Synthesis and biological evaluation of novel bifendate derivatives bearing 6,7-dihydro-dibenzo[c,e]azepine scaffold as potent P-glycoprotein inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.02.034

Overexpression of P-glycoprotein (P-gp) is one of the major problems in successful treatment of cancers. To find new P-gp inhibitors, a series of bifendate (DDB) derivatives bearing dibenzo[c,e]azepine scaffold were synthesized and evaluated. Compound 4i more potently reversed P-gp-mediated multidrug resistance (MDR) than DDB and verapamil (VRP) by blocking P-gp mediated drug efflux function and increasing drug accumulation in K562/A02 MDR cells, and persisted longer chemo-sensitizing effect (>24 h) than VRP (<6 h). Interestingly, unlike VRP, 4i showed no stimulation on the P-gp ATPase activity, suggesting it is not a substrate of P-gp. Given the low intrinsic cytotoxicity of 4i in vitro, it may represent a promising lead for developing therapeutics targeting P-gp-mediated MDR in combinational cancer chemotherapy.

Full text of DOI:10.1016/j.ejmech.2012.02.034

European Journal of Medicinal Chemistry 51 (2012) 137e144
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel bifendate derivatives bearing 6,
7-dihydro-dibenzo[c,e]azepine scaffold as potent P-glycoprotein inhibitors
,
b
,
,
,
,
c
a
,
,b
Xiaoke Gu a 1, Zhiguang Ren c 1, Xiaobo Tang a b, Hui Peng c , Qing Zhao , Yisheng Lai b, Sixun Peng a
,
**
Yihua Zhang a
,b,
*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, People’s Republic of China
c Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing 100850, People’s Republic of 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 in successful treatment of cancers.
To find new P-gp inhibitors, a series of bifendate (DDB) derivatives bearing dibenzo[c,e]azepine scaffold
were synthesized and evaluated. Compound 4i more potently reversed P-gp-mediated multidrug
resistance (MDR) than DDB and verapamil (VRP) by blocking P-gp mediated drug efflux function and
increasing drug accumulation in K562/A02 MDR cells, and persisted longer chemo-sensitizing effect
(>24 h) than VRP (<6 h). Interestingly, unlike VRP, 4i showed no stimulation on the P-gp ATPase activity,
suggesting it is not a substrate of P-gp. Given the low intrinsic cytotoxicity of 4i in vitro, it may represent
a promising lead for developing therapeutics targeting P-gp-mediated MDR in combinational cancer
chemotherapy.
Received 7 December 2011
Received in revised form
11 February 2012
Accepted 16 February 2012
Available online 25 February 2012
Keywords:
Bifendate
dibenzo[c,e]azepine
P-gp inhibitor
Multidrug resistance
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
interaction with anticancer drugs. Therefore, there is a clear
and urgent requirement for developing novel effective P-gp
The emergence of multidrug resistance (MDR) is regarded as the
major barrier to successful chemotherapy in cancer patients [1]. A
primary mechanism of MDR involves the overexpression of P-gp,
a member of ATP-binding cassette (ABC) transporter superfamily, in
the plasma membrane of drug-resistant cells [2]. P-gp mainly
utilizes energy derived from ATP hydrolysis to actively export
anticancer drugs out of tumor cells, leading to the reduction of
intracellular drug levels and consequent drug insensitivity. Direct
inhibition of P-gp with specific inhibitors is considered as a suitable
and attractive approach to overcome P-gp-mediated MDR [3], and
considerable attempts have been made to develop P-gp inhibitors
during past decades. However, until now, no drug of this class
has been approved for many reasons, such as low selectivity,
poor potency, inherent toxicity and/or adverse pharmacokinetic
inhibitors [4].
Nowadays, the strategies for generating such compounds have
been extended to the natural food, herbal extracts and their
synthetic derivatives [5], which are reported to be inhibitors of
one or more ABC drug efflux pumps, and usually are low in
toxicity and well tolerated in the human body [6,7]. Recently, it
has been demonstrated that lignans extracted from Schisandra
chinensis Baill (Wuweizi) and its components deoxyschizandrin
and schisandrol A have the ability to restore drug sensitivity at
non-toxic concentrations via direct interaction with P-gp [8,9].
Some other synthetic schisanderin derivatives exhibit the similar
effects as the natural products. Bifendate (DDB) is an analog of
schizandrin C [10], and has been widely used for the treatment of
chronic viral hepatitis B with few side effects for more than 20
years in China. Interestingly, recent investigations demonstrated
that DDB had MDR reversal activity in vitro and in vivo by
increasing intracellular accumulation of anticancer drugs and
promoting cancer cell apoptosis through inhibiting P-gp [11].
More importantly, DDB shows no pharmacokinetic interactions
with anticancer drugs [11]. However, DDB requires high doses to
reverse MDR, and the potency is lower as compared with the
classical P-gp inhibitor, verapamil (VRP). Therefore, development
* Corresponding author. State Key Laboratory of Natural Medicines, China
Pharmaceutical University, Nanjing 210009, People’s Republic of China.
Tel./fax: þ86 25 83271015.
** Corresponding author. Tel.: þ86 10 66932339; fax: þ86 10 68159436.
E-mail addresses: p_h2002@hotmail.com (H. Peng), zyhtgd@163.com (Y. Zhang).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.02.034

138
X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
of novel DDB derivatives with high chemo-sensitizing effects is of
great importance.
determined by MTS assay. A well-known anticancer drug Adria-
mycin (ADR) was used as the positive control. As shown in Table 1,
the target compounds displayed low intrinsic cytotoxicity since
their IC50 values were at a high micromolar level. As expected,
K562/A02 cells were significantly resistant to ADR: the IC50 value
To date, target-based drug design methods, such as virtual
screening or de novo design, are not suited for identification of new
P-gp inhibitors due to the low resolution of available crystal struc-
ture (only 3.8 Å) and the multispecific interaction patterns of P-gp
[12]. Our design strategy was based on structures-activity relation-
ship (SAR) studies of P-gp inhibitors as previously summarized in
literatures [3,5,13e16]. In brief, potent P-gp inhibitors were char-
acterized with two or three aromatic moieties, a tertiary nitrogen
atom, one to three hydrogen-bond acceptors, and/or one hydrogen-
bond donor. Keeping these in mind, and in view of the crucial role of
sixalkoxyl biphenyl moiety in DDB for pharmacological activity [17],
various N-substituted tertiary amines were respectively combined
to the sixalkoxyl biphenyl structure forming compounds (4aej) each
with a 6,7-dihydro-dibenzo[c,e]azepine ring to improve the P-gp
inhibitory effect and affinity of DDB with P-gp. And some of the
compounds (4aed) were further oxidized to the corresponding
amides (5aed) to examine the influence of amidation-modification
of the tertiary amine on P-gp inhibitory activity. Thus, fourteen
target compounds were designed and synthesized, and their ability
to interact with P-gp transporter in K562/A02 cells which over-
express P-gp was subsequently evaluated.
(46.69 mM) is 108-fold higher than that of parental non-resistant
cells. Interestingly, both K562 and K562/A02 cells were almost
equally insensitive to the target compounds, indicating that, unlike
ADR, they were not likely to be substrates of P-gp. Based on the
IC50s determined above, compounds at concentrations below IC10
were used in the following MDR reversal experiments.
3.1.2. Effect of the target compounds on Rh123 and ADR
accumulation
The effect of the target compounds (10 mM, IC10) on the intra-
cellular accumulation of Rhodamine 123 (Rh123, a fluorescent
substrate of P-gp) in K562/A02 cells was assayed by flow cytometry.
The classical P-gp inhibitor verapamil (VRP) was used as a positive
control. As shown in Fig. 1 A, treatment with VRP led to a significant
increase of intracellular Rh123 level in K562/A02 cells, the accu-
mulation fold change was 8.5 larger than that of vehicle control
(0.1% DMSO). Rh123 accumulation in the cells treated with 10 mM
DDB showed a slight increase and the accumulation fold change
was only 1.2, indicating that DDB had weak P-gp inhibitory effect,
which is consistent with previous report [11]. Interestingly, this
effect was enhanced greatly by the chemical modifications of DDB.
As observed in Fig.1 A, the Rh123 accumulation fold change of most
target compounds was significantly higher than that of DDB.
Compounds 4h and 4i obviously increased the intracellular Rh123
level in K562/A02 cells, and their accumulation fold change was
10.1 and 11.2, respectively, which was higher than that of VRP.
Compound 4d exhibited the same effect on Rh123 accumulation as
VRP.
In order to further investigate the effect of 4d, 4h and 4i on P-gp,
their dose-response effects on Rh123 accumulation in K562/A02
and parental sensitive K562 cells were respectively determined by
flow cytometry. As shown in Fig. 1 B, VRP, 4d, 4h and 4i produced
a significant increase in Rh123 accumulation in K562/A02 cells in
a dose-dependent manner, but not in P-gp-negative K562 cells,
suggesting that the effect of these compounds on Rh123 accumu-
lation is likely via inhibiting P-gp. Furthermore, ADR, another
substrate of P-gp, was used to test the inhibitory effect of 4i on P-gp
2. Chemistry
The synthetic routes of the target compounds 4aej and 5aed
are depicted in Scheme 1. The starting material DDB was treated
with excess LiAlH4 in THF to give the corresponding diol 2, which
was then converted to the methylsulfonic acid ester 3 by methyl-
sulfonyl chloride. The nucleophilic substitution of 3 by various
amines afforded the desired products 6,7-dihydro-dibenzo[c,e]
azepine (4aej). Further oxidation of 4aed with KMnO4 provided
the corresponding amides 5aed, respectively.
3. Results and discussion
3.1. Biological evaluation
3.1.1. Cytotoxicity assays
The cytotoxicities of the target compounds against K562 and its
drug-resistant subline K562/A02 cells which overexpress P-gp were
Scheme 1. Synthesis of the target compounds. Reagents and conditions: a) LiAlH4, THF, 0 ꢀC-rt, 4 h; b) methylsulfonyl chloride, Et3N, CH2Cl2, rt, 6 h; c) acetonitrile, Et3N, 40 ꢀC,
6e8 h; d) KMnO4, hexadecyl trimethyl ammonium bromide, CH2Cl2, reflux, 0.5e1 h.

X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
139
Table 1
5.0
cell killing. Anticancer drug ADR, a well-known P-gp substrate, was
also non-toxic to K562/A02 and K562 cells at 3.5 M and 0.05 M,
mM (high dose in this experiment), each only producing w10%
IC50 values of target compounds against K562 and K562/A02 cells.
Compound
IC50
(
m
M)a
Compound
IC50 (m
M)a
m
m
respectively (Fig. 3, green bar). However, its cytotoxicity against
K562/A02 cells was increased to various extents in the combination
with target compounds or VRP (Fig. 3 A). Noteworthily, 4h and 4i
significantly enhanced the sensitivity of K562/A02 cells to ADR at
K562/A02
K562
125
77.47
>100
>200
106
>100
140.3
45.36
K562/A02
K562
53.72
>200
>100
41.71
>100
43.48
0.43
4a
4b
4c
4d
4e
4f
117.2
>100
>100
111.9
87.64
>100
107
4i
4j
46.88
>200
>100
44.6
>100
53.37
46.69
5a
5b
5c
5d
ADR
2.5
mM concentration. The most active compound 4i showed potent
chemo-sensitizing effect even at 1.25
mM, because the survival rate
of K562/A02 cells treated with 4i or ADR alone was 98% and 95%,
respectively, while the survival rate of the cells treated by combi-
nation of 4i and ADR was dramatically reduced to 30%. Further-
more, the survival rate of K562/A02 cells was decreased to 8% and
4g
4h
34.11
a
IC50 values are expressed as means of triplicate experiments.
6% by combinational treatment of 2.5 mM or 5 mM 4i with ADR,
respectively (Fig. 3 A). On the other hand, VRP displayed much
poorer chemo-sensitizing effect compared with 4i under the same
conditions. Even if K562/A02 cells were treated with 5 mM VRP, the
survival rate was still higher than 50%. These results indicated that
the DDB derivatives, especially 4i, significantly potentiated the
cytotoxicity of ADR in K562/A02 cells in a dose-dependent manner.
Moreover, no such chemo-sensitizing effect was observed in P-gp-
negative K562 cells (Fig. 3 B), suggesting that the DDB derivatives
exert chemo-sensitizing effect via inhibition of P-gp function.
using the similar assay above. As shown in Fig. 1 C, 4i and VRP
increased the intracellular ADR level in K562/A02 cells in a dose-
dependent manner, but such effect was not observed in K562
cells. Collectively, both Rh123 and ADR accumulation fold changes
of 4i were obviously greater than that of DDB and VRP, suggesting
that 4i may be a potent P-gp inhibitor.
3.1.3. Inhibitory effect of 4i on P-gp-mediated Rh123 efflux function
To investigate if 4i can reverse P-gp-mediated drug efflux
function, intracellular Rh123 was photographed under a fluores-
cence microscope (Fig. 2 AeC) and determined using flow cytom-
etry (Fig. 2 D). As expected, the parental sensitive K562 cells
retained most of the fluorescence dye (Fig. 2 A1), while K562/A02
cells which overexpress P-gp effluxed the dye efficiently and there
was no fluorescence in the cells after 60-min incubation (Fig. 2 A2).
3.1.5. Effect of target compounds on the expression of mRNA and
protein levels of P-gp
It is known that P-gp-mediated MDR can be reversed either by
inhibiting transport function of P-gp or decreasing its expression
level [18]. Since we have already demonstrated the DDB derivatives
exert chemo-sensitizing effect via inhibition of P-gp function, we
next determined whether the active compounds 4d, 4h and 4i
exhibited an inhibitory effect on the expression of P-gp at mRNA
and protein levels, using RT-PCR and Western blot, respectively. As
shown in Fig. 4, no marked difference in P-gp expression at mRNA
(Fig. 4 A) or protein level (Fig. 4 B) was observed in K562/A02 cells
Interestingly, 4i significantly blocked the efflux in
a dose-
dependent manner, resulting in significant retention of Rh123 in
K562/A02 cells (Fig. 2 B1e3), and displayed much higher potency
than VRP at the same doses (Fig. 2 C1e3). As shown in Fig. 2 D,
intracellular Rh123 level in K562/A02 cells treated with 2.5 mM 4i
was almost the same as K562 cells, indicating 4i completely
inhibited P-gp efflux function (Fig. 2 D1). VRP, however, even up to
treated with target compounds (5 mM) for 72 h compared to vehicle
(0.1% DMSO). These results suggest that the DDB derivatives exert
the MDR reversal activity by inhibiting P-gp drug efflux function
and not by decreasing P-gp expression.
5 mM, could not achieve the similar effect as 4i (Fig. 2 D2). These
results supported the argument that 4i displayed stronger inhibi-
tory effect on P-gp-mediated drug efflux function than the classical
P-gp inhibitor VRP.
3.1.6. Duration of chemo-sensitizing effect of 4i toward ADR in
K562/A02 cells
3.1.4. Chemo-sensitizing effect of target compounds
Next, the duration of chemo-sensitizing effect of DDB derivative
4i was determined. Briefly, K562/A02 cells were incubated with 4i
(2.5 mM) or VRP (10 mM) for 24 h, and then each compound in the
To confirm the reversal effect of novel DDB derivatives on P-gp
mediated MDR, the cytotoxicity of ADR against K562/A02 cells and
K562 cells was evaluated in the presence or absence of the DDB
culture media was washed out, leaving cells with fresh media. ADR
at various concentrations was then added to the culture at 0, 6, 12,
or 24 h after the removal of the compounds to examine the dura-
tion of the MDR-reversal activity of 4i or VRP. In the absence of 4i or
derivatives at various concentrations (ch: 5
mM, cm: 2.5 mM and cl:
1.25 M) by MTS assay, using VRP as the positive control. As shown
m
in Fig. 3, the target compounds and VRP (ch, red bar) had little
inhibitory effect on the survival of K562/A02 and K562 cells at
C5
A
B
K562/A02
K562
K562/A02
K562
12
10
8
12
10
8
4
3
2
1
0
6
6
4
4
2
2
0
0
DDB
M). The relative values were identified by dividing the fluorescence
VRP
4h
4i
4d
VRP
4i
Fig. 1. The effect of the target compounds on the intracellular accumulation of Rh123 (0.5
m
M) or ADR (0.5
m
intensity of each measurement treated with compound or VRP by that of incubation with 0.1% DMSO. Data represents means ꢁ S.D. of three independent experiments.

140
X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
Fig. 2. Inhibitory effect of 4i on P-gp efflux function. (A) Intracellular Rh123 in K562 (A1) or K562/A02 (A2) cells was photographed under a fluorescence microscope. (BeC)
Intracellular Rh123 in K562/A02 cells treated with various concentrations of 4i (B1: 5.0 M, B2: 2.5 M, B3: 0.25 M) compared with VRP (C1: 5.0 M, C2: 2.5 M, C3: 0.25 M). (D)
m
m
m
m
m
m
Dose response of 4i or VRP in retaining Rh123 accumulation in K562/A02 cells. The purple line represents the level of Rh123 accumulation in K562 cells, serving as a control. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
K562
B120
K562/A02
A
100
80
60
40
20
0
100
80
60
40
20
0
control
ch
ADR
cl+ADR
cm+ADR
ch+ADR
control
ch
ADR
cl+ADR
cm+ADR
ch+ADR
4a 4d 4e 4g 4h
4i
4j VRP
4a 4d 4e 4g 4h
4i
4j VRP
Fig. 3. Chemo-sensitizing effect of the target compounds. The cytotoxicity of ADR against K562/A02 cells and K562 cells in the presence or absence of the target compounds at
various concentrations (cl: 1.25 M; cm: 2.5 M; ch: 5.0 M) was evaluated by MTS assay. The classical P-gp inhibitor VRP was used as a positive control. Survival rate was expressed
m
m
m
as percentage mean of cell growth ꢁ S.D. with respect to the control without any treatment of three independent experiments. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
VRP, the IC50s were 33.59, 35.15, 54.11, and 51.67
m
M when ADR was
some compounds (e.g., VRP) also stimulate the basal ATPase
activity, thus behaving like a substrate or competitive inhibitor for
the pumps [3]. Therefore, the MDR-reversal effect is exerted only at
a higher concentration which might easily reach toxic ranges,
added at 0, 6, 12, or 24 h to the culture, respectively (Fig. 5). As
observed previously, both 4i and VRP significantly sensitized K562/
A02 cells to ADR right after they were removed, the IC50s of ADR
were 1.78 and 12.92
mM, respectively (Fig. 5 A). And IC50s of ADR
were 26.06, 51.05 and 51.40
mM when ADR was added at 6, 12 and
24 h after the removal of VRP, respectively. The IC50s were almost
equal to those obtained in the control group which was not exposed
to any compounds. In sharp contrast, 4i showed significant reversal
activity even at 24 h after exposure (Fig. 5 D). The IC50s of ADR were
2.47, 5.03, and 10.29 mM when ADR was added to the cells at 6, 12,
and 24 h after removal of 4i. Hence, one conclusion can be drawn
from these results: 4i exhibited potent chemo-sensitizing effect,
which persisted for much longer time (>24 h) compared with
positive control VRP (<6 h) after removal of the agent from the
culture.
Fig. 4. Effects of active compounds on P-gp mRNA and protein expression level. (A)
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) Effect of target compounds on the expression of P-
gp at protein level. Independent experiments were performed at least three times, and
a representative experiment is shown.
3.1.7. Effect of 4i on P-gp ATPase activity
Since efflux of drug by P-gp normally requires energy from ATP
hydrolysis by the ATPase, which is generally stimulated in the
presence of P-gp substrate. Among the identified reversal agents,

X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
141
D
A
C
0 h
12 h
B100
24 h
6 h
4i
VRP
PBS
4i
4i
VRP
PBS
4i
VRP
PBS
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
VRP
PBS
80
60
40
20
0
0.3 0.6 0.9 1.2 1.5 1.8 2.1
0.3 0.6 0.9 1.2 1.5 1.8 2.1
0.3 0.6 0.9 1.2 1.5 1.8 2.1
0.3 0.6 0.9 1.2 1.5 1.8 2.1
ADR (LogµM)
ADR (Log µM)
ADR (Log µM)
ADR (Log µM)
Fig. 5. Duration of chemo-sensitizing effect of 4i and VRP toward ADR in K562/A02 cells after incubation and subsequent washout. Cell proliferation was determined by MTS assay.
Data represents means ꢁ SE of triplicate determinations.
leading to a serious side effect [19]. To learn whether 4i is
a substrate of P-gp, its effect on P-gp ATPase activity was deter-
mined according to a previously described method [20]. In brief,
the activity of P-gp ATPase was measured in the presence or
absence of Na3VO4 (as a negative control), 4i, or VRP (as a positive
control). Thus, the luminescence was detected in a luminometer,
and the luminescence value of each sample represented its ATP
level, which is negatively correlated with the activity of P-gp
ATPase. As shown in Table 2, VRP caused a significant increase in
the activity of P-gp ATPase (P < 0.01) compared with the untreated
group, while 4i or Na3VO4 showed no stimulation of the P-gp
ATPase activity, indicating that 4i was not a substrate of P-gp.
precise SARs remain further investigation when more DDB deriv-
atives bearing a dibenzo[c,e]azepine moiety will be available in the
near future.
4. Conclusions
In summary, a series of novel DDB derivatives containing
dibenzo[c,e]azepine scaffold 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 target compounds showed potent
chemo-sensitizing effect on K562/A02 cells, and compounds 4h
and 4i more potently blocked the drug efflux function of P-gp and
increased the accumulation of drug in K562/A02 cells than VRP but
did not inhibit expression of P-gp at mRNA and protein level.
Additionally, the chemo-sensitizing effect of 4i persisted much
longer (>24 h) than VRP (<6 h). Interestingly, unlike VRP, 4i did not
stimulate the activity of P-gp ATPase, suggesting it is not a substrate
of P-gp. Given the low intrinsic cytotoxicity of 4i in vitro, it might
represent a promising lead for developing therapeutics targeting
P-gp-mediated MDR in combinational cancer chemotherapy.
Further intensive investigations are still in progress.
3.2. Structure-activity relationships
Based on the results above, structure-activity relationships
(SARs) of DDB derivatives could be drawn as follows: compounds
with the dibenzo[c,e]azepine scaffold more strongly reversed P-gp-
mediated MDR than DDB and VRP. The substitutes at nitrogen atom
in dibenzo[c,e]azepine significantly affected the intensity of P-gp
inhibitory effect. Compounds bearing a third benzene ring except 4f
showed more potent inhibitory effect on the drug efflux function of
P-gp than those bearing an alkyl group (4d, 4h and 4i vs. 4a, 4b, 4e
and 4g). Introducing an electron donating substitute to the third
benzene ring, such as methylenedioxy group, could increase the
intensity of P-gp inhibitory effect (4i vs. 4h). In addition, the length
of the carbon chain linked to the third benzene ring and nitrogen
atom also played a crucial role in inhibition of P-gp. The compounds
with a two-carbon chain showed higher inhibitory effect on P-gp
than the compounds with a zero- or one-carbon chain (4h vs. 4d,
4f). Interestingly, when dibenzo[c,e]azepine was oxidized to
dibenzo[c,e]azepine-5-one, the P-gp inhibitory activity was signif-
icantly reduced (5aed vs. 4aed). One plausible explanation is that
conversion of amine to amide might reduce the electron density of
nitrogen atom, leading to low affinity with P-gp and consequent
poor P-gp inhibitory effect. All these trends above are highly
consistent with SAR analyses previously reported [3,5,13e16],
and hence verify our previous presumption that the various
N-substituted tertiary amines play a significant role in the MDR
reversal activity of these novel DDB derivatives. However, the
5. Experimental protocols
5.1. Chemical analysis
Melting points were determined on a Mel-TEMP II melting point
apparatus and uncorrected.
All of the synthesized compounds were purified by column
chromatography (CC) on silica gel 60 (200e300 mesh) or thin layer
chromatography (TLC) on silica gel 60 F254 plates (250 mm;
QingdaoOcean Chemical Company, China). Subsequently, they were
routinely analyzed by IR (Shimadzu FTIR-8400S), 1H NMR and 13C
NMR (Bruker ACF-300Q, 300 MHz), and MS (HewlettePackard 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. Solutions after reactions and extractions were
concentrated using a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over anhy-
drous sodium sulfate.
Table 2
Effect of 4i on P-gp ATPase activity.
5.2. Synthesis of 4,40-dimethoxy-5,6,50,60-dimethylenedioxy-2,20-
Compound
Concentration (
mM)
Luminescence
dibenzyl dimethanol 2
(relative light units)a
To a solution of DDB (4 g, 9.56 mmol) in dry THF (50 mL) at 0 ꢀC
was added LiAlH4 (1.09 g, 28.68 mmol) portionwise. The mixture
was stirred for 4 h at room temperature, cooled to 0 ꢀC, distilled
water was added slowly along the flask wall to the mixture until no
hydrogen generated. The mixture was filtered and washed with
CH2Cl2 several times. The filtrate was diluted with water and
extracted with CH2Cl2. The organic layer was washed with brine,
Untreated
VRP
Na3VO4
4i
0
200
200
40
242,328 ꢁ 1398
231,282 ꢁ 293*
281,688 ꢁ 439*
283,026 ꢁ 715*
*P < 0.01 vs. untreated group, determined by Student’s t test.
a
Relative light units represent the level of ATP in the sample, exhibiting a nega-
tive relationship with activity of P-gp ATPase. Data are expressed as means ꢁ SDs of
three separate experiments.

142
X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
dried with anhydrous sodium sulfate, filtered and evaporated in
vacuum to give the title compound as a white solid (2.9 g), yield:
85%; mp: 170e171 ꢀC (lit. [21]. 172e173 ꢀC). ESI-MS: m/z 385
[M þ Na]þ.
d
ppm): 3.25 (d, 1H, NCH2, J ¼ 16.2 Hz), 3.30 (d, 2H, NCH2,
J ¼ 12.6 Hz), 3.45 (d, 1H, NCH2, J ¼ 16.2 Hz), 3.57 (d, 2H, NCH2,
J ¼ 12.6 Hz), 3.79 (s, 3H, OCH3), 3.94 (s, 6H, 2 ꢂ OCH3), 5.96 (s, 2H,
OCH2O), 6.10 (s, 2H, OCH2O), 6.58 (s, 2H, 2 ꢂ AreH); 13C NMR
(CDCl3, 75 MHz,
d ppm): d 52.0, 55.4, 56.7, 101.7, 109.3, 110.6, 128.6,
5.3. Synthesis of 4,40-dimethoxy-5,6,50,60-dimethylenedioxy-2,20-
135.0, 142.9, 145.8, 171.1; IR (KBr, cmꢃ1):
n 3447, 2943, 2366, 1739,
dibenzyl dimethanesulfonate 3
1640, 1435,1384,1307,1169,1142, 1100,1042, 783, 668; ESI-MS: m/z
416 [M þ H]þ; HRMS (ESI m/z) for C21H22NO8 calcd 416.1345, found
416.1350 [M þ H]þ.
To a stirred solution of compound 2 (2 g, 5.5 mmol) in CH2Cl2
20 mL, triethylamine (2.33 ml, 16.5 mmol) was added followed by
the drop wise addition of methanesulfonyl chloride (1.28 ml,
16.5 mmol). The reaction mixture was stirred for 6 h at room
temperature. The progress of reaction was monitored by TLC. After
the completion of reaction, the reaction mixture was diluted with
water, extracted with CH2Cl2 and the organic layer was washed
with brine, dried with anhydrous sodium sulfate, filtered. Removal
of the solvent in vacuo gave the mesylated alcohol 3, which was
used as such in the next step.
5.4.4. 6-Benzyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4d)
The title compound was obtained starting from 3 and benzyl-
amine. As a white solid, yield: 81%; mp: 178e179 ꢀC. Analytical data
for 4d: 1H NMR (CDCl3, 300 MHz,
d ppm): 3.21 (d, 2H, NCH2,
J ¼ 12.5 Hz), 3.47 (d, 2H, NCH2, J ¼ 12.5 Hz), 3.59e3.71 (m, 2H,
NCH2), 3.94 (s, 6H, 2 ꢂ OCH3), 5.99 (s, 2H, OCH2O), 6.09 (s, 2H,
OCH2O), 6.53 (s, 2H, 2 ꢂ AreH), 7.19e7.44 (m, 5H, 5 ꢂ AreH); 13C
NMR (CDCl3, 75 MHz,
d ppm): d 54.9, 56.7, 59.7, 101.7, 109.2, 110.7,
5.4. General procedure for the preparation of 4aej
127.2, 128.5, 129.3, 129.4, 134.8, 138.9, 142.8, 145.7; IR (KBr, cmꢃ1):
n
3448, 2861, 2359, 1639, 1432, 1384, 1301, 1141, 1096, 1051, 747, 712;
ESI-MS: m/z 434 [M þ H]þ; HRMS (ESI m/z) for C25H24NO6 calcd
434.1604, found 434.1608 [M þ H]þ.
A solution of 3 (0.19 mmol), triethylamine (0.38 mmol) and
substituted amine (0.76 mmol) in acetonitrile (15 ml) was stirred at
40 ꢀC for 6e8 h, the reaction mixture was diluted with saturated
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 (CH2Cl2/EtOAc ¼ 10:1e5:1) to yield the
title compound, respectively.
5.4.5. 6-Propyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4e)
The title compound was obtained starting from 3 and n-pro-
pylamine. As a white solid, yield: 79%; mp: 138e140 ꢀC. Analytical
data for 4e: 1H NMR (CDCl3, 300 MHz,
d ppm): 0.97 (t, 3H, CH3,
J ¼ 7.2 Hz), 1.57e1.65 (m, 2H, CH3CH2), 2.42e2.59 (m, 2H, NCH2),
3.20 (d, 2H, NCH2, J ¼ 12.6 Hz), 3.54 (d, 2H, NCH2, J ¼ 12.6 Hz), 3.95
(s, 6H, 2 ꢂ OCH3), 5.99 (s, 2H, OCH2O), 6.09 (s, 2H, OCH2O), 6.57
5.4.1. 6-Ethyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz [c,e]azepine (4a)
The title compound was obtained starting from 3 and ethyl-
amine hydrochloride. As a white solid, yield: 87%; mp: 151e153 ꢀC.
(s, 2H, 2 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
d ppm): d 12.1, 21.0,
55.0, 56.8, 57.3, 101.7, 109.3, 110.6, 129.1, 134.9, 142.8, 145.7; IR (KBr,
Analytical data for 4a: 1H NMR (CDCl3, 300 MHz,
d
ppm): 1.06 (t, 3H,
cmꢃ1):
n 3588, 3568, 3448, 2949, 2867, 1641, 1489, 1466, 1432, 1384,
CH3, J ¼ 7.2 Hz), 2.67e2.85 (m, 2H, NCH2), 3.33 (d, 2H, NCH2,
1305, 1161, 1141, 1100, 1049, 753, 694; ESI-MS: m/z 386 [M þ H]þ;
HRMS (ESI m/z) for C21H24NO6 calcd 386.1604, found 386.1609
[M þ H]þ.
J ¼ 12.6 Hz), 3.73 (d, 2H, NCH2, J ¼ 12.6 Hz), 3.96 (s, 6H, 2 ꢂ OCH3),
6.01 (s, 2H, OCH2O), 6.10 (s, 2H, OCH2O), 6.64 (s, 2H, 2 ꢂ AreH); 13
C
NMR (CDCl3, 75 MHz,
d ppm): d 12.7, 48.8, 54.2, 56.8, 101.8, 109.6,
110.5, 129.2, 134.9, 142.9, 145.8; IR (KBr, cmꢃ1):
n
3447, 1635, 1437,
5.4.6. 6-Phenyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4f)
1381, 1308, 1142, 1101, 1045, 759, 641; ESI-MS: m/z 372 [M þ H]þ;
HRMS (ESI m/z) for C20H22NO6 calcd 372.1447, found 372.1452
[M þ H]þ.
The title compound was obtained starting from 3 and aniline.
As a white solid, yield: 81%; mp: 231e232 ꢀC. Analytical data for
4f: 1H NMR (CDCl3, 300 MHz,
d ppm): 3.83 (d, 2H, NCH2,
5.4.2. 6-Butyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz [c,e]azepine (4b)
The title compound was obtained starting from 3 and butyl-
amine. As a white solid, yield: 86%; mp: 144e146 ꢀC. Analytical data
J ¼ 12.9 Hz), 3.88 (s, 6H, 2 ꢂ OCH3), 4.32 (d, 2H, NCH2, J ¼ 12.9 Hz),
6.00 (s, 2H, OCH2O), 6.11 (s, 2H, OCH2O), 6.54 (s, 2H, 2 ꢂ AreH),
6.81e7.30 (m, 5H, 5 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
d ppm):
d
52.0, 56.7, 59.7, 101.8, 109.0, 110.4, 115.2, 118.5, 129.2, 129.6, 134.8,
for 4b: 1H NMR (CDCl3, 300 MHz,
d
ppm): 0.96 (t, 3H, CH3,
143.1, 145.7; IR (KBr, cmꢃ1):
n
3587, 3567, 3448, 2891, 1641, 1596,
J ¼ 7.2 Hz), 1.32e1.45 (m, 2H, CH2), 1.53e1.59 (m, 2H, CH2),
2.38e2.60 (m, 2H, NCH2), 3.18 (d, 2H, NCH2, J ¼ 12.5 Hz), 3.53 (d, 2H,
NCH2, J ¼ 12.5 Hz), 3.95 (s, 6H, 2 ꢂ OCH3), 5.99 (s, 2H, OCH2O), 6.09
(s, 2H, OCH2O), 6.56 (s, 2H, 2 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
1384, 1210, 1141, 1099, 1048, 753, 695; ESI-MS: m/z 420 [M þ H]þ;
HRMS (ESI m/z) for C24H22NO6 calcd 420.1447, found 420.1452
[M þ H]þ.
d
ppm):
d
14.1, 20.8, 30.1, 55.1, 56.7, 101.7, 109.2, 110.7, 129.2, 134.8,
5.4.7. 6-Methyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4g)
142.8, 145.7; IR (KBr, cmꢃ1):
n
3446, 3020, 2944, 2867, 2778, 1641,
1490, 1463, 1435, 1384, 1304, 1138, 1163, 1102, 1048, 756, 697; ESI-
MS: m/z 400 [M þ H]þ; HRMS (ESI m/z) for C22H26NO6 calcd
400.1760, found 400.1765 [M þ H]þ.
The title compound was obtained starting from 3 and methyl-
amine hydrochloride. As a white solid, yield: 85%; mp: 180e182 ꢀC.
Analytical data for 4g: 1H NMR (CDCl3, 300 MHz,
d ppm): 2.45
(s, 3H, CH3), 3.28 (d, 2H, NCH2, J ¼ 12.3 Hz), 3.46 (d, 2H, NCH2,
5.4.3. 6-(Methoxycarbonyl)methyl-3,9-dimethoxy-1,2-
methylenedioxy-10,11-methylenedi-oxy-6,7-dihydro-5H-dibenz[c,e]
azepine (4c)
The title compound was obtained starting from 3 and glycine
methyl ester hydrochloride. As a white solid, yield: 77%; mp:
156e158 ꢀC. Analytical data for 4c: 1H NMR (CDCl3, 300 MHz,
J ¼ 12.3 Hz), 3.95 (s, 6H, 2 ꢂ OCH3), 6.00 (s, 2H, OCH2O), 6.10 (s, 2H,
OCH2O), 6.60 (s, 2H, 2 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
d ppm):
d
42.7, 56.7, 56.8, 101.8, 109.4, 110.4, 128.4, 135.1, 142.9, 145.8; IR
(KBr, cmꢃ1):
n
3448, 2779, 1641, 1487, 1434, 1384, 1310, 1166, 1136,
1098, 1045, 706; ESI-MS: m/z 358 [M H]þ; HRMS (ESI m/z) for
C19H20NO6 calcd 358.1291, found 358.1295 [M þ H]þ.

X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
143
5.4.8. 6-Phenethyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4h)
(m, 1H, NCH2), 3.72e3.87 (m, 1H, NCH2), 3.83 (d, 1H, NCH2,
J ¼ 14.6 Hz), 3.96 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.30 (d,1H, NCH2,
J ¼ 14.6 Hz), 5.95e6.13 (m, 4H, 2 ꢂ OCH2O), 6.52 (s, 1H, AreH), 7.20
The title compound was obtained starting from 3 and 2-
phenethylamine. As a white solid, yield: 82%; mp: 138e140 ꢀC.
(s, 1H, AreH); 13C NMR (CDCl3, 75 MHz,
d ppm): d 13.5, 42.6, 50.9,
Analytical data for 4h: 1H NMR (CDCl3, 300 MHz,
d
ppm):
56.5, 57.0, 101.7, 102.0, 106.4, 108.7, 109.0, 109.7, 129.7, 133.1, 135.3,
2.65e2.85 (m, 2H, NCH2CH2), 2.86e2.93 (m, 2H, NCH2CH2), 3.26
(d, 2H, NCH2, J ¼ 12.5 Hz), 3.60 (d, 2H, NCH2, J ¼ 12.5 Hz), 3.93
(s, 6H, 2 ꢂ OCH3), 5.93 (s, 2H, OCH2O), 5.99 (s, 2H, OCH2O), 6.09
(s, 2H, OCH2O), 6.55 (s, 2H, 2 ꢂ AreH), 7.19e7.33(m, 5H, 5 ꢂ AreH);
136.7, 142.9, 143.2, 145.6, 147.2, 167.1; IR (KBr, cmꢃ1):
n
3587, 3567,
3448, 2985, 2920,1637,1419,1384,1316,1166,1140,1098,1045, 788,
756; ESI-MS: m/z 386 [M þ H]þ; HRMS (ESI m/z) for C20H20NO7
calcd 386.1240, found 386.1244 [M þ H]þ.
13C NMR (CDCl3, 75 MHz,
d ppm): d 35.0, 55.2, 56.7, 57.4,101.7,109.2,
110.7, 126.2, 128.5, 128.7, 129.2, 134.9, 140.3, 142.9, 145.7; IR (KBr,
5.5.2. 6-Butyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
cmꢃ1):
n
3567, 3448, 2808, 1639, 1487, 1434, 1384, 1304, 1136, 1107,
methylenedioxy-6,7-dihydro-dibenz[c,e] azepine-5-one (5b)
The title compound was obtained starting from 4b. As a white
solid, yield: 45%; mp: 162e163 ꢀC. Analytical data for 5b: 1H NMR
1048, 759, 700; ESI-MS: m/z 448 [M þ H]þ; HRMS (ESI m/z) for
C26H26NO6 calcd 448.1760, found 448.1765 [M þ H]þ.
(CDCl3, 300 MHz,
d
ppm): 0.93 (t, 3H, CH3, J ¼ 7.2 Hz), 1.23e1.35
5.4.9. 6-[2-(3,4-Methylenedioxy)phenyl]ethyl-3,9-dimethoxy-1,2-
methylenedioxy-10,11-methylenedioxy-6,7-dihydro-5H-dibenz[c,e]
azepine (4i)
The title compound was obtained starting from 3 and 3,4-
methylenedioxyphenethylamine hydrochloride. As a white solid,
yield: 81%; mp: 149e150 ꢀC. Analytical data for 4i: 1H NMR (CDCl3,
(m, 2H, CH2), 1.53e1.65 (m, 2H, CH2), 3.26e3.36 (m, 1H, NCH2),
3.66e3.75 (m, 1H, NCH2), 3.81 (d, 2H, NCH2, J ¼ 14.4 Hz), 3.95 (s, 3H,
OCH3), 3.97 (s, 3H, OCH3), 4.30 (d, 2H, NCH2, J ¼ 14.4 Hz), 5.94e6.12
(m, 4H, 2 ꢂ OCH2O), 6.52 (s, 1H, AreH), 7.23 (s, 1H, AreH); 13C NMR
(CDCl3, 75 MHz, d ppm): d 13.9, 20.1, 30.4, 47.6, 51.2, 56.5, 57.0, 101.7,
102.0, 106.5, 108.3, 108.9, 109.7, 129.8, 133.0, 135.3, 136.6, 143.2,
300 MHz,
d
ppm): 2.60e2.70 (m, 2H, NCH2CH2), 2.73e2.86 (m, 2H,
145.6, 147.1, 167.4; IR (KBr, cmꢃ1):
n
3567, 3448, 2955, 1641, 1618,
NCH2CH2), 3.24 (d, 2H, NCH2, J ¼ 12.5 Hz), 3.58 (d, 2H, NCH2,
1602,1426,1384,1310,1166,1139,1099,1045, 750, 697; ESI-MS: m/z
414 [M þ H]þ; HRMS (ESI m/z) for C22H24NO7 calcd 414.1553, found
414.1558 [M þ H]þ.
J ¼ 12.5 Hz), 3.94 (s, 6H, 2 ꢂ OCH3), 5.93 (s, 2H, OCH2O), 5.99 (s, 2H,
OCH2O), 6.09 (s, 2H, OCH2O), 6.55 (s, 2H,
2
ꢂ
AreH),
ppm):
6.64e6.78(m, 3H, 2 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
d
d
34.7, 55.3, 56.8, 57.6, 100.9, 101.8, 108.3, 109.1, 109.2, 110.7, 121.5,
5.5.3. 6-(Methoxycarbonyl)methyl-3,9-dimethoxy-1,2-
methylenedioxy-10,11-methylenedioxy-6,7- dihydro-dibenz[c,e]
azepine-5-one (5c)
129.2, 134.1, 134.9, 142.9, 145.8, 145.9, 147.7; IR (KBr, cmꢃ1):
n 3447,
2914, 2802, 1640, 1502, 1489, 1431, 1384, 1303, 1138, 1099, 1048,
1024, 750, 662; ESI-MS: m/z 492 [M þ H]þ; HRMS (ESI m/z) for
C27H26NO8 calcd 492.1658, found 492.1665 [M þ H]þ.
The title compound was obtained starting from 4c. As a white
solid, yield: 40%; mp: 187e189 ꢀC. Analytical data for 5c: 1H NMR
(CDCl3, 300 MHz,
d ppm): 3.75 (s, 3H, OCH3), 3.78e3.89 (m, 2H,
5.4.10. 6-Hydroxyethyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-5H-dibenz[c,e]azepine (4j)
NCH2), 3.95 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.52 (d, 1H, NCH2,
J ¼ 15.0 Hz), 4.75 (d, 1H, NCH2, J ¼ 15.0 Hz), 5.95e6.14(m, 4H,
2 ꢂ OCH2O), 6.48 (s, H, AreH), 7.26 (s, H, AreH); 13C NMR (CDCl3,
The title compound was obtained starting from 3 and ethanol-
amine. As a white solid, yield: 84%; mp: 100e101 ꢀC. Analytical data
75 MHz,
d ppm): d 48.7, 52.2, 52.3, 56.5, 57.0, 101.8, 102.1, 106.5,
for 4j: 1H NMR (CDCl3, 300 MHz,
d
ppm): 2.68e2.77 (m, 1H, NH2),
108.5, 109.1, 110.1, 128.4, 132.0, 135.4, 137.1, 143.0, 143.2, 145.8, 168.0,
2.95e3.03 (m, 1H, NH2), 3.37 (d, 2H, NCH2, J 12.6 Hz), 3.69 (d, 2H,
NCH2, J 12.6 Hz), 3.83 (t, 2H, OCH2, J 5.1 Hz), 3.96 (s, 6H, 2 OCH3),
6.01 (s, 2H, OCH2O), 6.10 (s, 2H, OCH2O), 6.63 (s, 2H, 2 AreH); 13C
169.8; IR (KBr, cmꢃ1):
n 3588, 3568, 3448, 2366, 1634, 1608, 1422,
1384, 1163, 1140, 1098, 1051, 671; ESI-MS: m/z 430 [M þ H]þ; HRMS
(ESI m/z) for C21H20NO9 calcd 430.1138, found 430.1143 [M þ H]þ.
NMR (CDCl3, 75 MHz,
d
ppm):
d
54.9, 56.5, 56.8, 58.5, 101.7, 109.3,
3588, 3568, 3448,
110.6, 128.7, 135.0, 142.9, 145.8; IR (KBr, cmꢃ1):
n
5.5.4. 6-Benzyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-dibenz[c,e] azepine-5-one (5d)
The title compound was obtained starting from 4d. As a white
solid, yield: 41%; mp: 170e171 ꢀC. Analytical data for 5d: 1H NMR
1638, 1384, 1307, 1145, 1095, 1042, 635; ESI-MS: m/z 388 [M þ H]þ;
HRMS (ESI m/z) for C20H22NO7 calcd 388.1396, found 388.1402
[M þ H]þ.
(CDCl3, 300 MHz,
d ppm): 3.75e3.86 (m, 1H, PhCH2), 3.81 (s, 3H,
5.5. General procedure for the preparation of 5aed
OCH3), 3.99 (s, 3H, OCH3), 4.26 (d, 1H, PhCH2, J ¼ 14.5 Hz), 4.59
(d, 1H, NCH2, J ¼ 14.8 Hz), 4.93 (d, 1H, NCH2, J ¼ 14.8 Hz), 5.96e6.10
(m, 4H, 2 ꢂ OCH2O), 6.14 (s,1H, AreH), 7.26 (s,1H, AreH), 7.28e7.35
To the solution of 4aed (0.70 mmol) in CH2Cl2 (10 mL) were
added hexadecyl trimethyl ammonium bromide (510 mg,
1.40 mmol) and KMnO4 (222 mg, 1.40 mmol). After reflux for
0.5e1 h, the reaction mixture was cooled to room temperature, and
then saturated aqueous NaHSO3 solution was added with vigorous
stirring. The reaction mixture was diluted with water, extracted
with CH2Cl2 and 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
(CH2Cl2/EtOAc ¼ 10:1e5:1) to yield 5aed, respectively, as a white
solid.
(m, 5H, 5 ꢂ AreH); 13C NMR (CDCl3, 75 MHz,
d ppm): d 50.3, 50.5,
56.6, 56.7, 101.7, 101.9, 106.4, 108.5, 109.1, 109.9, 127.5, 128.4, 128.6,
129.3, 132.4, 135.1, 137.3, 142.8, 143.2, 145.7, 147.0, 167.9; IR (KBr,
cmꢃ1):
n 3450, 1637, 1611, 1431, 1384, 1310, 1169, 1142, 1104, 1054,
753, 694; ESI-MS: m/z 448 [M þ H]þ; HRMS (ESI m/z) for C25H22NO7
calcd 448.1396, found 448.1402 [M þ H]þ.
5.6. Biological assays
5.6.1. Cytotoxicity assay
1 ꢂ 104 K562 and K562/A02 cells were seeded in 96-well plates
in RPMI-1640 and incubated for 24 h. The exponentially growing
cancer cells were incubated with various concentrations of
compounds for 72 h at 37 ꢀC (5% CO2, 95% humidity). After 72 h of
incubation, MTS was added directly to the cells. After additional
incubation for 3 h at 37 ꢀC, the absorbance at 490 nm was read on
5.5.1. 6-Ethyl-3,9-dimethoxy-1,2-methylenedioxy-10,11-
methylenedioxy-6,7-dihydro-dibenz[c,e] azepine-5-one (5a)
The title compound was obtained starting from 4a. As a white
solid, yield: 43%; mp: 125e127 ꢀC. Analytical data for 5a: 1H NMR
(CDCl3, 300 MHz,
d
ppm): 1.21 (t, 3H, CH3, J ¼ 7.2 Hz), 3.29e3.41

144
X. Gu et al. / European Journal of Medicinal Chemistry 51 (2012) 137e144
a
microplate reader (Thermo, USA). The IC50 values of the
and National Natural Science Foundation of China grant No.
30873083 and 81173082, respectively. We thank to Prof. Dong-
sheng Xiong (Institute of Hematology & Blood Diseases Hospital,
CAMS & PUMC, China) for providing K562 and K562/A02 cell
lines.
compounds for cytotoxicity were calculated by GraphPad Prism 3.0
software from the dose-response curves.
5.6.2. Flow cytometric analysis
1 ꢂ 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) for 1 h at 37 ꢀC, followed by addition of
Appendix A. Supplementary material
0.5 mM Rh123 or ADR and incubation for 30 min, respectively. The
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.02.034.
reaction was stopped by addition of ice-cold PBS and centrifugation,
washed with ice-cold PBSthree times, and subjected analysis by flow
cytometry. The relative values were identified by dividing the fluo-
rescence intensity of each measurement by that of vehicle control.
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K562/A02 cells were incubated with 5
or vehicle control for 48 h, respectively. Following incubation, the
cells were harvested and lyzed. The cell lysates (30 g) were sepa-
mM individual compound
m
rated by SDS-PAGE (12% gel) and transferred onto a PVDF membrane.
After blocked in a solution containing 5% non-fat milk in TBST buffer,
the target proteins were probed with anti-P-gp, anti-GAPDH,
respectively. Subsequently, the membranes were washed with TBST
buffer and incubated with horseradish peroxidaseeconjugated
secondary antibody. After additional washes with TBST buffer, the
proteineantibody complex were visualized by the enhanced Pho-
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K562/A02 cells were seeded in 6-well plates at a density of
5 ꢂ 105 cells and then cultured without or with target compounds
(5 mM) 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 and
data analysis were produced using Bio-Rad iQ5 software. The
relative mRNA level treated with inhibitors was expressed as fold
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5.6.5. Duration of the MDR-reversal activity of 4i
1 ꢂ 105 cells were plated in 96-well plates and cultured over-
night, followed by incubation with 4i (2.5 mM) or VRP (10 mM) for
24 h. Then, the cells were washed with PBS three times and
resuspended in complete medium. Various concentrations of ADR
was added to the culture at predefined intervals (0, 6, 12, and 24 h)
after removal of 4i or VRP, and incubated for an additional 72 h
(5% CO2, 95% humidity). MTS was added and absorbance at 490 nm
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Drug-stimulated activity of P-gp ATPase was detected by P-gp-
GloÔ assay system (Promega, USA). By following the user protocol
provided by the vender, the activity of P-gp ATPase was measured
in the presence or absence of 200
mM Na3VO4, 200 mM VRP (as
a positive reference), 40 M 4i. The luminescence of the sample
m
reflects the ATP level in the sample, which is negatively correlated
with the activity of P-gp ATPase and was detected in a luminometer
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Acknowledgements
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The work was financially supported by the Innovative Program
for Postgraduate Students of Jiangsu province (NO. CX10B-372Z)