (d, J = 8.3 Hz, 2H, 5,6-ArH), 7.71 (d, J =
15.7 Hz, 1H, β-H), 6.28 (d, J = 9.6 Hz, 1H, 5′-ArH), 5.10 (s, 1H, -CH=), 3.98 (s, 3H, -OCH3), 3.93

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(s, 3H, -OCH3), 3.17 (d, J = 6.1 Hz, 2H, -Ar-CH2), 1.70 (s, 3H, -CH3), 1.61 (s, 3H, -CH3); 13C
NMR (150 MHz, DMSO-d6) δ 192.8, 164.2, 163.2, 161.9, 140.1, 136.6, 131.9, 130.8, 130.6, 130.1,
126.9, 125.7, 123.1, 108.9, 106.1, 88.2, 56.8, 56.5, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for
C23H23F3O4 [M+H]+ 421.1626; Found: 421.1622.
4.3.12
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(4-trifluoromethylphenyl)pr
op-2-en-1-one (MC10)
Yellow solid, yield 58%; 1H NMR (600 MHz, DMSO-d6) δ 13.84 (s, 1H, 2′-OH), 7.96 (d, J = 15.7
Hz, 1H, α-H), 7.94 (d, J = 7.8 Hz, 2H, 3,5-ArH), 7.80 (d, J = 8.2 Hz, 2H, 2,6-ArH), 7.72 (d, J =
15.7 Hz, 1H, β-H), 6.29 (s, 1H, 5′-ArH), 5.10 (t, J = 7.2 Hz, 1H, -CH=), 3.99 (s, 3H, -OCH3), 3.93
(s, 3H, -OCH3), 3.17 (d, J = 7.1 Hz, 2H, -Ar-CH2), 1.71 (s, 3H, -CH3), 1.61 (s, 3H, -CH3); 13C
NMR (150 MHz, DMSO-d6) δ 192.8, 164.3, 163.2, 161.9, 139.9, 139.5, 130.9, 130.8, 129.4, 126.3,
126.3, 126.3, 123.0, 108.9, 106.1, 88.3, 56.8, 56.5, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for
C23H23F3O4 [M+H]+ 421.1626; Found: 421.1627.
4.3.13
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3-nitrophenyl)prop-2-en-1-
one (MC11)
Yellow solid, yield 61%; 1H NMR (400 MHz, CDCl3) δ 13.86 (s, 1H, 2′-OH), 8.46 (s, 1H, 2-ArH),
8.22 (d, J = 8.1 Hz, 1H, 4-ArH), 7.95 (d, J = 15.6 Hz, 1H, α-H), 7.85 (d, J = 7.7 Hz, 1H, -ArH),
7.73 (d, J = 15.6 Hz, 1H, β-H), 7.59 (t, J = 8.0 Hz, 1H, -ArH), 6.02 (s, 1H, 5′-ArH), 5.20 (t, J = 7.0
Hz, 1H, -CH=), 3.98 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3), 3.30 (d, J = 6.9 Hz, 2H, -Ar-CH2), 1.78
(s, 3H, -CH3), 1.68 (s, 3H, -CH3); 13C NMR (150 MHz, CDCl3) δ 192.2, 164.2, 163.9, 161.4,
148.7, 138.4, 137.6, 134.2, 131.6, 131.0, 129.9, 124.0, 122.5, 122.1, 110.2, 106.4, 86.5, 55.9, 55.6,
25.8, 21.4, 17.8. HRMS (ESI): Calcd for C22H23NO6 [M+H]+ 398.1603; Found: 398.1600.
4.3.14
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(2,3,4-trimethoxyphenyl)pr
op-2-en-1-one (MC12)
Yellow solid, yield 58%; 1H NMR (600 MHz, DMSO-d6) δ 14.11 (s, 1H, 2′-OH), 7.90 (d, J = 15.7
Hz, 1H, α-H), 7.85 (d, J = 15.7 Hz, 1H, β-H), 7.49 (d, J = 8.8 Hz, 1H, 6-ArH), 6.92 (d, J = 8.9 Hz,
1H, 5-ArH), 6.27 (s, 1H, 5′-ArH), 5.10 (t, J = 7.1 Hz, 1H, -CH=), 3.98 (s, 3H, -OCH3), 3.92 (s, 3H,

ACCEPTED MANUSCRIPT
-OCH3), 3.87 (s, 3H, -OCH3), 3.87 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.17 (d, J = 7.0 Hz, 2H,
-Ar-CH2), 1.70 (s, 3H, -CH3), 1.61 (s, 3H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 193.0, 163.7,
163.3, 161.7, 156.1, 153.5, 142.4, 137.8, 130.7, 126.5, 124.4, 123.2, 121.7, 109.0, 108.9, 106.1,
88.2, 61.8, 60.9, 56.7, 56.5, 56.4, 25.9, 21.5, 18.1. HRMS (ESI): Calcd for C25H30O7 [M+H]+
443.2070; Found: 443.2073.
4.3.15
(E)-1-(2-hydroxy-4,6-dimethoxy-3-(3-methylbut-2-en-1-yl)phenyl)-3-(3,4,5-trimethoxyphenyl)pr
op-2-en-1-one (MC13).
1
Yellow solid; yield: 63%; H NMR (400 MHz, CDCl3) δ 14.05 (s, 1H, 2′-OH), 7.77 (d, J = 15.5
Hz, 1H, α-H), 7.68 (d, J = 15.5 Hz, 1H, β-H), 6.83 (s, 2H, 2,6-ArH), 6.00 (s, 1H, 5′-ArH), 5.21 (s,
1H, -CH=), 3.94 (s, 3H, -OCH3), 3.91 (s, 6H, -OCH3), 3.90 (s, 6H, -OCH3), 3.30 (d, J = 6.7 Hz,
2H, -Ar-CH2), 1.78 (s, 1H, -CH3), 1.68 (s, 1H, -CH3); 13C NMR (150 MHz, DMSO-d6) δ 192.9,
163.9, 163.2, 161.7, 153.6, 142.7, 140.0, 131.0, 130.8, 127.4, 123.1, 108.9, 106.3, 106.1, 88.2,
60.6, 56.7, 56.4, 56.4, 25.9, 21.4, 18.1. HRMS (ESI): Calcd for C25H30O7 [M+H]+ 443.2070;
Found: 443.2071.
4.4 General procedure for chalcones MY1-2, MY4, MY6-8, MY10, MY12, 9
4.4.1 General procedure for compounds 5a-d
To a Schlenk tube were added Pd(OAc)2 (2.4 mg, 1 mol% Pd), the ligand (0.015 mmol, 1.5 mol%),
NaO-t-Bu (134 mg, 1.4 mmol) and degassed toluene (10 mL). The reaction vessel was fitted with
a rubber septum, evacuated, and back-filled with nitrogen. The Bromobenzene 4a-b (230 mg, 1
mmol) and the amines (1-methyl-piperazine, morpholine, 1-boc-piperazine, 1.2 mmol) were
successively added through the septum (solid aryl halides or amines were added after the addition
of NaO-t-Bu). The reaction mixture was stirred while heating at 100 °C. After 24 h, it was cooled
to ambient temperature. The mixture was diluted with water and extracted with ethyl acetate (3 ×
15 mL). The combined organic layer was washed with brine, dried over Na2SO4, and concentrated
under vacuum, the residue was chromatographed on silica gel to afford 5a-d, which could be used
directly for the next step without further purification [32].
4.4.2 General procedure for compounds 6a-d
.
To a solution of 5a-d (3.4 mmol) in CH2Cl2 (15 mL) at rt. respectively was added FeCl3 6H2O
(3.22 g, 3.5 equ.). The resulting yellow to amber colored suspension was stirred for 15 min and