A novel series of benzothiazepine derivatives as tubulin polymerization inhibitors with anti-tumor potency

Article abstract of DOI:10.1016/j.bioorg.2020.104585

In this work, a series of diaryl benzo[b][1,4]thiazepine derivatives D1-D36 were synthesized and screened as tubulin polymerization inhibitors with anti-tumor potency. They were designed by introducing the seven-member ring benzothiazepine as the linker f

Full text of DOI:10.1016/j.bioorg.2020.104585

Bioorganic Chemistry 108 (2021) 104585
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A novel series of benzothiazepine derivatives as tubulin polymerization
inhibitors with anti-tumor potency
,
,
Bin Wanga 1, Li-Ren Wanga 1, Lu-Lu Liua, Wei Wanga, Ruo-Jun Manb, Da-Jun Zhenga,
Yu-Shan Denga, Yu-Shun Yanga *, Chen Xua *, Hai-Liang Zhua
,
,
,*
a State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China
b Guangxi Biological Polysaccharide Separation, Purification and Modification Research Platform, Guangxi University for Nationalities, Nanning 530006, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work, a series of diaryl benzo[b][1,4]thiazepine derivatives D1-D36 were synthesized and screened as
tubulin polymerization inhibitors with anti-tumor potency. They were designed by introducing the seven-
member ring benzothiazepine as the linker for CA-4 modification for the first time. Among them, the hit com-
Benzothiazepine
Tubulin polymerization inhibitor
Anti-tumor
pound D8 showed potential on inhibiting the growth of several cancer cell lines (IC50 values: 1.48
μ
M for HeLa,
M for A549), being comparable with the positive controls
Colchicine and CA-4P. The calculated IC50 value of D8 as an tubulin polymerization inhibitor was 1.20 M. The
CA-4 modification
Mitotic catastrophe
Linker
1.47 μM for MCF-7, 1.52 μM for HT29 and 1.94 μ
μ
results of the flow cytometry assay revealed that D8 could induce the mitotic catastrophe and the death of living
cancer cells. D8 also indicated the anti-vascular activity. The possible binding pattern was implied by docking
simulation, inferring the possibility of introducing interactions with the nearby tubulin chain. Since the novel
structural trial has been conducted with preliminary discussion, this work might stimulate new ideas in further
modification of tubulin-related anti-cancer agents and therapeutic approaches.
1. Introduction
[13–15]. Cancer cells are usually more sensitive to mitotic catastrophe
because their polyploidy or aneuploidy were more prone to undergo
Cancer has been the leading cause of death all over the world for a
long time, thus the investigators have continuously seeking for potent
solutions for this poser [1]. For decades, researchers have attempted to
seek specific inhibitors of the key nodes in the pathways of cancer, and
the great efforts have led to positive feedback with many approved drugs
[2,3]. However, recent drug discovery inspired the interests on in-
hibitors for the allosteric sites or agonists with synergistic effects [4,5].
Now the hottest spots in this field seem to be Chimeric Antigen Receptor
T-Cell Immunotherapy (CAR-T) [6] and Programmed Cell Death Protein
1 (PD-1) [7]. Considering the adverse impact of both strategies [8,9],
many researchers have watched back to the existing mechanisms for
achieving the similar synergistic strategies, among which one is tubulin-
related functional molecules [10,11].
mitotic aberrations [16]. Moreover, in spite of other cell death process
which are induced by multifarious agents at high dose, mitotic catas-
trophe can be initiated by cytotoxic agents at low dose [17], therefore it
seems a potential therapeutic cut-in point [18].
Anchoring the tubulin-related mechanisms, two major investigating
directions were inducing or inhibiting the polymerization. Typical
agents of the former included Paclitaxel [19], whereas that of the latter
included Colchicine [20] and Vinblastine [21]. The binding site of
Colchicine has been most frequently studied due to less multidrug
resistance [22,23]. The clinical development indicated that the site was
potential for exploiting anti-cancer agents but Colchicine itself had
serious side effects [24]. Accordingly, Combretastain A-4 (CA-4), which
worked at the same site, attracted more attentions [25]. The following
tendency of modifying CA-4 resulted in a variety of functional mole-
cules, and some of them (including CA-4P [26], CA-1P [27], ZD6126
[28] and AVE8062 [29]) entered the clinical trials (Fig. 1A). As shown in
Fig. 1B, CA-4 analogues can be abstracted as two aromatic rings (Ring A
Tubulin-related functional molecules can cause remarkable variation
of microtubule dynamics, thus regulate the mitotic catastrophe process
[12]. In tumor events, mitotic catastrophe can regulate the onco-
suppressive procedure, and block the proliferation of living cells
* Corresponding authors.
E-mail addresses: ys_yang@nju.edu.cn (Y.-S. Yang), xuchn@nju.edu.cn (C. Xu), zhuhl@nju.edu.cn (H.-L. Zhu).
1
Both authors contributed equally to the work.
https://doi.org/10.1016/j.bioorg.2020.104585
Received 8 October 2020; Received in revised form 18 December 2020; Accepted 21 December 2020
Available online 8 January 2021
0045-2068/© 2020 Published by Elsevier Inc.

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
and Ring B) connected by Linker C. Additionally, one of the aromatic
rings should retain the 3,4,5-trimethoxy pattern. The modifications on
the linker brought several hit compounds with hetero-rings [30–38],
which inspired us to perform the design in this work (Fig. 1C). Similar to
previous series, a suitable linker should fix the cis-configuration of Ring
A and Ring B. Further, for the length of Linker C, when we compared
the previous designs with CA-4, the aliphatic chains [30,31] were
longer; the parallel rings [32,33] were shorter; while the conjugated
rings [34,35] enlarged the included angle between Ring A and Ring B.
Herein, we introduced the seven-member ring benzothiazepine as
Linker C for the first time, to perform a new trial of appropriate length
and angle. The selection of this kind of moiety was based on the inves-
tigation of the cyclic nitrogen-sulfur compounds with 5, 6 and 7-
membered rings [39–42]. Basically, one noteworthy point was that,
although the selected site allowed an outward-extending Linker C, the
actual impact of the steric benzothiazepine moiety was still uncertain to
some extent. The subsequent biological assay revealed that the hit
compound D8 could induce the mitotic catastrophe and the death of
living cancer cells. It might bring referable information of exploiting
tubulin-related anti-cancer agents, thus stimulate new ideas in both
structural evolution and therapeutic approaches.
synthesized and screened. Reports recommended that the 3,4,5-trime-
thoxy phenyl (TMP) moiety should act as Ring A or Ring B. There-
fore, considering both the requirement of structural diversity and the
difficulty of synthesis, we set the TMP as Ring A for D1-D18 and set it as
Ring B for D19-D29. Additionally, we involved D30-D36 with one of
the methoxyl group removed. All of them were synthesized and tested
for the first time. The general synthesis route was illustrated in Scheme
1. The synthesis consisted of two steps. Initially, aromatic ketone and
benzaldehydes with chosen substitutes were stirred at room temperature
with 50% NaOH solution to obtain various analogues of chalcones C1-
C36. Afterwards, 2-aminothiophenol was added to complete the cycli-
zation, resulting in the corresponding target compounds D1-D36. Sub-
sequent purification was conducted by recrystallisation to obtain the
refined compounds. All of the synthetic compounds gave satisfactory
analytical and spectroscopic data, which agreed with the structures in
Scheme 1. For developing anti-tubulin agents, the backbone in this work
was novel. Although using a ring to act as Linker C was reported,
introducing the seven-member ring benzothiazepine to perform a new
trial of appropriate length and angle was for the first time.
2.2. Biological activity
2. Results and discussion
2.2.1. The anti-proliferation assay
For anti-tumor agents, the anti-proliferation effect should be initially
evaluated. Since mitotic catastrophe occurred in various tumor classi-
fications, we included human breast adenocarcinoma cell line (MCF-7),
human epithelial cervical cancer cell line (HeLa), human colon cancer
2.1. Chemistry
Diaryl benzo[b][1,4]thiazepine derivatives D1-D36 were
Fig. 1. (A) Colchicine and analogues of CA-4 targeting tubulin at the Colchicine site; (B) Modification on the linker led to functional candidates; (C) The design
concept of this work, introducing benzothiazepine moiety as Linker C. s.
2

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
Scheme 1. The general synthesis route of D1-D36 in this series and the substitutes.
cell line (HT29), human non-small-cell lung cancer cell line (A549). In
consideration of cytotoxicity, we also chose human normal hepatocyte
line (L02). The results were shown in Table 1.
could be potent for all the tested cancer cells. Therefore, D8 was a quite
potential one due to the exhibited IC50 values (1.48 μM for HeLa, 1.47
μ
M for MCF-7, 1.52 μM for HT29 and 1.94 μM for A549). It was also
According to the situation of TMP group, the compounds were
divided into three patches (as Ring A for D1-D18; as Ring B for D19-
D29; not complete for D30-D36) and compared with each other. A
general tendency was that when the TMP group moved from Ring A to
Ring B, the anti-proliferation effect became less potency. Further
removing one methoxyl from the TMP led to a sharp decrease of their
activity against the growth of all the studied cell lines. This result agreed
with the previous demonstration that the TMP group was significant.
Moreover, when Linker C was not symmetrical, the TMP group showed
preference to act as the specific one of the two aromatic rings (in this
series was Ring A). After all, we were looking for a candidate which
comparable (basically the disparity was within one order of magnitude)
with the positive controls Colchicine and CA-4P. On the other hand, the
safety of D8 was attractive because its cytotoxicity was much lower than
either Colchicine or CA-4P. As we known, tubulin-related anti-cancer
agents usually exhibited high cytotoxicity, thus the new trial at Linker C
brought new information for the topic of high potency and low toxicity.
Further experiments were conducted on the top hit D8. Besides, we
chose MCF-7 in the subsequent cell-related experiments because D8 was
the most potent on it while the controls were less potent.
3

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
Table1
Table1 (continued)
In vitro anti-proliferation inhibitory activity and cytotoxcity of compounds D1-
D36.
GI50
(
μM)
CC50
L02
(μM)
HeLa
MCF-7
HT29
A549
GI50
(μM)
CC50
(μM)
Colchicine
CA-4P
0.71 ±
0.14
2.67 ±
0.55
1.94 ±
0.52
0.05 ±
0.01
0.02 ±
0.003
0.15 ±
0.04
HeLa
MCF-7
HT29
A549
L02
0.10 ±
0.02
0.62 ±
0.05
0.09 ±
0.02
0.16 ±
0.03
D1
3.10 ±
0.15
5.53 ±
0.15
16.5 ±
0.23
28.8 ±
0.12
7.27 ±
0.14
D2
5.17 ±
0.23
3.95 ±
0.33
30.5 ±
0.14
>100
8.21 ±
0.34
2.2.2. The tubulin polymerization inhibition assay
D3
4.65 ±
0.12
6.05 ±
0.46
20.3 ±
0.26
49.2 ±
0.40
9.37 ±
0.25
The tubulin polymerization inhibitory performance of D8 was eval-
uated with a Tubulin Polymerzation Kit including bovine brain tubulin
and corresponding buffer. As shown in Table 2, the calculated IC50 value
D4
4.77 ±
0.57
5.94 ±
0.86
3.98 ±
0.06
32.3 ±
0.61
9.13 ±
0.58
D5
2.49 ±
0.34
5.29 ±
0.46
14.5 ±
0.67
54.6 ±
0.15
7.79 ±
0.05
of D8 was 1.20 μM, which was slightly better than CA-4 but not that
D6
1.15 ±
0.15
4.34 ±
0.24
13.3 ±
0.74
28.4 ±
0.11
0.80 ±
0.32
potent as Colchicine. Anyway, D8 was potential due to its low cyto-
toxicity and possible modification on the introduced Ring C (also as the
linker).
D7
2.17 ±
0.22
3.25 ±
0.34
2.74 ±
0.57
9.11 ±
0.51
7.81 ±
0.12
D8
1.48 ±
0.12
1.47 ±
0.03
1.52 ±
0.07
1.94 ±
0.15
77.5 ±
0.36
2.2.3. The flow cytometry assay
D9
1.73 ±
0.14
5.63 ±
0.12
2.53 ±
0.26
5.17 ±
0.25
8.09 ±
0.89
Commonly, tubulin-related anti-cancer agents could induce the cell
apoptosis by blocking the cell cycle at G2/M phase. Accordingly, we
conducted the flow cytometry assay to check the cell apoptosis induction
and cell cycle blocking of the hit compound D8. MCF-7 cells incubated
with D8 were analyzed with Annexin V-FITC/ Propidium Iodide (PI)
system. As shown Fig. 2A&B, with the incubation time set as 48 h, when
D10
D11
D12
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
D29
D30
D31
D32
D33
D34
D35
D36
7.21 ±
0.50
26.7 ±
0.11
>100
>100
1.65 ±
0.70
1.08 ±
0.45
1.49 ±
0.03
10.9 ±
0.35
22.6 ±
0.12
2.89 ±
0.39
4.66 ±
0.41
9.46 ±
0.31
13.3 ±
0.44
27.2 ±
0.66
3.42 ±
0.06
the concentration of D8 increased (0, 0.375, 0.75, 1.5, 3.0 μM), the
31.5 ±
0.13
15.1 ±
0.46
74.6 ±
0.50
64.5 ±
0.95
99.5 ±
0.46
percentage of apoptotic cells was significantly elevated from 8.34% to
39.5%. A majority of the cells indicated early apoptosis. Besides, as
2.50 ±
0.21
8.39 ±
0.11
77.3 ±
0.61
>100
2.78 ±
0.54
shown Fig. 2C&D, with the concentration of D8 set as 1.5 μM, when the
1.94 ±
0.11
6.41 ±
0.20
14.3 ±
0.33
13.9 ±
0.35
2.28 ±
0.05
incubation time prolonged (0, 24, 48, 72 h), the percentage of apoptotic
cells also elevated from 8.48% to 24.8%. Still, a majority of the cells
indicated early apoptosis. Thus, D8 could induce cell apoptosis in MCF-7
cells in both dose-dependent and time-dependent manners.
9.69 ±
0.61
15.2 ±
0.36
14.7 ±
0.31
76.5 ±
0.20
9.69 ±
0.069
0.82 ±
0.35
0.51 ±
0.13
3.92 ±
0.13
12.9 ±
0.31
31.2 ±
0.11
The cell cycle arrest situations were also checked. As shown
Fig. 3A&B, MCF-7 cells were treated with various concentrations of D8
39.1 ±
0.10
80.1 ±
0.10
>100
>100
9.54 ±
0.96
at 0, 0.375, 0.75, 1.5, 3.0 μM for 48 h, the percentages of cells in the G2/
9.21 ±
0.07
2.75 ±
0.20
14.6 ±
0.20
48.2 ±
0.13
8.26 ±
0.65
M phase were 4%, 14.3%, 20.8%, 27.1%, 31.4%, respectively. Besides,
10.7 ±
0.08
1.38 ±
0.32
21.4 ±
0.41
30.3 ±
0.12
4.03 ±
0.14
as shown Fig. 3C&D, MCF-7 cells were treated with 1.5 μM of D8 for 0,
24, 48, 72 h, the percentages of cells in the G2/M phase were 2.7%,
14.3%, 22.3%, 30.9%, respectively. Therefore, D8 could effectively
induce cell cycle arrest in the G2/M phase and block cell mitosis in both
dose-dependent and time-dependent manners.
8.16 ±
0.60
2.46 ±
0.63
19.9 ±
0.21
18.0 ±
0.64
2.36 ±
0.04
5.99 ±
0.55
6.03 ±
0.53
20.3 ±
0.70
14.8 ±
0.04
3.24 ±
0.19
7.42 ±
0.41
28.7 ±
0.97
16.6 ±
0.60
16.6 ±
0.11
10.0 ±
0.57
2.2.4. Affection on tubulin dynamics, angiogenesis and cell cycle
>100
>100
>100
>100
92.6 ±
0.07
We conducted Western Blot assays to analysis the affection of D8 on
tubulin dynamics, angiogenesis and cell cycle (Fig. 4). In Fig. 4A,
compared with the control check, Colchiche and Paclitaxel obviously
induced the de-polymerization and polymerization of tubulin dynamics,
while D8 tended to cause the de-polymerization. Moreover, since
tubulin was associated with the angiogenesis, which commonly used the
VEGF (Vascular Endothelial Growth Factor)-VEGFR (Vascular Endo-
thelial Growth Factor Receptor) pair as a common positively-related
marker [43,44], we evaluated the potential anti-vascular activity of
compound D8 by checking the expression level of this pair. In Fig. 4B,
with the increase of D8 concentration, the level of VEGF decreased
remarkably and that of VEGFR decreased as well. Thus, D8 indicated
potent anti-vascular activity. Further, since the flow cytometry assay
7.67 ±
0.36
38.0 ±
0.36
25.3 ±
0.12
40.7 ±
0.12
5.21 ±
0.61
7.37 ±
0.41
23.5 ±
0.67
36.1 ±
0.30
46.1 ±
0.96
4.02 ±
0.99
7.07 ±
0.16
23.0 ±
0.57
>100
4.34 ±
0.61
6.58 ±
0.125
11.6 ±
0.54
84.7 ±
0.83
52.7 ±
0.22
>100
>100
>100
26.9 ±
0.10
>100
>100
49.6 ±
0.06
18.7 ±
0.31
>100
>100
20.7 ±
0.16
74.2 ±
0.21
9.96 ±
0.136
8.68 ±
0.56
18.9 ±
0.11
20.9 ±
0.57
71.4 ±
0.20
24.0 ±
0.50
90.2 ±
0.16
26.5 ±
0.35
65.6 ±
0.19
62.9 ±
0.54
36.5 ±
0.11
>100
16.9 ±
0.13
71.8 ±
0.25
72.9 ±
0.04
Table 2
The tubulin polymerization inhibitory activity of D8 and the controls.
18.9 ±
0.10
>100
>100
>100
31.3 ±
0.12
61.3 ±
0.25
9.49 ±
0.29
Compound
D8
CA-4
Colchicine
Vinorelbine
Paclitaxel
65.8 ±
0.14
33.1 ±
0.45
85.4 ±
0.34
14.5 ±
0.61
a
IC50
(μ
M)
1.20 ±
1.9 ±
0.50 ± 0.03
31.5 ± 2.8
–
0.09
0.12
25.2 ±
0.59
65.0 ±
0.17
35.5 ±
0.36
27.0 ±
0.99
a
Paclitaxel induced the polymerzation thus could not be calculated in this
assay.
4

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
Fig. 2. D8 induced cell apoptosis in MCF-7 cells in (A&B) dose-dependent and (C&D) time-dependent manners. (A) The result of MCF-7 treated with different
concentrations of D8 (0, 0.375, 0.75, 1.5, 3.0 M) for 48 h; (C) The result of MCF-7 treated with 1.5 M D8 for different periods (0, 24, 48, 72 h); (B&D) The apoptotic
ratios of MCF-7. Data were shown as mean ± SD of three independent experiments; **p < 0.01, ***p < 0.005.
μ
μ
indicated the cell cycle arrest in the G2/M phase, we checked the level of
several corresponding proteins after the incubation of D8. In Fig. 4C,
when the concentration of D8 increased, CDK1/CDC2, Cyclin A and
Cyclin D1 all showed lower expression level. This result agreed with the
cell cycle arrest capability of D8.
of multinuclear giant cells via mitotic catastrophe.
2.3. Molecular docking
Calculated by AutoDock 4.2 (The Scripps Research Institute) and
visualized by Discovery Studio Visualizer 2016 (Accelrys, Inc. San
Diego, CA). When choosing the crystal structure of tubulin, we selected
the relatively high-resolution Tubulin-Colchicine complex (PDB Code:
4O2B) [45] instead of the typical one (PDB Code: 1SA0) after a careful
comparison. The 2D binding pattern of D8 into tubulin and the 3D maps
of D8, Colchicine, CA-4 were depicted in Fig. 6. As shown in Fig. 6A, D8
retained significant interactions with the key residues including Ala180,
Cys241, Leu248, Ala250, Asp251, Leu255, Met259, Ala316 and Lys352
in comparison with Colchicine and CA-4. When we looked into the 3D
maps in Fig. 6B, we found that the TMP groups were all deep into the
binding site, however, the other ring(s) could stretch within the gap of
tubulins. D8 seemed slightly longer than the controls, therefore seemed
more potential for introducing interactions with another chain of
tubulin (Ala180, Val181). Generally speaking, the results indicated that
the volume of the bulky benzothiazepine moiety did not beyond the
boundary of the binding site. Besides, this new trial introduced a new
Ring C, thus subsequently brought the possibility of interchanges be-
tween the aromatic rings except the TMP. Besides, Ring B presented
2.2.5. The biological imaging assay
To check whether the anti-cancer effect of D8 was related to tubulin
polymerization and mitotic catastrophe, we performed the biological
imaging assay. Basically, interfering the tubulin-microtubule dynamics
might led to visible variation in cell morphology. Here the Cy3-labeled
goat anti-mouse IgG (H + L) was used as the tubulin antibody. The
staining patterns with Cy3 and DAPI were visualized under fluorescence
microscopy. As shown in Fig. 5, the original microtubules were thin fi-
bers in regular arranged pattern. When treated with the controls, the
morphology of the microtubules changed obviously. After the incuba-
tion with Colchicine, the microtubule fibers broke down, became shorter
and sparser. On the contrary, after the treatment with Paclitaxel, the
microtubules exhibited obvious polymerization with longer and thicker
bundles. The patterns in D8-treated groups were similar to that of
Colchicine-treated one. At low dose (0.1
arrest in mitotic process, thus led to the fragmentary and multi-polar
spindles. At high dose (0.5 M), D8 could even induce the formation
μM), D8 could induce the cells
μ
5

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
Fig. 3. D8 blocked the cell cycle at G2/M phase in MCF-7 cells in (A&B) dose-dependent and (C&D) time-dependent manners. (A) The result of MCF-7 treated with
different concentrations of D8 (0, 0.375, 0.75, 1.5, 3.0 μM) for 48 h; (C) The result of MCF-7 treated with 1.5 μM D8 for different periods (0, 24, 48, 72 h); (B&D) The
percentage sof cells in G2/M phase. Data were shown as mean ± SD of three independent experiments; ***p < 0.005.
Fig. 4. The affection analysis of D8 on (A) tubulin dynamics, (B) angiogenesis and (C) cell cycle. CK: Control check; S: Single; P: Polymer.
potential
π
-alkyl interactions with Ala180, Leu248, Ala250, and espe-
patterns and the biological activity would be investigated in the near
future due to the meticulous planning and heavy workload. As a possible
solution to improve the biological activity in this work was the opti-
mization on the substitutes on the newly formed Ring C, which seemed
cially, the o-chloro interacted with Ala250, Lys254. The 3D map also
presented the close distance between Ring B and the corresponding
α
-helix. The deeper connection between the differences in binding
6

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
Fig. 5. The images of MCF-7 cells from a confocal fluorescence microscope under different conditions. (a-c) Control; (d-f) 0.1
μ
M Paclitaxel; (g-i) 0.1
μM Colchicine;
(j-l) 0.1 μM D8; (m-o) 0.5 μM D8. Tubulins were labeled by Cy3 (Red) and nucleus were tagged by DAPI (blue). Scale bar: 20 μm.
Fig. 6. (A) The 2D docking model of compound D8 into tubulin. (B) The 3D docking models of D8, Colchicine, CA-4 into tubulin. The H-bonds were displayed as
green dotted lines. The π-related interactions were shown as pink dotted lines. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
quite close to the key residues of tubulin.
catastrophe and the death of living cancer cells. Docking simulation
implied the binding pattern of introducing interactions with the nearby
tubulin chain. This work might stimulate new ideas in both structural
evolution and therapeutic approaches for tubulin-related anti-cancer
agents.
3. Conclusions
In summary, a series of diaryl benzo[b][1,4]thiazepine derivatives
D1-D36 were synthesized and screened as tubulin polymerization in-
hibitors with anti-tumor potency. The seven-member ring benzothia-
zepine was introduced as the linker for CA-4 modification for the first
time. The hit compound D8 showed potential on inhibiting the growth
4. Experimental section
4.1. 1. Chemistry
of several cancer cell lines (IC50 values: 1.48
MCF-7, 1.52 M for HT29 and 1.94 M for A549), being comparable
with the positive controls Colchicine and CA-4P. The calculated IC50
value of D8 as an tubulin polymerization inhibitor was 1.20 M. The
flow cytometry assay revealed that D8 could induce the mitotic
μM for HeLa, 1.47 μM for
μ
μ
4.1.1. General
All purchased chemicals were used directly without purification. 1H
NMR and 13C NMR spectra were recorded on a Bruker Avance II 600
spectrometer and resolved with MestreNova software. Mass spectra
μ
7

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
were recorded on an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS.
59.20, 56.46. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C24H23FNO3S
424.1383, Found 424.1378.
4.1.2. General method of synthesizing (E)-Chalcone analogues (C1-C36)
According to the design concept, corresponding substituted aromatic
ketone (1 mmol) was added to 5 mL ethanol solution of corresponding
substituted aromatic formaldehyde (1 mmol). Subsequently, 50% NaOH
(0.5 mL) was added to the mixture. Thin layer chromatography was used
to check the completion of the reaction. The intermediate chalcone
analogues (C1-C36) were acquired after being filtered, washed by cold
ethanol and dried.
2-(p-Tolyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzo[b]
[1,4]thiazepine (D5)
White powder, yielding 64.5%, melt point 127–128 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.55 (d, J = 7.7 Hz, 1H, ArH), 7.52 (t, J = 7.6 Hz,
1H, ArH), 7.36 (s, 2H, ArH), 7.27 (d, J = 7.9 Hz, 1H, ArH), 7.23 (d, J =
8.0 Hz, 2H, ArH), 7.19 (t, J = 7.5 Hz, 1H, ArH), 7.23 (d, J = 7.9 Hz , 2H,
– –
–
ArH), 5.27 (dd, J = 12.2, 4.9 Hz, 1H, CH2 ), 3.86 (s, 6H, ( OCH3)2),
–
– –
3.76 (s, 3H, OCH3), 3.43 (dd, J = 13.0, 4.9 Hz, 1H, CH2 ), 2.86 (t, J
= 12.8 Hz, 1H,
S
CH ), 2.28 (s, 3H, CH3). 13C NMR (151 MHz,
– – –
–
4.1.3. General method of synthesizing diaryl benzo[b][1,4]thiazepine
derivatives (D1-D36)
DMSO‑d6) δ 168.84, 153.34, 152.57, 141.67, 140.77, 137.29, 135.27,
132.86, 130.39, 129.51, 126.48, 125.67, 125.38, 122.69, 105.41, 60.62,
60.01, 56.42, 21.13. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for
The solution of appropriate chalcone analogues (C1-C36, 1 mmol)
dissolved in ethanol in reflux condition. An equal equivalent of 2-amino-
thiophenol was added to the hot solution, and then concentrated hy-
drochloric acid was added dropwise. The reaction was kept continuing
for 4 h. The crude product was filtered, washed with cold ethanol, dried,
and recrystallized to obtain the target compounds D1-D36. The detailed
data were as follows.
C
25H26NO3S 420.1634, Found 420.1627.
2-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D6)
Yellow powder, yielding 58.6%, melt point 127–129 ◦C. 1H NMR
(400 MHz, DMSO‑d6) δ 7.57–7.51 (m, 2H, ArH), 7.37 (s, 2H, ArH),
7.30–7.26 (m, 3H, ArH), 7.20 (t, J = 7.6 Hz, 1H, ArH), 6.88 (d, J = 8.8
– –
Hz, 2H, ArH), 5.23 (dd, J = 12.2, 4.7 Hz, 1H, CH2 ), 3.86 (s, 6H,
2-Phenyl-4-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzo[b]
[1,4]thiazepine (D1)
–
– –
(
OCH3)2), 3.76 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.45 (d, J =
Light yellow powder, yielding 63.1%, melt point 149–151 ◦C. 1H
NMR (400 MHz, DMSO‑d6) δ 7.57 (d, J = 6.5 Hz, 1H, ArH), 7.53 (t, J =
7.6 Hz, 1H, ArH), 7.38 (s, 2H, ArH), 7.37–7.31 (m, 4H, ArH), 7.27 (d, J
= 7.8 Hz, 2H, ArH), 7.19 (t, J = 7.5 Hz, 1H, ArH), 5.25 (dd, J = 12.4, 4.8
13.9 Hz, 1H, CH2 ), 2.86 (t, J = 12.7 Hz, 1H,
CH ). 13C NMR
– – –
S
– –
(101 MHz, DMSO‑d6) δ 168.87, 153.37, 152.62, 141.65, 140.82, 137.23,
135.32, 132.83, 130.43, 129.47, 126.53, 125.62, 125.42, 122.74,
105.47, 60.62, 60.06, 56.46, 55.81. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C25H26NO4S 436.1583, Found 436.1579.
– –
–
–
Hz, 1H, CH2 ), 3.87 (s, 6H, –( OC–H3)2), 3.76 (s, 3H, OCH3), 3.49
S
(dd, J = 13.1, 4.9 Hz, 1H,
CH2 ), 2.88 (t, J = 12.8 Hz, 1H,
2-(3-Fluorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D7)
CH ). 13C NMR (101 MHz, DMSO‑d6) δ 168.82, 153.34, 152.62,
– – –
144.62, 140.75, 135.27, 132.82, 130.44, 129.03, 128.02, 126.58,
125.68, 125.39, 122.57, 105.39, 60.61, 60.13, 56.43. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C24H24NO3S 406.1477, Found 404.1465.
2-(4-Chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D2)
White powder, yielding 53.4%, melt point 130–132 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.59–7.55 (m, 2H, ArH), 7.43 (s, 2H, ArH), 7.38
(q, J = 7.9 Hz, 1H, ArH), 7.33 (d, J = 7.7 Hz, 1H, ArH), 7.23 (t, J = 7.4
Hz, 1H, ArH), 7.21 (d, J = 7.9 Hz, 1H, ArH), 7.17 (d, J = 10.1 Hz, 1H,
ArH), 7.12 (t, J = 8.5 Hz, 1H, ArH), 5.30 (dd, J = 12.5, 4.9 Hz, 1H,
Light yellow powder, yielding 68.4%, melt point 165–166 ◦C. 1H
NMR (400 MHz, DMSO‑d6) δ 7.56–7.52 (m, 2H, ArH), 7.42–7.36 (m, 6H,
ArH), 7.28 (d, J = 7.9 Hz, 1H, ArH), 7.19 (t, J = 7.5 Hz, 1H, ArH), 5.29
CH2 ), 3.88 (s, 6H, ( OCH3)2), 3.77 (s, 3H, OCH3), 3.58 (dd, J =
– –
–
– –
–
13.0, 4.7 Hz, 1H, CH2 ), 2.90 (t, J = 12.9 Hz, 1H,
CH ). 13C
– – –
S
NMR (151 MHz, DMSO‑d6) δ 188.24, 153.41, 153.37, 142.90, 142.60,
133.21, 131.27, 130.68, 127.32, 126.37, 126.21, 125.60, 123.73,
123.56, 122.97, 115.27, 106.75, 60.67, 56.73, 56.55. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C24H23FNO3S 424.1383, Found 424.1378.
2-(2-Chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D8)
– –
–
(dd, J = 12.4, 4.8 Hz, 1H, CH2 ), 3.87 (s, 6H, ( OCH3)2), 3.76 (s, 3H,
–
– –
OCH3), 3.49 (dd, J = 13.1, 4.9 Hz, 1H, CH2 ), 2.84 (t, J = 12.8 Hz,
1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 168.73, 153.35,
– – –
152.61, 143.51, 140.82, 135.28, 132.76, 132.44, 130.56, 128.96,
128.51, 125.74, 125.43, 122.19, 105.46, 60.62, 59.13, 56.46. HRMS
(ESI-TOF) m/z: [M + H]+ Calcd. for C24H23ClNO3S 440.1087, Found
440.1084.
Grey white powder, yielding 56.7%, melt point 139–140 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.65 (d, J = 7.68 Hz, 1H, ArH), 7.63 (d, J = 7.68
Hz, 1H, ArH), 7.55 (t, J = 7.7 Hz, 1H, ArH), 7.50 (d, J = 7.7 Hz, 1H,
ArH), 7.46 (s, 2H, ArH), 7.38–7.32 (m, 2H, ArH), 7.30 (d, J = 7.9 Hz, 1H,
ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 5.48 (dd, J = 13.0, 4.6 Hz, 1H,
2-(4-Bromophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D3)
Light yellow powder, yielding 69.3%, melt point 179–181 ◦C. 1H
NMR (600 MHz, DMSO‑d6) δ 7.55–7.52 (m, 4H, ArH), 7.38 (s, 2H, ArH),
7.31 (d, J = 8.5 Hz, 2H, ArH), 7.27 (d, J = 7.4 Hz, 1H, ArH), 7.19 (t, J =
– –
–
–
CH2 ), 3.88 (s, 6H, ( OCH3)2), 3.77 (s, 3H, OCH3), 3.55 (dd, J =
– –
13.1, 4.6 Hz, 1H, CH2 ), 2.77 (t, J = 13.1 Hz, 1H,
CH ). 13C
– – –
S
– –
7.5 Hz, 1H, ArH), 5.27 (dd, J = 12.4, 4.9 Hz, 1H, CH2 ), 3.87 (s, 6H,
NMR (151 MHz, DMSO‑d6) δ 168.28, 153.46, 152.67, 141.27, 140.90,
135.39, 132.58, 130.99, 130.70, 129.87, 129.77, 128.36, 128.15,
125.89, 125.65, 122.05, 105.24, 60.65, 60.23, 56.47. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C24H23ClNO3S 440.1087, Found 440.1082.
2-(3-Chlorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D9)
–
–
(
OCH3)2), 3.76 (s, 3H, OCH3), 3.48 (dd, J = 13.2, 4.9 Hz, 1H,
– –
CH2 ), 2.85 (t, J = 12.8 Hz, 1H,
CH ). 13C NMR (101 MHz,
– – –
S
DMSO‑d6) δ 168.72, 153.35, 152.59, 143.88, 140.83, 135.27, 132.75,
131.88, 130.56, 128.85, 125.73, 125.42, 122.19, 120.95, 105.46, 60.62,
59.18, 56.46. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C24H23BrNO3S
484.0582, Found 482.0586.
White powder, yielding 52.4%, melt point 132–134 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.58–7.57 (m, 2H, ArH), 7.45 (s, 2H, ArH), 7.41
(s, 1H, ArH), 7.37–7.33 (m, 3H, ArH), 7.31 (d, J = 7.4 Hz, 1H, ArH), 7.25
2-(4-Fluorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D4)
White powder, yielding 60.2%, melt point 138–140 ◦C. 1H NMR
(400 MHz, DMSO‑d6) δ 7.54 (dd, J = 15.3, 7.7 Hz, 2H, ArH), 7.42–7.36
(m, 4H, ArH), 7.28 (d, J = 7.8 Hz, 1H, ArH), 7.22–7.14 (m, 3H, ArH),
(t, J = 7.6 Hz, 1H, ArH), 5.31 (dd, J = 12.3, 4.9 Hz, 1H, CH2 ), 3.88
– –
–
–
(s, 6H, ( OCH3)2), 3.77 (s, 3H, OCH3), 3.61 (dd, J = 13.1, 5.0 Hz, 1H,
CH2 ), 2.92 (t, J = 12.8 Hz, 1H,
CH ). 13C NMR (151 MHz,
– – –
S
– –
– –
–
5.29 (dd, J = 12.4, 4.8 Hz, 1H, CH2
DMSO‑d6) δ 188.48, 158.65, 153.37, 142.33, 138.67, 133.59, 132.79,
130.61, 128.83, 127.12, 125.89, 123.44, 123.19, 122.73, 122.07,
121.09, 106.51, 60.65, 60.18, 56.63. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C24H23ClNO3S 440.1087, Found 440.1081.
–
), 3.87 (s,–6H, (–OCH ) ), 3.76
3 2
(s, 3H, OCH3), 3.49 (dd, J = 13.0, 4.9 Hz, 1H, CH2 ), 2.84 (t, J =
– – –
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 168.79,
12.8 Hz, 1H,
162.68, 161.06, 153.36, 152.56, 140.83, 135.28, 132.77, 130.51,
128.64, 128.58, 125.74, 125.44, 122.35, 115.81, 115.67, 105.46, 60.62,
2-(2-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-
8

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
dihydrobenzo[b][1,4]thiazepine (D10)
(600 MHz, DMSO‑d6) δ 7.68 (d, J = 2.2 Hz, 1H, ArH), 7.62 (d, J = 7.7 Hz,
1H, ArH), 7.60 (d, J = 8.5 Hz, 1H, ArH), 7.55 (t, J = 7.8 Hz, 1H, ArH),
7.46–7.45 (m, 3H, ArH), 7.31 (d, J = 7.9 Hz, 1H, ArH), 7.18 (t, J = 7.4
Yellow powder, yielding 58.8%, melt point 144–146 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.64 (d, J = 7.7 Hz, 1H, ArH), 7.54 (t, J = 7.7 Hz,
1H, ArH), 7.48 (s, 2H, ArH), 7.43 (d, J = 7.6 Hz, 1H, ArH), 7.35 (d, J =
7.8 Hz, 1H, ArH), 7.29–7.22 (m, 2H, ArH), 7.04 (d, J = 7.9 Hz, 1H, ArH),
– –
Hz, 1H, ArH), 5.43 (dd, J = 12.9, 4.6 Hz, 1H, CH2 ), 3.87 (s, 6H,
–
–
(
OCH3)2), 3.77 (s, 3H, OCH3), 3.57 (dd, J = 13.1, 4.6 Hz, 1H,
CH ). 13C NMR (151 MHz,
– – –
CH2 ), 2.78 (t, J = 13.1 Hz, 1H, S
– –
– –
6.94–6.92 (m, 1H, ArH), 5.44 (dd, J = 12.8, 4.5 Hz, 1H, CH2 ), 3.89
–
–
–
(s, 6H, ( OCH3)2), 3.81 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.53 (dd,
DMSO‑d6) δ 168.81, 153.43, 152.61, 149.99, 142.75, 137.61, 136.05,
135.62, 132.98, 131.89, 130.44, 129.97, 128.67, 128.36, 127.33,
126.49, 126.07, 125.69, 106.85, 60.69, 56.73. HRMS (ESI-TOF) m/z:
[M + H]+ Calcd. for C24H22Cl2NO3S 474.0698, Found 474.0693.
2-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D16)
J = 12.9, 4.4 Hz, 1H, CH2 ), 2.81 (t, J = 12.9 Hz, 1H,
CH ). 13C
– – –
S
– –
NMR (151 MHz, DMSO‑d6) δ 167.58, 153.93, 153.86, 141.02, 140.69,
135.02, 133.48, 131.07, 130.61, 128.77, 127.13, 125.89, 123.20,
122.73, 120.45, 112.85, 104.90, 60.68, 60.22, 56.59, 56.13. HRMS (ESI-
TOF) m/z: [M + H]+ Calcd. for C25H26NO4S 436.1583, Found 436.1579.
2-(4-(Methylthio)phenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D11)
Brown powder, yielding 65.4%, melt point 113–114 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.62 (d, J = 6.8 Hz, 1H, ArH), 7.56–7.54 (m, 1H,
ArH), 7.40 (s, 2H, ArH), 7.35 (d, J = 7.9 Hz, 1H, ArH), 7.25–7.23 (m, 1H,
ArH), 6.93 (s, 1H, ArH), 6.87 (s, 2H, ArH), 5.23 (dd, J = 11.5, 5.0 Hz,
White powder, yielding 57.5%, melt point 153–154 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.13 (d, J = 8.6 Hz, 2H, ArH), 7.65 (d, J = 7.7 Hz,
1H, ArH), 7.58 (t, J = 7.7 Hz, 1H, ArH), 7.42 (d, J = 8.6 Hz, 2H, ArH),
7.31 (d, J = 8.0 Hz, 1H, ArH), 7.28 (t, J = 7.6 Hz, 1H, ArH), 6.66 (s, 2H,
– –
–
–
1H, CH2 ), 3.86 (s, 6H, ( OCH3)2), 3.77 (s, 3H, OCH3), 3.73 (s, 3H,
–
–
– –
OCH3), 3.64 (s, 3H, OCH3), 3.49 (dd, J = 13.1, 4.8 Hz, 1H, CH2 ),
ArH), 5.13 (dd, J = 11.9, 5.2 Hz, 1H, CH2 ), 3.68 (s, 6H, ( OCH3)2),
CH ). 13C NMR (151 MHz, DMSO‑d6) δ
– – –
2.93 (t, J = 12.4 Hz, 1H, S
– –
–
– –
–
3.64 (s, 3H, OCH3), 3.55 (dd, J = 12.9, 4.4 Hz, 1H, CH2 ), 2.83 (t, J
167.72, 153.23, 151.72, 148.71, 144.84, 134.74, 133.80, 128.01,
127.61, 127.37, 127.01, 125.96, 124.00, 123.41, 122.93, 122.67,
121.40, 120.15, 106.66, 60.66, 56.62, 56.09. HRMS (ESI-TOF) m/z: [M
+ H]+ Calcd. for C26H28NO5S 466.1688, Found 466.1682.
= 12.9 Hz, 1H,
S
CH ), 2.55 (s, 3H, -SCH3). 13C NMR (151 MHz,
– – –
DMSO‑d6) δ 168.78, 153.38, 151.67, 146.64, 139.89, 134.89, 133.38,
131.34, 130.82, 129.26, 127.78, 125.89, 122.82, 119.15, 118.63,
117.59, 106.32, 60.64, 56.64, 56.60, 14.84. HRMS (ESI-TOF) m/z: [M +
H]+ Calcd. for C25H26NO3S2 452.1354, Found 450.1348.
2-(Naphthalen-2-yl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D17)
2-(4-(Trifluoromethyl)phenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-
dihydrobenzo[b][1,4]thiazepine (D12)
White powder, yielding 59.7%, melt point 133–135 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.89 (d, J = 8.2 Hz, 2H, ArH), 7.85 (s, 2H, ArH),
7.58 (d, J = 7.6 Hz, 1H, ArH), 7.55 (t, J = 7.7 Hz, 1H, ArH), 7.52–7.48
Light yellow powder, yielding 60.4%, melt point 168–169 ◦C. 1H
NMR (600 MHz, DMSO‑d6) δ 7.71 (d, J = 8.2 Hz, 2H, ArH), 7.58–7.55
(m, 4H, ArH), 7.43 (s, 2H, ArH), 7.34 (d, J = 7.7 Hz, 1H, ArH), 7.24–7.22
(m, 3H, ArH), 7.41 (s, 2H, ArH), 7.31 (d, J = 7.8 Hz, 1–H, ArH–), 7.21 (t, J
= 7.6 Hz, 1H, ArH), 5.42 (dd, J = 12.2, 4.8 Hz, 1H, CH2 ), 3.86 (s,
– –
– –
6H, ( OCH3)2), 3.76 (s, 3H, OCH3), 3.59 (dd, J = 13.1, 4.9 Hz, 1H,
(m, 1H, ArH), 5.39 (dd, J = 12.5, 4.8 Hz, 1H, CH2 ), 3.88 (s, 6H,
(
OCH3)2), 3.77 (s, 3H, OCH3), 3.61 (d, J = 13.1, 1H, CH2 ), 2.93
CH ). 13C NMR (151 MHz,
– – –
CH2 ), 3.04 (t, J = 12.8 Hz, 1H, S
–
–
– –
– –
(t, J = 12.9 Hz, 1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ
DMSO‑d6) δ 169.03, 153.36, 152.52, 141.68, 140.85, 135.34, 133.14,
132.88, 132.83, 130.50, 128.80, 128.32, 127.95, 126.86, 126.54,
125.76, 125.46, 125.27, 124.63, 122.60, 105.53, 60.62, 60.27, 56.48.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C28H26NO3S 456.1634,
Found 456.1628.
– – –
168.85, 153.44, 152.65, 142.69, 142.33, 139.23, 135.26, 133.12,
130.02, 128.45, 127.45, 126.85, 126.60, 123.77, 123.08, 106.82, 60.68,
60.56, 56.73. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C25H23F3NO3S
474.1351, Found 474.1346.
2-(Furan-2-yl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzo
[b][1,4]thiazepine (D13)
2-(4-(Benzyloxy)phenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D18)
Grey white powder, yielding 56.7%, melt point 135–137 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.61 (s, 1H, ArH), 7.55–7.52 (m, 1H, ArH), 7.47
(d, J = 7.6 Hz, 1H, ArH), 7.45 (s, 2H, ArH), 7.33 (d, J = 7.9 Hz, 1H, ArH),
7.19 (t, J = 7.6 Hz, 1H, ArH), 6.39–6.38 (m, 1H, ArH), 6.25 (d, J = 3.2
White powder, yielding 58.6%, melt point 126–128 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.57 (d, J = 7.6 Hz, 1H, ArH), 7.55 (t, J = 7.7 Hz,
1H, ArH), 7.44–7.38 (m, 6H, ArH), 7.33 (d, J = 8.8 Hz, 2H, ArH), 7.26
(d, J = 8.7 Hz, 2H, ArH), 7.23 (t, J = 7.6 Hz, 1H, ArH), 6.96 (d, J = 8.8
– –
– –
Hz, 2H, ArH), 5.23 (dd, J = 12.2, 4.9 Hz, 1H, CH2 ), 5.10 (s, 2H,
Hz, 1H, ArH), 5.34 (dd, J = 12.4, 5.2 Hz, 1H, CH2 ), 3.88 (s, 6H,
–
–
– – –
CH2), 3.87 (s, 6H, ( OCH3)2), 3.77 (s, 3H, OCH3), 3.51 (dd, J =
(
OCH3)2), 3.77 (s, 3H, OCH3), 3.61 (dd, J = 13.0, 5.0 Hz, 1H,
CH2 ), 2.89 (t, J = 12.8 Hz, 1H,
S
CH ). 13C NMR (151 MHz,
CH ). 13C
– – –
13.1, 4.8 Hz, 1H, CH2 ), 2.87 (t, J = 12.7 Hz, 1H, S
– –
– – –
– –
DMSO‑d6) δ 167.81, 153.43, 153.38, 153.27, 144.43, 142.49, 141.47,
135.47, 133.13, 132.86, 127.96, 126.90, 125.26, 124.70, 122.62,
106.71, 106.17, 60.68, 56.59. HRMS (ESI-TOF) m/z: [M + H]+ Calcd.
for C22H22NO4S 396.1270, Found 396.1263.
NMR (151 MHz, DMSO‑d6) δ 188.19, 160.87, 153.37, 144.25, 142.26,
137.16, 133.72, 131.36, 128.94, 128.43, 128.27, 128.04, 119.93,
115.65, 106.50, 69.86, 60.64, 56.64. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C31H30NO4S 512.1895, Found 512.1890.
2-(Thiophen-2-yl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D14)
4-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D19)
Light yellow powder, yielding 62.5%, melt point 125–126 ◦C. 1H
NMR (600 MHz, DMSO‑d6) δ 7.54–7.51 (m, 2H, ArH), 7.41 (d, J = 5.0
Hz, 1H, ArH), 7.37 (s, 2H, ArH), 7.26 (d, J = 8.1 Hz, 1H, ArH), 7.18 (t, J
= 7.6 Hz, 1H, ArH), 7.05 (d, J = 3.3 Hz, 1H, ArH), 6.97–6.95 (m, 1H,
White powder, yielding 66.6%, melt point 136–137 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.14 (d, J = 8.2 Hz, 2H, ArH), 7.65 (d, J = 7.6 Hz,
1H, ArH), 7.60 (d, J = 8.0 Hz, 2H, ArH), 7.54 (t, J = 7.6 Hz, 1H, ArH),
7.28 (d, J = 7.8 Hz, 1H, ArH), 7.22 (t, J = 7.5 Hz, 1H, ArH), 6.67 (s, 2H,
– –
–
– –
– – –
ArH), 5.17 (dd, J = 11.8, 4.9 Hz, 1H, CH2 ), 3.69 (s, 6H, ( OCH3)2),
ArH), 5.60 (dd, J = 11.9, 5.3 Hz, 1H, CH2 ), 3.87 (s, 6H, ( OCH3)2),
–
– – –
3.63 (s, 3H, OCH3), 3.37 (dd, J = 13.2, 5.0 Hz, 1H, CH2 ), 2.90 (t, J
3.76 (s, 3H, OCH3), 3.62 (dd, J = 13.1, 5.3 Hz, 1H, CH2 ), 2.76 (t, J
= 13.4 Hz, 1H,
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 168.41,
= 12.6 Hz, 1H,
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 168.95,
– – –
S
– – –
S
153.36, 153.22, 149.28, 140.83, 137.13, 133.71, 132.73, 131.38,
129.36, 128.90, 128.44, 128.26, 128.22, 128.07, 108.11, 106.50, 60.67,
56.65. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C22H22NO3S2
412.1041, Found 412.1035.
153.12, 152.19, 140.00, 137.25, 136.72, 135.43, 132.17, 130.64,
130.13, 126.03, 125.60, 125.40, 122.95, 103.95, 60.44, 56.47, 56.17.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C24H23ClNO3S 440.1087,
Found 440.1080.
2-(2,4-Dichlorophenyl)-4-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D15)
4-(4-Bromophenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D20)
Yellow powder, yielding 63.1%, melt point 116–118 ◦C. 1H NMR
White powder, yielding 58.2%, melt point 148–150 ◦C. 1H NMR
9

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
– –
ArH), 5.10 (dd, J = 11.9, 5.1 Hz, 1H, CH2 ), 4.13 (q, J = 7.0 Hz, 2H,
(600 MHz, DMSO‑d6) δ 8.06 (d, J = 8.6 Hz, 2H, ArH), 7.74 (d, J = 8.6 Hz,
2H, ArH), 7.65 (d, J = 7.6 Hz, 1H, ArH), 7.54 (t, J = 7.7 Hz, 1H, ArH),
7.28 (d, J = 6.8 Hz, 1H, ArH), 7.22 (t, J = 7.5 Hz, 1H, ArH), 6.67 (s, 2H,
–
–
–
CH2), 3.69 (s, 6H, ( OCH3)2), 3.63 (s, 3H, OCH3), 3.43 (dd, J =
– –
– – –
S CH ), 1.37 (t,
13.2, 5.1 Hz, 1H, CH2 ), 2.92 (t, J = 12.6 Hz, 1H,
– –
–
–
ArH), 5.17 (dd, J = 11.9, 5.2 Hz, 1H, CH2 ), 3.69 (s, 6H, ( OCH3)2),
J = 7.0 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO‑d6) δ 168.53,
161.50, 153.12, 152.99, 140.27, 137.20, 135.33, 130.52, 130.06,
129.83, 125.31, 125.28, 122.80, 114.87, 103.91, 63.84, 60.43, 56.16,
15.03. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C26H28NO4S
450.1739, Found 450.1732.
–
– –
3.63 (s, 3H, OCH3), 3.36 (dd, J = 13.2, 5.2 Hz, 1H, CH2 ), 2.89 (t, J
– – –
= 12.4 Hz, 1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 168.95,
153.12, 151.97, 140.04, 137.24, 135.44, 133.95, 133.93, 130.82,
130.76, 130.64, 126.03, 125.43, 123.03, 116.23, 116.08, 103.94, 60.44,
60.31, 56.17. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C24H23BrNO3S
484.0582, Found 484.0576.
4-(4-Nitrophenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D26)
4-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D21)
Yellow powder, yielding 53.6%, melt point 164–165 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.38–8.35 (m, 4H, ArH), 7.68 (d, J = 8.6 Hz, 1H,
ArH), 7.57 (t, J = 7.7 Hz, 1H, ArH), 7.33 (d, J = 8.7 Hz, 1H, ArH),
7.26–7.24 (m, 1H, ArH), 6.68 (s, 2H, ArH), 5.23 (dd, J = 12.1, 5.2 Hz,
White powder, yielding 63.6%, melt point 125–126 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.21–8.18 (m, 2H, ArH), 7.66 (d, J = 7.6 Hz, 1H,
ArH), 7.56–7.53 (m, 1H, ArH), 7.37 (t, J = 8.8 Hz, 2H, ArH), 7.29 (d, J =
7.7 Hz, 1H, ArH), 7.22 (d, J = 7.5 Hz, 1H, ArH), 6.67 (s, 2H, ArH), 5.16
– –
–
–
1H, CH2 ), 3.70 (s, 6H, ( OCH3)2), 3.63 (s, 3H, OCH3), 3.50 (dd, J
CH ). 13C
– – –
S
– –
= 12.9, 4.9 Hz, 1H, CH2 ), 2.95 (t, J = 12.5 Hz, 1H,
– –
–
– –
(dd, J = 11.9, 5.2 Hz, 1H, CH2 ), 3.69 (s, 6H, ( OCH3)2), 3.63 (s, 3H,
NMR (151 MHz, DMSO‑d6) δ 164.76, 153.59, 153.11, 152.06, 144.72,
139.97, 137.25, 135.36, 132.61, 131.48, 130.60, 128.82, 125.76,
125.52, 123.38, 121.50, 104.06, 60.45, 56.60, 56.16. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C24H23N2O5S 451.1327, Found 451.1321.
4-(Thiophen-2-yl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
–
OCH3), 3.39 (dd, J = 13.2, 5.2 Hz, 1H, CH2 ), 2.90 (t, J = 12.6 Hz,
1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) 167.58, 153.58, 152.88,
– – –
145.54, 140.34, 138.51, 136.78, 131.67, 130.94, 129.34, 129.15,
125.90, 121.36, 120.43, 107.15, 60.61, 56.62, 56.60. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C24H23FNO3S 424.1383, Found 424.1386.
4-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihydrobenzo[b]
[1,4]thiazepine (D22)
drobenzo[b][1,4]thiazepine (D27)
Yellow powder, yielding 58.6%, melt point 136–137 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.85 (d, J = 5.0 Hz, 1H, ArH), 7.81 (d, J = 3.4 Hz,
1H, ArH), 7.65 (d, J = 8.5 Hz, 1H, ArH), 7.51 (t, J = 7.6 Hz, 1H, ArH),
7.23 (dd, J = 10.0, 3.7 Hz, 2H, ArH), 7.19 (t, J = 7.5 Hz, 1H, ArH), 6.70
White powder, yielding 59.3%, melt point 139–141 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.04 (d, J = 8.2 Hz, 2H, ArH), 7.65 (d, J = 8.8 Hz,
1H, ArH), 7.56–7.53 (m, 1H, ArH), 7.36 (d, J = 8.1 Hz, 2H, ArH), 7.30
(d, J = 7.7 Hz, 1H, ArH), 7.22 (d, J = 7.6 Hz, 1H, ArH), 6.66 (s, 2H, ArH),
– –
(s, 2H, ArH), 5.16 (dd, J = 11.5, 5.3 Hz, 1H, CH2 ), 3.70 (s, 6H,
–
–
(
OCH3)2), 3.63 (s, 3H, OCH3), 3.38 (dd, J = 13.1, 5.1 Hz, 1H,
– –
–
– –
5.14 (dd, J = 11.9, 5.2 Hz, 1H, CH2
CH ). 13C NMR (151 MHz,
– – –
CH2 ), 2.88 (t, J = 12.2 Hz, 1H, S
–
), 3.68 (s,–6H, (–OCH ) ), 3.63
3 2
(s, 3H, OCH3), 3.39 (dd, J = 13.1, 5.2 Hz, 1H, CH2 ), 2.91 (t, J =
DMSO‑d6) δ 164.76, 153.11, 152.06, 144.71, 139.97, 137.25, 135.36,
132.62, 131.48, 130.60, 128.82, 125.76, 125.52, 123.38, 104.06, 60.45,
56.60, 56.16. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C22H22NO3S2
412.1041, Found 412.1038.
– – –
–
12.6 Hz, 1H,
S
CH ), 2.41 (s, 3H, CH3). 13C NMR (151 MHz,
DMSO‑d6) δ 168.81, 153.13, 152.89, 139.87, 138.70, 137.29, 135.53,
130.71, 130.17, 129.94, 129.68, 128.79, 127.60, 125.68, 123.77,
120.60, 103.96, 60.44, 56.17, 21.58. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C25H26NO3S 420.1634, Found 420.1626.
4-(3,4-Dimethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D28)
4-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D23)
Yellow powder, yielding 57.5%, melt point 189–190 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.70 (d, J = 2.0 Hz, 1H, ArH), 7.66 (d, J = 8.5 Hz,
1H, ArH), 7.63 (d, J = 7.6 Hz, 1H, ArH), 7.52 (t, J = 7.6 Hz, 1H, ArH),
7.26 (d, J = 7.9 Hz, 1H, ArH), 7.52 (t, J = 7.5 Hz, 1H, ArH), 7.08 (d, J =
8.5 Hz, 1H, ArH), 6.67 (s, 2H, ArH), 5.11 (dd, J = 11.8, 5.1 Hz, 1H,
White powder, yielding 57.1%, melt point 124–126 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.21 (s, 2H, ArH), 7.69 (d, J = 7.6 Hz, 1H, ArH),
7.59 (t, J = 7.6 Hz, 1H, ArH), 7.41 (s, 1H, ArH), 7.30 (s, 1H, ArH), 7.14
(d, J = 8.8 Hz, 2H, ArH), 6.66 (s, 2H, ArH), 5.16 (dd, J = 11.8, 5.0 Hz,
– –
–
–
CH2 ), 3.86 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.69 (s, 6H,
– –
–
–
– –
– –
( OCH3)2), 3.63 (s, 3H, OCH3), 3.33 (dd, J = 13.0, 5.2 Hz, 1H,
1H, CH2 ), 3.89 (s, 3H, OCH3), 3.69 (s, 6H, ( OCH3)2), 3.63 (s, 3H,
–
1H,
– –
OCH3), 3.52 (dd, J = 13.2, 5.1 Hz, 1H, CH2 ), 2.97 (t, J = 12.5 Hz,
CH ). 13C NMR (151 MHz,
– – –
CH2 ), 2.86 (t, J = 12.6 Hz, 1H, S
S
CH ). 13C NMR (151 MHz, DMSO‑d6) 168.27, 153.15, 152.16,
DMSO‑d6) δ 168.67, 153.12, 152.84, 152.20, 149.26, 140.17, 137.21,
135.31, 130.52, 130.14, 125.40, 125.33, 122.86, 122.06, 111.55,
110.18, 103.95, 60.53, 60.43, 56.16, 55.91. HRMS (ESI-TOF) m/z: [M +
H]+ Calcd. for C26H28NO5S 466.1688, Found 466.1682.
– – –
149.29, 143.37, 139.95, 137.30, 135.50, 130.73, 129.35, 126.42,
125.58, 124.24, 122.88, 103.93, 60.54, 60.44, 56.50, 56.19. HRMS (ESI-
TOF) m/z: [M + H]+ Calcd. for C25H26NO4S 436.1583, Found 436.1579.
4-(4-(Methylthio)phenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D24)
4-(4-(Benzyloxy)phenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D29)
Light yellow powder, yielding 61.5%, melt point 141–143 ◦C. 1H
NMR (600 MHz, DMSO‑d6) δ 8.10 (d, J = 8.6 Hz, 2H, ArH), 7.67 (d, J =
7.7 Hz, 1H, ArH), 7.56 (t, J = 7.6 Hz, 1H, ArH), 7.41 (d, J = 8.6 Hz, 2H,
ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.24 (t, J = 7.6 Hz, 1H, ArH), 6.66
Yellow powder, yielding 62.3%, melt point 116–117 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.19 (d, J = 8.7 Hz, 2H, ArH), 7.68 (d, J = 7.7 Hz,
1H, ArH), 7.59–7.57 (m, 1H, ArH), 7.50 (d, J = 7.3 Hz, 2H, ArH), 7.43 (t,
J = 7.4 Hz, 2H, ArH), 7.40–7.35 (m, 2H, ArH), 7.28 (t, J = 7.4 Hz, 1H,
ArH), 7.20 (d, J = 8.9 Hz, 2H, ArH), 6.66 (s, 2H, ArH), 5.27 (s, 2H,
– –
(s, 2H, ArH), 5.15 (dd, J = 11.9, 5.2 Hz, 1H, CH2 ), 3.69 (s, 6H,
–
–
– –
–
– –
(
OCH3)2), 3.63 (s, 3H, OCH3), 3.46–3.43 (m, 1H, CH2 ), 2.95 (t,
CH2), 5.16 (dd, J = 11.9, 5.2 Hz, 1H, CH2 ), 3.68 (s, 6H,
J = 12.8 Hz, 1H,
CH ), 2.57 (s, 3H, -SCH3). 13C NMR (151 MHz,
(
OCH3)2), 3.63 (s, 3H, OCH3), 3.35 (dd, J = 13.0, 5.1 Hz, 1H,
– – –
S
–
–
– –
DMSO‑d6) 170.77, 153.14, 152.88, 139.87, 137.30, 135.53, 130.71,
130.52, 129.15, 126.60, 125.76, 125.69, 123.75, 108.25, 103.97, 60.44,
60.24, 56.18, 14.62. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for
CH2 ), 2.88 (t, J = 12.3 Hz, 1H,
CH ). 13C NMR (151 MHz,
– – –
S
DMSO‑d6) δ 162.72, 161.50, 153.14, 152.88, 144.15, 137.36, 137.07,
136.93, 132.27, 131.41, 129.02, 128.99, 128.53, 128.51, 128.33,
128.31, 128.23, 115.36, 104.00, 70.01, 60.45, 56.60. HRMS (ESI-TOF)
m/z: [M + H]+ Calcd. for C31H30NO4S 512.1895, Found 512.1890.
2-(4-Chlorophenyl)-4-(3,4-dimethoxyphenyl)-2,3-dihy-
C
25H26NO3S2 452.1354, Found 452.1346.
4-(4-Ethoxyphenyl)-2-(3,4,5-trimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D25)
Yellow powder, yielding 61.3%, melt point 139–140 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 8.06 (d, J = 8.9 Hz, 2H, ArH), 7.62 (d, J = 8.8 Hz,
1H, ArH), 7.51 (t, J = 8.3 Hz, 1H, ArH), 7.23 (d, J = 7.9 Hz, 1H, ArH),
7.16 (t, J = 8.1 Hz, 1H, ArH), 7.05 (d, J = 8.9 Hz, 2H, ArH), 6.67 (s, 2H,
drobenzo[b][1,4]thiazepine (D30)
Yellow powder, yielding 63.6%, melt point 121–122 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.86 (s, 1H, ArH), 7.82 (d, J = 8.4 Hz, 1H, ArH),
7.62–7.56 (m, 2H, ArH), 7.43 (d, J = 7.8 Hz, 1H, ArH), 7.38 (dd, J =
10

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
22.5, 8.6 Hz, 4H, ArH), 7.28 (t, J = 7.2 Hz, 1H, ArH), 7.13 (d, J = 8.6 Hz,
7.19 (t, J = 7.5 Hz, 1H, ArH), 7.12 (d, J = 8.5 Hz, 1H, ArH), 5.41 (dd, J =
– –
1H, ArH), 5.25 (dd, J = 12.6, 4.9 Hz, 1H, CH2 ), 3.88 (d, J = 10.0 Hz,
– – –
13.0, 4.5 Hz, 1H, CH2 ), 3.86 (d, J = 9.0 Hz, 6H, ( OCH3)2), 3.50
–
– –
– –
(dd, J = 13.0, 4.6 Hz, 1H, CH2 ), 2.82 (t, J = 13.1 Hz, 1H,
6H, ( OCH3)2), 3.61 (dd, J = 12.9, 4.3 Hz, 1H, CH2 ), 2.92 (t, J =
– – –
12.9 Hz, 1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 170.82,
CH ). 13C NMR (151 MHz, DMSO‑d6) 170.81, 152.51, 149.38,
– – –
S
149.52, 142.83, 135.69, 132.74, 132.19, 130.90, 130.41, 129.02,
128.79, 128.59, 128.53, 126.42, 112.01, 60.23, 56.55, 56.48. HRMS
(ESI-TOF) m/z: [M + H]+ Calcd. for C23H21ClNO2S 410.0981, Found
410.0978.
141.06, 135.40, 131.14, 130.69, 129.86, 129.75, 128.31, 127.93,
125.81, 125.59, 122.17, 121.95, 111.76, 110.21, 60.23, 56.17, 55.98.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C23H21ClNO2S 410.0981,
Found 410.0976.
2-(4-Bromophenyl)-4-(3,4-dimethoxyphenyl)-2,3-dihydrobenzo
[b][1,4]thiazepine (D31)
2-(3-Chlorophenyl)-4-(3,4-dimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D36)
Yellow powder, yielding 56.1%, melt point 118–120 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.89 (s, 1H, ArH), 7.84 (d, J = 8.4 Hz, 1H, ArH),
7.59 (d, J = 7.4 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 2H, ArH), 7.46 (d, J =
7.6 Hz, 1H, ArH), 7.30 (d, J = 8.4 Hz, 3H, ArH), 7.14 (d, J = 8.5 Hz, 1H,
Yellow powder, yielding 57.4%, melt point 121–122 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.83 (s, 1H, ArH), 7.79 (d, J = 8.4 Hz, 1H, ArH),
7.57 (t, J = 6.7 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 2H, ArH), 7.40 (d, J =
8.0 Hz, 1H, ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.26 (t, J = 7.4 Hz, 1H,
ArH), 7.12 (d, J = 8.5 Hz, 1H, ArH), 5.23 (dd, J = 12.6, 4.9 Hz, 1H,
– –
ArH), 5.25 (dd, J = 12.6, 4.8 Hz, 1H, CH2 ), 3.89 (d, J = 9.9 Hz, 6H,
–
– –
– – –
CH2 ), 3.88 (d, J = 11.0 Hz, 6H, ( OCH3)2), 3.57 (dd, J = 12.8, 4.2
(
OCH3)2), 3.67–3.60 (m, 1H, CH2 ), 2.93 (t, J = 12.9 Hz, 1H,
S
CH ). 13C NMR (151 MHz, DMSO‑d6) δ 149.44, 143.51, 135.55,
CH ). 13C NMR (151
– – –
Hz, 1H, CH2 ), 2.90 (t, J = 12.9 Hz, 1H, S
– – –
– –
132.31, 131.94, 131.69, 131.24, 130.79, 128.84, 127.67, 127.23,
126.03, 121.16, 111.87, 58.66, 56.40, 56.25. HRMS (ESI-TOF) m/z: [M
+ H]+ Calcd. for C23H21BrNO2S 454.0476, Found 454.0470.
4-(3,4-Dimethoxyphenyl)-2-(4-fluorophenyl)-2,3-dihydrobenzo
[b][1,4]thiazepine (D32)
MHz, DMSO‑d6) δ 168.55, 152.73, 149.43, 141.13, 135.26, 131.22,
130.55, 129.99, 129.70, 128.45, 128.02, 125.93, 125.62, 122.26,
121.93, 110.33, 60.16, 56.12, 56.02. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C23H21ClNO2S 410.0981, Found 410.0975.
Yellow powder, yielding 53.6%, melt point 152–153 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.84 (s, 1H, ArH), 7.80 (d, J = 8.4 Hz, 1H, ArH),
7.61–7.56 (m, 2H, ArH), 7.42 (d, J = 7.8 Hz, 1H, ArH), 7.39 (dd, J = 8.7,
5.5 Hz, 2H, ArH), 7.28 (t, J = 7.4 Hz, 1H, ArH), 7.17 (t, J = 8.8 Hz, 2H,
ArH), 7.13 (d, J = 8.5 Hz, 1H, ArH), 5.26 (dd, J = 12.5, 4.9 Hz, 1H,
4.2. Biological assay
4.2.1. Anti-proliferation assay
Human breast adenocarcinoma cell line MCF-7, human epithelial
cervical cancer cell line HeLa, human colon cancer cell line HT29,
human non-small-cell lung cancer cell line A549 and human normal
hepatocyte line L02 were purchased from American Type Culture
Collection (ATCC). The other cell lines grew in DMEM (Hyclone). All
media was supplemented with 10% foetal bovine serum (FBS, BI), 2
mmol/L of L-glutamine, 100 units/mL of penicillin–streptomycin
(Sigma-Aldrich), 100 mg/mL streptomycin (Hyclone) and incubated at
37 ◦C in a water saturated atmosphere containing 5% CO2.
– –
–
CH2 ), 3.88 (d, J = 10.1 Hz, 6H, ( OCH3)2), 3.58 (dd, J = 12.9, 4.4
– –
Hz, 1H, CH2 ), 2.92 (t, J = 12.9 Hz, 1H,
CH ). 13C NMR (151
– – –
S
MHz, DMSO‑d6) δ 164.64, 152.97, 152.11, 144.61, 139.92, 137.15,
135.28, 132.74, 131.48, 130.68, 128.65, 125.76, 125.63, 123.27,
104.16, 60.31, 56.22, 56.05. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for
C
23H21FNO2S 394.1277, Found 394.1268.
4-(3,4-Dimethoxyphenyl)-2-(p-tolyl)-2,3-dihydrobenzo[b][1,4]
thiazepine (D33)
The anti-proliferative activities of the prepared compounds against
the cultured cells were evaluated using a standard MTT (3-(4,5-dime-
thylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)-based colorimetric
assay with some modification. After the incubation with the above
condition for 24 h, cells were treated with the target compounds at
Yellow powder, yielding 54.7%, melt point 115–116 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.90 (s, 1H, ArH), 7.84 (d, J = 8.1 Hz, 1H, ArH),
7.60 (t, J = 6.6 Hz, 2H, ArH), 7.48 (d, J = 7.6 Hz, 1H, ArH), 7.31 (t, J =
7.4 Hz, 1H, ArH), 7.21 (d, J = 8.1 Hz, 2H, ArH), 7.14 (dd, J = 12.6, 8.3
– –
Hz, 3H, ArH), 5.19 (dd, J = 12.4, 4.9 Hz, 1H, CH2 ), 3.89 (d, J = 12.0
0.001 μM, 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM and 500 μM in the
–
– –
Hz, 6H, ( OCH3)2), 3.62 (dd, J = 12.4, 3.4 Hz, 1H, CH2 ), 2.96 (t, J
presence of 10% FBS for 48 h. Afterwards, cell viability was assessed by
the conventional MTT reduction assay carried out strictly according to
the manufacturer’s instructions (Sigma-Aldrich). The absorbance
(OD570) was read on LX300 Epson Diagnostic micro-plate reader. Three
replicate wells were used for each concentration and each assay was
measured three times.
– – –
–
= 12.8 Hz, 1H,
S
CH ), 2.28 (s, 3H, CH3). 13C NMR (151 MHz,
DMSO‑d6) δ 170.82, 149.53, 149.01, 140.94, 137.64, 135.71, 130.76,
129.57, 129.44, 129.25, 128.41, 126.47, 112.07, 60.23, 56.55, 56.48,
19.03. HRMS (ESI-TOF) m/z: [M + H]+ Calcd. for C24H24NO2S
390.1528, Found 390.1521.
4-(3,4-Dimethoxyphenyl)-2-(3-methoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D34)
4.2.2. The tubulin polymerization inhibition
Yellow powder, yielding 55.4%, melt point 132–133 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.91 (s, 1H, ArH), 7.85 (d, J = 8.1 Hz, 1H, ArH),
7.64 (d, J = 6.9 Hz, 1H, ArH), 7.61 (t, J = 7.7 Hz, 1H, ArH), 7.48 (d, J =
7.4 Hz, 1H, ArH), 7.32 (t, J = 7.4 Hz, 1H, ArH), 7.24 (t, J = 8.1 Hz, 1H,
ArH), 7.15 (d, J = 8.6 Hz, 1H, ArH), 6.93–6.88 (m, 2H, ArH), 6.85 (dd, J
This assay was conducted with a Tubulin Polymerzation Kit (Cyto-
skeleton, Inc. Denver, CO) including bovine brain tubulin (0.4 mg, 97%
pure) and corresponding buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM
EGTA, and 1 mM GTP). D8 and the controls (CA-4, Colchicine, Vinor-
elbine, Paclitaxel) were added respectively in the system with 100 μL of
– –
= 7.9, 2.0 Hz, 1H, ArH), 5.19 (dd, J = 12.4, 4.9 Hz, 1H, CH2 ), 3.90
general tubulin buffer. The pH condition was set as 6.9, the temperature
was 37 ◦C, and the absorbance at 340 nm was monitored every 1 min for
20 min on a Tecan Microplate Reader. The IC50 was defined as the
compound concentration that inhibited the extent of polymerization by
50% after 20 min incubation.
–
– –
–
(d, J = 9.6 Hz, 6H, ( OCH3)2), 3.71 (s, 3H, OCH3), 3.64 (dd, J = 12.4,
3.6 Hz, 1H, CH2 ), 2.96 (t, J = 12.8 Hz, 1H,
CH ). 13C NMR
– – –
S
(151 MHz, DMSO‑d6) δ 170.82, 167.56, 159.73, 149.52, 145.36, 135.72,
130.84, 130.20, 128.28, 126.44, 125.62, 118.68, 113.64, 112.45,
112.05, 111.72, 60.23, 56.48, 55.48. HRMS (ESI-TOF) m/z: [M + H]+
Calcd. for C24H24NO3S 406.1477, Found 406.1473.
4.2.3. Cell apoptosis and cell cycle analysis
Approximately 105 per well cells were cultured in a 24-well plate and
allowed to adhere. Subsequently, the medium was replaced with fresh
culture medium containing compound D8 at final concentrations of 0,
2-(2-Chlorophenyl)-4-(3,4-dimethoxyphenyl)-2,3-dihy-
drobenzo[b][1,4]thiazepine (D35)
Yellow powder, yielding 58.2%, melt point 156–157 ◦C. 1H NMR
(600 MHz, DMSO‑d6) δ 7.77 (s, 1H, ArH), 7.74 (d, J = 8.5 Hz, 1H, ArH),
7.62–7.56 (m, 2H, ArH), 7.53 (t, J = 7.6 Hz, 1H, ArH), 7.50 (d, J = 7.6
Hz, 1H, ArH), 7.38–7.31 (m, 2H, ArH), 7.30 (d, J = 7.9 Hz, 1H, ArH),
0.375, 0.75, 1.5 and 3.0 μM. Non-treated wells received an equivalent
volume of ethanol (<0.1%). After 48 h, cells were trypsinized, washed in
PBS and centrifuged at 2000 rpm for 5 min. The pellet was resuspended
11

B. Wang et al.
Bioorganic Chemistry 108 (2021) 104585
in 500
μ
L staining solution (containing 5
μ
L AnnexinV-FITC and 5
μ
L PI
eliminated from the protein and the polar hydrogen was added to the
proteins. The ligands except the control (Colchicine) were retained. The
results were visualized by Discovery Studio Visualizer 2016 (Accelrys,
Inc. San Diego, CA) consulting the reference [46].
(5 mg/mL) in Binding Buffer), mixed gently and incubated for 15 min at
room temperature assay dilution buffer in dark. The samples were then
analyzed by a FACSCalibur flow cytometer (Becton Dickinson, US).
Time-dependent protocol was similar to the process above with con-
centration of D8 set as 1.5
μM and incubation time varying (0, 24, 48,72
4.4. Statistical analysis
h).
Cells were plated in 6-well plates (106 per well) and incubated at
Prism 7.0 (GraphPad, USA) software was used to perform statistical
analysis. Data were expressed as means ± standard error. Differences
were analyzed according to the one-way ANOVA followed by Dunnett’s
multiple comparisons test or Student’s unpaired t-test for two-group
comparison in appropriate condition. As a presentation format, p <
0.05, p < 0.01 and p < 0.001 were considered statistically significant
and tagged by *, ** and ***, respectively.
◦
37 C or 24 h. Exponentially growing cells were then incubated with
compound D8 at different concentrations (0, 0.375, 0.75, 1.5 and 3.0
μ
M). After 48 h, cells were centrifuged at 1500 rpm at 4 ◦C for 5 min,
◦
fixed in 70% ethanol at 4 C for at least 12 h and subsequently resus-
pended in phosphate buffered saline (PBS) containing 0.1 mg/mL RNase
A and 5 mg/mL PI. The cellular DNA content was measured by flow
cytometry for cell cycle distribution analysis, plotting at least 10,000
events per sample. The percentage of cells in the subG0/G1, G0/G1, S
and G2/M phases of the cell cycle were determined using Flowjo 7.6.1
software. The time-dependent assay used the similar protocol with 1.5
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
μ
M of D8.
4.2.4. Western Blot
Total proteins were extracted from MCF-7 (human breast cancer
cells) after treated with RIPA lysis buffer containing 1% PMSF and
centrifuged at 16,000 g at 4 ◦C for 15 min using Eppendorf 5810R
centrifuge. Protein concentrations were measured using the BCA Protein
Acknowledgment
This work is supported by the Guangxi Natural Science Foundation
under Grant No. 2019AC20294 (GK AD20159070), the National Un-
dergraduate Training Program for Innovation and Entrepreneurship
(No. 202010284046X).
Assay kit (#23227 Thermo Fisher Scientific, USA). Approximately 80 μg
of total protein were separated by 10% SEMS-polyacrylamide gels and
transferred to PVDF membranes. After blocked with 5% defatted milk
solution, the membranes were incubated with primary antibodies, such
as Cyclin A (#AF0142, rabbit polyclonal IgG, 1: 1000 dilution); CDK1/
CDC2 (#DF6024, rabbit polyclonal IgG, 1: 1000 dilution), VEGFR1
(#AF6204, rabbit polyclonal IgG, 1: 1000 dilution), VEGFA (#AF5131,
rabbit polyclonal IgG, 1: 1000 dilution), β-Actin (#66009-1-Ig, mouse
polyclonal IgG, 1:20000 dilution) and GAPDH (#60004-1-1g, mouse
polyclonal IgG, 1:20000 dilution) at 4 ◦C overnight. After thrice washing
in TBST for each 5 min, the membranes were incubated with HRP-
conjugated secondary antibodies (#SA00001-1, #SA00001-2, 1: 5000
dilution) at room temperature for 2 h. The detection was performed by
an enhanced chemo-luminescent reagent (Thermo Fisher Scientific,
USA) according to the manufacturer’s instructions. The bands were then
recorded by a digital camera (Tanon 5200, Shanghai, China). Finally,
the results were analyzed with Image J Software (National Institutes of
Health, BetheSEMa, Maryland, USA), and all the targeted proteins were
normalized to GAPDH and β-Actin.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104585.
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