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

Article abstract of DOI:10.1016/j.ejmech.2018.01.019

As a continuation of previous research, fifteen bifendate derivatives bearing 6,7-dihydro-dibenzo [c,e]azepine scaffold were synthesized and evaluated as P-gp-medicated multidrug resistance (MDR) reversal agents. Biological evaluation indicated that compounds 6k and 9c more potently reversed P-gp-mediated MDR than bifendate and verapamil (VRP) by blocking P-gp mediated drug efflux function and not by decreasing P-gp expression in K562/A02 MDR cells. Interestingly, wound-healing and chamber migration assay showed that 6k and 9c could significantly attenuate the migration of MDA-MB-231 cells. Notably, 6k and 9c could markedly suppress the invasive activity of MDA-MB-231 cells, thus displayed potential anti-metastasis activity. Preliminary mechanism studies indicated that the anti-metastasis activity of 6k and 9c was associated with their inhibitory effect on the activity and expression of MMP-2 and MMP-9. These results, together with the MDR reversal results indicated that compounds 6k and 9c might be promising leads for developing novel anti-cancer agents with P-gp and tumor metastasis inhibitory activities.

Full text of DOI:10.1016/j.ejmech.2018.01.019

Accepted Manuscript
Synthesis and biological evaluation of bifendate derivatives bearing 6,7-dihydro-
dibenzo[c,e]azepine scaffold as potential P-glycoprotein and tumor metastasis
inhibitors
Xiaoke Gu, Yanfei Jiang, Yingying Qu, Jing Chen, Dingding Feng, Chenglin Li,
Xiaoxing Yin
PII:
S0223-5234(18)30019-9
10.1016/j.ejmech.2018.01.019
EJMECH 10096
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 October 2017
Revised Date: 28 December 2017
Accepted Date: 5 January 2018
Please cite this article as: X. Gu, Y. Jiang, Y. Qu, J. Chen, D. Feng, C. Li, X. Yin, Synthesis and
biological evaluation of bifendate derivatives bearing 6,7-dihydro-dibenzo[c,e]azepine scaffold as
potential P-glycoprotein and tumor metastasis inhibitors, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.01.019.
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ACCEPTED MANUSCRIPT
Synthesis
and
biological
evaluation
of
bifendate
derivatives
bearing
6,7-dihydro-dibenzo[c,e]azepine scaffold as potential P-glycoprotein and tumor metastasis
inhibitors
Xiaoke Gua, 1, Yanfei Jianga, 1, Yingying Qua, Jing Chena, Dingding Fenga, Chenglin Lia, , Xiaoxing Yina,
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Jiangsu Center for the Collaboration and
Innovation of Cancer Biotherapy, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China
Graphical Abstract
Compounds 6k and 9c exhibited significantly anti-MDR and anti-metastasis activities.
Corresponding authors. Tel.: +86-516-83372005; fax: +86-516-83372005; e-mail: yinxx@xzhmu.edu.cn (X. Y.)
Tel.: +86-516-83262009; fax: +86-516-83262630; e-mail: licl@xzhmu.edu.cn (C. L.)
1 These two authors contributed equally to this work.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of bifendate derivatives bearing 6,7-dihydro-dibenzo[c,e]azepine
scaffold as potential P-glycoprotein and tumor metastasis inhibitors
Xiaoke Gua, 1, Yanfei Jianga, 1, Yingying Qua, Jing Chena, Dingding Fenga, Chenglin Lia, , Xiaoxing Yina,
aJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Jiangsu Center for the Collaboration and
Innovation of Cancer Biotherapy, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China
Abstract
As a continuation of previous research, fifteen bifendate derivatives bearing 6,7-dihydro-dibenzo
[c,e]azepine scaffold were synthesized and evaluated as P-gp-medicated multidrug resistance (MDR)
reversal agents. Biological evaluation indicated that compounds 6k and 9c more potently reversed
P-gp-mediated MDR than bifendate and verapamil (VRP) by blocking P-gp mediated drug efflux function
and not by decreasing P-gp expression in K562/A02 MDR cells. Interestingly, wound-healing and chamber
migration assay showed that 6k and 9c could significantly attenuate the migration of MDA-MB-231 cells.
Notably, 6k and 9c could markedly suppress the invasive activity of MDA-MB-231 cells, thus displayed
potential anti-metastasis activity. Preliminary mechanism studies indicated that the anti-metastasis activity
of 6k and 9c was associated with their inhibitory effect on the activity and expression of MMP-2 and
MMP-9. These results, together with the MDR reversal results indicated that compounds 6k and 9c might
be promising leads for developing novel anti-cancer agents with P-gp and tumor metastasis inhibitory
activities.
Keywords: bifendate; dibenzo[c,e]azepine; P-gp inhibitor; multidrug resistance; metastasis.
1. Introduction
Multidrug resistance (MDR) is regarded as the leading cause for the failure of chemotherapy in cancer
patients [1-3]. A well-established mechanism underlying MDR phenotype is the overexpression of ATP
binding cassette (ABC) transporter superfamily [4-6]. Among these ABC transporters, P-glycoprotein
Corresponding authors. Tel.: +86-516-83372005; fax: +86-516-83372005; e-mail: yinxx@xzhmu.edu.cn (X. Y.)
Tel.: +86-516-83262009; fax: +86-516-83262630; e-mail: licl@xzhmu.edu.cn (C. L.)
1 These two authors contributed equally to this work.
1

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(P-gp) is the best known and most important mediator of MDR, which can efflux various chemotherapeutic
drugs out of tumor cells, leading to the decrease of intracellular drug levels and consequent drug
insensitivity. Development of P-gp inhibitors is considered as a suitable and attractive strategy to overcome
P-gp-mediated MDR [7-10]. As for this aspect, considerable attempts have been made to exploit P-gp
inhibitor during past decades. However, no drug has been approved in clinic due to the poor potency,
inherent toxicity and/or adverse pharmacokinetic interaction with anticancer drugs. Therefore, there is a
clear and urgent requirement for developing novel P-gp inhibitors with safety and effective profiles.
Currently, natural products and their derivatives have been identified as novel and effective P-gp
inhibitors by virtue of their advantages, such as low toxicity, few unfavorable pharmacokinetic
interactions, etc [11-13]. Bifendate, an analogue of schisandrin C, can reverse P-gp-mediated MDR in
vitro and in vivo by inhibiting P-gp, and more importantly, there was little pharmacokinetic interaction
issue between bifendate and co-administered drugs, at least adriamycin (ADR) [14, 15]. Recently, we
have described a series of bifendate derivatives bearing dibenzo[c,e]azepine scaffold as novel P-gp
inhibitors [16]. Among the synthesized compounds, ZG1142 (Fig. 1) more potently reversed
P-gp-mediated MDR than bifendate and classical P-gp inhibitor verapamil (VRP) through blocking P-gp
efflux function, and persisted long chemo-sensitizing effect. More importantly, ZG1142 showed no
stimulation on the P-gp ATPase activity, indicating that it is not a substrate of P-gp [16].
Structure-activity (SAR) relationships showed that the third benzene ring (A) was important for the P-gp
inhibitory effect, and the length of the linker between nitrogen atom and the third benzene could
significantly affect the P-gp inhibitory effects [16]. Previously SAR summarized in literatures showed
that potent P-gp inhibitors were characterized with two or three aromatic moieties, a linker containing a
tertiary nitrogen atom, hydrogen-bond acceptors and/or donors[17-19]. With these in mind and
considering that a variable alkylphenyl linker incorporating an amide moiety is benefit for the P-gp
inhibitory activities [20-24], we designed a new series of molecules (6a-l, Fig. 1) where the alkyl moiety
between nitrogen atom and the third benzene of previous series was substituted with alkyl linkers
incorporating an amide bond. In additional, the presence of a reverse-amide-bond (i.e., from NHCO to
CONH) in the molecule may significantly affect the bioactivity of the target compound [25, 26]. Toward
this end, three compounds (9a-c, Fig. 1) with a reverse-amide-bond were prepared. Thus, fifteen
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compounds were synthesized, and their ability to overcome P-gp-mediated MDR was subsequently
assessed.
Figure 1. Chemical structure of ZG1142 and the target compounds (6a-l, 9a-c)
2. Chemistry
The synthetic routes of the target compounds 6a-l are depicted in Scheme 1. Compounds 2 and 3 were
prepared starting from bifendate according to literature procedures [16]. Treatment of various diamines
with Boc2O afforded the mono-Boc-protected amines derivatives 1a-b, which were directly reacted with 3
to produce the corresponding 6,7-dihydro-dibenzo[c,e]azepine intermediates 4a-b, respectively. After the
deletion of Boc group, compounds 5a-b were subsequently condensed with various aromatic acids to
produce the target compounds 6a-l, respectively.
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Scheme 1. Synthesis of the target compounds 6a-l. Reagents and conditions: a) Boc2O, CHCl3, 0 °C-rt, 24
h; b) LiAlH4, THF, 0 °C-rt, 4 h; c) methylsulfonyl chloride, Et3N, CH2Cl2, rt, 6 h; d) 1a-b, acetonitrile,
Et3N, 40 °C, 6-8 h; e) HCl/EtOH, 0 °C-rt, 12 h; f) RCOOH, HATU, Et3N, DMF, rt, 6-8 h.
The synthetic routes of compounds 9a-c are depicted in Scheme 2. Boc-β-Ala was condensed with
various aromatic amines in the presence of HATU to afford the Boc-protected intermediates 7a-c,
respectively. After the deletion of Boc group, compounds 8a-b were subsequently reacted with 3 to afford
the target compounds 9a-c, respectively.
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Scheme 2. Synthesis of the target compounds 9a-c. Reagents and conditions: a) RNH2, HATU, EtN3, DMF,
rt, 6-8 h; b) CF3COOH, CH2Cl2, 0 °C-rt, 4-6 h; c) 8a-c, acetonitrile, Et3N, 40 °C, 6-8 h.
3. Results and discussion
3.1. Biological evaluation
3.1.1 Effect of the target compounds on Rh123 accumulation
It is well-known that P-gp transporter can efflux P-gp substrate chemotherapeutic drugs out of tumor
cells, leading to the decrease of intracellular drug concentration and consequent drug insensitivity. Thus,
flow cytometric analysis was firstly employed to assess the inhibitory effect of target compounds on P-gp
by examining the intracellular accumulation of rhodamine 123 (Rh123, a P-gp fluorescent substrate) in
P-gp-overexpressing K562/A02 cells. Classical P-gp inhibitor verapamil (VRP) was used as a positive
control. As shown in Figure 2A, Rh123 level in K562/A02 cells treated with VRP was significantly
increased, for the Rh123 accumulation fold change was 7.15. While, bifendate showed much weaker P-gp
inhibitory effect than VRP, which is consistent with the previous report [15]. Interestingly, the target
compounds, especially 6j, 6k and 9c, significantly increased the intracellular Rh123 level in K562/A02
cells. Clearly, compound 9c was the most potent one, and the value of Rh123 accumulation fold change
was 9.32, which was even higher than that of positive control VRP and ZG1142.
Subsequently, the dose-response effects of the most active compounds 6j, 6k, 9c and VRP (10, 3.3, 0.33
µM) on Rh123 accumulation in K562/A02 and parental sensitive K562 cells were further investigated by
flow cytometry. As was expected, 6j, 6k, 9c and VRP can significantly augment Rh123 accumulation in
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K562/A02 cells at a dose-dependent manner, while this effect was not detected in P-gp-negative K562 cells
(Fig. 2B), suggesting that such effect on Rh123 amassment in K562/A02 cells is likely achieved by
inhibiting P-gp.
Figure 2. The effect of the target compounds (A: at the dose of 10 µM; B: at the dose of 10, 3.3 and 0.3 µM,
respectively) on the intracellular accumulation of Rh123 (0.5 µM). The relative values were identified by
dividing the fluorescence 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.
3.1.2 Cytotoxicity assays
Considering that a perfect P-gp inhibitor should overcome MDR at non-toxic doses [27, 28], we next
determined the intrinsic cytotoxicity of the target compounds against K562/A02 (Table 1) and parental
sensitive K562 cells (Table 2) by MTS assay using adriamycin (ADR) as the positive control. As was
expected, K562/A02 cells overexpressing P-gp were insensitive to ADR (a substrate of P-gp), for the IC50
value of ADR for K562/A02 was 25.04 µM (Table 1), which was 64-fold higher than that for K562 cells
(0.39 µM, Table 2). As shown in Table 1, most of the target compounds displayed slight intrinsic
cytotoxicity (IC50 = 26.24 ~ 72.93 µM) against K562/A02 cells in vitro, and several compounds exhibited
even higher cytotoxicity (IC50 = 11.47 ~ 23.31 µM). While, all the target compounds displayed relatively
higher intrinsic cytotoxicity in K562 cells than that in K562/A02 cells (Table 2). Based on the IC50 values
mentioned above, compounds at the concentration below IC10 were selected in the following MDR reversal
experiments.
Table 1. IC50 values of target compounds against K562/A02 cells.
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Compounds IC50 (µM) a Compounds IC50 (µM) a Compounds
IC50 (µM) a
72.93 ± 3.42
43.21 ± 3.12
69.86 ± 2.90
25.04 ± 1.47
>100
26.24 ± 0.53
28.30 ± 1.99
31.84 ± 0.86
23.31 ± 2.23
18.36 ± 0.97
11.47 ± 0.05
34.47 ± 1.18
28.99 ± 0.89
63.92 ± 1.32
65.84 ± 3.21
47.22 ± 2.05
28.24 ± 0.63
6a
6b
6c
6d
6e
6f
6g
6h
6i
9a
9b
9c
6j
ADR
ZG1142
6k
6l
a IC50 values are expressed as means of triplicate experiments.
Table 2. IC50 values of target compounds against K562 cells.
Compounds IC50 (µM) a Compounds IC50 (µM) a Compounds
IC50 (µM) a
23.05 ± 2.64
29.82 ± 3.72
34.78 ±1.37
0.39 ± 0.04
51.35 ± 0.42
10.96 ± 3.61
14.11 ± 1.89
11.82 ± 1.18
8.18 ± 1.16
5.85 ± 1.36
5.07 ± 1.18
13.67 ± 0.93
12.54 ± 2.25
15.03 ± 3.74
30.49 ± 1.74
16.87 ± 2.47
14.44 ±2.01
6a
6b
6c
6d
6e
6f
6g
6h
6i
9a
9b
9c
6j
ADR
ZG1142
6k
6l
a IC50 values are expressed as means of triplicate experiments.
3.1.3 MDR reversal effects of 6k and 9c
To confirm whether compounds 6k and 9c can reverse P-gp mediated MDR, we next determined the
cytotoxicity of ADR against K562/A02 cells in the presence or absence of the active compounds (6k, and
9c) at non-toxic concentration (2.0 µM) by MTS assay, and VRP (2.0 µM) was selected as the positive
control. Meanwhile, ZG1142, a potent P-gp inhibitor of bifendate derivative reported by us [16], was also
selected as a positive control. As summarized in Table 3, IC50 value of ADR against K562/A02 was 24.44
µM, indicating that ADR alone exhibited poor inhibitory effect on the survival of K562/A02 cells.
Interestingly, when ADR was combined with compounds 6k, 9c and VRP, the IC50 values of ADR was
decreased to 6.40, 4.24 and 13.45 µM, respectively, indicating that all the compounds could sensitize
K562/A02 cells to ADR at different levels. Clearly, 9c displayed the most potent chemo-sensitizing effect.
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The reversal fold (RF) of 9c was 5.76, which was significantly higher than that of positive control VRP (RF
= 1.82) and ZG1142 (RF = 3.99) at the same dose.
Table 3. Chemo-sensitizing effect of the target compounds
Compound
6k + ADR
IC50 (µM) a
6.40 ± 0.60
4.24 ± 0.29
6.13 ± 1.04
RF b
3.82
5.76
3.99
Compound
VRP + ADR
ADR
IC50 (µM) a
13.45 ± 0.87
24.44 ± 1.76
RF b
1.82
/
9c + ADR
ZG1142 + ADR
a The cytotoxicity of ADR against K562/A02 cells in the presence or absence of the target compounds (2.0
µM) was evaluated by MTS assay.
b RF: Reversal fold (RF) refers to fold-change in drug sensitivity. RF = (IC50 of ADR without target
compound)/(IC50 of ADR with target compound)
3.1.4 Effect of active compounds 6k and 9c on P-gp expression and function
It is believed that P-gp-mediated MDR can be overcame either by reducing P-gp expression and/or
inhibiting its function [29]. To understand the potential molecular mechanisms underlying the action of 6k
and 9c, we firstly tested the effect of 6k and 9c on P-gp expression at mRNA and protein levels using
RT-PCR and Western blot, respectively. Data showed that treatment with 6k and 9c did not significantly
change P-gp expression at mRNA or protein level in K562/A02 cells (shown in the Supplementary
Material). These data suggested that compounds 6k and 9c may exert P-gp-mediated MDR reversal activity
by inhibiting P-gp efflux function and not by decreasing P-gp expression.
3.1.5 Anti-migratory and anti-invasive activities of 6k and 9c
Besides MDR, migration and invasion are also major causes of mortality in patients with cancer [30, 31].
Recent research showed that Schisandrin B (structure was shown in the Supplementary Material), a natural
product with alkoxyl biphenyl scaffold, could attenuate cancer invasion and metastasis via inhibiting
epithelial-mesenchymal transition [32]. In addition, in early studies, sun et al. found that bifendate could
suppress the invasion of tumor cells [33]. With these in mind and considering that active compounds 6k
and 9c shared the similar alkoxyl biphenyl scaffold as bifendate, we examined whether compounds 6k and
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9c could inhibit the migration and invasion of tumor cells. Due to the highly metastatic and invasive
capacity, MDA-MB-231 cells (human breast carcinoma cells), were selected as models in this study.
Firstly, the effects of compounds 6k and 9c on the motility properties of MDA-MB-231 cells were
determined by wound-healing assay and chamber migration assay, respectively [34]. As shown in Figure 3,
at doses below IC50 (IC50 values of 6k and 9c were shown in the Supplementary Material), 6k and 9c
significantly suppressed the migration of MDA-MB-231 cells across the wounded space in a dose
depended manner. The inhibitory effect on cell mobility was subsequently confirmed by the chamber
migration assay (Fig. 4). Exposure to 6k and 9c led to an obviously delay in cell migration up to 65.7% and
54.8%, respectively.
Figure 3. Inhibitory Effects of 6k and 9c on MDA-MB-231 cells migration in vitro. (A) Compounds
inhibit migration of cells across the wounded space. Cell monolayer was wounded by a 200 µL pipette tip
followed by treatment with various concentrations (5, 15, and 45 µM) of compound for 24 h. And distance
of the wound edge was measured before and after the treatment. Image magnification: × 100. (B)
Quantification of the relative migration. Relative migration (%) were identified by dividing the migration
distance of MDA-MB-231 cells treated with 6k or 9c by that of control group. **P < 0.01 represents
significant difference from the control group.
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Figure 4. Effects of 6k and 9c on the mobility of MDA-MB-231 cells in vitro. (A) 6k and 9c inhibit cell
migration. Cells were cultured with indicated concentrations of compounds for 24 h, and then seeded in the
upside of transwell. After incubation for 4 h, the downward side of the membrane was stained with crystal
violet. Image magnification: × 200. (B) Quantification of the migrated cells. Migration cells (% of control)
were identified by dividing the number of the migration MDA-MB-231 cells treated with 6k or 9c by that
of control group. **P < 0.01 represents significant difference from the control group.
Next, the anti-invasive activities of 6k and 9c were determined by invasion assay. In the absence of
compound 6k or 9c, most MDA-MB-231 cells were able to penetrate through the matrigel-coated filters,
indicating its high invasive capability. After exposure to 6k or 9c, the number of MDA-MB-231 cells able
to penetrate through the matrigel-coated filters was significantly decreased (Fig. 5), suggesting the invasive
activity of MDA-MB-231 cells was markedly suppressed.
Figure 5. Effects of 6k and 9c on the invasion of MDA-MB-231 cells in vitro. (A) 6k and 9c inhibit cell
invasion. After treatment with different concentrations of 6k and 9c for 24 h, the cells were seeded in the
upside of transwell pre-coated with matrigel, and incubated for 24 h. Then, the invaded cells were
measured by crystal violet staining. Image magnification: × 200. (B) Quantification of the invaded cells.
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Invasion cells (% of control) were identified by dividing the number of the invasion MDA-MB-231 cells
treated with 6k or 9c by that of control group. **P < 0.01 represents significant difference from the control
group.
3.1.6 Effect of 6k and 9c on the activity and protein expression of MMP-2 and MMP-9
It is well-known that matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play critical
roles in invasion and metastasis of malignant cells [35]. To probe the potential anti-invasive mechanisms,
the effects of 6k and 9c on the activity and protein expression of MMP-2 and MMP-9 in MDA-MB-231
cells were determined by gelatin zymography assay and western blot, respectively. As shown in Figure 6,
6k and 9c significantly decreased the gelatinolytic activity of MMP-2 and MMP-9 in a dose dependent
manner (Fig. 6A). Quantification analysis showed that treatment with 6k or 9c, even at the lowest
concentration (5 µM), reduced MMP-2 activity to 59.1% and 52.8%, respectively, and MMP-9 activity was
decreased to 58.2% and 52.6%, respectively. In addition, 6k and 9c could significantly suppress the protein
expression of MMP-2 and MMP-9 in a dose dependent manner (Fig. 6B). These data suggested that the
anti-metastatic activity of 6k and 9c was associated with their inhibitory effect on MMP-2 and MMP-9
activity and expression.
Figure 6. Effects of 6k and 9c on the activity and expression of MMP-2 and MMP-9 in MDA-MB-231
cells. (A) 6k and 9c inhibit the activity of MMP-2/9 in MDA-MB-231 cells. Cells were treated with
indicated concentrations of 6k and 9c for 24 h. The culture supernatants were collected, and then MMP-2/9
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activity was determined by gelatin zymography assay. (B) 6k and 9c suppress the protein expression of
MMP-2/9. MDA-MB-231 cell lysates were subjected to immunoblotting with antibodies against MMP-2
and MMP-9. **P < 0.01 represents significant difference from the control group.
3.2. Structure-Activity Relationships
Based on the results above, structure-activity relationships (SARs) of the target compounds on P-gp
inhibitory effects could be summarized as follows: The length of the linker between the benzene ring and
nitrogen atom in azepine ring can significantly affect P-gp inhibitory effect. Compounds with a four atoms
chain displayed higher inhibitory effect on P-gp than that with an eight atoms chain (6i vs. 6a, 6j vs. 6b, 6k
vs. 6c, 6l vs. 6d). Interestingly, introducing methoxyl substitutes to the third benzene ring could
significantly increase the intensity of P-gp inhibition (6k vs. 6j vs. 6i, 6c vs. 6b vs. 6a). P-gp inhibitory
effect can enhanced by changing methoxy or methyl into chlorine atom in the third benzene ring,
unfortunately, the intrinsic cytotoxicity was also increased (6b vs. 6f, 6d vs. 6e). Notably, when
amide-bond was reversed (from NHCO to CONH) in the molecule, the P-gp inhibitory intensity was
significantly increased (9a vs. 6i, 9c vs. 6j), which was consistent with previously reported that a
reverse-amide-bond can significantly affect the bioactivity of the target compounds. One plausible
explanation is that reverse of amide might affect the affinity between target compounds with P-gp, and
consequent weak P-gp inhibitory intensity. However, the precise SARs remain further investigation when
more derivatives of this kind will be available in near future.
4. Conclusions
In summary, fifteen novel bifendate derivatives containing dibenzo[c,e]azepine scaffold were
synthesized and evaluated as P-gp-medicated MDR reversal agents. Among them, compounds 6k and 9c
more potently increased the accumulation of P-gp substrate in K562/A02 cells than VRP by inhibiting P-gp
efflux function instead by decreasing P-gp protein expression levels. Notably, wound-healing and chamber
migration assay showed that 6k and 9c could significantly attenuate the motility of MDA-MB-231 cells.
More importantly, 6k and 9c could markedly suppress the invasive activity of MDA-MB-231 cells, thus
displayed potential anti-metastasis activity. Preliminary mechanism studies indicated that the
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anti-metastasis activity of 6k and 9c were associated with their inhibitory effect on MMP-2 and MMP-9
activity and expression. These results, together with the MDR reversal results suggested that compounds 6k
and 9c might be promising leads for the development novel anti-cancer agents with P-gp and tumor
metastasis inhibitory activities.
5. Experimental protocols
5.1. Chemical analysis
All of the synthesized compounds were purified by column chromatography on silica gel 60 (200–300
mesh) or thin layer chromatography (TLC) on silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical
Company, China). Melting points were measured on a YRT-3 melting point apparatus and uncorrected.
Subsequently, all of them were analyzed by IR (Thermofisher Nicolet is10), 1HNMR and 13C NMR (JEOL,
400 MHz), and MS (Agilent 1290/6460 LC/MSD spectrometer) routinely. All solvents were reagent grade
and, when necessary, were purified and dried by standards methods.
5.2. Synthesis of 1a-b
Ethane diamine (6 g, 0.1 mol) or hexamethylenediamine (12 g, 0.1 mol) was dissolved in CHCl3 (100
mL) and cooled to 0 °C. Boc2O (4.6 mL, 0.01 mol) was dispersed in CHCl3 (100 mL) and the mixture was
added to the above reaction solution slowly. Then, the solution was stirred for 24 h at room temperature.
After the completion of reaction, the mixture was evaporated in vacuo and the residue was diluted with
water and extracted with CHCl3. The organic layer was washed sequentially with saturated NaHCO3
solution and brine then dried with sodium sulfate, filtered and evaporated in vacuo to give corresponding
crude compounds 1a-b (92%), respectivley, which were used without further purification.
5.3. Synthesis of 5a-b
Et3N (1.2 mL, 5eq) was added to the stirred solution of compound 3 and 1a (2eq) in acetonitrile at room
temperature under a nitrogen atmosphere. The reaction mixture was stirred and refluxed for 5 h at 60 °C.
After the completion of reaction, the mixture was evaporated in vacuo and the residue was diluted with
water and extracted with ethyl acetate. The organic layer was washed sequentially with saturated NaHCO3
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solution and brine, then dried with sodium sulfate, filtered and evaporated in vacuo to give corresponding
crude compound 4a. And then the Boc-protection group is removed using hydrochloric acid in ethanol to
afford compound 5a, which was used without further purification. The title crude compound 5b was
obtained starting from 3 and 1a.
5.4. General procedure for the preparation of 6a-l
A solution of 5a or 5b, triethylamine and substituted benzoic acid in DMF was stirred at 0 °C, then
HATU was added to the solution. The reaction mixture was stirred at 0 °C for 1 h, then 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 to yield the title compounds, respectively.
5.4.1.N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)hexyl)-2-methoxybenzamide (6a)
The title compound was obtained starting from 5a and 2-methoxybenzoic acid. As a yellow solid, yield:
64.2%; mp: 129.8-131.6 °C. Analytical data for 6a: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.42-1.59 (m, 4H),
1.60-1.72 (m, 2H), 1.85-2.00 (m, 2H), 3.03-3.33 (m, 2H), 3.39-3.52 (m, 2H), 3.93 (s, 6H, 2 × Ar-OCH3),
3.98 (s, 3H, Ar-OCH3), 4.09-4.24 (m, 2H), 6.05 (d, 2H, -OCH2O-, J = 1.4 Hz), 6.14 (d, 2H, -OCH2O-, J =
1.4 Hz), 6.85 (s, 2H, 2 × Ar-H), 6.95-7.01 (m, 2H, 2 × Ar-H), 7.43 (m, 1H, Ar-H), 7.91-7.93 (m, 1H, Ar-H),
8.08 (t, 1H, -NH-, J = 5.7 Hz); 13C NMR (CDCl3, 100 MHz, δ ppm): 24.7, 25.8, 26.0, 29.1, 39.5, 54.3，54.4,
56.1 (2C, 2 × ArCH2N), 57.0 (2C, 2 × OCH3), 102.5 (2C, 2 × -OCH2O-), 110.2, 111.4, 111.6 (2C, 2 ×
Ar-C), 121.1, 121.2 (2C, 2 × Ar-C), 131.7 (2C, 2 × Ar-C), 133.2 (2C, 2 × Ar-C), 137.0 (2C, 2 × Ar-C),
143.8 (2C, 2 × Ar-C), 146.4 (2C, 2 × Ar-C), 157.7, 166.0 (-CONH-); IR (KBr, cm-1): ν 3398, 2943, 2360,
1644, 1600, 1544, 1496, 1439, 1384, 1311, 1148, 1102, 1058, 923, 841; ESI-MS: m/z: 577.2 [M+H]+.
5.4.2.N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)hexyl)-3,4-dimethoxybenzamide (6b)
14

ACCEPTED MANUSCRIPT
The title compound was obtained starting from 5a and 3,4-dimethoxybenzoic acid. As a yellow solid,
yield: 60.4%; mp: 129.8-131.6 °C. Analytical data for 6b: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.39-1.49
(m, 4H), 1.57-1.68 (m, 2H), 1.70-1.90 (m, 2H), 2.86-3.11 (m, 2H), 3.36-3.54 (m, 4H), 3.87 (s, 3H, OCH3),
3.88 (s, 3H, OCH3), 3.91 (s, 6H, 2 × -OCH3), 3.97 (s, 1H), 4.00 (s, 1H), 6.02 (d, 2H, -OCH2O-, J = 1.2 Hz),
6.12 (d, 2H, Ar-OCH2O-, J = 1.2 Hz), 6.74 (s, 2H, Ar-H), 6.83 (d, 1H, Ar-H, J = 8.0 Hz), 7.29-7.35 (m, 2H,
Ar-H); 13C NMR (CDCl3, 100 MHz, δ ppm): 26.7, 26.9, 27.0, 29.5, 40.0, 54.8, 54.9 (2C, 2 × OCH3), 56.1
(2C, 2 × ArCH2N), 56.9 (2C, 2 × OCH3), 102.0 (2C, 2 × -OCH2O-), 109.7 (2C, 2 × Ar-C), 110.3, 110.5,
110.6 (2C, 2 × Ar-C), 119.3 (2C, 2 × Ar-C), 127.4 (2C, 2 × Ar-C), 135.4 (2C, 2 × Ar-C), 143.1 (2C, 2 ×
Ar-C), 145.9 (2C, 2 × Ar-C), 149.0, 151.7, 167.3 (-CONH-); IR (KBr, cm-1): ν 3413,2939, 2360, 2341,
1639, 1617, 1582, 1507, 1437, 1311, 1230, 1147, 1101, 1048, 922, 844; ESI-MS: m/z: 607.2 [M+H]+.
5.4.3.N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)hexyl)-3,4,5-trimethoxybenzamide (6c)
The title compound was obtained starting from 5a and 3,4,5-trimethoxybenzoic acid. As a yellow solid,
yield: 61.6%; mp: 134.2-136.1 °C. Analytical data for 6c: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.35-1.50
(m, 4H), 1.55-1.69 (m, 2H), 1.71-1.98 (m, 2H), 2.17-2.37 (m, 1H), 3.00-3.13 (m, 1H), 3.20-3.48 (m, 4H),
3.81-3.86 (m, 12H, 4 × Ar -OCH3), 3.97 (s, 3H, Ar-OCH3), 4.00-4.05 (m, 1H), 4.29-4.33 (m, 1H), 6.04 (dd,
2H, -OCH2O-, J = 6.4, 1.2 Hz), 6.13 (dd, 2H, -OCH2O-, J = 9.2, 1.2 Hz), 6.81 (d, 2H, 2 × Ar-H, J = 4.4 Hz),
7.01 (s, 2H, 2 × Ar-H), 8.75 (s, 1H, -NH-); 13C NMR (CDCl3, 100 MHz, δ ppm): 24.2, 25.6, 25.7, 28.5,
40.3, 53.5, 54.3, 54.9, 56.3 (2C, 2 × ArCH2N), 56.7, 57.2, 61.0, 102.5 (2C, 2 × -OCH2O-), 104.5 (2C, 2 ×
Ar-C), 110.0, 110.3, 111.2, 111.8, 120.0, 122.4, 129.0, 137.1, 137.3, 140.9, 143.7, 143.9, 146.3, 146.6,
153.2 (2C, 2 × Ar-C), 168.4 (-CONH-); IR (KBr, cm-1): ν 3414, 2942, 2360, 2341, 1643, 1617, 1584, 1499,
1437, 1383, 1311, 1235, 1148, 1102, 1058, 922, 844; ESI-MS: m/z: 637.2 [M+H]+.
5.4.4.N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)hexyl)-4-methylbenzamide (6d)
The title compound was obtained starting from 5a and 4-methylbenzoic acid. As a yellow solid, yield:
50.2%; mp: 101.6-103.2 °C. Analytical data for 6d: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.36-1.48 (m, 4H),
15

ACCEPTED MANUSCRIPT
1.56-1.62 (m, 4H), 2.37 (s, 3H, -CH3), 2.44-2.66 (m, 2H), 3.20 (d, 2H, J = 12.4 Hz), 3.41-3.46 (m, 2H),
3.57 (d, 2H, J = 12.8 Hz), 3.92 (s, 6H, 2 × Ar-OCH3), 5.97 (d, 2H, -OCH2O-, J = 1.2 Hz), 6.08 (d, 2H,
-OCH2O-, J = 1.2 Hz), 6.56 (s, 2H, 2 × Ar-H), 7.20 (d, 2H, 2 × Ar-H, J = 8.0 Hz), 7.64 (d, 2H, 2 × Ar-H, J
= 8 Hz); 13C NMR (CDCl3, 100 MHz, δ ppm): 21.5, 26.9, 27.2, 27.5, 29.66, 40.0, 55.0 (2C, 2 × ArCH2N),
55.2, 56.8 (2C, 2 × OCH3), 101.9 (2C, 2 × -OCH2O-), 109.4 (2C, 2 × Ar-C), 110.6 (2C, 2 × Ar-C), 126.9
(2C, 2 × Ar-C), 128.4 (2C, 2 × Ar-C), 129.3 (2C, 2 × Ar-C), 131.9, 135.1 (2C, 2 × Ar-C), 141.9, 143.0 (2C,
2 × Ar-C), 145.8 (2C, 2 × Ar-C), 167.6 (-CONH-); IR (KBr, cm-1): ν 3413, 2931, 2360, 2341, 1639, 1616,
1541, 1506, 1435, 1374, 1306, 1143, 1099, 1050, 925, 842; ESI-MS: m/z: 561.2 [M+H]+.
5.4.5.4-chloro-N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1
,2-e]azepin-7(8H)-yl)hexyl)benzamide (6e)
The title compound was obtained starting from 5a and 4-chlorobenzoic acid. As a yellow solid, yield:
52.3%; mp: 75.5-76.8 °C. Analytical data for 6e: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.39-1.45 (m, 4H),
1.59-1.76 (m, 4H), 2.50-2.74 (m, 2H), 3.28 (d, 2H, J = 12.5 Hz), 3.39-3.51 (m, 2H), 3.62 (d, 2H, J = 12.4
Hz), 3.93 (s, 6H, 2 × Ar-OCH3), 5.99 (d, 2H, -OCH2O-, J = 1.3 Hz), 6.09 (d, 2H, -OCH2O-, J = 1.4 Hz),
6.59 (s, 2H, 2 × Ar-H), 7.36-7.41 (m, 2H, 2 × Ar-H), 7.70-7.76 (m, 2H, 2 × Ar-H); 13C NMR (CDCl3, 100
MHz, δ ppm): 26.6, 26.9, 27.0, 29.4, 40.0, 54.7 (3C, 3 × NCH2), 56.9 (2C, 2 × OCH3), 102.0 (2C, 2 ×
-OCH2O-), 109.9 (2C, 2 × Ar-C), 110.5 (2C, 2 × Ar-C), 128.5 (2C, 2 × Ar-C), 128.9 (2C, 2 × Ar-C), 133.1
(2C, 2 × Ar-C), 135.4 (2C, 2 × Ar-C), 137.6, 137.7, 143.1 (2C, 2 × Ar-C), 145.9 (2C, 2 × Ar-C), 166.6
(-CONH-); IR (KBr, cm-1): ν 3413, 2932, 2359, 2341, 1638, 1540, 1488, 1434, 1384, 1306, 1144, 1098,
1051, 925, 846; ESI-MS: m/z: 581.1 [M+H]+.
5.4.6.3,4-dichloro-N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]ben
zo[1,2-e]azepin-7(8H)-yl)hexyl)benzamide (6f)
The title compound was obtained starting from 5a and 3,4-dichlorobenzoic acid. As a yellow solid, yield:
53.4%; mp: 85.5-88.3 °C. Analytical data for 6f: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.40-1.53 (m, 4H),
1.62-1.74 (m, 2H), 1.77-1.97 (m, 2H), 2.77-2.96 (m, 2H), 3.41-3.52 (m, 4H), 3.88 (d, 2H, J = 12.8 Hz),
3.96 (s, 6H, 2 × Ar-OCH3), 6.03 (d, 2H, -OCH2O-, J = 1.2 Hz), 6.13 (d, 2H, -OCH2O-, J = 1.2 Hz), 6.72 (s,
16

ACCEPTED MANUSCRIPT
2H, 2 × Ar-H), 7.49 (d, 1H, Ar-H, J = 8.4 Hz), 7.755-7.78 (m, 1H, Ar-H), 8.04 (d, 1H, Ar-H, J = 2.0 Hz);
13C NMR (CDCl3, 100 MHz, δ ppm): 25.1, 25.9, 26.2, 28.8, 39.8, 53.7, 53.9 (2C, 2 × ArCH2N), 57.1 (2C, 2
× OCH3), 102.3 (2C, 2 × -OCH2O-), 110.3 (2C, 2 × Ar-C), 111.0 (2C, 2 × Ar-C), 123.9, 126.6, 129.6 (2C,
2 × Ar-C), 130.5, 132.9, 134.5, 135.6, 136.4 (2C, 2 × Ar-C), 143.4 (2C, 2 × Ar-C), 146.3 (2C, 2 × Ar-C),
165.5 (-CONH-); IR (KBr, cm-1): ν 3415, 2933, 2360, 2341, 1637, 1540, 1489, 1435, 1384, 1310, 1148,
1100, 1044, 923, 840; ESI-MS: m/z: 615.1 [M+H]+.
5.4.7.2,4-dichloro-N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]ben
zo[1,2-e]azepin-7(8H)-yl)hexyl)benzamide (6g)
The title compound was obtained starting from 5a and 2,4-dichlorobenzoic acid. As a yellow solid, yield:
49.0%; mp: 70.2-72.3 °C. Analytical data for 6g: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.39-1.52 (m, 4H),
1.61-1.67 (m, 2H), 1.82-1.98 (m, 2H), 2.86-3.01 (m, 2H), 3.41-3.46 (m, 4H), 3.90 (s, 6H, 2 × Ar-OCH3),
4.00-4.08 (m, 2H), 6.01 (d, 2H, -OCH2O-, J = 1.6 Hz), 6.11 (d, 2H, -OCH2O-, J = 1.2 Hz), 6.68 (s, 2H,
Ar-H), 7.13-7.18 (m, 1H, Ar-H), 7.38 (d, 1H, Ar-H, J = 2.0 Hz), 7.54 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-H); 13C
NMR (CDCl3, 100 MHz, δ ppm): 24.9, 26.0, 26.4, 28.8, 39.8, 53.3, 53.5 (2C, 2 × ArCH2N), 57.0 (2C, 2 ×
OCH3), 102.2 (2C, 2 × -OCH2O-), 110.4 (2C, 2 × Ar-C), 111.0 (2C, 2 × Ar-C), 126.6, 127.5, 129.8, 130.0
(2C, 2 × Ar-C), 130.9, 131.1, 134.0, 136.4 (2C, 2 × Ar-C), 143.4 (2C, 2 × Ar-C), 146.3 (2C, 2 × Ar-C),
165.6 (-CONH-); IR (KBr, cm-1): ν 3413, 2936, 2360, 1640, 1616, 1555, 1490, 1436, 1383, 1309, 1146,
1101, 1046, 923, 845; ESI-MS: m/z: 615.1 [M+H]+.
5.4.8.N-(6-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)hexyl)thiophene-2-carboxamide (6h)
The title compound was obtained starting from 5a and thiophene-2-carboxylic acid. As a yellow solid,
yield: 65.8%; mp: 84.7-86.4 °C. Analytical data for 6h: 1H NMR (CDCl3, 400 MHz, δ ppm): 1.33-1.46 (m,
4H), 1.52-1.67 (m, 4H), 2.40-2.59 (m, 2H), 3.17 (d, 2H, J = 12.4 Hz), 3.42 (m, 2H), 3.51 (d, 2H, J = 12.4
Hz), 3.92 (s, 6H, 2 × Ar-OCH3), 5.96 (d, 2H, -OCH2O-, J = 1.4 Hz), 6.07 (d, 2H, -OCH2O-, J = 1.4 Hz),
6.54 (s, 2H, 2 × Ar-H), 7.03-7.05 (m, 1H), 7.42-7.44 (m, 1H), 7.48-7.50 (m, 1H); 13C NMR (CDCl3, 100
MHz, δ ppm): 26.9, 27.3, 27.7, 29.7, 40.0, 55.0 (2C, 2 × ArCH2N), 55.1, 56.8 (2C, 2 × OCH3), 101.8 (2C, 2
17

ACCEPTED MANUSCRIPT
× -OCH2O-), 109.3 (2C, 2 × Ar-C), 110.7 (2C, 2 × Ar-C), 127.7, 127.9, 129.0, 129.8 (2C, 2 × Ar-C), 134.9
(2C, 2 × Ar-C), 139.3, 142.9 (2C, 2 × Ar-C), 145.8 (2C, 2 × Ar-C), 162.0 (-CONH-); IR (KBr, cm-1): ν
3413, 2932, 2360, 2341, 1637, 1617, 1541, 1506, 1432, 1373, 1304, 1209, 1142, 1098, 1050, 924, 844;
ESI-MS: m/z: 553.1 [M+H]+.
5.4.9.N-(2-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]azep
in-7(8H)-yl)ethyl)-2-methoxybenzamide (6i)
The title compound was obtained starting from 5b and 2-methoxybenzoic acid. As a yellow solid, yield:
59.3%; mp: 74.9-76.3 °C. Analytical data for 6i: 1H NMR (DMSO, 400 MHz, δ ppm ): 2.49-2.51 (m, 2H),
3.14-3.22 (m, 2H), 3.29-3.41 (m, 2H), 3.64-3.83 (m, 2H), 3.90 (s, 6H, 2 × Ar-OCH3), 3.92 (s, 3H,
Ar-OCH3), 6.09 (d, 2H, -OCH2O-, J = 0.8 Hz), 6.13 (d, 2H, -OCH2O-, J = 0.8 Hz), 6.96-7.02 (m, 1H,
Ar-H), 7.05-7.09 (m, 1H, Ar-H), 7.17-7.21 (m, 1H, Ar-H), 7.49-7.55 (m, 1H, Ar-H), 7.84-7.88 (m, 1H,
Ar-H), 8.54-8.57 (m, 1H, Ar-H); 13C NMR (DMSO, 100 MHz, δ ppm): 35.5, 38.8, 52.9, 56.5 (2C, 2 ×
ArCH2N), 57.1 (2C, 2 × OCH3), 102.7 (2C, 2 × -OCH2O-), 110.6 (2C, 2 × Ar-C), 112.7 (2C, 2 × Ar-C),
121.1 (2C, 2 × Ar-C), 122.6, 131.2 (2C, 2 × Ar-C), 133.3 (2C, 2 × Ar-C), 136.7 (2C, 2 × Ar-C), 143.3 (2C,
2 × Ar-C), 146.5 (2C, 2 × Ar-C), 157.7, 166.3 (-CONH-); IR (KBr, cm-1): ν 3412, 2908, 1643, 1619, 1545,
1496, 1444, 1396, 1312, 1243, 1172, 1107, 1047, 918, 839; ESI-MS: m/z: 521.2 [M+H]+.
5.4.10.N-(2-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]aze
pin-7(8H)-yl)ethyl)-3,4-dimethoxybenzamide (6j)
The title compound was obtained starting from 5b and 3,4-dimethoxybenzoic acid. As a yellow solid,
yield: 56.1%; mp: 90.6-92.4 °C. Analytical data for 6j: 1H NMR (CDCl3, 400 MHz, δ ppm): 2.64-2.96 (m,
2H), 3.27 (d, 2H, J = 12.4 Hz), 3.51-3.60 (m, 2H), 3.61-3.70 (m, 2H), 3.95 (s, 12H, 4 × Ar-OCH3), 6.00 (s,
2H, -OCH2O-), 6.10 (s, 2H, -OCH2O-), 6.57 (s, 2H, 2 × Ar-H), 6.88 (d, 1H, Ar-H, J = 7.7 Hz), 7.01 (dd, 1H,
Ar-H, J = 1.3, 0.8 Hz), 7.30-7.39 (m, 1H, Ar-H), 7.49 (s, 1H, Ar-H); 13C NMR (CDCl3, 100 MHz, δ ppm):
37.3, 53.8, 54.9 (2C, 2 × OCH3), 56.1 (2C, 2 × ArCH2N), 56.9 (2C, 2 × OCH3), 101.9 (2C, 2 × -OCH2O-),
109.3 (2C, 2 × Ar-C), 110.3, 110.7 (2C, 2 × Ar-C), 110.8, 119.5, 127.3, 128.6 (2C, 2 × Ar-C), 135.1 (2C, 2
× Ar-C), 143.0 (2C, 2 × Ar-C), 145.9 (2C, 2 × Ar-C), 149.0, 151.8, 167.3 (-CONH-); IR (KBr, cm-1): ν
18

ACCEPTED MANUSCRIPT
3413, 2937, 1639, 1604, 1584, 1544, 1506, 1433, 1383, 1305, 1229, 1182, 1097, 1047, 924, 856; ESI-MS:
m/z: 551.2 [M+H]+.
5.4.11.N-(2-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]aze
pin-7(8H)-yl)ethyl)-3,4,5-trimethoxybenzamide (6k)
The title compound was obtained starting from 5b and 3,4,5-trimethoxybenzoic acid. As a yellow solid,
yield: 53.2%; mp: 122.1-123.8 °C. Analytical data for 6k: 1H NMR (CDCl3, 400 MHz, δ ppm): 2.88-3.13
(m, 2H), 3.44 (d, 2H, J = 12.8 Hz), 3.73-3.82 (m, 4H), 3.89 (s, 3H, Ar-OCH3), 3.94 (s, 6H, 2 × Ar-OCH3),
3.96 (s, 6H, 2 × Ar-OCH3), 6.02 (s, 2H, -OCH2O-), 6.12 (s, 2H, -OCH2O-), 6.65 (s, 2H, 2 × Ar-H), 7.22 (s,
2H, 2 × Ar-H); 13C NMR (CDCl3, 100 MHz, δ ppm): 36.5, 54.0, 54.7 (2C, 2 × ArCH2N), 56.5 (2C, 2 ×
OCH3), 57.0 (2C, 2 × OCH3), 61.0, 102.2 (2C, 2 × -OCH2O-), 104.8 (2C, 2 × Ar-C), 110.2 (2C, 2 × Ar-C),
110.5 (2C, 2 × Ar-C), 125.9, 129.4 (2C, 2 × Ar-C), 135.9 (2C, 2 × Ar-C), 140.9, 143.3 (2C, 2 × Ar-C),
146.2 (2C, 2 × Ar-C), 153.2 (2C, 2 × Ar-C), 167.5 (-CONH-); IR (KBr, cm-1): ν 3413, 2357, 1639, 1617,
1585, 1538, 1497, 1434, 1335, 1309, 1232, 1126, 1100, 1050, 923, 856; ESI-MS: m/z: 581.2 [M+H]+.
5.4.12N-(2-(4,10-dimethoxy-6H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1,2-e]aze
pin-7(8H)-yl)ethyl)-3,4,5-trimethoxybenzamide (6l)
The title compound was obtained starting from 5b and 4-methylbenzoic acid. As a yellow solid, yield:
52.4%; mp: 94.7-96.6 °C. Analytical data for 6l: 1H NMR (CDCl3, 400 MHz, δ ppm): 2.40 (s, 3H, Ar-CH3),
2.62-2.73 (m, 1H), 2.86-2.93 (m, 1H), 3.23-3.29 (m, 2H), 3.51-3.58 (m, 2H), 3.62-3.69 (m, 2H), 3.95 (s, 6H,
2 × Ar-OCH3), 6.00 (d, 2H, -OCH2O-, J = 1.4 Hz), 6.10 (d, 2H, -OCH2O-, J = 1.5 Hz), 6.57 (s, 2H, 2 ×
Ar-H), 7.23-7.26 (m, 2H, Ar-H), 7.71-7.75 (m, 2H, Ar-H); 13C NMR (CDCl3, 100 MHz, δ ppm): 21.6, 37.2,
53.7, 54.9 (2C, 2 × ArCH2N), 56.8 (2C, 2 × OCH3), 101.9 (2C, 2 × -OCH2O-), 109.2 (2C, 2 × Ar-C), 110.6
(2C, 2 × Ar-C), 127.1 (2C, 2 × Ar-C), 128.6 (2C, 2 × Ar-C), 129.3 (2C, 2 × Ar-C), 131.7, 135.1 (2C, 2 ×
Ar-C), 142.0, 143.0 (2C, 2 × Ar-C), 145.9 (2C, 2 × Ar-C), 167.7 (-CONH-); IR (KBr, cm-1): ν 3413, 2940,
2359, 2341, 1638, 1616, 1489, 1435, 1384, 1305, 1143, 1098, 1049; ESI-MS: m/z: 505.2 [M+H]+.
5.5 Synthesis of 8a-c
19

ACCEPTED MANUSCRIPT
A solution of Boc-beta-alanine, triethylamine and substituted aniline in DMF was stirried at 0 °C, then
HATU was added to the solution. The reaction mixture was stirred at room temperature for 1 h, then
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 vacuo to give corresponding crude
compound 7a-c, respectively. The crude product was purified by column chromatography to yield the title
compounds, respectively. And then the Boc-protection group is removed using trifluoroacetic acid (TFA) in
dichloromethane to afford compound 8a-c, which was used in the next without other purification.
5.6 Synthesis of 9a-c
Et3N (0.5 mL, 5eq) was added to the stirred solution of compound 3 (100 mg) and 8a, 8b or 8c (2eq) in
acetonitrile at room temperature under a nitrogen atmosphere. The reaction mixture was stirred and
refluxed for 5 h at 60 °C. After the completion of reaction, the mixture was evaporated in vacuo and the
residue was diluted with water and extracted with ethyl acetate. The organic layer was washed sequentially
with saturated NaHCO3 solution and brine then dried with sodium sulfate, filtered and evaporated in
vacuum. The crude product was purified by column chromatography to yield the title compound,
respectively
5.6.1.3-(4,10-dimethoxy-6,8-dihydro-7H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1
,2-e]azepin-7-yl)-N-(2-methoxyphenyl)propanamide (9a)
The title compound was obtained starting from 3 and 3-amino-N-(2-methoxyphenyl) propanamide. As a
white solid, yield: 55.1%; mp: 190.2-191.8 °C. Analytical data for 9a: 1H NMR (CDCl3, 400 MHz, δ ppm):
2.52-3.12 (m, 4H), 3.33 (d, J = 11.2 Hz, 2H), 3.61 (m, 5H), 3.94 (s, 6H, 2 × Ar-OCH3), 6.02 (s, 2H,
-OCH2O-), 6.12 (s, 2H, -OCH2O-), 6.58 (s, 2H, 2 × Ar-H), 6.81-6.89 (m, 1H, Ar-H), 6.97-7.03 (m, 2H, 2 ×
Ar-H), 8.38-8.48 (m, 1H, Ar-H), 11.11 (s, 1H,-NH-); 13C NMR (CDCl3, 100 MHz, δ ppm): 34.0, 51.1, 54.6,
55.7 (2C, 2 × ArCH2N), 56.9 (2C, 2 × OCH3), 102.0 (2C, 2 × -OCH2O-), 109.4 (2C, 2 × Ar-C), 110.1 (2C,
2 × Ar-C), 110.6 (2C, 2 × Ar-C), 120.3, 121.1, 123.5, 128.5 (2C, 2 × Ar-C), 135.1 (2C, 2 × Ar-C), 143.0
(2C, 2 × Ar-C), 145.9 (2C, 2 × Ar-C), 148.6, 170.6 (-CONH-); IR (KBr, cm-1): ν 3413, 2926, 2360, 1641,
1602, 1544, 1489, 1431, 1366, 1305, 1237, 1181, 1096, 1044, 918, 832; ESI-MS: m/z: 521.1 [M+H]+.
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5.6.2.3-(4,10-dimethoxy-6,8-dihydro-7H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1
,2-e]azepin-7-yl)-N-(4-methoxyphenyl)propanamide (9b)
The title compound was obtained starting from 3 and3-amino-N-(4-methoxyphenyl) propanamide. As a
white solid, yield: 70.8%; mp: 77.6-78.5 °C. Analytical data for 9b: 1H NMR (CDCl3, 400 MHz, δ ppm):
2.52-2.68 (m, 2H ), 2.75-3.05 (m, 2H), 3.30 (d, 2H, J = 12.4 Hz), 3.59 (d, 2H, J = 8.4 Hz), 3.80 (s, 3H,
Ar-OCH3), 3.95 (s, 6H, 2 × Ar-OCH3), 6.02 (d, 2H, -OCH2O-, J = 0.8 Hz), 6.12 (d, 2H, -OCH2O-, J = 1.2
Hz), 6.56 (s, 2H, 2 × Ar-H), 6.85 (d, 2H, 2 × Ar-H, J = 8.9 Hz), 7.47 (d, 2H, 2 × Ar-H, J = 8.9 Hz), 11.07 (s,
13
1H, -NH-); C NMR (CDCl3, 100 MHz, δ ppm): 33.2, 50.6, 54.2 (2C, 2 × ArCH2N), 55.6, 56.9 (2C, 2 ×
OCH3), 102.0 (2C, 2 × -OCH2O-), 109.1 (2C, 2 × Ar-C), 110.6 (2C, 2 × Ar-C), 114.2 (2C, 2 × Ar-C), 121.1
(2C, 2 × Ar-C), 128.4 (2C, 2 × Ar-C), 132.2, 135.2 (2C, 2 × Ar-C), 143.1 (2C, 2 × Ar-C), 146.0 (2C, 2 ×
Ar-C), 155.9, 170.3 (-CONH-); IR (KBr, cm-1): ν 3413, 2939, 1639, 1616, 1548, 1511, 1488, 1433, 1304,
1242, 1142, 1097, 1045, 924, 828; ESI-MS: m/z: 521.1 [M+H]+.
5.6.3.3-(4,10-dimethoxy-6,8-dihydro-7H-[1,3]dioxolo[4',5':3,4]benzo[1,2-c][1,3]dioxolo[4',5':5,6]benzo[1
,2-e]azepin-7-yl)-N-(3,4-dimethoxyphenyl)propanamide (9c)
The title compound was obtained starting from 3 and 3-amino-N-(3,4-dimethoxyphenyl)propanamide. As a
white solid, yield: 85.7%; mp: 166.8-168.6 °C. Analytical data for 9c: 1H NMR (CDCl3, 400 MHz, δ ppm):
2.57-2.72 (m, 2H), 2.79-3.10 (m, 2H), 3.35 (d, 2H, J = 12.4 Hz), 3.63 (d, 2H, J = 12.8 Hz), 3.87 (s, 3H,
Ar-OCH3), 3.89 (s, 3H, Ar-OCH3), 3.95 (s, 6H, 2 × Ar-OCH3), 6.02 (d, 2H, -OCH2O-, J = 1.4 Hz), 6.12 (d,
2H, -OCH2O-, J = 1.4 Hz), 6.59 (d, 2H, 2 × Ar-H, J = 2.3 Hz), 6.76-6.81 (m, 1H, Ar-H), 6.85-6.88 (m, 1H,
Ar-H), 7.48 (d, 1H, Ar-H, J = 2.3 Hz), 11.02 (s, 1H, -NH-); 13C NMR (CDCl3, 100 MHz, δ ppm): 33.3, 50.5,
54.2 (2C, 2 × ArCH2N), 56.0, 56.2, 57.0 (2C, 2 × OCH3), 102.0 (2C, 2 × -OCH2O-), 104.6, 109.5 (2C, 2 ×
Ar-C), 110.6 (2C, 2 × Ar-C), 111.2, 111.4, 127.9 (2C, 2 × Ar-C), 132.7, 135.4 (2C, 2 × Ar-C), 143.2 (2C, 2
× Ar-C), 145.4, 146.1 (2C, 2 × Ar-C), 149.1, 170.2 (-CONH-); IR (KBr, cm-1): ν 3413, 2939, 2360, 2341,
1637, 1616, 1558, 1514, 1436, 1304, 1214, 1148, 1103, 1053, 924; ESI-MS: m/z: 551.1 [M+H]+.
5.7. Biological assays
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5.7.1. Cytotoxicity assay
1×104 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 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.
5.7.2. 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) for 1 h at 37 °C, followed by addition of 0.5 µM Rh123
or ADR and incubation for 30 min, respectively. The reaction was stopped by addition of ice-cold PBS and
centrifugation, washed with ice-cold PBS three times, and subjected analysis by flow cytometry. The
relative values were identified by dividing the fluorescence intensity of each measurement by that of
vehicle control.
5.7.3 Wound healing assay
MDA-MB-231 cells were seeded in 6-well plates until confluence. The monolayer of cells was wounded
by scraping with a sterile 200 µL pipette tip. After rinsing, each culture twice with PBS to remove detached
cells and the addition of compound 6k or 9c (5, 15 and 45 µM) in DMEM medium containing 10% FBS,
photographs of the wounded area were immediately taken at the time of wounding and thereafter 24 h with
an inverted microscope (Olympus) equipped with a digital camera (Nikon, Japan, Tokyo) to determine the
relative distance of cell migration.
5.7.4 Chamber migration and invasion assay
In chamber migration assay, transwell system (Corning Costar, Cambridge, MA) was utilized.
MDA-MB-231 cells with or without the target compound 6k or 9c (5, 15, or 45 µM, 24 h) were suspended
in serum-free medium, and then seeded into the upper compartment, while the medium containing 10%
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fetal bovine serum was added in the lower compartment. After incubation for 4 h, the non-migrated cells on
the upper side of the membrane were removed with a cotton swab. The migrated cells on the bottom
surface were fixed with 100% methanol and stained with 0.5% crystal violet. The migrated cells were
quantified by manual counting and three randomly chosen fields were analyzed for each group.
Invasion assays were performed with chambers as described previously [34]. The assay was performed
essentially as the chamber migration assays above except that transwell chambers were initially coated with
matrigel (40 µg/100 µL/chamber) at 37°C for 1 h. After incubation for 24 h, the invaded cells were stained
and analyzed as conducted above.
5.7.5 Gelatin zymography assay
Cells were treated with or without isorhamnetin (10, 20, or 40 µM) in serum-free medium for 24 h, and
then the supernatants were collected. Gelatin zymography assay was performed according to previous
method [34]. After treatment, enzyme-digested regions were observed as white bands against blue
background. The zones of enzymatic activity were seen as negatively stained bands.
5.7.6. Western blot analysis
Cells were treated with various concentrations of 6k or 9c (5, 15, or 45 µM) for 24 h, then collected and
lysed. Western blot analysis was conducted according to previous methods [34]. The membranes were
exposed to BCIP/NBT alkaline phosphatase color developing reagent for 15 min. All blots were stripped
and reprobed with polyclonal anti-GAPDH to verify equal protein loading.
Acknowledgements
The work was financially supported by National Natural Science Foundation of China (Grant No.
81402789 and 81402969), Natural Science Foundation of Jiangsu Province (Grant No. BK20171179), the
Foundation for Jiangsu Six Talent Peaks Program (Grant No. 2017-YY-039) and Jiangsu Provincial 333
High-level Talents Cultivation Project.
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Highlights
1. Novel bifendate derivatives were synthesized as anti-MDR and anti-metastasis agents.
2. 6k and 9c exhibited high MDR reversal effect by inhibiting P-gp function.
3. 6k and 9c markedly suppressed the migration and invasive activity of tumor cells.
4. 6k and 9c significantly inhibited MMP-2 and MMP-9 activity and expression in vitro.