Synthesis and biological evaluation of bifendate-chalcone hybrids as a new class of potential P-glycoprotein inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.02.050

Overexpression of P-glycoprotein (P-gp) is one of the major problems to successful cancer chemotherapy. To find novel effective P-gp inhibitors, a series of bifendate-chalcone hybrids were synthesized and evaluated. Among them, the most active compound 8g

Full text of DOI:10.1016/j.bmc.2012.02.050

Bioorganic & Medicinal Chemistry 20 (2012) 2540–2548
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of bifendate–chalcone hybrids
as a new class of potential P-glycoprotein inhibitors
Xiaoke Gu a,b, Zhiguang Ren c,d, Xiaobo Tang a,b, Hui Peng c, , Yuanfang Ma d, Yisheng Lai a,b, Sixun Peng a,b
,
⇑
Yihua Zhang a,b,
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
c Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing 100850, PR China
d Institute of Immunology, Medical School of Henan University, Kaifeng 475001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Overexpression of P-glycoprotein (P-gp) is one of the major problems to successful cancer chemotherapy.
To find novel effective P-gp inhibitors, a series of bifendate–chalcone hybrids were synthesized and eval-
uated. Among them, the most active compound 8g had little intrinsic cytotoxicity (IC50 > 200 lM), and
could increase accumulation of Rhodamine 123 in K562/A02 cells more potently than bifendate and
verapamil (VRP) by inhibiting P-gp efflux function. And 8g displayed potent chemo-sensitizing effect
and persisted for much longer time (>24 h) compared with VRP (<6 h). In addition, 8g, unlike VRP,
showed no stimulation on the P-gp ATPase activity, suggesting it is not a P-gp substrate. Therefore, 8g
may represent a promising lead to develop MDR reversal agents for cancer chemotherapy.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 15 January 2012
Revised 21 February 2012
Accepted 22 February 2012
Available online 3 March 2012
Keywords:
P-gp inhibitor
Bifendate
Chalcone
Multidrug resistance
Cancer chemotherapy
1. Introduction
have turned to natural products and their synthetic derivatives,
which are inhibitors of one or more ABC drug efflux pumps, and
Multidrug resistance (MDR) is regarded as a leading cause for
failure of cancer chemotherapy.1 The mechanisms underlying
MDR are complicated, one of them is the overexpression of
P-glycoprotein (P-gp, MDR1/ABCB1), a member of ATP-binding
cassette (ABC) transporter superfamily, which pumps antineoplas-
tic drugs out of the tumor cells, lowering intracellular drug levels,
and consequently leading to drug insensitivity.2 Coadministration
of the anticancer drugs with a P-gp inhibitor is considered to be
an attractive approach to overcome P-gp-mediated MDR.3 Indeed,
during past decades a large amount of compounds have been eval-
uated as P-gp inhibitors, including the third generation inhibitors
zosuqidar, tariquidar and elacridar. Although they have showed
potent P-gp inhibitory effects in animals, their clinical efficacy is
disappointing for many reasons, such as low selectivity, poor
\potency, inherent toxicity and/or adverse pharmacokinetic inter-
action with anticancer drugs.4 So far, no drugs of this class have en-
tered market, therefore, there is an unmet requirement for
developing novel effective P-gp inhibitors.5 Presently, researchers
usually low in toxicity and well tolerated in human body.6–8
Flavonoids, which exist abundantly in edible plants and many
folk-medicines, have already received considerable attention as
ABC transporter inhibitors.6 Chalcones, prominent secondary
metabolites precursors of flavonoids, displayed similar inhibitory
effect on P-gp function as flavonoids.9,10
We previously described that a series of bifendate derivatives are
more potent than bifendate and verapamil (VRP) in reversing P-gp
mediated MDR,11 and the sixalkoxyl biphenyl moiety in bifendate
played a crucial role in its biological activities.12 Keeping these in
mind, and in view of that two aromatic rings joined by a a,b-unsat-
urated carbonyl system in chalcones also played significant effects
in their pharmacological activities, including P-gp inhibitory
effect,13,14 we hypothesized that bifendate scaffold hybridized with
chalcone moiety might enhance the P-gp inhibitory effect of
bifendate. Therefore, in this paper, we firstly synthesized a series
of bifendate–chalcone hybrids (8a–i); additionally, considering that
introduction of nitrile group to the molecule could enhance MDR
reversal effects of P-gp inhibitors,15 we prepared bifendate–chal-
cone hybrids (11a–c) containing a nitrile substituent in the
a,
b-unsaturated moiety. Thus, twelve target compounds (Fig. 1) were
synthesized, and their ability to reverse P-gp-mediated MDR was
subsequently evaluated.
⇑
Corresponding authors. Tel.: +86 10 66932339; fax: +86 10 68159436 (H.P.);
tel./fax: +86 25 83271015 (Y.Z.).
E-mail addresses: p_h2002@hotmail.com (H. Peng), zyhtgd@163.com (Y. Zhang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.050

X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
2541
Treatment of 3 with imidazole in the presence of 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-di-
meth ylamino-pyridine (DMAP) in CH2Cl2 afforded amide 4, which
was then reduced by NaBH4 in THF to give the corresponding
hydroxylate 5. Direct oxidation of 5 with pyridinium chlorochro-
mate (PCC) provided aldehyde 6. Treatment of 6 with various
substituted acetophenones 7a–i through BF3-catalyzed Claisen–
Schmidt reaction afforded 8a–i, respectively. The synthetic routes
of the target compounds 11a–c are shown in Scheme 2. Briefly, Cla-
isen-type condensation between the methyl benzoate 9a–c and
acetonitrile gave
a-cyano ketones 10a–c, which was treated with
6 via -proline-catalyzed Aldol reaction provided 11a–c, respec-
L
tively. All the chemical structures of the target compounds are
listed in Table 1.
3. Results and discussion
3.1. Biological evaluation
Figure 1. Chemical structures of bifendate, chalcone, and bifendate–chalcone
hybrids.
3.1.1. Cytotoxicity assay
Since an ideal P-gp inhibitor should reverse MDR at non-toxic
concentrations,17 the intrinsic cytotoxicity of the target compounds
against parental sensitive K562 cells and K562/A02 cells which
overexpress P-gp (induced by adriamycin)18 was determined by
MTS assay. Anticancer drug adriamycin (ADR) was served as the
2. Chemistry
The synthetic routes of the target compounds 8a–i are depicted
in Scheme 1. Compounds 1–3 were prepared starting from
bifendate by three steps according to literature procedures.16
Scheme 1. Synthesis of the target compounds 8a–i. Reagents and conditions: (a) 10% NaOH, reflux, 5 h; (b) (Ac)2O, reflux, 8 h; (c) CH3OH, reflux, 3 h; (d) imidazole, EDCI,
DMAP, CH2Cl2, rt, 2 h; (e) NaBH4, THF/H2O, 0 °C, 1 h; (f) PCC, CH2Cl2, 0 °C, rt, 1 h; (g) BF3–Et2O, 1,4-dioxane, 115 °C, 4–5 h.
Scheme 2. Synthesis of the target compounds 11a–c. Reagents and conditions: (a) acetonitrile, 60% NaH, 80 °C, 3 h; (b) L-proline, ethanol, rt, 10 h.

2542
X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
Table 1
possessed little (IC50 > 100 lM) or slight (IC50 = 37.2–73.37 lM)
Structures of the target compounds
intrinsic cytotoxicity in vitro, indicating that this kind of com-
pounds are suitable candidates for the development of P-gp inhibi-
tors. And in the following MDR reversal experiments, compounds
were tested at concentrations below IC10
.
3.1.2. Effect of the target compounds on Rh123 accumulation
It is known that P-gp transporter, which overexpresses on the
plasma membrane of drug-resistant cells, can rapidly efflux Rhoda-
mine 123 (Rh123, a fluorescent substrate of P-gp) before it enters
the cells, resulting in a reduction in the fluorescent signal due to
a decrease of Rh123 in the intracellular accumulation. Therefore,
we firstly examined the effect of the target compounds (10 lM)
Compound
R0
R substituents: A-ring
on the intracellular accumulation of Rh123 in K562/A02 cells by
flow cytometry. The classical P-gp inhibitor verapamil (VRP) was
employed as a positive control. As shown in Figure 2A, treatment
20
30
40
50
8a
H
H
H
H
H
with VRP (10 lM) significantly increased Rh123 level in K562/
8b
8c
8d
8e
8f
8g
8h
8i
11a
11b
11c
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
OH
OCH3
H
OCH3
H
H
OCH3
NO2
OCH3
CH3
OCH3
H
H
H
H
OCH3
CH3
OCH3
H
H
H
A02 cells, showing the value of Rh123 accumulation fold change
was 8.1. In comparison, the value of Rh123 accumulation fold
change of bifendate was only 1.2, suggesting that bifendate dis-
played much lower P-gp inhibitory effect than VRP, which is con-
sistent with the previous report.19 Interestingly, this effect was
obviously strengthened by bifendate–chalcone hybrids. As shown
in Figure 2A, the Rh123 accumulation fold changes of most target
compounds were significantly higher than that of bifendate. Nota-
bly, 8d, 8f and 8g dramatically increased the intracellular Rh123
level in K562/A02 cells, and the values of Rh123 accumulation fold
change were 8.3, 9.2 and 9.1, respectively, which were even higher
than that of VRP.
Next, we further investigated the dose response effects of active
compounds 8d, 8f, 8g and 8h on Rh123 accumulation in K562/A02
and parental sensitive K562 cells by flow cytometry. As expected,
VRP, 8d, 8f, 8g and 8h significantly increased Rh123 accumulation
in K562/A02 cells in a dose-dependent manner, while such effect
was not observed in P-gp-negative K562 cells (Fig. 2B), suggesting
that the effect of these compounds on Rh123 accumulation is likely
via inhibiting P-gp function.
OCH3
OCH3
OCH3
CH3
H
H
H
H
H
CN
CN
CN
H
OCH3
OCH3
Table 2
IC50 values of target compounds against K562 and K562/A02 cells in vitro by MTS
assay
a
a
Compound
IC50
(l
M)
Compound
IC50
(
l
M)
K562/A02
K562
K562/A02
K562
8a
8b
8c
8d
8e
8f
>100
>100
>100
>200
>100
>200
>200
>100
>100
>100
>200
>100
>200
>200
8h
8i
>200
>100
37.21
48.36
38.94
>100
46.69
171.8
73.37
>100
61.27
60.29
>100
0.43
11a
11b
11c
Bifendate
8g
ADRb
3.1.3. Inhibitory effect of 8g on P-gp efflux function
To further support the above presumption, the ability of 8g to
inhibit P-gp efflux function was assayed by detecting intracellular
Rh123 according to a previously described method with minor
modification.20 As shown in Figure 3A, the parental sensitive
K562 cells retained most of the fluorescence dye Rh123 (Fig. 3
A1), while K562/A02 cells, which overexpress P-gp, could pump
the Rh123 out efficiently since little Rh123 was observed in
K562/A02 cells after 60-min incubation (Fig. 3 A2). However, the
amount of Rh123 in K562/A02 cells treated with 8g was signifi-
cantly increased in a dose-dependent manner (Fig. 3 B1–3), and
a
IC50 values represent the concentration which results in a 50% decrease in cell
growth after 72 h incubation. IC50 values are expressed as means of triplicate
experiments.
b
ADR was used as a positive control.
positive control. As shown in Table 2, K562/A02 cells were signifi-
cantly resistant to ADR (a substrate of P-gp). The IC50 value of ADR
for K562/A02 cells (46.69 lM) was 108-fold higher than that for
K562 cells (0.43
lM). Interestingly, most of the target compounds
A12
B
9
K562/A02
8
K562
10
8
**
**
7
6
5
4
3
2
1
0
**
**
**
6
*
*
4
*
2
0
VRP
8d
8f
8g
8h
Figure 2. The effect of the target compounds on the intracellular accumulation of Rh123 (0.5
lM). The Rh123 accumulation fold change values were identified by dividing the
fluorescence intensity obtained from each measurement by that of control cells (0.1% DMSO) in each cell group, respectively. Data represent means S.D. of three
independent experiments. ⁄P <0.05, ⁄⁄P <0.01 versus bifendate group.

X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
2543
Figure 3. Inhibitory effect of 8g on P-gp efflux function. K562 cells (A1) or K562/A02 cells (A2, B–C) were incubated with 0.5
lM Rh123 in the presence (B–C) or absence (A) of
8g (B1: 0.33 lM, B2: 3.3 lM, B3: 10 lM) or VRP (C1: 0.33 lM, C2: 3.3 lM, C3: 10 lM) for 30 min. After treatment, the cells were washed twice with PBS and resuspended in
medium, and then incubated at 37 °C for an additional 60 min to allow efflux of the dye. Intracellular Rh123 was then observed and photographed under a fluorescence
microscope. (D) Dose–response of 8g (D1) or VRP (D2, a positive control) in retaining Rh123 accumulation in K562/A02 cells. Intracellular Rh123 was determined by
measuring the cell-associated fluorescence using flow cytometry. The purple line represents the level of Rh123 accumulation in K562 cells. 0.1% DMSO was used as vehicle
control.
8g displayed much higher potency than VRP at the same doses
(Fig. 3 C1–3). Similar results were observed through flow cytome-
try assay. As shown in Figure 3D, the Rh123 level in K562/A02 cells
treated with 0.1% DMSO was much lower than that in K562 cells,
however, it was obviously increased by treatment with 8g in a
A02 and K562 cells was evaluated in the presence or absence of
the hybrids at various concentrations (CH: 10 M, CM: 5 M and
CL: 2.5 M) by MTS assay, using VRP as the positive control. As
shown in Figure 4, both VRP and the target compounds (CH, red
bar) at 10 M displayed little inhibitory effect (<10%) on the sur-
vival of K562/A02 and K562 cells. And anticancer drug ADR was
non-toxic to K562/A02 and K562 cells at 3.5 M and 0.2 M,
l
l
l
l
dose-dependent manner. And treatment with 8g at 3.3
lM could
achieve the similar effect as VRP at 10 M (Figure 3D). These
l
l
l
results verified our assumption that 8g could effectively inhibit
P-gp efflux function, and its potency is much higher than the
classical P-gp inhibitor VRP under the same conditions.
respectively (Fig. 4, green bar). However, combination treatment
with ADR and the target compounds or VRP decreased the survival
rate of K562/A02 cells to various extents (Fig. 4A). Notably, 8d, 8f,
and 8g exhibited potent chemo-sensitizing effect, and 8g was the
most active. It can be seen from Fig. 4A that 90% or 95% of K562/
A02 cells were still surviving by treatment with ADR or 8g alone,
however, the survival rate of the cells was dramatically reduced
3.1.4. Chemo-sensitizing effect of target compounds
Subsequently, we determined chemo-sensitizing effect of the
target compounds. Briefly, the cytotoxicity of ADR against K562/
K562/A02
K562
A
B
120
100
80
60
40
20
0
120
100
control
control
80
60
40
20
0
CH
CH
ADR
ADR
*
*
*
CL+ADR
CM+ADR
CH+ADR
CL+ADR
CM+ADR
*
*
*
*
*
*
** CH+ADR
**
**
**
**
**
8d
8f
8g
8h
VRP
8d
8f
8g
8h
VRP
Figure 4. Chemo-sensitizing effect of the active compounds. Drug-resistant K562/A02 (A) and parental K562 (B) cells were treated without or with 3.5
lM and 0.2 lM ADR,
respectively, combined with three concentrations (CL: 2.5 M; CM: 5 M and CH: 10 M) of active compound or VRP for 72 h. The survival rates of the cells were determined
l
l
l
by MTS assay, and expressed as percentage mean of cell growth S.D. with respect to the control without any treatment of three independent experiments. The classical P-gp
inhibitor VRP was used as a positive control. ⁄P <0.05, ⁄⁄P <0.01 versus ADR treatment alone group.

2544
X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
0 h
6 h
12 h
24 h
A
B
C 100
D 100
100
80
60
40
20
0
100
80
8g
8g
8g
8g
VRP
PBS
VRP
PBS
VRP
PBS
80
60
40
20
0
80
60
40
20
0
VRP
PBS
60
40
20
0
ADR 1(L.2og
µ
1.5M)
A0D.9R (Log1µ.5M)1.8
A0D.9R (Log1µ.5M)1.8
0.3
0.9 1.2
0.6ADR (Log 1µ.M5 )1.8
2.1
0.3 0.6 0.9
1.8 2.1
0.3 0.6
1.2
2.1
0.3 0.6
1.2
2.1
Figure 5. Duration of chemo-sensitizing effect of 8g and VRP toward ADR in K562/A02 cells after incubation and subsequent washout. K562/A02 cells were incubated with
10 M 8g or VRP for 24 h, and then each compound in the culture media was washed out, and the cells were transferred to compound-free fresh media. Various
l
concentrations of ADR was then added to the culture at different time points 0 h (A), 6 h (B), 12 h (C), or 24 h (D) after the removal of the compounds, and incubated for
additional 72 h. Cell proliferation was determined by MTS assay. Data represents means S.E. of triplicate determinations.
to 46%, 20% and 5% by combination treatment with ADR (3.5
l
M)
points 6, 12, and 24 h after 8g was removed, its IC50s were 3.57,
5.80, and 17.04 M, respectively. These data indicated that 8g dis-
and 8g at 2.5, 5 and 10 M, respectively. On the other hand, VRP
l
l
displayed much weaker chemo-sensitizing effect compared with
8g under the same conditions. Even if K562/A02 cells were treated
with VRP at 10 lM, the survival rate was still higher than 32%.
played potent chemo-sensitizing effect and persisted for much
longer time (>24 h) compared with positive control VRP (<6 h)
after they were removed from the culture.
Obviously, 8g was more potent than the classical P-gp inhibitor
VRP, and such chemo-sensitizing effect was not observed in
P-gp-negative K562 cells (Fig. 4B), suggesting that bifendate–chal-
cone hybrids may exert chemo-sensitizing effect by inhibition of
P-gp.
3.1.6. Effect of target compounds on the expression of P-gp at
mRNA and protein levels
It is believed that inhibiting transport function of P-gp and/or
decreasing its expression level could reverse P-gp-mediated
MDR.21 We next examined whether these active compounds, hy-
brids 8d, 8f and 8g, could decrease the expression of P-gp at mRNA
and protein levels by RT-PCR and Western blot analyses, respec-
tively. It can be seen from Figure 6 that there was no marked
changes in P-gp expression at mRNA (Fig. 6A) or protein level
(Fig. 6B) in K562/A02 cells treated with target compounds
3.1.5. Duration of chemo-sensitizing effect of 8g toward ADR in
K562/A02 cells
Next, we selected the most active compound 8g to determine
the duration of such chemo-sensitizing effect according to the pro-
cedure previously described.20 Firstly, K562/A02 cells were incu-
bated with 10
l
M 8g or VRP for 24 h, and then each compound
(10 lM) for 72 h compared with the vehicle control (0.1% DMSO).
in the culture media was washed out, and the cells were trans-
ferred to compound-free fresh media. Various concentrations of
ADR was then added to the culture at different time points (0, 6,
12, or 24 h) after the removal of the compounds, and incubated
for additional 72 h. Cell proliferation was determined by MTS assay
to examine the duration of the MDR-reversal activity of 8g or VRP.
As shown in Figure 5, the IC50s of ADR towards K562/A02 cells
which were in the absence of treatment with 8g or VRP, were
These results suggest that the bifendate–chalcone hybrids may re-
verse P-gp-mediated MDR by inhibiting P-gp efflux function and
not by decreasing P-gp expression.
3.1.7. Effect of 8g on P-gp ATPase activity
P-gp normally requires energy from ATP hydrolysis by the
ATPase, and this enzyme is typically activated for transportation
by P-gp substrates. Some identified reversal agents (e.g., VRP) also
stimulated the basal ATPase activity, thus behaving like a substrate
or competitive inhibitor for the pumps,22 namely, it competes with
the cytotoxic drugs for efflux by the P-gp. Therefore, a high concen-
tration of such drug is necessary to maintain the MDR-reversal
effect. In this case, it might easily reach toxic ranges, leading to a
serious side effect.23 Therefore, the effect of 8g on P-gp ATPase
activity was determined to examine whether it is a P-gp substrate
according to a previously described method.24 Briefly, the activity
of P-gp ATPase was measured in the presence or absence of Na3VO4
(as an inhibitor control), 8g, or VRP (as a substrate control). Then,
the luminescence of each sample was detected in a luminometer.
33.59, 35.15, 54.11, and 51.67 lM at the following time points 0,
6, 12, or 24 h, respectively. Both 8g and VRP significantly sensitized
K562/A02 cells to ADR right after they were removed, the IC50s of
ADR were 2.15 and 12.92
ADR were 26.06, 51.05 and 51.40
l
M, respectively (Fig. 5. A). The IC50s of
M, respectively, when addition
l
was at the time points 6, 12 and 24 h after the removal of VRP.
These IC50s were almost equal to those obtained in the control
group (PBS) which was not exposed to any compounds, suggesting
that the chemo-sensitizing effect of VRP may last no more than 6 h.
In sharp contrast, 8g exhibited significant reversal activity even at
24 h after exposure. When ADR was added to the cells at the time
1.2
A
B
1
0.8
0.6
0.4
0.2
0
DMSO VRP
8d
8g
8f
Figure 6. (A) Effects of active compounds on P-gp mRNA level. The relative P-gp mRNA level treated with compounds was expressed as fold change of the control cultures (in
the presence of 0.1% DMSO). Data shown are mean S.D. from three independent experiments. (B) Effects of active compounds on P-gp protein expression level. Independent
experiments were performed at least three times by Western blot, and a representative experiment is shown.

X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
2545
Table 3
compounds were purified by column chromatography (CC) on sil-
ica gel (200–300 mesh). Analytical TLC was performed on GF/UV
254 plates and the chromatograms were visualized under UV light
at 254 and 365 nm. Subsequently, all the compounds were rou-
tinely analyzed by IR (Shimadzu FTIR-8400S), 1H NMR and 13C
NMR (Bruker ACF-300Q, 300 MHz), and MS (Hewlett–Packard
1100 LC/MSD spectrometer). High resolution mass spectra (HRMS)
were recorded on Agilent technologies LC/MSD TOF. All solvents
were reagent grade and, when necessary, were purified and dried
by standards methods. Compounds 1–3 were synthesized as previ-
ously described.16 Compounds 7a–i and 9a–c were commercially
available.
Effect of 8g on P-gp ATPase activity
Drug
Concentration (
lM)
Luminescencea (relative light units)
Untreated
VRP
Na3VO4
8g
0
200
200
40
242,328 1398
231,282 293*
281,688 439*
271,290 2480*
a
Relative light units represent the level of ATP in the sample, exhibiting a neg-
ative relationship with activity of P-gp ATPase. Data are expressed as means S.D.s
of three separate experiments.
*
p <0.01 versus untreated group, determined by Student’s t test.
And, the luminescence value of each sample represents its ATP
level, which is negatively correlated with the activity of P-gp ATP-
ase. As shown in Table 3, VRP caused a significant increase in the
activity of P-gp ATPase (P <0.01) compared with the untreated
group, while 8g or Na3VO4 showed no stimulation of the P-gp ATP-
ase activity, indicating that 8g was not a P-gp substrate.
5.2. Synthesis of methyl 50-(1H-imidazole-1-carbonyl)-7,70-
dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate 4
To a solution of compound 3 (2 g, 5.0 mmol) in dry CH2Cl2
(50 mL), imidazole (404 mg, 5.9 mmol), EDCI (1.1 g, 5.9 mmol)
and DMAP (61 mg, 0.5 mmol) was added, and the mixture was stir-
red at room temperature for 2 h. The reaction solution was diluted
with water and extracted with CH2Cl2. The organic layer was
washed sequentially with 1 N HCl, saturated NaHCO3 solution
and brine, then dried with sodium sulfate, filtered and evaporated
in vacuo to give corresponding crude compound 4 (98%), which
was used in the next without other purification.
3.2. Structure–activity relationships
As mentioned above, the bifendate–chalcone hybrids exhibited
potent P-gp inhibitory effect, and several compounds possessed
activity comparable to or even stronger than classical P-gp inhibitor
VRP. It was obvious that R substitutes at benzene ring (A-ring) of
chalcone moiety (Table 1) played an important role in the
P-gp-mediated MDR reversal activity. Firstly, the compounds with
a methoxy group in the A-ring usually showed more potent
inhibitory effect than those bearing a hydroxy or nitro group (8c
vs 8b and 8i). Secondly, compounds with a methoxy group at the
ortho or meta position of the A-ring exhibited better activity than
those with a para-methoxy group (8d vs 8c, 8f vs 8e). Thirdly, chang-
ing the methoxy group into methyl group could further improved
the P-gp inhibitory activity (8g vs 8e, 11b vs 11a), and the com-
pound 8g was the most active. Fourthly, when a nitrile group was
5.3. Synthesis of methyl 50-(hydroxymethyl)-7,70-dimethoxy-
{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate 5
A water solution of NaBH4 (332 mg, 8.8 mmol) was dropped to
the stirred solution of compound 4 (2 g, 4.4 mmol) in THF 30 mL at
0 °C. The reaction mixture was stirred for 1 h at room temperature.
After the completion of reaction, 10% aqueous HCl was added slowly
to the mixture until no hydrogen generated. The mixture was fil-
tered and washed with CH2Cl2 several times. The filtrate was diluted
withwater and extracted with CH2Cl2. The organic layer was washed
with brine, dried with anhydrous sodium sulfate, filtered and evap-
orated in vacuum. The crude product was purified by chromatogra-
phy (PE/EtOAc = 5:3) to afford the title compound 5 (1.6 g). As a
white solid, yield: 92%; mp: 126–128 °C. Analytical data for 5: 1H
NMR (CDCl3, 300 MHz, d ppm): 3.71 (s, 3H, COOCH3), 3.95 (s, 3H, Ar-
OCH3), 3.97 (s, 3H, ArOCH3), 4.35 (d, 1H, CH2OH, J = 12 Hz), 4.40 (d,
1H, CH2OH, J = 12 Hz), 5.91–6.04 (m, 4H, 2 ꢀ OCH2O), 6.78 (s, 1H,
Ar-H), 7.33 (s, 1H, Ar-H). ESI-MS m/z: 413 [M+Na]+.
introduced into the
a,b-unsaturated moiety of the hybrids, the
P-gp inhibitory activity was significantly reduced (8c vs 11a, 8h vs
11c), probably because the introduction of nitrile group might
change the electron density distribution of the hybrids, thus
decreasing the affinity with P-gp. However, the precise SARs could
be drawn only when further investigation will be completed.
4. Conclusions
In summary, a series of novel bifendate–chalcone hybrids were
synthesized and their inhibitory effect on P-gp was evaluated using
the classical P-gp inhibitor VRP as a positive control. Most of the tar-
get compounds exhibited strong P-gp inhibitory effect than the lead
compound bifendate, and compounds 8d, 8f and 8g more potently
reversed P-gp-mediated MDR than VRP through blocking the drug
efflux function of P-gp. Further study showed that 8g did not inhibit
expression of P-gp at mRNA and protein levels. Additionally, the
chemo-sensitizing effect of 8g persisted much longer (>24 h) than
that of VRP (<6 h). And, unlike VRP, 8g showed no stimulation of
the P-gp ATPase activity, suggesting it was not a P-gp substrate. Be-
5.4. Synthesis of methyl 50-formyl-7,70-dimethoxy-{4,40-
bibenzo[d][1,3]dioxole}-5-carboxylate 6
Compound 5 (1.5 g, 3.8 mmol) was dissolved in CH2Cl2 (30 mL),
and cooled to 0 °C. PCC (900 mg, 4.2 mmol) was added and the
solution was stirred for 1 h at rt. Then, the mixture was filtered
through Celite, and washed with CH2Cl2. The solvent was removed
under reduced pressure and the crude product was purified by
flash chromatography (PE/EtOAc = 3:1) gave the title compound 6
(1.46 g). As a white solid, yield: 98%; mp: 179–181 °C. Analytical
data for 6: 1H NMR (CDCl3, 300 MHz, d ppm): 3.68 (s, 3H, COOCH3),
3.98 (s, 3H, ArOCH3), 3.99 (s, 3H, ArOCH3), 6.01–6.10 (m, 4H,
2 ꢀ OCH2O), 7.32 (s, 1H, Ar-H), 7.42 (s, 1H, Ar-H); 13C NMR (CDCl3,
75 MHz, d ppm): d 52.1 (COOCH3), 56.5 (OCH3), 56.8 (OCH3), 102.6
(OCH2O), 102.7 (OCH2O), 107.7, 108.2, 111.9, 114.1, 124.5, 128.7,
138.3, 139.9, 143.2, 143.7, 147.0, 148.1, 166.1 (COOCH3), 189.5
sides, the intrinsic cytotoxicity of 8g was very low (IC50 > 200 lM) in
vitro, therefore, 8g might represent a promising lead to develop
P-gp-mediated MDR reversal agents in cancer chemotherapy.
5. Experimental protocols
5.1. Chemical analysis
(CHO); IR (KBr, cmꢁ1):
m 3007, 2903, 2358, 1710, 1682, 1632,
1579, 1484, 1455, 1416, 1406, 1352, 1306, 1255, 1156, 1100,
1039, 928, 858, 757; ESI-MS m/z: 411 [M+Na]+; HRMS (ESI m/z)
for C19H16O9Na calcd 411.0692, found 411.0696 [M+Na]+.
Melting points were determined on a Mel-TEMP II melting
point apparatus which was uncorrected. All of the synthesized

2546
X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
5.5. General procedure for the preparation of 8a–i
BF3–Et2O was added (35.6 L, 0.13 mmol) to the stirred solution
111.9, 112.2, 113.8 (2 ꢀ ArC), 121.6 (CH@CHCO), 124.4, 128.4,
130.7 (2 ꢀ ArC), 131.2, 136.8, 138.6, 142.0 (C H@CHCO), 143.1,
143.4, 147.2, 147.8, 163.1, 166.1 (COOCH3), 188.7 (CH@CHCO); IR
l
of compound 6 (100 mg, 0.26 mmol) and various substituted ace-
tophenones 7a–i (0.28 mmol) in dry 1,4-dioxane at room temper-
ature under a nitrogen atmosphere. The reaction mixture was
stirred and refluxed for 4–5 h at 115 °C and cooled to room tem-
perature. And then, the reaction mixture was diluted with satu-
rated aqueous NaHCO3 and extracted with EtOAc. The organic
layer was washed with brine, dried with anhydrous sodium sulfate,
filtered and evaporated in vacuum. The crude product was purified
by column chromatography (PE/EtOAc = 10:1–1:1) to yield the title
compounds, respectively.
(KBr, cmꢁ1):
m 2946, 1713, 1655, 1635, 1603, 1582, 1432, 1417,
1352, 1320, 1250, 1171, 1101, 1042, 926, 828, 759; ESI-MS: m/z
521 [M+H]+, 543 [M+Na]+; HRMS (ESI m/z) for C28H25O10 calcd
521.1448, found 521.1450 [M+H]+.
5.5.4. (E)-Methyl 7,70-dimethoxy-50-(3-(2-methoxyphenyl)-3-
oxoprop-1-en-1-yl)-{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate
(8d)
The title compound was obtained starting from 6 and 20-methoxy-
acetophenone. As a yellow solid, yield: 63%; mp: 74–76 °C. Analytical
data for 8d: 1H NMR (CDCl3, 300 MHz, d ppm): 3.66 (s, 3H, OCH3),
3.93 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 5.92–6.00
(m, 4H, 2 ꢀ OCH2O), 6.90–6.98 (m, 2H, 2 ꢀ Ar-H), 7.03 (s, 1H, Ar-H),
7.10 (d, 1H, CH@CHCO, J = 15.7 Hz), 7.30–7.46 (m, 4H, CH@CHCO,
3 ꢀ Ar-H); 13C NMR (CDCl3, 75 MHz, d ppm): d 52.0 (COOCH3), 55.6
(OCH3), 56.5 (OCH3), 56.7 (OCH3), 102.1 (OCH2O), 102.4 (OCH2O),
106.0, 110.0, 111.5, 111.8, 111.2, 120.5 (CH@CHCO), 124.3, 126.5,
128.2, 129.3, 130.0, 132.4, 136.7, 138.4, 141.9 (CH@CHCO), 142.9,
143.3, 147.0, 147.7, 157.8, 166.1 (COOCH3), 193.0 (CH@CHCO); IR
5.5.1. (E)-Methyl 7,70-dimethoxy-50-(3-oxo-3-phenylprop-1-en-
1-yl)-{4,40-bibenzo[d][1,3] dioxole}-5-carboxylate (8a)
The title compound was obtained starting from 6 and acetophe-
none. As a yellow solid, yield: 70%; mp: 141–143 °C. Analytical
data for 8a: 1H NMR (CDCl3, 300 MHz, d ppm): 3.67 (s, 3H,
COOCH3), 3.99 (s, 3H, ArOCH3), 4.00 (s, 3H, ArOCH3), 5.96–6.08
(m, 4H, 2 ꢀ OCH2O), 7.08 (s, 1H, Ar-H), 7.25 (d, 1H, CH@CHCO,
J = 15.0 Hz), 7.41 (s, 1H, Ar-H), 7.44–7.48 (m, 2H, Ar-H), 7.54 (d,
1H, CH@CHCO, J = 15.1 Hz), 7.52–7.58 (m, 1H, Ar-H), 7.88 (d, 2H,
(KBr, cmꢁ1):
m 2938, 1720, 1635, 1598, 1582, 1484, 1416, 1352,
Ar-H, J = 7.8 Hz); 13C NMR (CDCl3, 75 MHz,
d
ppm):
d
52.1
1322, 1290, 1245, 1173, 1100, 1042, 926, 757; ESI-MS: m/z 521
[M+H]+, 543 [M+Na]+; HRMS (ESI m/z) for C28H25O10 calcd
521.1448, found 521.1449 [M+H]+.
(COOCH3), 56.6 (OCH3), 56.7 (OCH3), 102.2 (OCH2O), 102.5
(OCH2O), 106.3, 110.0, 111.8, 112.3, 121.7 (CH@CHCO), 124.3,
128.1, 128.4 (2 ꢀ ArC), 128.5 (2 ꢀ ArC), 132.6, 136.9, 138.2, 138.5,
142.9 (C H@CHCO), 143.0, 143.4, 147.1, 147.8, 166.1 (COOCH3),
5.5.5. (E)-Methyl 50-(3-(2,4-dimethoxyphenyl)-3-oxoprop-1-en-
1-yl)-7,70-dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-
190.5 (CH@CHCO); IR (KBr, cmꢁ1):
m 2945, 2368, 1718, 1654,
1636, 1584, 1447, 1432, 1416, 1353, 1173, 1100, 1042, 933, 757;
ESI-MS: m/z 491.1 [M+H]+; HRMS (ESI m/z) for C27H23O9 calcd
491.1342, found 491.1344 [M+H]+.
carboxylate (8e)
The title compound was obtained starting from 6 and 20,40-dime-
thoxyacetophenone. As a yellow solid, yield: 41%; mp: 86–88 °C.
Analytical data for 8e: 1H NMR (CDCl3, 300 MHz, d ppm): 3.66 (s,
3H, OCH3), 3.83 (s, 6H, 2 ꢀ OCH3), 3.85 (s, 3H, OCH3), 3.98 (s, 6H,
2 ꢀ OCH3), 5.96–6.01 (m, 4H, 2 ꢀ OCH2O), 6.44 (d, 1H, Ar-H,
J = 2.0 Hz), 6.49–6.52 (m, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.25 (d, 1H,
CH@CHCO, J = 15.6 Hz), 7.38 (s, 1H, Ar-H), 7.41 (d, 1H, CH@CHCO,
J = 15.6 Hz). 7.61 (d, 1H, Ar-H, J = 8.6 Hz); 13C NMR (CDCl3,
75 MHz, d ppm): d 52.0 (COOCH3), 55.5 (OCH3), 55.6 (OCH3), 56.5
(OCH3), 56.7 (OCH3), 98.7, 102.0 (OCH2O), 102.4 (OCH2O), 105.0,
106.1, 110.2, 111.8, 112.1, 122.3, 124.4, 126.7 (CH@CHCO), 128.7,
132.6, 136.5, 138.5, 140.4 (CH@CHCO), 142.9, 143.3, 147.1, 147.8,
5.5.2. (E)-Methyl 50-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-
yl)-7,70-dimethoxy-{4,40-bibenzo [d][1,3]dioxole}-5-carboxylate
(8b)
The title compound was obtained starting from 6 and 40-
hydroxyacetophenone. As a yellow solid, yield: 72%; mp: 248–
250 °C. Analytical data for 8b: 1H NMR (DMSO, 300 MHz, d ppm):
3.57 (s, 3H, COOCH3), 3.95 (s, 3H, ArOCH3), 3.99 (s, 3H, ArOCH3),
5.89–6.07 (m, 4H, 2 ꢀ OCH2O), 6.86 (s, 1H, Ar-H), 6.89 (s, 1H, Ar-
H), 7.28–7.45 (m, 3H, 2ꢀAr-H, CH@CHCO), 7.70 (d, 1H, CH@CHCO,
J = 15.6 Hz), 7.97 (d, 2H, Ar-H, J = 7.8 Hz), 10.39 (s, 1H, OH); 13C
NMR (DMSO, 75 MHz, d ppm): d 51.9 (COOCH3), 56.4 (OCH3),
56.5 (OCH3), 101.9 (OCH2O), 102.4 (OCH2O), 106.2, 109.4, 110.9,
111.9, 115.2 (2 ꢀ ArC), 121.1 (CH@CHCO), 124.4, 127.6, 129.1,
131.0 (2 ꢀ ArC), 136.2, 137.8, 140.2 (C H@CHCO), 142.5, 143.0,
146.5, 147.5, 162.1, 165.5 (COOCH3), 186.9 (CH@CHCO); IR (KBr,
160.2, 164.8, 166.1 (COOCH3), 190.4 (CH@CHCO); IR (KBr, cmꢁ1):
2359, 1718, 1636, 1608, 1412, 1383, 1355, 1326, 1253, 1163,
1097, 1040, 926, 665; ESI-MS: m/z 551 [M+H]+, 573 [M+Na]+; HRMS
(ESI m/z) for C29H27O11 calcd 551.1553, found 551.1556 [M+H]+.
m
5.5.6. (E)-Methyl 50-(3-(2,5-dimethoxyphenyl)-3-oxoprop-1-en-
1-yl)-7,70-dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-
cmꢁ1):
m 3244, 2359, 1708, 1635, 1605, 1586, 1558,1635,
1442,1431, 1236, 1209, 1165, 1134, 1040, 927, 841; ESI-MS: m/z
507.0 [M+H]+; HRMS (ESI m/z) for C27H23O10 calcd 507.1291, found
507.1293 [M+H]+.
carboxylate (8f)
The title compound was obtained starting from 6 and 20,50-dime-
thoxyacetophenone. As a yellow solid, yield: 58%; mp: 65–67 °C.
Analytical data for 8f: 1H NMR (CDCl3, 300 MHz, d ppm): 3.66 (s,
3H, COOCH3), 3.77 (s, 6H, 2 ꢀ ArOCH3), 3.97 (s, 6H, 2 ꢀ ArOCH3),
5.90–6.04 (m, 4H, 2 ꢀ OCH2O), 6.78–6.84 (m, 1H, Ar-H), 6.94–6.98
(m, 1H, Ar-H), 7.03 (s, 2H, Ar-H), 7.15 (d, 1H, CH@CHCO,
J = 15.0 Hz), 7.44–7.48 (m, 2H, Ar-H), 7.34 (d, 1H, CH @CHCO,
J = 15.1 Hz), 7.37 (s, 1H, Ar-H); 13C NMR (CDCl3, 75 MHz, d ppm): d
52.0 (COOCH3), 55.8 (OCH3), 56.4 (OCH3), 56.5 (OCH3), 56.7
(OCH3), 102.1 (OCH2O), 102.5 (OCH2O), 106.0, 109.9, 111.7, 113.2,
114.4, 118.4 (CH@CHCO), 124.3, 126.3, 128.2, 129.8, 136.7, 138.4,
141.9 (CH@CHCO), 142.9, 143.3, 147.1, 147.7, 152.2, 153.5, 166.1
5.5.3. (E)-Methyl 7,70-dimethoxy-50-(3-(4-methoxyphenyl)-3-
oxoprop-1-en-1-yl)-{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate
(8c)
The title compound was obtained starting from 6 and 40-methoxy-
acetophenone. As a yellow solid, yield: 81%; mp: 172–174 °C. Analyt-
ical data for 8c: 1H NMR (CDCl3, 300 MHz, d ppm): 3.67 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 5.95–6.02
(m, 4H, 2 ꢀ OCH2O), 6.92 (d, 2H, 2 ꢀ Ar-H, J = 8.7 Hz), 7.07 (s, 1H,
Ar-H), 7.27 (d, 1H, CH@CHCO, J = 15.6 Hz), 7.41 (s, 1H, Ar-H), 7.53
(d, 1H, CH@CHCO, J = 15.6 Hz), 7.91 (d, 2H, 2 ꢀ Ar-H, J = 8.7 Hz); 13
C
(COOCH3), 192.5 (CH@CHCO); IR (KBr, cmꢁ1):
m 2359, 1636, 1413,
NMR (CDCl3, 75 MHz, d ppm): d 52.1 (COOCH3), 55.5 (OCH3), 56.7
1384, 1171, 1102, 1048, 763; ESI-MS: m/z 551.0 [M+H]+; HRMS
(ESI m/z) for C29H27O11 calcd 551.1553, found 551.1556 [M+H]+.
(OCH3), 56.8 (OCH3), 102.2 (OCH2O), 102.6 (OCH2O), 106.3, 110.1,

X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
2547
5.5.7. (E)-Methyl 50-(3-(2,4-dimethylphenyl)-3-oxoprop-1-en-1-
yl)-7,70-dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate
(8g)
added and extracted with EtOAc, and the organic layer was washed
with brine, dried with anhydrous sodium sulfate, filtered and
evaporated in vacuum to give the corresponding title compounds
10a–c, which were carried onto the next step without further
purification.
The title compound was obtained starting from 6 and 20,
40-dimethylacetophenone. As a yellow solid, yield: 68%; mp:
76–78 °C. Analytical data for 8g: 1H NMR (CDCl3, 300 MHz, d
ppm): 2.33 (s, 6H, 2 ꢀ CH3), 3.67 (s, 3H, OCH3), 3.97 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 5.93–6.01 (m, 4H,
2 ꢀ OCH2O), 6.91 (d, 1H, CH@CHCO, J = 15.9 Hz), 6.92–7.02 (m,
3H, 3 ꢀ Ar-H), 7.22 (d, 1H, CH@CHCO, J = 15.9 Hz), 7.24–7.28 (m,
1H, Ar-H), 7.37 (s, 1H, Ar-H); 13C NMR (CDCl3, 75 MHz, d ppm): d
20.3 (CH3), 21.3 (CH3), 52.0 (COOCH3), 56.5 (OCH3), 56.7 (OCH3),
102.2 (OCH2O), 102.5 (OCH2O), 105.9, 109.9, 111.8, 112.2, 124.3,
125.8 (CH@CHCO), 126.1, 127.9, 128.4, 132.0, 136.1, 136.9, 137.2,
138.4, 140.6, 143.0, 143.4, 143.6 (CH@CHCO), 147.1, 147.6, 166.0
5.7. General procedure for the preparation of 11a–c
To the solution of 6 (100 mg, 0.26 mmol) and 10a–c (0.31 mmol)
in ethanol (15 mL) were added L-proline (12 mg, 0.1 mmol). The
mixture was stirred for 10 h at rt. After the completion of reaction,
the mixture was evaporated under reduced pressure and the resi-
due was purified by column chromatography (PE/EtOAc = 15:1–
5:1) to yield the title compounds, respectively.
(COOCH3), 195.9 (CH@CHCO); IR (KBr, cmꢁ1):
m
2918, 1719, 1636,
5.7.1. (E)-Methyl 50-(2-cyano-3-(4-methoxyphenyl)-3-oxoprop-
1-en-1-yl)-7,70-dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-
carboxylate (11a)
1583, 1432, 1416, 1354, 1173, 1101, 1042, 936, 821, 762; ESI-
MS: m/z 519 [M+H]+, 541 [M+Na]+; HRMS (ESI m/z) for C29H27O9
calcd 519.1655, found 519.1656 [M+H]+.
The title compound was obtained starting from 6 and 10a. As a
yellow solid, yield: 87%; mp: 76–78 °C. Analytical data for 11a: 1
H
5.5.8. (E)-Methyl 7,70-dimethoxy-50-(3-oxo-3-(3,4,5-
NMR (CDCl3, 300 MHz, d ppm): 3.69 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 3.97 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 5.92–6.08 (m, 4H,
2 ꢀ OCH2O), 6.89 (d, 2H, 2 ꢀ Ar-H, J = 8.4 Hz), 7.36 (s, 1H, Ar-H),
7.73 (s, 2H, 2 ꢀ Ar-H), 7.76 (s, 1H, Ar-H), 7.98 (s, 1H, CH@CCN);
13C NMR (CDCl3, 75 MHz, d ppm): d 21.6 (CH3), 52.2 (COOCH3),
55.5 (OCH3), 56.6 (OCH3), 56.7 (OCH3), 102.3 (2 ꢀ OCH2O), 108.2,
108.7 (CH@CHCO), 109.7 (CN), 112.1, 113.7 (2 ꢀ ArC), 114.3,
117.6, 124.4, 125.1, 128.5, 131.6 (2 ꢀ ArC), 132.3, 138.4, 139.0,
143.3, 143.4, 143.8, 147.1, 147.9, 152.6 (CH@CHCO), 163.6, 165.9
trimethoxyphenyl)prop-1-en-1-yl)-{4,40-
bibenzo[d][1,3]dioxole}-5-carboxylate (8h)
The title compound was obtained starting from 6 and 30,40,
50-trimethoxyacetophenone. As a yellow solid, yield: 61%; mp:
80–82 °C. Analytical data for 8h: 1H NMR (CDCl3, 300 MHz,
d ppm): 3.68 (s, 3H, OCH3), 3.90 (s, 6H, 2 ꢀ OCH3), 3.91 (s,
3H, OCH3), 3.98 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 5.98–6.02 (m,
4H, 2 ꢀ OCH2O), 7.05 (s, 1H, Ar-H), 7.15 (s, 2H, 2 ꢀ Ar-H), 7.19 (d,
1H, CH@CHCO, J = 15.5 Hz), 7.41 (s, 1H, Ar-H), 7.61 (d, 1H, CH
@CHCO, J = 15.5 Hz); 13C NMR (CDCl3, 75 MHz, d ppm): d 52.0
(COOCH3), 56.4 (2 ꢀ OCH3), 56.7 (OCH3), 56.8 (OCH3), 60.9
(OCH3), 102.1 (OCH2O), 102.5 (OCH2O), 106.2 (2 ꢀ ArC), 107.7,
110.2, 111.9, 112.0, 121.2 (CH@CHCO), 124.3, 128.2, 133.6, 136.9,
138.5, 142.9 (CH@CHCO), 143.0, 143.3, 146.3, 147.3, 147.7, 153.1
(COOCH3), 187.8 (CH@CHCO); IR (KBr, cmꢁ1):
m 2914, 2365, 1714,
1637, 1602, 1410, 1384, 1354, 1254, 1174, 1101, 1039, 924, 750;
ESI-MS: m/z 546 [M+H]+, 568 [M+Na]+; HRMS (ESI m/z) for
C
29H24NO10 calcd 546.1400, found 546.1403 [M+H]+.
5.7.2. (E)-Methyl 50-(2-cyano-3-oxo-3-(p-tolyl)prop-1-en-1-yl)-
7,70-dimethoxy-{4,40-bibenzo[d][1,3]dioxole}-5-carboxylate
(11b)
(2 ꢀ ArC), 166.0 (COOCH3), 188.8 (CH@CHCO); IR (KBr, cmꢁ1):
m
3004, 2930, 1719, 1635, 1577, 1432, 1414, 1348, 1321, 1192,
1173, 1158, 1127, 1100, 1043, 926, 836, 751; ESI-MS: m/z 581
[M+H]+, 603 [M+Na]+; HRMS (ESI m/z) for C30H29O12 calcd
581.1659, found 581.1663 [M+H]+.
The title compound was obtained starting from 6 and 10b. As a
yellow solid, yield: 88%; mp: 133–135 °C. Analytical data for 11b:
1H NMR (CDCl3, 300 MHz, d ppm): 2.41 (s, 3H, CH3), 3.69 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 5.92–6.08 (m, 4H,
2 ꢀ OCH2O), 7.20 (d, 2H, 2 ꢀ Ar-H, J = 8.0 Hz), 7.36 (s, 1H, Ar-H),
7.60 (d, 2H, 2 ꢀ Ar-H, J = 8.0 Hz). 7.67 (s, 1H, Ar-H), 8.01 (s, 1H, CH
@CCN); 13C NMR (CDCl3, 75 MHz, d ppm): d 21.6 (CH3), 52.1
(COOCH3), 56.6 (OCH3), 56.7 (OCH3), 102.5 (OCH2O), 102.6
(OCH2O), 108.4, 108.7 (CH@CHCO), 108.8 (CN), 112.1, 114.4,
117.4, 124.4, 125.0, 129.0 (2 ꢀ ArC), 129.1 (2 ꢀ ArC), 133.4, 138.4,
139.1, 143.3, 143.4, 143.8, 147.1, 147.8, 153.1 (CH@CHCO), 165.9
5.5.9. (E)-Methyl 7,70-dimethoxy-50-(3-(4-nitrophenyl)-3-
oxoprop-1-en-1-yl)-{4,40-bibenzo[d] [1,3]dioxole}-5-
carboxylate (8i)
The title compound was obtained startingfrom 6 and 40-nitroace-
tophenone. As a yellow solid, yield: 45%; mp: 183–185 °C. Analytical
data for 8i: 1H NMR (CDCl3, 300 MHz, d ppm): 3.69 (s, 3H, OCH3), 4.00
(s, 3H, OCH3), 4.01 (s, 3H, OCH3), 6.01 (s, 4H, 2 ꢀ OCH2O), 7.08 (s, 1H,
Ar-H), 7.17 (d, 1H, CH@CHCO, J = 15.7 Hz), 7.42 (s, 1H, Ar-H), 7.54 (d,
1H, CH@CHCO, J = 15.6 Hz), 7.98 (d, 2H, 2 ꢀ Ar-H, J = 8.1 Hz), 8.28 (d,
2H, 2 ꢀ Ar-H, J = 8.7 Hz); 13C NMR (CDCl3, 75 MHz, d ppm): d 52.1
(COOCH3), 56.7 (2 ꢀ OCH3), 102.3 (OCH2O), 102.5 (OCH2O), 106.6,
109.7, 111.8, 111.8, 112.7, 120.9 (CH@CHCO), 123.7 (2 ꢀ ArC),
124.3, 127.5, 129.3 (2 ꢀ ArC), 137.6, 138.5, 143.1, 1432, 143.4,
144.9 (C H@CHCO),147.8, 149.9, 150.6, 166.0 (COOCH3), 193.0
(COOCH3), 189.2 (CH@CHCO); IR (KBr, cmꢁ1):
m 2946, 1715, 1637,
1604, 1576, 1466, 1433, 1327, 1268, 1245, 1174, 1097, 1062,
1041, 930, 832, 751; ESI-MS: m/z 530 [M+H]+, 552 [M+Na]+; HRMS
(ESI m/z) for C29H24NO9 calcd 530.1451, found 530.1453 [M+H]+.
5.7.3. (E)-Methyl 50-(2-cyano-3-oxo-3-(3,4,5-
trimethoxyphenyl)prop-1-en-1-yl)-7,70-dimethoxy-{4,40-
bibenzo[d][1,3]dioxole}-5-carboxylate (11c)
(CH@CHCO); IR (KBr, cmꢁ1):
m 2359, 1706, 1636, 1575, 1384, 1322,
1175, 1097, 1048, 926, 840, 706; ESI-MS: m/z 536 [M+H]+, 558
[M+Na]+; HRMS (ESI m/z) for C27H22NO11 calcd 536.1193, found
536.1195 [M+H]+.
The title compound was obtained starting from 6 and 10c. As a
yellow solid, yield: 35%; mp: 82–84 °C. Analytical data for 11c: 1
H
NMR (CDCl3, 300 MHz, d ppm): 3.69 (s, 3H, OCH3), 3.87 (s, 6H,
2 ꢀ OCH3), 3.92 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.04 (s, 3H,
OCH3), 5.92–6.05 (m, 4H, 2 ꢀ OCH2O), 7.08 (s, 2H, 2 ꢀ Ar-H), 7.36
(s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 8.06 (s, 1H, CH@CCN); 13C NMR
(CDCl3, 75 MHz, d ppm): d 52.1 (COOCH3), 56.4 (2 ꢀ OCH3), 56.6
(OCH3), 56.7 (OCH3), 61.0 (OCH3), 102.5 (OCH2O), 102.6 (OCH2O),
107.0 (2 ꢀ ArC), 107.6 (CH@CHCO), 107.7 (CN), 108.5, 112.1,112.2,
118.0, 124.4, 124.9, 130.9, 138.4, 139.4, 143.3, 143.4, 143.8, 147.1,
5.6. General procedure for the preparation of 10a–c
To the solution of 9a–c (1.5 mmol) in CH3CN (15 mL) were
added 60% NaH (120 mg, 3.0 mmol). The mixture was stirred for
3 h at 80 °C. Then, water (10 mL) was added, and the mixture
was concentrated by rotary evaporation. Additional water was

2548
X. Gu et al. / Bioorg. Med. Chem. 20 (2012) 2540–2548
147.8, 153.0 (CH@CHCO), 153.6 (2 ꢀ ArC), 165.9 (COOCH3), 187.7
(CH@CHCO); IR (KBr, cmꢁ1):
2942, 2371, 1718, 1636, 1583,
and data analysis were produced using Bio-Rad iQ5 software. The
relative mRNA level treated with inhibitors was expressed as fold
change of the control (in the presence of 0.1% DMSO).
m
1415, 1384, 1356, 1331, 1174, 1127, 1102, 1036, 930, 755; ESI-
MS: m/z 606 [M+H]+, 628 [M+Na]+; HRMS (ESI m/z) for
C
31H28NO12 calcd 606.1612, found 606.1615 [M+H]+.
5.8.6. P-gp ATPase assay
Drug-stimulated activity of P-gp ATPase was detected by
P-gp-Glo™ assay system (Promega, USA). By following the user
protocol provided by the vender, the activity of P-gp ATPase was
5.8. Biological assays
5.8.1. Cytotoxicity assay
measured in the presence or absence of 200
lM Na3VO4, 200 lM
1 ꢀ 104 K562 and K562/A02 cells were grown in 96-well micro-
titer plates and incubated for 24 h. Various concentrations of com-
pounds diluted with medium were added into the wells. And the
exponentially growing cancer cells were incubated for 72 h at
37 °C (5% CO2, 95% humidity). Then, MTS was added directly to
the cells. After additional incubation for 3 h at 37 °C, the absor-
bance at 490 nm was read on a microplate reader (Thermo, USA).
The IC50 values of the compounds for cytotoxicity were calculated
by GraphPad Prism 3.0 software from the dose–response curves.
VRP (as a positive reference), 40 M 8g. The luminescence was de-
tected in a luminometer (Perkin–Elmer TD-20, USA).
l
Acknowledgments
The work was financially supported by the Innovative Program
for Postgraduate Students of Jiangsu province (NO. CX10B-372Z)
and National Natural Science Foundation of China Grant No.
30873083 and 81173082, respectively. We thank to Professor
Dongsheng Xiong (Institute of Hematology & Blood Diseases Hos-
pital, CAMS & PUMC, China) for providing K562 and K562/A02 cell
lines.
5.8.2. Flow cytometric analysis
1 ꢀ 106 K562/A02 or K562 cells in culture were pre-incubated
with different concentrations of the target compounds, VRP or
vehicle control (0.1% DMSO) at 37 °C for 1 h, followed by addition
References and notes
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l
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5.8.5. RT-PCR analysis
K562/A02 cells were seeded in 6-well plates at a density of
5 ꢀ 105 cells and then cultured without or with 10
lM target com-
pounds for 48 h. Total RNA was extracted and then reverse tran-
scripted from mRNA to cDNA using the RT-PCR kit (Promega, WI,
USA). The PCR profile was as follows: 10 min at 95 °C, followed
by 30 cycles of 30 s at 95 °C and 1 min at 60 °C. The standard curve