Design, synthesis and biological evaluation of novel 6-phenyl-1,3a,4,10b-tetrahydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one derivatives as potential BRD4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115601

Bromodomain-containing protein 4 (BRD4) is a key epigenetic regulator in cancer, and inhibitors targeting BRD4 exhibit great anticancer activity. By replacing the methyltriazole ring of the BRD4 inhibitor I-BET-762 with an N-methylthiazolidone heterocyclic ring, fifteen novel BRD4 inhibitors were designed and synthesized. Compound 13f had a hydrophobic acetylcyclopentanyl side chain, showing the most potent BRD4 inhibitory activity in the BRD4-BD1 inhibition assay (IC₅₀ value of 110 nM), it also significantly suppressed the proliferation of MV-4-11 cells with high BRD4 level (IC₅₀ value of 0.42 μM). Furthermore, the potent apoptosis-promoting and G0/G1 cycle-arresting activity of compound 13f were indicated by flow cytometry. As the downstream-protein of BRD4, c-Myc was in significantly low expression by compound 13f treatment in a dose-dependent manner. All the findings supported that this novel compound 13f provided a perspective for developing effective BRD4 inhibitors.

Full text of DOI:10.1016/j.bmc.2020.115601

Bioorganic&MedicinalChemistry28(2020)115601
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel 6-phenyl-1,3a,4,10b-
tetrahydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one derivatives as potential
BRD4 inhibitors
Qifei Lia,1, Jieming Lia,1, Yan Caia, Yuxing Zoua, Bin Chena, Feng Zoua, Jiaxian Moa, Ting Hana,
⁎
⁎
Weiwei Guoa, Wenlong Huanga,b, Qianqian Qiua, , Hai Qiana,b,
a Center of Drug Discovery, State Key Laboratory of Natural Medicine, China Pharmaceutical University, Nanjing 210009, PR China
b Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bromodomain-containing protein 4 (BRD4) is a key epigenetic regulator in cancer, and inhibitors targeting BRD4
exhibit great anticancer activity. By replacing the methyltriazole ring of the BRD4 inhibitor I-BET-762 with an N-
methylthiazolidone heterocyclic ring, fifteen novel BRD4 inhibitors were designed and synthesized. Compound
13f had a hydrophobic acetylcyclopentanyl side chain, showing the most potent BRD4 inhibitory activity in the
BRD4-BD1 inhibition assay (IC50 value of 110 nM), it also significantly suppressed the proliferation of MV-4-11
cells with high BRD4 level (IC50 value of 0.42 μM). Furthermore, the potent apoptosis-promoting and G0/G1
cycle-arresting activity of compound 13f were indicated by flow cytometry. As the downstream-protein of BRD4,
c-Myc was in significantly low expression by compound 13f treatment in a dose-dependent manner. All the
findings supported that this novel compound 13f provided a perspective for developing effective BRD4 in-
hibitors.
BRD4 inhibitors
Anti-tumor
Apoptosis
Anti-proliferation
1. Introduction
applicable to the development of small molecule inhibitors.
A pair of bromodomains (BD1 and BD2) are contained in the BRD4
The bromodomain and extra-terminal domain (BET) protein family
includes BRD2, BRD3, BRD4 and bromodomain testis-specific protein
(BRDT). The BET protein consists of two N-terminal bromodomains and
one C-terminal domain (ET), and their sequences are highly conserved.1
Among them, BRD2 functions through the E2F transcription factor
pathway;2 BRD3 helps in recruiting hematopoietic transcription factor,
GATA1, by regulating the maturation of red blood cells, mega-
karyocytes and mast cells.3,4 BRD4 is currently the most widely studied
protein of BET family, and it is involved in many human diseases, in-
cluding cancer, inflammation, cardiovascular disease, central nervous
system (CNS) disease and human immunodeficiency virus (HIV) infec-
tion.5–8 BRD4 is a critical mediator of transcriptional elongation, and it
influences mitotic progression.9–11 The abnormal expression of BRD4
will cause the activation of the downstream gene c-Myc, which could
lead to the occurrence of cancers such as acute leukemia, lymphoma,
melanoma and so on.12 Therefore, BRD4 has been developed as ther-
apeutic target for research on cancer drug, and such research is
structure, each of which is consisted of a conserved fold and two loop
regions.1 Although BD1 and BD2 possess a high similarity in sequence
of substrate-binding site, some functions of BD1 and BD2 are different.
For example, BD2 have wide effects towards other acetylated sub-
strates, whereas BD1 is inclined to only discerning H4 acetylation
marks.13 Therefore, BD1 is the main targeted binding site for BRD4
inhibitors.14,15 BRD4 inhibitor (+)-JQ-1 and I-BET-762 are potent
BRD4 inhibitors. Provided by PDB data, the core structures of the two
potent inhibitors can form essential hydrogen bonds with the residue
Tyr 97 in BRD4 in Fig. 1 (PDB ID: 4c66 and 3mxf). Additionally, the co-
crystal structure of BRD4 bromodomain 1 (BRD4-BD1) and histone
peptide H4K8acK12ac indicates that acetylated lysine is recognized by
a central hydrophobic cavity, and hydrogen bond interaction is formed
between the acetylcarbonyl oxygen atom and the conserved Tyr97 via
water molecules.16 Therefore, Tyr 97 residue is an important binding
site for BRD4-BD1.
As shown in Fig. 2, the researchers have made some structural
⁎ Corresponding authors at: Center of Drug Discovery, State Key Laboratory of Natural Medicine, China Pharmaceutical University, Nanjing 210009, PR China (Hai
Qian).
E-mail addresses: qianqianqiu_cpu@126.com (Q. Qiu), qianhai24@163.com (H. Qian).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115601
Received 23 January 2020; Received in revised form 16 June 2020; Accepted 16 June 2020
Availableonline20June2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
Fig. 1. (A) Hydrogen bonds formed between I-BET-762 inhibitor and residue Tyr 97 via water molecule; (B) hydrogen bonds formed between (+)-JQ-1 inhibitor and
residue Tyr 97 via water molecule.
Fig. 2. Design of target compounds.
modifications to the BRD4 inhibitor I-BET-762. It has been reported by
Hewitt, et al. that the BRD4 inhibitory activity was significantly im-
proved when the methyltriazole ring of the lead compound I-BET-762
was substituted with methylisoxazole ring (Fig. 2A).17,18 In addition, Li,
et al. has reported that on the basis of retaining the predominant ske-
letal structure of benzoazepine and replacing methyltriazole with N-
methylpyridone, compounds with more potent BRD4 inhibitory activity
were obtained (Fig. 2B).19 Therefore, the methyltriazole ring of com-
pound I-BET-762 could be further modified, and these modifications
had little impact on target- selectivity between BRD4 and other BRD
proteins. Since the acetylated lysine (KAc) binding site was the most
important part in the design of BRD4 inhibitors, interacting with the
acetylated lysine binding site have been used for developing several
kinds of specific small molecular BRD4 inhibitors.20–24 It has been re-
ported that the 2-thiazolidinone structure could well simulate the
acetylated lysine structure, and it acted as an effective BRD4 mimic
group.25,26 Thus, the 2-thiazolidinone was a new strategy to replace the
methyltriazole ring of compound I-BET-762, which was different from
the previous studies to replace the methyltriazole ring of compound I-
BET-762 with N-methylpyridone or methylisoxazole ring.
was carried out. Based on the structure-activity relationship of I-BET-
762, the methyltriazole moiety was replaced with a methylthiazolidone
moiety to maintain the interaction with the residue Tyr 97, which could
keep selective inhibition of BRD4-BD1 instead of BRD4-BD2.
Consequently, a series of biological evaluation demonstrated that
compound 13f did exhibit more powerful BRD4 inhibitory activity than
I-BET-762, and its activity was comparable to (+)-JQ-1. Compound 13f
was shown to be potent BRD4 inhibitor with an IC50 value of 110 nM,
and it had an IC50 value of 0.42 μM against MV-4-11 cell anti-
proliferation. It provided a structural perspective for developing novel
effective BRD4 inhibitors.
2. Results and discussion
2.1. Synthesis
Synthesis of 6-phenyl-1,3a,4,10b-tetrahydro-2H-benzo[c]thiazolo
[4,5-e]azepin-2-one derivatives was shown in Scheme 1. Compounds
11, 12, 13a-l and 15 were synthesized using 2-bromo-5-methox-
ybenzaldehyde (1) and 2,4-thiazolidinedione (6) as starting materials,
and the final product was obtained through ten steps of reaction. First,
commercially available 2-bromo-5-methoxybenzaldehyde (1) reacted
with 4-chlorophenyl magnesium bromide (2) to prepare 3, and the
intermediate 3 was oxidized to 4. Alcohol borate reacted with 4 to
Here we synthesized a series of new scaffold compounds based on
the structure of I-BET-762. The BRD proteins and their binding site
were different, and a lot of research has been done on the BRD4 binding
site. On the basis of this binding site, a reasonable compound design
2

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Bioorganic&MedicinalChemistry28(2020)115601
Scheme 1. Reagents and conditions: (i) anhydrous THF, 0℃→r.t, 2 h; (ii) dry DCM, pyridinium chlorochromate, r.t, 1.5 h; (iii) Bis(pinacolato)diborane, dioxane,
KOAc, Pd(dppf)Cl2, 95 ℃, 2.5 h; (iv) MeI, K2CO3, DMF, 75℃, 2 h; (v) POCl3, DMF, 90-115℃, 4 h; (vi) NaH, Triethyl phosphonoacetate, THF, 0 ℃→r.t; (vii) I-5,
Na2CO3, dioxane/H2O, Pd(PPh3)4, 90 ℃, 20 h; (viii) Ammonium formate, EtOH, reflux; (ix) 1 N NaOH, THF, r.t; (x) ROH/RNH2, EDC·HCl, DMAP, r.t; (xi) NaBH4,
MeOH, r.t; (xii) K2CO3, EtOH, r.t for 24 h.
produce intermediate 5. Commercially available thiazolidine-2,4-dione
(6) was methylated to obtain intermediate 7, and then Vilsmeir for-
mylation was used for 7 to get 8. Compound 8 reacted with triethyl-
phosphonoacetate to obtain 9, and then 9 reacted with intermediate 5
through Suzuki coupling reaction to obtain 10. Target compound 11
was synthesized via 10 reacting with ammonium formate to close the
ring. Finally, 11 was hydrolyzed by NaOH, producing target compound
12 which was substituted with different types of amines to produce the
target products, 13a ~ 13 k. In addition, compound 10 was reduced by
sodium borohydride to obtain compound 14, and then 14 was reacted
under alkaline conditions to obtain target compound 15.
applied as positive controls. As shown by the IC50 values in Table 1, all
compounds had relatively better inhibition activities than I-BET-762.
Particularly, the IC50 value of compounds 13f and 13j was 0.11 μM and
0.16 μM, respectively. When X was nitrogen atom, the compounds’
inhibitory activities were better than those when X was oxygen atom. In
addition, polar groups, such as hydroxyl, were introduced into the side
chain leading to decreased inhibitory activities for BRD4-BD1. The R
side chain was substituted with acetylcyclopentylamino giving com-
pound 13f which had the strongest inhibitory activity for BRD4-BD1
among all the compounds. Acetyl t-butylamine side chain had sig-
nificantly better inhibitory activity for BRD4-BD1 than acetobutylamine
side chain comparing the results for compound 13j (0.16 μM) with that
for 13 l (1.02 μM). This indicated that the introduction of long fatty side
chains was not beneficial to improve the inhibitory activity. Further-
more, compound with acetylarylamine side chain (13g) had better ac-
tivity than others with acetylheterocyclic aliphatic amine side chains
(13b, 13c, 13d).
2.2. Biological evaluations
2.2.1. Inhibitory activity against BRD4
To probe the inhibition activities of the designed compounds tar-
geting BRD4, time-resolved fluorescence resonance energy transfer (TR-
FRET) binding assay was used, and (+)-JQ-1 and I-BET-762 were
In summary, the inhibition activities of compounds towards BRD4
3

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
Table 1
Table 2
Structures and inhibitory activities of compounds for BRD4-BD1.
Anti-proliferation activity of target compounds against MV-4-11 and HL-60
cells.
Compound
HL-60
MV-4-11
IC50 (μM)a
IC50 (μM)a
11
6.21
NTb
0.26
0.88
2.86
1.78
0.94
1.12
0.91
0.51
0.42
0.67
1.44
0.70
0.64
1.06
3.48
0.92
0.16
1.23
0.13
12
0.17
0.08
0.05
0.14
0.12
0.08
0.06
0.03
0.08
0.07
0.02
0.11
0.12
0.04
0.05
0.06
13a
12.85
7.62
10.37
8.69
4.56
5.52
7.74
12.36
8.46
5.41
9.05
NTb
1.07
13b
13c
0.63
0.42
13d
13e
0.36
0.21
0.39
0.33
Compound
R
X
BRD4-BD1 Inhibitory Activity IC50 (μM)a
13f
13g
11
N
0.42
0.87
0.74
0.48
0.54
0.43
0.14
0.06
0.08
0.05
0.02
0.06
0.04
0.02
13h
13i
0.48
0.94
0.22
0.36
13j
12
N
N
N
N
N
N
13k
13l
13a
13b
13c
13d
13e
15
5.44
3.46
NTb
0.24
0.18
(+)-JQ-1
I-BET-762
a
The values were reproduced in three assays, the values were expressed as
SD.
Not tested.
the means
b
compounds could also suppress the proliferation of HL-60 cells at some
extent, which was significantly weaker than that of MV-4-11 cells.
BRD4 has higher expression in MV-4-11 cells than that in HL-60, so MV-
4-11 cells were more sensitive to these designed BRD4 inhibitors. All in
all, compound 13e and 13f had better anti-proliferation against MV-4-
11 cells.
13f
N
N
N
0.11
0.20
0.68
0.01
0.01
0.03
13 g
13 h
2.2.3. Cell cycle analysis
Flow cytometry analysis was utilized to explore the effect of com-
pound 13f on the cell cycle in MV-4-11 cells. As shown in Fig. 3,
compound 13f and (+)-JQ-1 could significantly arrest MV-4-11 cells in
G1 phase in a dose-dependent manner. The cell-cycle arrest potency of
compound 13f was inferior to (+)-JQ-1 at the same concentration.
13i
N
N
N
0.21
0.16
0.51
0.02
0.01
0.04
13j
2.2.4. Cell apoptosis
13 k
BRD4 inhibitors could induce apoptosis.27 Therefore, the apoptosis-
promoting effect of compound 13f in MV-4-11 cells were determined
using flow cytometry. The results in Fig. 4 showed that compound 13f
and (+)-JQ-1 could lead to MV-4-11 cell apoptosis at different drug
concentrations. The total number of cells undergoing apoptosis sig-
nificantly increased with the increase of concentration, suggesting that
compound 13f could effectively induce apoptosis of MV-4-11 cells in a
dose-dependent manner.
13 l
15
N
O
1.02
0.48
0.1
0.02
(+)-JQ-1
I-BET-762
–
–
–
–
0.074
0.32
0.002
0.07
a
Each value was reproduced in three assays, the values were expressed as
2.2.5. Western blot assay
the means
SD.
It has been reported that BET bromodomain proteins can act as
regulatory factors for c-Myc.28,29 To further evaluate the as-synthesized
compound, western blotting was carried out to determine c-Myc ex-
pression. Compound 13f and the positive control (+)-JQ-1 were in-
vestigated. Fig. 5 showed that compound 13f and (+)-JQ-1 could well
inhibit the expression of c-Myc protein. The higher was concentration
of the compound, the lower was expression. (+)-JQ-1 had better potent
ability to inhibit c-Myc expression at the same concentration.
could be improved by introducing a hydrophobic R side chain with a
small structure, or retaining an aromatic R side chain that did not have
a hydrophilic group.
2.2.2. Inhibition of cell growth
It is well known that BRD4 is a recognized target for treating leu-
kemia. Therefore, the anti-proliferative effects of the target compounds
against MV-4-11 (biphenotypic B myelomonocytic leukemia) cell line
and HL-60 (acute promyelocytic leukemia) cell line were evaluated in
vitro. As shown in Table 2, most of the compounds showed potent in-
hibition effect against MV-4-11 cells. Half of the compounds had better
inhibition effect against MV-4-11 cell line than the positive control, I-
BET762. In the anti-proliferation against MV-4-11 cells, compound 13e,
13f, 13g, 13i and 13j were a little inferior to (+)-JQ-1. These
2.2.6. Molecular docking assay
To explore the interaction between compound 13f and BRD4,
Schrödinger software was employed to analyze the possible binding
modes. Fig. 6 showed that Pi interactions were formed between the
thiazolidinone ring of compound 13f and Trp81. Meanwhile, com-
pound 13f had hydrogen-bond interactions with Asp 145 through water
molecule. Moreover, the acetylcyclopentylamine side chain of
4

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
Fig. 3. Effects of compound 13f and (+)-JQ-1 on MV-4-11 cell cycle analysis: (A) control; (B) compound 13f (0.5 μM); (C) compound 13f (5 μM); (D) (+)-JQ-1
(0.5 μM); (E) (+)-JQ-1 (5 μM); (F) cell cycle analysis, the values were expressed as the means SD.
compound 13f was embed with cavity of BRD4-BD1 and formed hy-
drogen-bond interactions with Tyr 97. These results showed that the
compounds had relatively good binding ability to BRD4-BD1 as de-
signed.
(300 MHz). Chemical shifts were showed as d H part per million (ppm)
relative to tetramethyl silane (TMS) as internal standard. Hertz (Hz)
expressed coupling constants (J values). Liquid chromatography-mass
spectrometer (LC-MS) spectra were operated on Waters ACQUITY
UPLC-TQD in ESI mode. Column chromatography silica gel (200–300
mesh) was used to purify the compounds. The purification process was
monitored by thin layer chromatography which was performed on GF/
UV 254 plates and were visualized using UV light at 254 and 365 nm.
The purity of compounds was assessed by HPLC (> 95%).
3. Conclusions
In summary, the BRD4 inhibitor, I-BET-762, was chosen as the core
structure to design and construct fifteen novel BRD4 inhibitors, and
their biological activities were evaluated. The results indicated that
these compounds had potent BRD4 inhibitory activity. Particularly, the
BRD4 inhibitory activity of compound 13f (with an IC50 value of
110 nM) was superior to that of I-BET-762. Furthermore, the as-syn-
thesized compounds exhibited better anti-proliferation activity in MV-
4-11 cells than HL-60 cell, suggesting that the designed compounds
might be more sensitive to MV-4-11 than to HL-60 cells. Cell cycle
analysis revealed that compound 13f significantly arrested MV-4-11
cells in G1 phase in a dose-dependent manner. Apoptosis analysis and
western blotting assay showed that compound 13f induced cell apop-
tosis by down-regulating c-Myc expression. Moreover, the docking re-
sults showed that compound 13f could form hydrogen-bond interac-
tions with residue Tyr 97 of BRD4, further indicating that the designed
compounds had better binding affinity to BRD4-BD1. In conclusion, the
thiazolidinone structure could well simulated the structure of acety-
lated lysine25,26 and improve BRD4 inhibitory activity. Compound 13f
could be considered as a promising BRD4 inhibitor candidate for further
development.
4.2. General procedure for the synthesis of 11,12 13a–13 l and 15
4.2.1. Preparation of 3
Compound 1 (15.18 g, 70.59 mmol) was dissolved in anhydrous
tetrahydrofuran (100 mL) followed by slowly adding compound 2 (1 M)
in tetrahydrofuran (85 mL, 84.71 mmol) at 0 °C under nitrogen pro-
tection. The system was then stirred at room temperature for 2 h.
Saturated ammonium chloride solution (70 mL) was added at low
temperature with stirring for 30 min. The mixture was filtered by celite
and washed with ethyl acetate. The organic layers were combined,
washed with brine and dried over anhydrous sodium sulfate. The or-
ganic layer was evaporated and the residue was separated by silica gel
column chromatography to obtain 3 (18.5 g) as a white solid (yield:
80%).
4.2.2. Preparation of 4
Compound 3 (18.47 g, 56.38 mmol) was dissolved in dry di-
chloromethane (150 mL), and pyridinium chlorochromate (15.80 g,
73.29 mmol) was added under nitrogen protection. The system was
stirred at room temperature for 1.5 h. The mixture was filtered by celite
and washed with dichloromethane. The organic layers were combined,
washed with saturated sodium bicarbonate and saturated brine, dried
over anhydrous sodium sulfate. The organic layer was evaporated and
the residue was separated by silica gel column chromatography to ob-
tain 4 (16.2 g) as a white solid (yield: 88%). 1H NMR (300 MHz, CDCl3)
4. Experimental section
4.1. Methods and materials
All materials, reagents and solvents were obtained from commercial
sources. They were utilized without further purification. The 1H NMR
spectra and 13C NMR spectra were recorded on Bruker AV300
5

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Bioorganic&MedicinalChemistry28(2020)115601
Fig. 4. Effects of compound 13f and (+)-JQ-1 on MV-4-11 cell apoptosis: (A) control; (B)compound 13f (0.5 μM); (C) compound 13f (5 μM); (D) (+)-JQ-1 (0.5 μM);
(E) (+)-JQ-1 (5 μM); (F) cell apoptosis analysis, the values were expressed as the means SD.
δ: 7.76 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.8 Hz, 1H), 7.49 – 7.39 (m,
2H), 6.91 (dd, J = 8.8, 3.0 Hz, 1H), 6.89 – 6.83 (m, 1H), 3.81 (s, 3H).
organic layers were combined, washed with saturated brine, dried over
anhydrous sodium sulfate and filtered with celite. The organic layer
was evaporated and the residue was separated by silica gel column
chromatography to obtain 5 (4.2 g) as a yellow solid (yield: 72%).1H
NMR (300 MHz, CDCl3) δ: 7.80 – 7.65 (m, 3H), 7.38 (d, J = 8.4 Hz,
2H), 7.06 – 6.91 (m, 2H), 3.83 (s, 3H), 1.08 (s, 12H).
4.2.3. Preparation of 5
Under nitrogen, compound 4 (5.08 g, 15.60 mmol), biboronic acid
pinacol borate (5.94 g, 23.40 mmol), potassium acetate (6.13 g,
62.41 mmol), and [1,1′-bis (Diphenylphosphino) ferrocene] palladium
dichloride (0.688 g, 0.94 mmol) were dissolved in 1,4-dioxane (85 mL).
The system was stirred at 95 °C for 2.5 h, which was then diluted with
water (60 mL) and extracted with ethyl acetate (3 × 60 mL). The
4.2.4. The preparation of 7
The 2,4-thiazolidinedione (8.4 g, 71.72 mmol) compound was dis-
solved in DMF (70 mL), anhydrous potassium carbonate (19.82 g,
Fig. 5. Effect of compound 13f on c-Myc expression; (A) representative western blotting band of c-Myc in MV-4-11cells; (B) band intensity of c-Myc expression
analysis for three independent experiments. **P < 0.01, ***P < 0.001 vs control group, #P < 0.05, ##P < 0.01 vs 0.5 μM group.
6

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
Fig. 6. Docking assay for compound 13f with BRD4-BD1. (A) Pi interactions formed between the thiazolidinone ring of compound 13f and Trp81, hydrogen-bond
interactions with Asp 145 and Tyr 97. (B) The acetylcyclopentylamine side chain of compound 13f was embed with cavity of BRD4-BD1 and formed hydrogen-bond
interactions with Tyr 97. (C) Compound 13f was bound with cavity of BRD4-BD1.
46.0 mmol) was added under nitrogen protection, and methyl iodide
(6.64 mL, 0.11 mol) was then added. The system was stirred at 75 °C for
2 h. The mixture was filtered by celite and washed with ethyl acetate.
The organic layers were combined, washed with brine and dried over
anhydrous sodium sulfate. The organic layer was evaporated and the
residue was separated by silica gel column chromatography to obtain 7
(6.5 g) as a white solid (yield: 69%). 1H NMR (300 MHz, CDCl3) δ: 3.97
(s, 2H), 3.13 (s, 3H).
65%). 1H NMR (300 MHz, CDCl3) δ: 7.66 (d, J = 8.5 Hz, 2H), 7.40 (d,
J = 8.5 Hz, 2H), 7.32 (d, J = 8.5 Hz, 1H), 7.19 (dd, J = 8.5, 2.5 Hz,
1H), 7.13 – 7.05 (m, 2H), 5.61 (d, J = 15.3 Hz, 1H), 4.17 – 4.10 (m,
2H), 3.91 (s, 3H), 3.12 (s, 3H), 1.26 – 1.22 (m, 3H).
4.2.8. Preparation of target compound 11
Compound 10 (2.80 g, 6.11 mmol) was dissolved in ethanol (50 mL)
followed by adding ammonium formate (3.86 g, 61.15 mmol) under
nitrogen protection. The reaction was heated for 24 h. The mixture was
filtered by celite and washed with ethyl acetate. The organic layers
were combined, washed with brine and dried over anhydrous sodium
sulfate. The organic layer was evaporated and the residue was sepa-
rated by silica gel column chromatography to obtain 11 (0.9 g) as a
white solid (yield: 32%). 1H NMR (300 MHz, CDCl3) δ: 7.92 (d,
J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.3 Hz, 1H),
7.21 (s, 1H), 7.05 (dd, 1H), 5.36 (t, J = 6.8 Hz, 1H), 4.07 (q,
J = 7.3 Hz, 2H), 3.90 (s, 3H), 3.00 (d, J = 6.8 Hz, 2H), 2.51 (s, 3H),
1.20 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ: 170.66, 169.37,
167.44, 160.97, 141.16, 138.50, 137.04, 132.36, 130.73, 129.22,
128.70, 123.92, 115.61, 114.97, 108.60, 95.77, 60.42, 55.40, 35.18,
26.91, 13.57; LC-MS (ESI) m/z: 457.5 [M + H] +; Elemental Analysis
for C23H22ClN2O4S: C, 60.32; H, 4.84; N, 6.12; Found: C, 60.36; H, 4.85;
N, 6.09;
4.2.5. Preparation of 8
Compound 7 (4.5 g, 34.31 mmol) was dissolved in phosphorus
oxychloride (9.57 mL, 0.10 mol) at 0 °C followed by slowly adding DMF
(5.28 mL, 68.62 mmol). The system was stirred at 90 °C for 3 h, and
then at 115° C for 1 h. The reaction solution was poured into an ice-
water mixture, stirred, and extracted with ethyl acetate (3 × 60 mL).
The organic layers were combined, washed with saturated brine, dried
over anhydrous sodium sulfate and filtered with suction. The organic
layer was evaporated and the residue was separated by silica gel
column chromatography to obtain 8 (3.8 g) as a yellow solid (yield:
62%). 1H NMR (300 MHz, CDCl3) δ: 9.84 (s, 1H), 3.45 (s, 3H).
4.2.6. Preparation of 9
Sodium hydride (1.62 g, 40.54 mmol, 60%) was added into anhy-
drous tetrahydrofuran (70 mL) at 0 °C followed by adding triethyl
phosphorylacetate (8.04 mL, 40.54 mmol) under nitrogen protection.
The mixture was stirred at low temperature for 30 min. Compound 8
(3.60 g, 20.27 mmol) was added into the system. The mixture was
stirred at room temperature for 1 h. The system was quenched with
saturated ammonium chloride, and the reaction solution was con-
centrated. The system was added with 40 mL water and extracted with
ethyl acetate (3 × 50 mL). The organic layers were combined, washed
with brine and dried over anhydrous sodium sulfate. The organic layer
was evaporated and the residue was separated by silica gel column
4.2.9. Preparation of target compound 12
Compound 11 (3.40 g, 7.44 mmol) was dissolved in tetrahydrofuran
(60 mL), and then 1 N aqueous solution of sodium hydroxide (0.89 g,
22.32 mL) was added dropwise. The reaction was stirred at room
temperature for 4 h. The solution was neutralized with 1 N hydrochloric
acid aqueous solution and extracted with dichloromethane
(3 × 50 mL). The organic layer was evaporated and the residue was
separated by silica gel column chromatography to obtain 12 (2.5 g) as a
white solid (yield: 78%). 1H NMR (300 MHz, CDCl3) δ: 12.39 (s, 1H),
8.05 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.53 (d, J = 8.5 Hz,
1H), 7.36 (dd, J = 8.5, 2.5 Hz, 1H), 7.20 – 7.16 (m, 1H), 5.26 (m, 1H),
3.89 (s, 3H), 2.96– 2.94 (m, 2H), 2.37 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ: 170.86, 167.95, 161.06, 141.29, 138.62, 137.45, 132.90,
130.56, 129.18, 128.81, 123.81, 115.81, 108.78, 95.77,54.44, 37.78,
34.09, 29.19; LC-MS (ESI) m/z: 429.4 [M + H] +; Elemental Analysis
for C21H18ClN2O4S: C, 58.67; H, 4.22; N, 6.52; Found: C, 58.69; H, 4.26;
N, 6.49;
chromatography to obtain 9 (18.5 g) as a yellow solid (yield: 56%). 1
H
NMR (300 MHz, CDCl3) δ: 7.68 (d, J = 15.5 Hz, 1H), 5.83 (d,
J = 15.5 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 3.41 (s, 3H), 1.32 (t,
J = 7.1 Hz, 3H).
4.2.7. Preparation of 10
Compound 5 (5.40 g, 14.49 mmol) and compound 9 (3.59 g,
14.49 mmol) were added in a 250 mL double-necked round-bottomed
flask,
and
dissolved
into
1,4-dioxane
(80
mL).
Tetratriphenylphosphonium (0.67 g, 0.58 mmol) and aqueous solution
of sodium carbonate (1.5 M, 20 mL) were added into the system under
nitrogen protection. After the addition, the system was transferred to an
oil bath and heated to reflux for about 20 h. The mixture was diluted
with water and extracted with ethyl acetate (3 × 60 mL). The organic
layer was evaporated, and the residue was separated by silica gel
column chromatography to obtain 10 (4.3 g) as a yellow solid (yield:
4.2.10. Preparation of target compound 13a
Compound 12 (0.3 g, 0.70 mmol), ethylamine (54.1 μL, 0.84 mmol),
EDC · HCl (0.16 g, 0.84 mmol) and DMAP (85.46 mg, 0.70 mmol) were
dissolved in dichloride (20 mL). The reaction was stirred at room
temperature for 12 h. The reaction solution was diluted with water and
extracted with dichloromethane (20 mL × 3). The organic layers were
combined, washed with brine and dried over anhydrous sodium sulfate.
7

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
The organic layer was evaporated and the residue was separated by
silica gel column chromatography to obtain target compound 13a
(0.2 g) as a white solid (yield: 63%). 1H NMR (300 MHz, CDCl3) δ: 7.90
(dd, J = 8.5, 1.9 Hz, 2H), 7.53 (dd, J = 8.5, 2.0 Hz, 2H), 7.42 (dd,
J = 8.2, 1.9 Hz, 1H), 7.21 (s, 1H), 7.04 (d, J = 8.3 Hz, 1H), 5.50 (s,
1H), 5.37 (t, J = 5.9 Hz, 1H), 3.89 (s, 3H), 3.19 (t, J = 10.7 Hz, 2H),
2.87 (d, J = 8.6 Hz, 2H), 2.51 (s, 3H), 1.07 (q, J = 7.2 Hz, 3H).
(13e). White solid, yield 66%, m.p.164–166 °C; 1H NMR (300 MHz,
CDCl3) δ: 7.88 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.41 (d,
J = 8.3 Hz, 1H), 7.19 (d, J = 1.8 Hz, 1H), 7.02 (dd, J = 8.2, 1.9 Hz,
1H), 6.18 (s, 1H), 5.36 (t, J = 6.5 Hz, 1H), 3.86 (s, 3H), 2.80 (m, 2H),
2.46 (s, 3H), 0.64 – 0.37(m, 4H); 13C NMR (75 MHz, CDCl3) δ: 170.88,
169.66, 167.58, 161.03, 141.01, 138.53, 137.20, 132.27, 130.60,
130.43, 129.18, 128.77, 123.96, 123.89, 116.26, 116.22, 115.71,
108.76, 95.81, 55.43, 37.27, 26.99, 22.20, 5.86; LC-MS (ESI) m/z:
468.4 [M + H] +; Elemental Analysis for C24H23ClN3O3S : C, 61.47; Cl,
7.56; N, 8.96; Found: C, 61.45; Cl, 7.52; N, 8.98;
4.2.11. Compound 13b–13l were synthesized via similar procedures as for
compound 13a
4.2.11.1. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-ethylacetamide (13a). White
solid, yield 63%. 1H NMR (300 MHz, CDCl3) δ: 7.90 (dd, J = 8.5,
1.9 Hz, 2H), 7.53 (dd, J = 8.5, 2.0 Hz, 2H), 7.42 (dd, J = 8.2, 1.9 Hz,
1H), 7.21 (s, 1H), 7.04 (d, J = 8.3 Hz, 1H), 5.50 (s, 1H), 5.37 (t,
J = 5.9 Hz, 1H), 3.89 (s, 3H), 3.19 (t, J = 10.7 Hz, 2H), 2.87 (d,
J = 8.6 Hz, 2H), 2.51 (s, 3H), 1.07 (q, J = 7.2 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ: 170.90, 167.92, 161.06, 141.26, 138.56, 137.28,
4.2.11.6. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-cyclopentylacetamide
(13f). White solid, yield 64%, m.p. 168–170 °C; 1H NMR (300 MHz,
CDCl3) δ: 7.89(dd, J = 8.3, 2.2 Hz, 2H), 7.52 (dd, J = 8.3, 2.2 Hz, 2H),
7.41 (dd, J = 8.1, 2.3 Hz, 1H), 7.20 (s, 1H), 7.03 (d, J = 8.2 Hz, 1H),
5.61 (s, 1H), 5.35 (d, J = 2.5 Hz, 1H), 4.07 (s, 1H), 3.88 (d, J = 2.4 Hz,
3H), 2.84 (d, J = 4.3 Hz, 2H), 2.50 (d, J = 2.4 Hz, 3H), 1.87 (s, 2H),
1.56 (s, 4H), 1.25 (s, 2H); 13C NMR (75 MHz, CDCl3) δ:171.27, 168.00,
161.54, 141.65, 139.08, 137.73, 133.42, 131.14, 129.67, 129.27,
124.29, 116.55, 116.15, 109.27, 96.31, 55.94, 51.31, 38.14, 32.98,
27.53, 23.63; LC-MS (ESI) m/z: 496.5 [M + H] +; Elemental Analysis
for C26H27ClN3O3S: C, 62.83; H, 5.48; N, 8.45; Found: C, 62.85; H, 5.52;
N, 8.41;
132.90, 130.66, 129.18, 128.81, 123.81, 115.81, 108.78, 55.44, 37.72,
+
34.07, 29.19, 27.00, 14.17; LC-MS (ESI) m/z: 456.4 [M + H]
;
Elemental Analysis for C23H23ClN3O3S: C, 60.45; H, 5.07; N, 9.20;
Found: C, 60.41; H, 5.03; N, 9.24;
4.2.11.2. 6-(4-chlorophenyl)-8-methoxy-1-methyl-4-(2-morpholino-2-
oxoethyl)-1,4-dihydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one
(13b). White solid, yield 67%, m.p. 145–147 °C. 1H NMR (300 MHz,
CDCl3) δ: 8.03 – 7.86 (m, 2H), 7.60 – 7.49 (m, 2H), 7.48 – 7.38 (m, 1H),
7.22 (s, 1H), 7.10 – 6.98 (m, 1H), 5.47 (d, J = 2.7 Hz, 1H), 3.90 (d,
J = 2.6 Hz, 3H), 3.61 (s, 4H), 3.53 (s, 2H), 3.40 (s, 2H), 3.09 – 2.94 (m,
2H), 2.52 (d, J = 2.5 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ: 170.68,
167.32, 161.02, 141.15, 138.62, 137.13, 131.57, 130.70, 129.19,
4.2.11.7. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-phenylacetamide (13 g). White
solid, yield 72%, m.p.182–184 °C; 1H NMR (300 MHz, CDCl3) δ: 8.05 –
7.76 (m, 3H), 7.45 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.33 –
6.95 (m, 4H), 6.65 (d, J = 6.8 Hz, 2H), 5.47 (s, 1H), 3.86 (s, 3H), 2.94
(s, 2H), 2.52 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.14, 167.11,
161.04, 140.97, 138.78, 138.70, 136.24, 131.09, 130.91, 130.28,
128.76, 123.85, 116.14, 115.65, 108.69, 95.86, 66.19, 66.05, 55.41,
+
45.52, 41.63, 34.59, 27.01; LC-MS (ESI) m/z: 498.5 [M + H]
;
129.08, 128.64, 124.49, 123.22, 118.91, 117.16, 116.55, 108.80,
+
Elemental Analysis for C25H25ClN3O4S: C, 60.18; H, 5.05; N, 8.42;
Found: C, 60.23; H, 5.01; N, 8.46;
95.83, 55.75, 37.75, 27.22; LC-MS (ESI) m/z: 504.5 [M + H]
;
Elemental Analysis for C27H23ClN3O3S : C, 64.22; H, 4.59; N, 8.32;
Found: C, 64.18; H, 4.55; N, 8.36;
4.2.11.3. 6-(4-chlorophenyl)-8-methoxy-1-methyl-4-(2-oxo-2-(pyrrolidin-
1-yl)ethyl)-1,4-dihydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one
4.2.11.8. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-(4-fluorophenyl)acetamide
(13 h). White solid, yield 60%, m.p.174–176 °C; 1H NMR (300 MHz,
CDCl3) δ: 8.04 – 7.76 (m, 3H), 7.52 (d, J = 7.3 Hz, 2H), 7.45 (d,
J = 7.8 Hz, 1H), 7.31 – 6.97 (m, 3H), 6.61 (d, J = 6.8 Hz, 2H), 5.46 (s,
1H), 3.81 (s, 3H), 2.96 (s, 2H), 2.54 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ: 170.16, 167.10, 167.03, 161.04, 156.31, 140.97, 138.70, 136.27,
135.16, 131.09, 131.02, 130.26, 130.20, 129.06, 124.46, 120.73,
120.62, 117.07, 116.51, 115.34, 115.27, 115.04, 108.82, 95.84,
55.74, 37.65, 27.19; LC-MS (ESI) m/z: 522.5 [M + H] +; Elemental
Analysis for C27H22ClFN3O3S : C, 62.01; H, 4.24; N, 8.03; Found: C,
62.03; H, 4.26; N, 8.07;
(13c). White solid, yield 64%, m.p. 152–154 °C. 1H NMR (300 MHz,
CDCl3) δ: 7.90 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.42 (d,
J = 8.3 Hz, 1H), 7.20 (d, J = 1.8 Hz, 1H), 7.03 (dd, J = 8.2, 1.9 Hz,
1H), 5.52 (t, J = 6.5 Hz, 1H), 3.89 (s, 3H), 3.47 – 3.33 (m, 4H), 2.97 (d,
J = 6.5 Hz, 2H), 2.51 (s, 3H), 2.00 – 1.89 (m, 2H), 1.86 – 1.74 (m, 2H);
13C NMR (75 MHz, CDCl3) δ:170.68, 167.71, 166.99, 160.98, 141.25,
138.62, 137.05, 130.91, 130.79, 129.22, 128.71, 123.94, 116.72,
115.64, 108.62, 95.93, 55.40, 46.13, 45.32, 36.05, 26.95, 25.53,
23.80; LC-MS (ESI) m/z: 482.5 [M + H] +; Elemental Analysis for
C
25H25ClN3O3S: C, 62.17; H, 5.22; N, 8.70; Found: C, 62.15; H, 5.22; N,
8.74;
4.2.11.4. 6-(4-chlorophenyl)-8-methoxy-1-methyl-4-(2-oxo-2-(piperidin-
1-yl)ethyl)-1,4-dihydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one
4.2.11.9. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-(prop-2-yn-1-yl)acetamide
(13i). White solid, yield 58%, m.p.142–144 °C. 1H NMR (300 MHz,
CDCl3) δ: 7.92 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.44 (d,
J = 8.2 Hz, 1H), 7.25 (s, 1H), 7.06 (d, J = 6.3 Hz, 1H), 6.32 (s, 1H),
5.40 (t, J = 6.7 Hz, 1H), 4.01 – 3.92 (m, 2H), 3.91 (s, 3H), 2.90 (d,
J = 6.7 Hz, 2H), 2.51 (s, 3H), 2.19 (s, 1H); 13C NMR (75 MHz, CDCl3) δ:
171.00, 168.01, 167.47, 161.08, 141.01, 138.56, 137.25, 132.89,
130.60, 129.24, 128.80, 123.94, 115.81, 115.70, 108.80, 95.81,
78.76, 71.16, 55.44, 37.13, 28.76, 27.03; LC-MS (ESI) m/z: 466.4
[M + H] +; Elemental Analysis for C24H21ClN3O3S : C, 61.73; H,
4.53; N, 9.00; Found: C, 61.75; H, 4.56; N, 9.04;
(13d). White solid, yield 64%, m.p. 160–162 °C. 1H NMR (300 MHz,
CDCl3) δ: 7.96 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.46 (d,
J = 8.4 Hz, 1H), 7.22 (d, J = 1.8 Hz, 1H), 7.15 (dd, J = 8.4, 1.8 Hz,
1H), 5.56 (t, J = 6.5 Hz, 1H), 3.81 (s, 3H), 3.55 – 3.36 (m, 4H), 2.94 (d,
J = 6.5 Hz, 2H), 2.53 (s, 3H), 2.14 – 1.81 (m, 4H), 1.87 – 1.76 (m, 2H);
13C NMR (75 MHz, CDCl3) δ: 169.98, 167.32, 166.35, 161.02, 141.10,
138.68, 136.22, 131.15, 130.25, 129.06, 124.42, 117.74, 116.49,
108.75, 95.94, 55.77, 45.88, 42.06, 34.44, 27.20, 25.87, 25.16,
23.88; LC-MS (ESI) m/z: 496.5 [M + H] +; Elemental Analysis for
C
26H27ClN3O3S : C, 62.83; H, 5.48; N, 8.45; Found: C, 62.79; H, 5.44; N,
8.46
4.2.11.10. N-(tert-butyl)-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-
oxo-1,4-dihydro-2H-benzo[c]-thiazolo[4,5-e]azepin-4-yl)acetamide
(13j). White solid, yield 67%; m.p.152–154 °C; 1H NMR (300 MHz,
4.2.11.5. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-cyclopropylacetamide
8

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
CDCl3) δ: 7.87 (dd, J = 8.4, 2.0 Hz, 2H), 7.48 (dd, J = 8.5, 2.1 Hz, 2H),
7.39 (dd, J = 8.2, 2.0 Hz, 1H), 7.18 (s, 1H), 7.00 (dd, J = 6.3, 2.0 Hz,
1H), 5.58 (s, 1H), 5.42 – 5.28 (m, 1H), 3.84 (d, J = 2.2 Hz, 3H), 2.78
(d, J = 5.9 Hz, 2H), 2.46 (d, J = 2.1 Hz, 3H), 1.21 (d, J = 2.2 Hz, 9H);
13C NMR (75 MHz, CDCl3) δ: 170.70, 167.62, 167.29, 161.03, 141.11,
138.61, 137.13, 132.31, 130.69, 129.20, 128.73, 123.85, 116.45,
115.71, 108.73, 95.83, 55.41, 50.82, 38.19, 28.14, 26.98; LC-MS
(ESI) m/z: 484.5 [M + H] +; Elemental Analysis for C25H27ClN3O3S:
C, 61.91; H, 5.61; N, 8.66; Found: C, 61.94; H, 5.65; N, 8.68;
NMR (300 MHz, CDCl3) δ: 7.97 (dd, J = 8.5, 1.9 Hz, 2H), 7.54 (dd,
J = 8.5, 2.0 Hz, 2H), 7.49 (dd, J = 8.2, 1.9 Hz, 1H), 7.21 (s, 1H), 7.11
(d, J = 8.3 Hz, 1H), 5.50 (s, 1H), 5.36 (t, J = 5.9 Hz, 1H), 3.86 (s, 3H),
3.17 (t, J = 10.7 Hz, 2H), 2.87 (d, J = 8.6 Hz, 2H), 2.51 (s, 3H), 1.05
(q, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 170.93, 167.95,
161.08, 141.24, 138.58, 137.28, 132.92, 130.66, 129.18, 128.85,
123.87, 115.83, 108.78, 55.42, 37.72, 34.05, 29.19, 27.06, 14.15; LC-
MS (ESI) m/z: 460.4 [M + H] +; Elemental Analysis for C23H23ClNO5S :
C, 59.93; H, 5.03; N, 3.04; Found: C, 59.95; H, 5.05; N, 3.08;
4.2.11.11. 2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-dihydro-
2H-benzo[c]thiazolo[4,5-e]azepin-4-yl)-N-(4-hydroxyphenyl)acetamide
(13 k). White solid, yield 48%, m.p.169–170 °C; 1H NMR (300 MHz,
CDCl3) δ: 8.01 – 7.77 (m, 3H), 7.48 (d, J = 7.3 Hz, 2H), 7.40 (d,
J = 7.8 Hz, 1H), 7.31 – 6.97 (m, 4H), 6.61 (d, J = 6.8 Hz, 2H), 5.45 (s,
1H), 3.85 (s, 3H), 2.95 (s, 2H), 2.50 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 170.12, 167.17, 166.37, 161.03, 153.28, 140.99, 138.70, 136.26,
131.09, 130.70, 130.45, 130.27, 129.07, 124.47, 120.71, 117.47,
4.3. Biological evaluations
4.3.1. BRD4 inhibition TR-FRET assay
The BRD4-BD1 TR-FRET Assay Kit (Item No. 600520) from Cayman
(Ann Arbor, MI, USA) was used to perform the BRD4 inhibition assay.
All assays were carried out in 384 well plates. Concentrations of com-
pounds were set as 20 μΜ, 10 μΜ, 5 μΜ, 1 μΜ, 0.5 μΜ, 0.2 μΜ, 0.1 μΜ,
0.05 μΜ and 0.01 μΜ. First, compounds (5 μL) containing assay buffer
was added into plate, BRD4-BD1 Europium Chelate in 10 μL of assay
buffer was then added into 5 μL of compounds. After a 15-minute room
temperature incubation, 5 μL of BRD4-BD1 Ligand/APC acceptor mix-
ture was added. The plates were incubated at room temperature for
60 min plates. PerkinElmer multimode plate reader with TR-FRET
technique was used to read the plates under 340 nm excitation and
670 nm emission/620 nm emission. The BRD4 inhibition rate was
calculated by the absorbance between the negative control and the
compound group. IC50 was calculated using the BRD4 inhibition rate
data.
116.53, 115.47, 115.19, 114.97, 108.81, 95.85, 55.74, 37.65, 27.20;
+
LC-MS (ESI) m/z: 520.4 [M
+
H]
; Elemental Analysis for
C
27H23ClN3O4S: C, 62.24; H, 4.45; N, 8.07; Found: C, 62.28; H, 4.48;
N, 8.12;
4.2.11.12. N-butyl-2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-1,4-
dihydro-2H-benzo[c]thiazolo-[4,5-e]azepin-4-yl)acetamide (13 l). White
solid, yield 68%; m.p.170–172 °C; 1H NMR (300 MHz, CDCl3) δ: 7.91
(d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.1 Hz, 1H),
7.22 (s, 1H), 7.05 (d, J = 6.0 Hz, 1H), 5.77 (s, 1H), 5.39 (t, J = 5.8 Hz,
1H), 3.90 (s, 3H), 3.15 (d, J = 5.6 Hz, 2H), 2.88 (d, J = 6.7 Hz, 2H),
2.51 (s, 3H), 1.33 (m, 4H), 0.96 – 0.73 (m, 3H); 13C NMR (75 MHz,
CDCl3) δ: 171.35, 168.55, 168.01, 161.53, 141.61, 139.04, 137.71,
133.12, 131.13, 129.68, 129.27, 124.34, 116.70, 116.18, 109.25,
96.30, 55.93, 39.37, 38.14, 31.42, 27.50, 19.96, 13.69; LC-MS (ESI)
m/z: 520.4 [M + H] +; Elemental Analysis for C25H27ClN3O3S: C,
61.91; H, 5.61; N, 8.66; Found: C, 61.93; H, 5.61; N, 8.69;
4.3.2. Cell growth inhibition assay
In all assays, MV-4-11 and HL-60 leukemia cells were cultured in
IMDM modified with 10% FBS and 1% penicillin–streptomycin at 37 °C
condition in Thermo CO2 incubators. In all assays, cells were collected
in the logarithmic growth phase, and 100 μL of medium containing
10,000 cells were seeded in 96-well cell culture plates per well. After
12 h of culture, 10 μL of compounds with different concentrations were
added to each cell well. After 72 h of culture, 5 μL of CCK-8 was added
into each well. After 1–4 h culture, PerkinElmer multimode plate reader
was used to read the plate at 450 nm. IC50 value was calculated by the
data using GraphPad Prism 7 software.
4.2.11.13. Ethyl
(E)-3-(4-(2-((4-chlorophenyl)(hydroxy)methyl)-4-
methoxyphenyl)-3-methyl-2-oxo-2,3-dihydrothiazol-5-yl)acrylate
(14). Compound 10 (1.20 g, 2.62 mmol) was dissolved in methanol
(40 mL) followed by adding sodium borohydride (0.20 g, 5.24 mmol)
under nitrogen protection. The reaction was stirred at room
temperature for 6 h. The reaction was quenched with saturated
4.3.3. Apoptosis and cell cycle Analysis assay
ammonium
chloride
and
extracted
with
dichloromethane
In all assays, MV-4-11 leukemia cells were cultured in IMDM
modified with 10% FBS and 1% penicillin–streptomycin at 37 °C con-
dition in Thermo CO2 incubators. Flow cytometry was used to analyze
cycle and apoptosis of cell which was treated by compounds. In this
assay, cells were collected in the logarithmic growth phase, and 2 mL of
medium containing 1 × 106 cells were seeded in 6-well cell culture
plates per well followed by adding the test compound. After 12 h of
culture, flow cytometry was used to analyze the effects of cycle and
apoptosis on cell. Annexin V-FITC Apoptosis Detection Kit and Cell
Cycle Detection Kit were obtained from KeyGEN BioTECH.
(3 × 30 mL). The organic layers were combined, washed with brine,
dried over anhydrous sodium sulfate. The organic layer was evaporated
and the residue was separated by silica gel column chromatography to
obtain target compound 14 (0.8 g) as a white solid (yield: 66%). 1H
NMR (300 MHz, DMSO‑d6) δ: 7.49–7.48 (d, J = 3 Hz, 1H), 7.26–7.23
(m, 3H), 7.05–7.02 (m, 3H), 6.76–6.71 (d, J = 15 Hz, 1H), 6.15–6.14
(d, J = 3 Hz, 1H), 5.68–5.63 (d, J = 15 Hz, 1H), 5.57–5.55 (d,
J = 6 Hz, 1H), 4.12–4.06 (q, J = 6 Hz, 2H), 3.91 (s, 3H), 2.36 (s, 3H),
1.21–1.16 (t, J = 6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ: 171.35,
168.55, 168.01, 161.53, 141.61, 139.04, 137.71, 133.12, 131.13,
129.68, 129.27, 124.34, 116.70, 116.18, 109.25, 96.30, 55.93, 39.37,
4.3.4. Western blotting
38.14, 31.42, 27.50, 19.96, 13.69; LC-MS (ESI) m/z: 484.5 [M + H] +
;
In all assays, MV-4-11 leukemia cells were cultured in IMDM
modified with 10% FBS and 1% penicillin–streptomycin at 37 °C con-
dition in Thermo CO2 incubators. Cells were collected in the loga-
rithmic growth phase, which were treated with compounds for another
4 h. The cells were lysed at 4 °C and placed in 1.5 mL centrifuge tubes to
further determine the corresponding protein concentration. A 10% se-
paration adhesive and a 4% concentration adhesive were prepared, and
the SDS-polyacrylamide gel was set in an electrophoresis tank for
sample loading, electrophoresis, and protein transfer. Subsequently, the
PVDF membrane was washed 3 times with TBST for 10 min each. The
PVDF membrane was immersed in a 5% non-fat milk TBST solution,
then placed on a shaker at room temperature, and shaken slowly for 2 h
4.2.11.14. Ethyl
2-(6-(4-chlorophenyl)-8-methoxy-1-methyl-2-oxo-
1,2,4,6-tetrahydrobenzo[5,6]oxepino-[4,3-d]thiazol-4-yl)acetate
(15). Compound 14 (0.8 g, 1.74 mmol) was dissolved in ethanol
(30 mL) followed by adding potassium carbonate (0.48 g, 3.48 mmol)
under nitrogen protection. The reaction was stirred at room
temperature for 24 h. The mixture was filtered by celite and washed
with ethyl acetate. The organic layers were combined, washed with
brine, dried over anhydrous sodium sulfate. The organic layer was
evaporated and the residue was separated by silica gel column
chromatography to obtain 3 (0.45 g) as a white solid (yield: 56%). 1H
9

Q. Li, et al.
Bioorganic&MedicinalChemistry28(2020)115601
to block non-specific protein binding sites. The PVDF membrane was
transferred overnight with the primary antibody diluent at 4 °C. After
the reaction, the PVDF membrane was washed 3 times with TBST for
10 min each. Although the secondary antibody was incubated only 2 h
before development.
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Schrödinger 2017 was used for the molecular docking assay. The
structure of BRD4 protein (PDB ID: 4O74) was obtained from the
Protein Data Bank. It was prepared, optimized and minimized using
relevant module by Schrodinger software. Receptor Grid Generation
Module was generated to dock the compound which was prepared using
the LigPrep module. Finally, the calculation was performed based on
the Schrödinger XP precision, and the structure of lowest-energy was
shown as the final result.
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Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET)
Proteins. J Med Chem. 2016;59:1634–1641.
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evaluation of novel selective BRD4-BD1 inhibitors. Bioorg Med Chem.
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Declaration of Competing Interest
17. Hewitt MC, Leblanc Y, Gehling VS, et al. Development of methyl isoxazoleazepines as
inhibitors of BET. Bioorg Med Chem Lett. 2015;25:1842–1848.
18. Albrecht BK, Gehling VS, Hewitt MC, et al. Identification of a Benzoisoxazoloazepine
Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a
Candidate for Human Clinical Trials. J Med Chem. 2016;59:1330–1339.
19. Li J, Wang P, Zhou B, et al. Development of 4,5-dihydro-benzodiazepinone deriva-
tives as a new chemical series of BRD4 inhibitors. Eur J Med Chem.
2016;121:294–299.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
20. Taylor AM, Vaswani RG, Gehling VS, et al. Discovery of Benzotriazolo[4,3-d][1,4]
diazepines as Orally Active Inhibitors of BET Bromodomains. ACS Med Chem Lett.
2016;7:145–150.
Acknowledgments
We than the National Science Foundation of China (No. 81872733
and No. 81803353) and the Natural Science Foundation of Jiangsu
Province (No. BK20191482) for support of this work.
21. Ouyang L, Zhang L, Liu J, et al. Discovery of a Small-Molecule Bromodomain-
Containing Protein 4 (BRD4) Inhibitor That Induces AMP-Activated Protein Kinase-
Modulated Autophagy-Associated Cell Death in Breast Cancer. J Med Chem.
2017;60:9990–10012.
22. Hu J, Wang Y, Li Y, et al. Structure-based optimization of a series of selective BET
inhibitors containing aniline or indoline groups. Eur J Med Chem. 2018;150:156–175.
23. Hu J, Tian CQ, Damaneh MS, et al. Structure-Based Discovery and Development of a
Series of Potent and Selective Bromodomain and Extra-Terminal Protein Inhibitors. J
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