Discovery of Novel Benzothiazepinones as Irreversible Covalent Glycogen Synthase Kinase 3β Inhibitors for the Treatment of Acute Promyelocytic Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.0c02254

Recently, irreversible inhibitors have attracted great interest in antitumors due to their advantages of forming covalent bonds to target proteins. Herein, some benzothiazepinone compounds (BTZs) have been designed and synthesized as novel covalent GSK-3β inhibitors with high selectivity for the kinase panel. The irreversible covalent binding mode was identified by kinetics and mass spectrometry, and the main labeled residue was confirmed to be the unique Cys14 that exists only in GSK-3β. The candidate 4-3 (IC50 = 6.6 μM) showed good proliferation inhibition and apoptosis-inducing ability to leukemia cell lines, low cytotoxicity on normal cell lines, and no hERG inhibition, which hinted the potential efficacy and safety. Furthermore, 4-3 exhibited decent pharmacokinetic properties in vivo and remarkably inhibited tumor growth in the acute promyelocytic leukemia (APL) mouse model. All the results suggest that these newly irreversible BTZ compounds might be useful in the treatment of cancer such as APL.

Full text of DOI:10.1021/acs.jmedchem.0c02254

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Article
Discovery of Novel Benzothiazepinones as Irreversible Covalent
Glycogen Synthase Kinase 3β Inhibitors for the Treatment of Acute
Promyelocytic Leukemia
⊥
⊥
Peng Zhang, Zhihui Min, Yang Gao, Jiang Bian, Xin Lin, Jie He, Deyong Ye, Yilin Li, Chao Peng,
Yunfeng Cheng,* and Yong Chu*
Cite This: J. Med. Chem. 2021, 64, 7341−7358
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*
sı Supporting Information
ABSTRACT: Recently, irreversible inhibitors have attracted great
interest in antitumors due to their advantages of forming covalent
bonds to target proteins. Herein, some benzothiazepinone compounds
(
BTZs) have been designed and synthesized as novel covalent GSK-3β
inhibitors with high selectivity for the kinase panel. The irreversible
covalent binding mode was identified by kinetics and mass
spectrometry, and the main labeled residue was confirmed to be the
unique Cys14 that exists only in GSK-3β. The candidate 4-3 (IC = 6.6
5
0
μM) showed good proliferation inhibition and apoptosis-inducing
ability to leukemia cell lines, low cytotoxicity on normal cell lines, and
no hERG inhibition, which hinted the potential efficacy and safety.
Furthermore, 4-3 exhibited decent pharmacokinetic properties in vivo
and remarkably inhibited tumor growth in the acute promyelocytic
leukemia (APL) mouse model. All the results suggest that these newly irreversible BTZ compounds might be useful in the treatment
of cancer such as APL.
INTRODUCTION
amino acid sequence identity in 85% overall and 98%
especially within their kinase domains. Recent studies have
confirmed that GSK-3β is the dominant subtype both
physiologically and pathologically; therefore, the research
work of GSK-3 mainly focuses on the β subtype.
■
9
The malignant tumor remains the second leading cause of
death worldwide, and aberrant protein kinase regulation is
believed to be responsible for the occurrence of various
cancers. Consequently, kinases have been very promising
targets for anticancer drugs. However, most of the reported
protein kinase inhibitors are reversible ATP competitive
inhibitors. Due to the abundance of kinases and their high
sequence conservation in the ATP-binding site, the discovery
of potential drugs with high kinase selectivity has proved
extremely challenging.
GSK-3β is critical in the regulation of transcription factors
and signaling pathways that modulates gene transcription, cell
10−14
growth, differentiation, and apoptosis.
Thus, inhibition of
GSK-3β might offer a new potential treatment for severe
1
5−18
human diseases, such as Alzheimer’s disease,
diabetes,
type II
19,20
21
22,23
chronic inflammatory disease, and cancer.
Irreversible covalent kinase inhibitors have drawn increasing
attention mainly due to their advantages over reversible non-
covalent inhibitors, including (1) higher binding affinity to
target, (2) longer interaction time with kinase, (3) higher
therapeutic index, and (4) prevention of emergence of drug
Lately, emerging data revealed that GSK-3β might be a novel
modulator of antitumor immune responses and is thought to
be a novel checkpoint regulator for cancer, which further
highlights the potential applications of GSK-3β inhibitors in
cancer therapy.
To the best of our knowledge, though lots of small
24−27
1
−3
resistance.
To date, two covalent Bruton’s tyrosine kinase
(
BTK) inhibitors of Ibrutinib and Acalabrutinib and two
molecules with different structures have been published to
covalent epidermal growth factor receptor (EGFR) inhibitors
of Afatinib and Osimertinib have been approved by the FDA in
the treatment of B-cell malignancies and non-small cell lung
carcinoma (NSCLC) (Figure 1), thus resulting in the
Received: December 29, 2020
Published: May 24, 2021
4
−7
development of more covalent kinase inhibitors.
Glycogen synthase kinase-3 (GSK-3) is a highly conserved
8
serine/threonine-protein kinase. In mammals, GSK-3 has two
highly related subtypes named GSK-3α and GSK-3β that share
©
2021 American Chemical Society
https://doi.org/10.1021/acs.jmedchem.0c02254
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Figure 1. Representative covalent kinase inhibitors approved by the FDA.
2
8
29
inhibit GSK-3β, only thiadiazolidinones (TDZDs) and
In the previous work, we found that benzothiazepinones
(BTZs) were a novel family of GSK-3β inhibitors. After that,
further investigations were performed over this skeleton to
study the influence of structural modifications on biological
activity. After an extensive study among different substituent
groups on the core BTZ ring, herein we describe a successful
strategy of introducing an electrophilic warhead into the core
skeleton to build a new type of BTZ compounds as highly
selective irreversible GSK-3β inhibitors. These new com-
pounds were biophysically identified to work in an irreversible
covalent manner and showed a good inhibitory activity to
GSK-3β at the sub-micromolar level. They also showed good
selectivity to GSK-3α and other kinases. Moreover, we assayed
and discussed the proliferation inhibition and apoptosis-
inducing ability of these compounds on some cancer and
normal cell lines. Finally, we evaluated in vivo therapeutic
efficacy in a localized leukemia xenograft mouse model. The
30,31
halomethyl ketones
as irreversible inhibitors with 1−10 μM (Figure 2). One
have been reported to inhibit GSK-3β
Figure 2. Novel irreversible GSK-3β inhibitors.
compound of TDZDs named tideglusib (NP-12) is undergoing
phase IIb clinical trials in Europe for the evaluation of curative
effects on orphan tauopathy and Alzheimer’s disease.
3
2−34
Scheme 1. Reagents and Conditions: (a) Malonic Acid, Piperidine, Pyridine, Reflux, 4 h; (b) 2-Aminothiophenol, TBAF·
H O, 80 °C, 24 h; and (c) Acrolyl Chloride, DIPEA, DCM, rt, 24 h
3
2
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Article
Figure 3. Compounds for identification of acryloyl attaching position and reactive mode.
3
6
observed results, including good features of pharmacokinetics
PKs) and hERG value, suggested that the newly irreversible
BTZ compounds might be potentially powerful candidates
with good safety for the treatment of cancer such as acute
promyelocytic leukemia (APL).
reported assay. Briefly, after co-incubation of GSK-3β,
substrate GS-2 peptide, and ATP in the presence/absence of
the test compounds, the activity was calculated based on the
unreacted ATP left in solution by the Kinase-Glo luminescent
kinase assay. The members of this new family showed good
GSK-3β inhibitory activities between 2.5 and 20 μM. Nearly all
of them except compounds substituted with groups of 4-CF3-
Ph, 4-morpholino-Ph, hydrogen, or methyl (4-10, 4-16 ∼ 4-
(
CHEMISTRY
■
The general synthesis of target compounds 4 is outlined in
Scheme 1. The intermediate acids 2-1 to 2-16 were
synthesized based on Knoevenagel condensation, in which a
mixture of propanedioic acid and different aromatic form-
aldehydes 1-1 to 1-16 was heated to reflux in the presence of a
base such as piperidine or pyridine. Compounds 2-17 and 2-18
were purchased commercially. An optimized reaction was
applied to prepare the key intermediates 3, in which cyclization
of 2-aminobenzenethiol with various acids 2 was performed at
1
8) showed the IC values for GSK-3β around 5 μM, which is
nearly equal to that of the best reported irreversible inhibitors,
such as TDZD-8.
Structure−Activity Relationship Studies. A preliminary
structure−activity relationship (SAR) was analyzed according
to the preceding in vitro data (Table 1). The acryloyl group
substituted in the N5 position of the BTZ ring seemed to be
critical for activity retention because the acryloyl moiety could
act as a Michael addition acceptor to form a covalent bond
with a specific cysteine residue of the targeted protein (4-1 vs
50
8
0 °C without any solvent but tetra-n-butylammonium fluoride
35
(
TBAF). Finally, benzothiazepinone analogues 3-1 to 3-18
5
, 6, and 7). Besides, the nature and size of C2-R groups
should also be crucial for activity. The inhibition dramatically
were successfully obtained from recrystallization in accepted
yields. Based on the acylation of 3 with acryloyl chloride
catalyzed by DIPEA, the object BTZ compounds 4 were
obtained, in which the acryloyl group was introduced to the
framework in 5-position.
Table 1. Inhibition of BTZs to GSK-3β and Cancer Cells
Molt4
HL60
GSK-3β a NB4 IC
IC
IC
Raji IC
50
c
5
0
50
(μM)
50
(μM)
c
c
c
compd.
IC50 (μM)
(μM)
(μM)
RESULTS AND DISCUSSION
■
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
-1
-2
-3
-4
-5
-6
-7
-8
4.9
6.6
6.6
6.7
4.0
2.5
6.4
4.8
6.2
15
2.8
6.0
7.5
6.2
5.8
15
765.3
556.72
85.92
75.58
82.36
77.52
80.27
106.35
87.61
83.16
76.31
84.43
79.64
91.50
88.72
97.54
65.03
565.4
729.84
695.47
97.38
86.3
659.35
74.05
28.45
18.39
47.06
50.39
51.28
75.53
43.25
47.60
50.28
89.56
34.84
84.50
56.34
56.25
581.36
837.05
Design and Identification of BTZ Compounds as
Potent GSK-3β Inhibitors. Because the acryloyl group is a
good warhead used in covalent drugs, we introduce it into our
BTZ skeleton to make an irreversible mode (Figure 3). We
first synthesized compounds 4-1, 6, and 7 to check the best
binding position. Both compounds 6 and 7 showed no
inhibition to GSK-3β at even 100 μM concentration, while 4-1
showed good inhibition at an IC50 value of 4.9 μM, which
suggested that the direct introduction of the acryloyl group at
the 5-position of the BTZ ring should be very crucial to
maintain the inhibitory activity. To further examine the
influence mode of the acryloyl moiety in 4-1, compound 5
with the corresponding saturated propionyl moiety was
synthesized. The biological test showed that no GSK-3β
inhibitory activity was observed for compound 5 at even a high
concentration of 100 μM, which indicated that the covalent
reaction could happen at the carbon−carbon double bond of
the acryloyl group in compound 4-1.
35.52
19.56
22.62
31.65
33.84
59.37
37.28
32.94
38.55
35.21
24.41
37.65
62.37
57.68
42.37
22.37
226.51
95.71
93.14
91.43
126.04
94.35
92.07
106.04
96.38
101.75
106.87
115.06
117.66
97.95
44.76
198.52
-9
-10
-11
-12
-13
-14
-15
-16
-17
-18
17
20
1.4
b
TDZD-8
Based on that results, we designed and synthesized some
analogues of 4-1, in which a variety of aryl, substituted aryl,
and alkyl groups were substituted in the 2-position of the BTZ
ring (Scheme 1). The GSK-3β inhibitory activities of the newly
prepared BTZ compounds were evaluated by a non-radioactive
method described in our previous work according to the
a
IC , the mean value of at least two separate determinations, each
determination was means of triplicate experiments. TDZD-8, the
reference compound. IC , the mean value of at least three
independent experiments, each determination was means of triplicate
experiments.
5
0
b
c
5
0
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Table 2. GSK-3α Inhibitory Activity of BTZ Compounds at 100 μM
compd.
4-3
4-4
4-6
4-7
4-8
4-10
20.0
4-11
32.0
4-13
50.7
4-14
92.9
4-15
13.4
%
inhibition
87.3
46.7
61.8
33.4
30.0
a
Table 3. Inhibitory Activity (% Inhibition) of Representative Compounds at 100 μM against Several Protein Kinases
b
c
d
e
compd.
CK2
PKA
Flt-1
KDR
4-1
4-2
4-4
Su11248
BBIBW2992
Dasatinib
AP24534 (nM)
0
22
0
3.1
18.8
26.3
0
7.8
15.4
21.9
0


1.7
0
8
7
7.4
0
88.3
14
11.1
0
5.5
1
73.9
0
12.2
53.8










89

88.1









94.8



1.7
0.7










74.8

FGFR1
c-Kit
0

26.9
6.5
2.7
13.0
0
0
0
0
39.8
49.4
71.4
70.6

PDGFR-α
PDGFR-β
EPH-A2
EGFR
RET
ErbB4
C-Src
ABL

97
0
0
0


94
93.2


a
b
Corresponding data is the IC50 value and the symbol “″ indicates no test operated. Su11248 has been used as a reference inhibitor for
c
d
PDGFR-β, KDR, Flt-1, and c-Kit. BIBW2992 has been used as a reference inhibitor for ErbB4 and EGFR. Dasatinib has been used as a reference
e
inhibitor for C-Src, ABL, and EPH-A2. AP24534 has been used as a reference inhibitor for FGFR1 and KDR.
decreased (4-17 and 4-18) as the R is hydrogen or methyl,
while increased greatly when aromatic groups were introduced
into. Both electron-donating and electron-withdrawing groups
substituted at the aromatic ring could inhibit the enzymatic
activity at the low-micromole level (<10 μM) except the
morpholino substituent, suggesting that a relatively large
hydrophobic pocket could exist in the enzyme.
Scheme 2. Reagents and Conditions: MeOH, H O, rt, 24 h,
Yield: 64%
2
Validation of Selectivity. Selectivity is very important for
kinase inhibitor development. Recent studies have suggested
that toxicities may be linked to the inhibition of both GSK3
paralogs. To examine the selectivity of these new BTZs to
GSK-3 isoforms, they were tested against GSK-3α by mobility
shift assay at ChemPartner (Shanghai, China). The results
clearly showed that the peaks of two hydrogens from olefin in
4
-2 disappeared in the spectrum of compound 8 (Figure 4).
As a representative, the kinetic features of compound 4-2
(Table 2) indicated that most of them showed little inhibitory
activity even at a very high concentration of 100 μM, which
suggested their good sub-type specificity to GSK-3.
were studied for exploration of the mechanism of these new
BTZ derivatives. First, it was checked whether a competitive
relationship exists between 4-2 and ATP. The measurement
was carried out for 4-2 to inhibit the enzyme at 2 and 4 μM
separately while keeping the GS-2 concentration constant with
different ATP concentrations. The results showed that 4-2 acts
in a non-ATP competitive manner (Figure 5A). Second,
similar tests were performed to see if 4-2 competes with GS-2.
Its inhibition was measured at the same concentrations of 2
and 4 μM separately, while in turn keeping the ATP
concentration constant with different GS-2 concentrations.
The results indicate that compound 4-2 acts in a non-GS-2
competitive manner (Figure 5B).
The interaction characteristics between 4-2 and the target
enzyme were further investigated. Normally, different
incubation times do not affect the inhibition of reversible
inhibitors, but for an irreversible inhibitor, its inhibition
percentage increases as the co-incubation time with the
enzyme increases. As shown in Figure 5C, the inhibition
increased with more pre-incubation time, which indicates 4-2
irreversibly inhibiting GSK-3β.
Three key compounds were further evaluated for their
selectivity on 14 tyrosine and serine/threonine kinases, which
are key regulatory factors in the signaling process of tumor
pathogenesis. A high concentration of 100 μM was applied for
three compounds in all tests, which were performed by ELISA
for tyrosine kinases and by Kinase Profiler service of Millipore
Corporation (Dundee, UK) for serine/threonine kinases (CK2
and PKA), respectively. The results (Table 3) showed that the
selected compounds nearly displayed no inhibition to the
whole tested kinases. All results to some extent demonstrate
that these BTZ compounds might be highly selective to GSK-
3
β as we had expected.
Validation of Target Engagement and Binding
Mechanism. To test the reactivity of BTZ compounds to
thiol groups, we analyzed the reaction of 4-2 with 2-
mercaptoethanol by stirring them at room temperature in a
mixture of MeOH and H O for 24 h (Scheme 2). 2-
2
Mercaptoethanol is a simple compound that could mimic the
reactivity of cysteine. The reaction afforded a 64% yield of the
To confirm the binding mode, the mass spectrometry (MS)
analyses proceeded after co-incubation of compound 4-3 with
1
S-alkylated product 8, which was identified by H NMR. It
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1
Figure 4. H NMR spectra of compounds 4-2 and 8.
Figure 5. Double-reciprocal plot of kinetic data for the GSK-3β assay at different 4-2 concentrations. (A) ATP concentrations varied from 0.5 to 8
μM in the final reaction mixture. The GS-2 concentration was kept at 6.25 μM constant in all of the tests. (B) GS-2 concentrations varied from 0.78
to 12.5 μM in the final reaction mixture. The ATP concentration was kept 2 μM constant in all of the tests. (C) Time-dependent inhibition to
GSK-3β for 4-2. Each point was the mean of two separate experiments, which were performed in triplicate.
the GSK-3β enzyme under the same condition for activity test.
According to mass results, only the mass corresponding to
GSK-3β (48919.9907 Da) was detected when using only pure
enzyme without 4-3, whereas it showed a mass peak of
Therefore, we chose NB4 and NB4-R1 cells as the subject of
further study for three candidates of 4-3, 4-4, and 4-12.
Similarly, various cell lines were exposed to different
concentrations of 4-3, 4-4, and 4-12 (0−100 μM) for 24−48
h. Cell viability was determined by the CCK-8 assay. All the
three inhibitors showed marked growth inhibition to NB4 and
NB4-R1 cells in a dose-dependent manner (Figure 8A,B).
With the increase of concentrations, the inhibition activity of
4-3 was higher than that of the other two inhibitors 4-4 and 4-
12 with IC of 19.56, 22.62, and 24.41 μM in NB4 cells at 24
4
9247.9014 Da after adding inhibitor 4-3, which confirms the
formation of a covalent bond between 4-3 and the enzyme
(
Figure 6).
Subsequent protease digestion and MS/MS experiments
identified the modified peptides and localized the sites of
modification predominately to Cys14 and slightly to Cys199
5
0
(Figure 7). Cumulatively, these results provide strong evidence
h and with IC of 26.42, 48.27, and 93.06 μM in NB4-R1 cells
50
that 4-3 is an irreversible, covalent inhibitor and that unique
Cys14 of GSK-3β is the main labeled site.
at 24 h, respectively, which suggested that 4-3 displayed the
strongest proliferation inhibitory effects on both NB4 and
NB4-R1 cells. Furthermore, the proliferation inhibitory effects
of 4-3 were not significantly different at 24 and 48 h in NB4
(Figure 8C) and NB4-R1 cells (Figure 8D), respectively.
Accordingly, we chose 24 h as a subsequent time point.
For evaluating the possible cytotoxicity, we examined the
affection of the three inhibitors to human normal liver cells
Validation in Cells. Inspired by the good activity and
selectivity of BTZs to the enzyme, especially the irreversible
action mode shown in these compounds, we further conducted
a cell-based screening to evaluate their effects on the viability
of cancer cells. Various patient-derived cell lines were exposed
to GSK-3β inhibitors in different concentrations (0−1000 μM)
for 24 h. The values of IC50 were determined by the CCK-8
assay. The results listed in Table 1 showed that these
compounds inhibited NB4 cells greater than the other three
cell lines such as Molt4, HL-60, and lymphoma cell Raji.
(LO ) and human umbilical vein endothelial cells (HUVECs).
2
The data exhibited that the viability percentages of the two cell
lines were over 80% compared to the control after incubated
with three compounds (0−50 μM) for 24 h (Figure 8E,F).
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Figure 6. TOF MS spectra. (A) Pure GSK-3β enzyme (MW = 48919.99) without 4-3 as a control showed a 48919.9907 mass peak. (B) GSK-3β
with inhibitor 4-3 (MW = 327.37) showed a 49247.9014 mass peak corresponding to the C−S bond covalently formed between inhibitor 4-3 and
the GSK-3β enzyme.
Figure 7. Higher-energy collisional dissociation MS/MS spectrum of GSK-3β peptide-modified 4-3. The above listed are predicted b- and y-type
ions (not including all) and below listed is the peptide sequence. C* means the cysteine labeled by 4-3. (A) The data was recorded on the [M +
2
+
H] ion at m/z 1279.5638 of the GSK-3β peptide TTSFAES(C*)KPVQQPSAFGSMK (residues 7−27) harboring a modification(C H FNO S)
1
8
14
2
3
+
on Cys14. The spec count value is 12. (B) The data was recorded on the [M + H] ion at m/z 1014.5118 of the GSK-3β peptide
DIKPQNLLLDPDTAVLKL(C*)DFGSAK (residues 181−205) harboring a modification (C H FNO S) on Cys199. The spec count value is 1.
1
8
14
2
When the inhibition concentration reached 100 μM, there still
had above 70% cell viability for 4-3 in both HUVEC and LO2
cells, which indicated that 4-3 had little cytotoxicity.
Next, apoptotic cells were determined by Annexin V/
propidium iodide (PI) staining and flow cytometric analysis.
The percentage of apoptosis in NB4 and NB4-R1 cells treated
with three inhibitors for 24 h was all significantly increased in a
dose-dependent pattern when compared to the control with p
indicated 4-3 processing, the apoptosis percentages of NB4
cells were obviously reduced (about 2−4 fold change)
compared with the corresponding cells that were unpretreated
(14.56% ± 1.15 vs 11.95% ± 0.97 at 12.5 μM, p < 0.05,
48.70% ± 2.58 vs 18.62% ± 2.75 at 25 μM, 81.40% ± 3.52 vs
19.88% ± 2.26 at 50 μM, and 90.60% ± 1.55 vs 24.98% ± 3.04
at 100 μM, p < 0.01, respectively). A similar tendency was
observed in NB4-R1 cells, which also presented a marked
reduction of apoptosis percentages when pretreated for Z-
VAD-FMK at the same time (Figure 9E,F). Thus, the results
confirmed that caspase participated in the 4-3-induced cell
apoptosis.
<
0.01, and the highest percentage of apoptosis among the
three inhibitors was promoted by 4-3 (Figure 9A,B).
Moreover, the percentage of apoptosis induced by 4-3 in
NB4 cells was higher than that in NB4-R1 cells, which was
consistent with the CCK-8 result above.
To assess whether caspase would take part in the apoptosis
induced by 4-3, Z-VAD-FMK, an irreversible pan-caspase
inhibitor with good cell permeability was selected to block
caspase activation of apoptotic cells. As shown in Figure 9C,D,
pretreated with 10 μM of Z-VAD-FMK for 4 h before the
In summary, candidate 4-3 produced stronger growth
inhibition to NB4 and NB4-R1 cells than other cell lines
such as Molt4, HL-60, and Raji cells (Figure 10A). As is well
known, lithium chloride (LiCl), as a classical GSK-3β inhibitor,
had been much reported to have potent anti-leukemic activity
37
in MLL-associated leukemia and in AML- or RA-sensitive
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Figure 8. Proliferation inhibition of three GSK-3β inhibitors in NB4 and NB4-R1 cells and their cytotoxicity to normal cell lines by the CCK-8
assay. (A) Proliferation inhibition rate of NB4 cells after exposure to three inhibitors at various concentrations for 24 h. (B) Proliferation inhibition
rate of NB4-R1 cells after exposure to three inhibitors at various concentrations for 24 h. Inhibition rate of NB4 cells (C) and NB4-R1 cells (D)
after exposure to 4-3 at various concentrations for 24 and 48 h. Viability of two normal cell lines HUVECs (E) and LO cells (F) after exposure to
2
three inhibitors at various concentrations for 24 h. Data are presented as mean ± standard error of the mean (SEM) of three independent
experiments and significant differences are indicated as *P < 0.01 vs control.
3
8
APL cells. Under the same conditions, we assessed the
inhibition percentages of LiCl to NB4 and NB4-R1 cells by the
CCK-8 test. Compared with the control group, LiCl showed
more significant inhibitory effects on NB4 cells than on NB4-
R1 cells, and the IC50 value at 24 h was 5.32 mM and 42.50
mM for NB4 and NB4-R1 cells, respectively (Figure 10B).
Exhilaratingly, the inhibition of 4-3 is stronger than LiCl 270-
fold on NB4 and 1600-fold on NB4-R1 cells, respectively,
indicating that 4-3 might be a potentially powerful candidate
for leukemia treatment.
To assign the observed results to GSK-3β, 4-3 was treated
with NB4 and NB4-R1 cells to check its effects on glycogen
synthase (GS) phosphorylation. GS is a well-known down-
stream physiological target that can be directly phosphorylated
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Figure 9. Induction of cell apoptosis by three inhibitors of GSK-3β. Apoptosis of NB4 cells (A) and NB4-R1 cells (B) exposed to different
concentrations of three inhibitors for 24 h. Apoptotic cells were detected by a flow cytometer using Annexin V/PI double staining. (C)
Representative scatter plot for a flow cytometer. 4-3 induced apoptosis in NB4 cells in combination with Z-VAD-FMK (irreversible pan-caspase
inhibitor) in a dose-dependent manner. (D) Percentages of apoptotic NB4 cells treated with indicated inhibitor 4-3 or in combination with Z-
VAD-FMK for 24 h. (E,F) Representative scatter plots for flow cytometer in NB4-R1 cells. Apoptosis of NB4-R1 cells exposed to different
concentrations of 4-3 or in combination with Z-VAD-FMK for 24 h. The data shown are representative of values from at least three independent
experiments (mean ± SEM; ** P < 0.05; * P < 0.01 vs control).
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Figure 10. Cell proliferation and viability of 4-3 and LiCl. (A) IC50 of 4-3 for several tumor cells. (B) Proliferation inhibition of NB4 and NB4-
R1cells exposed to different concentrations of LiCl for 24 h by the CCK-8 assay. The data shown are representative of values from at least three
independent experiments (mean ± SEM; *, P < 0.01 vs control).
Figure 11. (A) The western blot analysis of p-GS and GS expression of NB4 and NB4-R1 cells was obtained after 24 h of treatment with 4-3, and
the representative bands were shown. (B) Values denoted the relative protein intensity as ratios of p-GS to GS. Data were represented as means ±
SEM (n = 3), and significant differences were indicated as **, P < 0.05; *, P < 0.01.
by GSK-3β and thereby inactivated. The phospho-GS (p-GS)
was detected by western blotting after 4-3 co-incubated with
cells for 24 h (Figure 11A). The bands were quantified by gray
analysis, and the data showed that 4-3 decreased the ratio of p-
GS/GS both in two cells in a dose-dependent manner (Figure
The APL mouse model was selected for the PD study. Balb/
c nude mice bearing subcutaneous xenografts of NB4 cells
were treated with 4-3 (15 mg/kg/d) for 2 weeks, and the
effects of 4-3 on the growth of xenografts of human APL NB4
cells were evaluated. The results showed that 4-3 inhibited
tumor growth by 75.97% relative to vehicle control (Figure
1
1B), indicating that the inhibition was produced by targeting
12A). In addition, the mice treated with 4-3 had mild weight
GSK-3β in both cells.
loss compared with control (Figure 12B). The treated mice
were killed at the end of therapy, and dissection of tumor tissue
displayed that 4-3-treated mice had much smaller tumor
masses compared with control (Figure 12C). The gross tumor
tissues were presented in Figure 12D. These results indicated
that GSK-3β inhibition by 4-3 had antitumor activity and
therapeutic efficacy in localized leukemia xenograft models.
In the hERG assay, the calculated IC value of 4-3 was 37.5
50
μM, and it suggests that 4-3 is not an inhibitor to the hERG
channel showing good heart safety (Supporting Information,
Figure S1).
Mouse PK Properties and Pharmacodynamic Studies.
Based on its good activity in the cellular assay and unique
mode of action, compound 4-3 was further profiled in vivo PKs
and pharmacodynamics (PDs). After intraperitoneal injection
CONCLUSIONS
■
(
i.p.) of 4-3 at a dose of 15 mg/kg in mice, the compound
Irreversible GSK-3β covalent inhibitors should be clinically
more therapeutic benefits to overcome the resistance. Herein,
we found a new kind of irreversible GSK-3β inhibitors with the
advantage of high selectivity over kinases. Their IC50 values
were mostly around 5 μM, which is comparable to the well-
known covalent GSK-3β inhibitor TDZD-8 undergoing clinical
trials. The covalent binding mode was confirmed biophysically
by kinetic and MS studies, where incubation of recombinant
GSK-3β kinase with 4-3 resulted in an addition of the expected
molecular weight. Furthermore, the labeled cysteines of Cys14
and Cys199 in GSK-3β were identified by MS/MS analysis.
Subsequently, we investigated the inhibitors for their effects
on human APL cell lines followed in a xenograft model.
demonstrated good PK properties (Figure S2), with long T1/2
of 14.2 h, high AUC values, and maximum concentration
(Cmax) was reached quickly at 1.0 h (Table 4).
Table 4. PK Parameters of 4-3 Following a Single
Administration to Mice
dose
T
C
AUC
AUC
inf
(ng/mL·h)
T1/2
d
max
max
(ng/mL)
last
c
a
b
(
mg/kg) route (h)
(ng/mL·h)
(h)
1
5
i.p.
1
515
3503.42
10838.33
14.2
a
b
Time for peak plasma concentrations. Peak plasma concentrations.
c
d
Area under the concentration−time curve. Terminal half-life.
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Figure 12. In vivo activity of 4-3. (A). 4-3 inhibition of localized growth in NB4 cells. NB4 cells were transplanted into the right flanks of nude
mice, which were intraperitoneally injected daily with 4-3 (15 mg/kg/d) for 2 weeks. Tumor volumes were measured (mean ± SEM, n = 5; *, P <
0
.01 vs control). (B). The body weight of mice recorded during the treatment period. Data were recorded as the mean ± SEM, n = 5; **, P < 0.05
vs control. (C). Weights of tumors dissected from mice treated with 4-3 or control (mean ± SEM, n = 5; *, P < 0.01 vs control). (D). The gross
subcutaneous xenografts of NB4 cells were determined.
Interestingly, our inhibitors exerted anti-proliferation and
inducing apoptosis not only on ATRA-sensitive APL cells
but also on ATRA-resistant cells in a dose-dependent manner
with less cytotoxicity in cells. The western blotting analysis
assigned the cellular activity to the GSK-3β inhibition. The
potential mechanism of inducing apoptosis possibly could be
related to the caspase cascade, which confirmed a previous
study of ATP-competitive GSK-3β inhibitor on B- and T-ALL
cell lines. Among the inhibitors, compound 4-3 showed
much more sensitivity to NB4 and NB4-R1 cells than to
Molt4, HL-60, and Raji cells. Therefore, we selected NB4 and
NB4-R1 cells as the main subject for further study.
were close to or even higher than that of classical As O -
2 3
42
induced NB4 cells reported in previous study and particularly
43
on ATRA-resistant NB4-R1 cells. Moreover, 4-3 had good
PK features in mice with rapid onset, high exposure, and long
duration. In APL xenograft nude mice, 4-3 developed lower
tumor volume than control and no significant weight loss.
Moreover, 4-3 showed low cytotoxicity on normal cell lines
and no inhibition in the hERG test. All the results illustrated
the potential safety and therapeutic efficacy of 4-3 in leukemia.
Since 4-3 represents a new scaffold and interaction unreported
before, it should be a promising candidate to offer more
therapeutic possibilities due to the advantages of irreversible
covalent inhibition, such as high activity, good safety, and high
selectivity.
3
9
LiCl is a classical GSK-3β inhibitor with potent anti-
37
leukemic activity in MLL-associated leukemia and in AML-
38
or RA-sensitive APL cells, but much serious side effects from
GSK-3 has two highly conserved isoforms α and β. Recent
studies suggested that toxicities may be linked to the inhibition
of both GSK3 paralogs. In our study, 4-3 was confirmed to
bind mainly to the unique residues of Cys14 and slightly to
Cys199 of GSK-3β in covalent modes with spec count values
of 12 and 1, respectively. It roughly indicated that the binding
40,41
high-dosage use limited its clinical application.
Our results
confirmed that 4-3 could depress NB4 and NB4-R1 cell
proliferation at low μM level while LiCl worked at high mM
level, suggesting that 4-3 is much superior to LiCl in the
treatment of leukemia. Moreover, the benefits from 4-3 in cells
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of 4-3 on Cys14 is 12-fold stronger than that on Cys199.
Because the unique Cys14 residue only exists in β isoform and
not in α isoform for GSK-3, and the Cys199 residue was also
replaced by other residues in other structurally related kinases,
such as the CDK family, the specific binding mechanism to
Cys14 and Cys199 well explained the selectivity of 4-3 to both
GSK-3α and other related kinases in our test. These results can
also be expected for the high selectivity of other BTZ
compounds, which had been partly confirmed by the measured
results.
3-Phenylacrylic Acid (2-2). Prepared using the general procedure
starting from benzaldehyde. White solid. Yield: 91%. mp: 134.6−
135.7 °C.
3
-(4-Fluorophenyl)acrylic Acid (2-3). Prepared using the general
procedure starting from 4-fluorobenzaldehyde. White solid. Yield:
8%. mp 205.8−207.4 °C.
-(4-Chlorophenyl)acrylic Acid (2-4). Prepared using the general
8
3
procedure starting from 4-chlorobenzaldehyde. White solid. Yield:
95%. mp 247.1−250.5 °C.
3-(3-Chlorophenyl)acrylic Acid (2-5). Prepared using the general
procedure starting from 3-chlorobenzaldehyde. White solid. Yield:
9
4%. mp 179.1−182.5 °C.
-(2-Chlorophenyl)acrylic Acid (2-6). Prepared using the general
procedure starting from 2-chlorobenzaldehyde. White solid. Yield:
3%. mp 212.5−214.2 °C.
-(4-Bromophenyl)acrylic Acid (2-7). Prepared using the general
Recently, computational tools have emerged to accelerate
the rational discovery of new inhibitors. Wagner rationally
designed the isoform selectivity based on a single amino acid
difference in the ATP-binding domain of GSK-3α (Glu196)
3
9
3
44
and GSK-3β (Asp133). Liu proposed a strategy based on the
procedure starting from 4-bromobenzaldehyde. White solid. Yield:
85%. mp 255.9−257.6 °C.
3-(4-Methylphenyl)acrylic Acid (2-8). Prepared using the general
procedure starting from 4-methyl benzaldehyde. White solid. Yield:
assessment of pK values to screen reactive amino acid residues
a
45
for designing targeted covalent kinase inhibitors. These
studies demonstrate the feasibility of isoform-selective
inhibition. According to that way, much work is worth doing
in our future study, such as confirming the functional
selectivity or activity of Cys14 and Cys199 residues via point
mutations, careful assessments of the reactivities for targetable
residues in GSK-3 by calculating their pKa values, and
prediction of the binding modes through docking or molecular
dynamics simulations. The deep understanding of the
biochemical functions, topological features, and interaction
modes would help to bind specifically to Cys14 or other
unique cysteine residues by fine modification of the structures
and reactivities of our compounds, which can predictably
improve the paralog selectivity and off-target inhibition against
other kinases.
9
1%. mp 208.2−211.3 °C.
-(4-Methoxyphenyl)acrylic Acid (2-9). Prepared using the general
procedure starting from 4-methoxybenzaldehyde. White solid. Yield:
3%. mp 218.9−220.5 °C.
-(4-Trifluoromethylphenyl)acrylic Acid (2-10). Prepared using
3
9
3
the general procedure starting from 4-trifluoromethylbenzaldehyde.
White solid. Yield: 88%. mp 208.9−210.6 °C.
3-(2-Trifluoromethylphenyl)acrylic Acid (2-11). Prepared using
the general procedure starting from 2-trifluoromethylbenzaldehyde.
White solid. Yield: 91%. mp 205.4−206.2 °C.
3
-(4-Methoxycarbonylphenyl)acrylic Acid (2-12). Prepared using
the general procedure starting from 4-trifluoromethylbenzaldehyde.
White solid. Yield: 75%. mp 246.2−247.9 °C.
3
-(4-Nitrophenyl)acrylic Acid (2-13). Prepared using the general
procedure starting from 4-nitrobenzaldehyde. White solid. Yield: 83%.
mp 287.5−290.6 °C.
3
-(4-Cyanophenyl)acrylic Acid (2-14). Prepared using the general
EXPERIMENTAL SECTION
■
procedure starting from 4-cyanobenzaldehyde. White solid. Yield:
7%. mp 253.3−255.8 °C.
3
Chemistry. All chemical reagents and solvents used in experiments
were purchased from commercial suppliers and used without further
purification. The silica gel (200−300 mesh) used for column
chromatography and the thin-layer chromatograph used to monitor
the progress of the reaction were purchased from Qingdao Haiyang
8
-([1,1′-Biphenyl]-4-yl)acrylic Acid (2-15). Prepared using the
general procedure starting from [1,1′-biphenyl]-4-carbaldehyde.
White solid. Yield: 75%. mp 224.6−227.8 °C.
3
-(4-Morpholinophenyl)acrylic Acid (2-16). Prepared using the
1
Chemical Co., Ltd. H NMR spectra were performed on the Mercury
general procedure starting from 4-morpholinobenzaldehyde. White
solid. Yield: 75%. mp 244.7−246.3 °C.
1
3
Plus 400 spectrometer at 400 MHz and C NMR spectra at 100 or
51 MHz, respectively. Chemical shifts (δ) are reported in parts per
1
General Procedure for the Synthesis of 2,3-Dihydro-2-sub-
stituted-1,5-benzothiazepin-4(5H)-one. A mixture of 2-aminothio-
phenol (4 mmol), unsaturated acid 2 (2 mmoL), and TBAF (0.2
mmol, 10 mol %) was heated to 80 °C under stirring for 24 h. After
cooling, 30 mL of dichloromethane was added. The mixture was
washed with aq HCl (2 N), aq Na CO (saturated), and brine. Then,
million (ppm) relative to internal tetramethylsilane, and J values are
reported in hertz. The low-resolution mass spectra (LC−MS) were
recorded using the Agilent LC−MS 1100 instrument with an ESI
mass selective detector. HRMS (EI) experiments were performed
using an AB 5600+ Q-TOF spectrometer. Melting points were
determined by the SGW X-4 thermometer and were uncorrected. The
purity of final compounds was analyzed by high-performance liquid
chromatography (HPLC) (Hewlett-Packard 1100), with the purity of
all compounds being over 95%. The HPLC instrument was equipped
with a photodiode array detector using a Zorbax RP-18 column (5
μm, 4.6 mm × 250 mm, reverse phase column). The mobile phases A
and B were acetonitrile and the solution of 10 mM ammonium acetate
in purified water (pH = 6.0), respectively.
General Procedure for the Synthesis of 3-Substituted Acrylic
Acid. A mixture of substituted carbaldehyde (200 mmol),
propenedioic acid (20.8 g, 200 mmol) in a solution of pyridine (10
mL 120 mmol), and piperidine (1 mL) was warmed at reflux for 2 h.
The resultant solution was poured into 2 M HCl aq. and then cooled
to room temperature. The present solid was collected by filtration,
washed with water, and recrystallized from ethanol/water.
2
3
the organic layer was dried over MgSO and concentrated. The crude
product residue was purified by chromatography on silica gel to afford
pure product 3.
2,3-Dihydro-2-(2-thienyl)-1,5-benzothiazepin-4(5H)-one (3-1).
Prepared using the general procedure starting from 3-(2-thienyl)-
4
acrylic acid (2-1). Yield: 70%. Off-white solid. mp 172.6−173.4 °C.
1
H NMR (CDCl ): δ 8.18 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.45−
3
7.41 (m, 1H), 7.25−7.19 (m, 4H), 6.95−6.91 (m, 2H), 5.16 (dd, J =
1
3
8.0 Hz, 4.0 Hz, 1H), 2.94−2.82 (m, 2H). C NMR (100 MHz,
CDCl ): δ 171.1, 146.5, 135.8, 129.9, 126.2, 126.1, 125.1, 124.1,
3
123.4, 122.5, 76.6, 76.4, 76.2, 47.9, 41.6, 29.1. ESI-MS (positive),
262.0 (M + 1) . HRMS (EI) m/z: calcd for C H NOS (M ),
262.0355; found, 262.0367.
2,3-Dihydro-2-phenyl-1,5-benzothiazepin-4(5H)-one (3-2). Pre-
+
+
1
3
11
2
pared using the general procedure starting from 3-phenylacrylic acid
1
3
-(2-Thienyl)acrylic Acid (2-1). Prepared using the general
procedure starting from 2-thenaldehyde. Yellow solid. Yield: 85%.
mp 158.2−159.6 °C.
(2-2). Yield: 82%. White solid. mp 177.6−179.9 °C. H NMR
(CDCl ): δ 8.30 (s, 1H), 7.42−7.17 (m, 9H), 4.87−4.91 (m, 1H),
3
2.94−2.79 (m, 2H). ESI-MS (positive), 256.1 (M + 1).
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1
2
,3-Dihydro-2-(4-fluorophenyl)-1,5-benzothiazepin-4(5H)-one
solid. mp 137.9−139.7 °C. H NMR (DMSO-d ): δ 8.35 (s, 1H),
6
(
3-3). Prepared using the general procedure starting from 3-(4-
7.66 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 14.4,
7.7 Hz, 3H), 7.30−7.16 (m, 2H), 4.92 (dd, J = 10.7, 5.9 Hz, 1H),
fluorophenyl)acrylic acid (2-3). Yield: 55%. White solid. mp 182.9−
1
1
3
1
1
5
84.0 °C. H NMR (CDCl ): δ 9.99 (s, 1H), 7.54 (d, J = 8.0 Hz,
3
2.96−2.75 (m, 2H). C NMR (100 MHz, CDCl ): δ 171.2, 146.4,
3
H), 7.47−7.43 (m, 1H), 7.39−7.35 (m, 2H), 7.21−7.11 (m, 4H),
1
40.7, 135.3, 129.9, 126.3, 126.2, 125.4, 125.2, 122.8, 51.9, 40.5. ESI-
1
3
.01 (dd, J = 8.0, 4.0 Hz, 1H), 2.69−2.48 (m, 2H). C NMR (100
+
MS (positive), 323.9 (M + 1) . HRMS (EI) m/z: calcd for
C H F NOS (M ), 324.0664; found, 324.0661.
,3-Dihydro-2-(2-trifluoromethylphenyl)-1,5-benzothiazepin-
(5H)-one (3-11). Prepared using the general procedure starting from
MHz, CDCl ): δ 171.5, 162.4, 160.8, 140.7, 138.5, 135.2, 129.7,
+
3
1
6
12 3
1
27.6, 127.5, 126.2, 125.8, 122.7, 115.1, 114.9, 51.8, 40.9, 29.1. ESI-
MS (positive), 273.9 (M + 1) . HRMS (EI) m/z: calcd for
2
+
4
+
C H FNOS (M ), 274.0696; found, 274.0696.
1
5
2
12
3-(2-trifluoromethylphenyl)acrylic acid (2-11). Yield: 80%. Off-white
,3-Dihydro-2-(4-chlorophenyl)-1,5-benzothiazepin-4(5H)-one
1
solid. mp 131.2−134.2 °C. H NMR (DMSO-d ): δ 8.43 (s, 1H),
6
(
3-4). Prepared using the general procedure starting from 3-(4-
7
.63 (d, J = 7.5 Hz, 1H), 7.45−7.42 (m, 2H), 7.37 (dd, J = 7.1, 2.0
chlorophenyl)acrylic acid (2-4). Yield: 71%. White solid. mp 205.4−
Hz, 1H), 7.25−7.18 (m, 4H), 5.45 (dd, J = 10.5, 6.3 Hz, 1H), 2.90−
1
2
5
(
1
07.9 °C. H NMR (CDCl ): δ 10.00 (s, 1H), 7.55−7.14 (m, 8H),
13
3
2
1
4
.78 (m, 2H). C NMR (100 MHz, CDCl ): δ 171.4, 140.9, 139.6,
1
3
3
.01 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 2.70−2.48 (m, 2H). C NMR
35.4, 131.6, 129.7, 129.2, 128.2, 126.9, 126.7, 126.1, 125.6, 122.7,
100 MHz, CDCl ): δ 171.5, 141.1, 140.7, 135.2, 133.0, 129.8, 128.3,
+
3
8.1, 38.9. ESI-MS (positive), 323.9 (M + 1) . HRMS (EI) m/z:
27.2, 126.2, 125.6, 122.7, 51.9, 40.7, 29.1. ESI-MS (positive), 289.9
M + 1) . HRMS (EI) m/z: calcd for C H ClNOS (M ), 290.0401;
+
+
+
calcd for C16
H
12
F
3
NOS (M ), 324.0664; found, 324.0664.
(
15 12
2
,3-Dihydro-2-(4-methoxycarbonylphenyl)-1,5-benzothiazepin-
(5H)-one (3-12). Prepared using the general procedure starting from
-(4-methoxycarbonylphenyl)acrylic acid (2-12). Yield: 57%. White
found, 290.0403.
2
4
,3-Dihydro-2-(3-chlorophenyl)-1,5-benzothiazepin-4(5H)-one
3
(
3-5). Prepared using the general procedure starting from 3-(3-
1
solid. mp 184.2−186.7 °C. H NMR (CDCl ): δ 8.83 (br s, 1H), 7.98
3
chlorophenyl)acrylic acid (2-5). Yield: 64%. White solid. mp 200.9−
1
(d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.44−7.21 (m, 5H),
2
1
5
1
1
02.2 °C. H NMR (CDCl ): δ 8.62 (s, 1H), 7.66 (d, J = 7.6 Hz,
3
4
.92 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.90 (s, 3H), 2.90−2.78 (m, 2H).
H), 7.44 (t, J = 7.6 Hz, 1H), 7.30−7.19 (m, 6H), 4.84 (dd, J = 10.9,
1
3
1
3
C NMR (100 MHz, CDCl ): δ 171.9, 166.0, 147.6, 140.9, 135.2,
.8 Hz, 1H), 2.91−2.75 (m, 2H). C NMR (100 MHz, CDCl ): δ
3
3
1
5
29.8, 129.6, 129.0, 126.1, 125.8, 125.3, 122.8, 76.7, 76.4, 76.2, 52.3,
1.5, 40.5. ESI-MS (positive), 314.0 (M + 1) . HRMS (EI) m/z:
S (M ), 314.0845; found, 314.0847.
2,3-Dihydro-2-(4-nitrophenyl)-1,5-benzothiazepin-4(5H)-one (3-
3). Prepared using the general procedure starting from 3-(4-
71.4, 144.6, 140.7, 135.3, 133.9, 129.8, 129.5, 127.4, 126.2, 126.1,
+
+
25.4, 124.0, 122.7, 51.9, 40.7. ESI-MS (positive), 289.9 (M + 1) .
HRMS (EI) m/z: calcd for C H ClNOS (M ), 290.0401; found,
+
+
calcd for C17
H
15NO
1
5
12
3
2
90.0398.
,3-Dihydro-2-(2-chlorophenyl)-1,5-benzothiazepin-4(5H)-one
3-6). Prepared using the general procedure starting from 3-(2-
chlorophenyl)acrylic acid (2-6). Yield: 74%. White solid. mp 188.7−
1
2
(
nitrophenyl)acrylic acid (2-13). Yield: 21%. White solid. mp 188.2−
1
190.3 °C. H NMR (CDCl ): δ 8.03−7.59 (m, 4H), 7.46−7.17 (m,
3
1
1
89.6 °C. H NMR (CDCl ): δ 9.07 (s, 1H), 7.61 (dd, J = 13.1, 7.8
9H), 7.42−7.17 (m, 9H), 5.62−5.47 (m, 2H), 4.91−4.87 (m, 1H),
3
+
Hz, 3H), 7.54−7.43 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 7.4
3.01−2.85 (m, 2H). ESI-MS (positive), 390.9 (M + 1) .
Hz, 2H), 5.32 (dd, J = 10.9, 5.5 Hz, 1H), 2.96−2.86 (m, 1H), 2.81
2,3-Dihydro-2-(4-cyanophenyl)-1,5-benzothiazepin-4(5H)-one
1
3
(
dd, J = 12.5, 5.5 Hz, 1H). C NMR (100 MHz, CDCl ): δ 171.4,
(3-14). Prepared using the general procedure starting from 3-(4-
3
1
1
41.7, 141.0, 135.2, 131.9, 129.9, 127.6, 127.1, 126.2, 125.5, 125.1,
cyanophenyl)acrylic acid (2-14). Yield: 25%. White solid. mp 195.5−
+
1
24.4, 122.9, 46.9, 40.7. ESI-MS (positive), 289.9 (M + 1) . HRMS
197.1 °C. H NMR (CDCl ): δ 10.03 (s, 1H), 8.13 (d, J = 8.0 Hz,
3
+
(EI) m/z: calcd for C H ClNOS (M ), 290.0401; found, 290.0411.
1H), 8.05−8.02 (m, 3H), 7.53−7.45 (m, 6H), 7.24−7.17 (m, 2H),
1
5
12
1
3
2
,3-Dihydro-2-(4-bromophenyl)-1,5-benzothiazepin-4(5H)-one
5.09 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 2.79−2.48 (m, 2H). C NMR
(3-7). Prepared using the general procedure starting from 3-(4-
(
100 MHz, CDCl ): δ 170.1, 165.9, 152.5, 146.4, 141.4, 135.0, 134.2,
3
bromophenyl)acrylic acid (2-7). Yield: 68%. White solid. mp 169.2−
1
31.8, 129.7, 127.2, 126.4, 125.7, 125.3, 124.5, 123.0, 122.6, 122.2,
1
1
8
70.8 °C. H NMR (DMSO-d ): δ 10.00 (s, 1H), 7.48−7.14 (m,
+
6
51.7, 40.6. ESI-MS (positive), 280.1 (M + 1) . HRMS (EI) m/z:
calcd for C H N OS (M ), 281.0743; found, 281.0746.
1
3
H), 5.01 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 2.69−2.48 (m,2H). C NMR
+
1
6
12
2
(100 MHz, CDCl ): δ 171.1, 141.6, 140.6, 135.3, 131.3, 129.8, 127.6,
3
2,3-Dihydro-2-([1,1′-biphenyl]-4-yl)-1,5-benzothiazepin-4(5H)-
1
26.2, 125.7, 122.7, 121.1, 51.8, 40.6. ESI-MS (positive), 334.0, 336.0
one (3-15). Prepared using the general procedure starting from 3-
+
+
(
M + 1, M + 3) . HRMS (EI) m/z: calcd for C H BrNOS (M ),
15 12
([1,1′-biphenyl]-4-yl)acrylic acid (2-15). Yield: 57%. White solid. mp
3
33.9896; found, 333.9900.
,3-Dihydro-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one
3-8). Prepared using the general procedure starting from 3-(4-
methylphenyl)acrylic acid (2-8). Yield: 65%. Off-white solid. mp
1
1
(
2
(
89.6−191.0 °C. H NMR (CDCl ): δ 7.95 (d, J = 8.3 Hz, 2H),7.59
3
2
d, J = 7.5 Hz, 1H), 7.55−7.47 (m, 1H), 7.41−7.31 (m, 2H), 7.26 (s,
H), 7.17 (s, 1H), 4.73 (dt, J = 15.0, 7.5 Hz, 1H), 3.90 (s, 3H), 2.80
dd, J = 10.3, 8.5 Hz, 2H). ESI-MS (positive), 332.1 (M + 1) .
(
+
1
1
7
7
3
1
5
75.6−179.1 °C. H NMR (DMSO-d ): δ 8.06 (s, 1H), 7.65 (d, J =
6
2
,3-Dihydro-2-(4-morpholinophenyl)-1,5-benzothiazepin-4(5H)-
.7 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.26−7.20 (m, 3H), 7.17 (d, J =
one (3-16). Prepared using the general procedure starting from 3-(4-
.9 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 4.86 (dd, J = 10.6, 5.8 Hz, 1H),
morpholinophenyl)acrylic acid (2-16). Yield: 38%. White solid. mp
1
3
.79 (s, 3H), 2.90−2.75 (m, 2H). C NMR (100 MHz, CDCl ): δ
1
3
2
13.8−215.8 °C. H NMR (CDCl ): δ 8.15 (s, 1H), 7.65 (d, J = 7.7
3
71.5, 158.5, 140.6, 135.3, 134.9, 129.5, 126.9, 126.2, 122.6, 113.5,
4.7, 52.1, 41.0, 29.1. ESI-MS (positive), 270.1 (M + 1) . HRMS (EI)
Hz, 1H), 7.42 (t, J = 7.6 Hz, 1H), 7.30−7.11 (m, 4H), 6.84 (d, J = 8.6
Hz, 2H), 4.85 (dd, J = 10.7, 5.7 Hz, 1H), 3.84 (t, J = 7.6 Hz, 4H),
+
+
m/z: calcd for C H NOS (M ), 270.0947; found, 270.0949.
16
15
3
.14 (t, J = 7.6 Hz, 4H), 2.91−2.82 (m, 1H), 2.79 (dd, J = 12.4, 5.8
2
,3-Dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-
13
Hz, 1H). C NMR (100 MHz, CDCl ): δ 171.6, 150.1, 140.6, 135.3,
3
one (3-9). Prepared using the general procedure starting from 3-(4-
methoxlphenyl)acrylic acid (2-9). Yield: 70%. Off-white solid. mp
1
33.9, 129.4, 126.7, 126.3, 126.0, 122.6, 115.0, 66.2, 52.2, 48.5, 40.9.
+
1
ESI-MS (positive), 341.0 (M + 1) . HRMS (EI) m/z: calcd for
C H N O S (M ), 341.1318; found, 341.1319.
1
7
62.7−163.9 °C. H NMR (DMSO-d ): δ 8.35 (s, 1H), 7.65 (d, J =
6
+
.7 Hz, 1H), 7.42 (t, J = 7.1 Hz, 1H), 7.25−7.11 (m, 6H), 4.86 (dd, J
19 20
2
2
2
,3-Dihydro-1,5-benzothiazepin-4(5H)-one (3-17). Prepared
=
11.1, 5.6 Hz, 1H), 2.94−2.84 (m, 1H), 2.79 (dd, J = 12.5, 5.6 Hz,
1
3
using the general procedure starting from acrylic acid (2-17). White
1
1
4
H), 2.32 (s, 3H). C NMR (100 MHz, CDCl ): δ 171.7, 140.7,
3
1
solid. Yield: 31%. mp 214.3−216.8 °C. H NMR (CDCl ): δ 7.83 (s,
39.9, 137.0, 135.3, 129.5, 128.9, 126.1, 126.0, 125.6, 122.6, 52.4,
3
+
1H), 7.62−7.58 (d, J = 1.4 Hz, 1H), 7.38−7.34 (m, 1H), 7.19−7.15
0.9, 20.5. ESI-MS (positive), 286.1 (M + 1) . HRMS (EI) m/z:
+
(
m, 1H), 7.11−7.08 (d, J = 7.9 Hz, 1H), 3.47−3.43 (m, 2H), 2.65−
calcd for C H NO S (M ), 308.0716; found, 308.0725.
16
15
2
13
2
,3-Dihydro-2-(4-trifluoromethylphenyl)-1,5-benzothiazepin-
2.61 (m, 2H). C NMR (100 MHz, CDCl ): δ 173.1, 140.8, 134.9,
3
4
(5H)-one (3-10). Prepared using the general procedure starting from
129.2, 126.3, 125.9, 122.7, 76.6, 76.4, 76.2, 33.7, 32.9. ESI-MS
+
3-(4-trifluoromethylphenyl)acrylic acid (2-10). Yield: 30%. Off-white
(positive), 180.1 (M + 1) .
7
352
https://doi.org/10.1021/acs.jmedchem.0c02254
J. Med. Chem. 2021, 64, 7341−7358

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2
,3-Dihydro-2-methyl-1,5-benzothiazepin-4(5H)-one (3-18). Pre-
2,3-dihydro-2-(4-bromophenyl)-1,5-benzothiazepin-4(5H)-one (3-7).
1
pared using the general procedure starting from 3-methylacrylic acid
Yield: 46%. White solid. mp 117.0−118.8 °C. H NMR (CDCl ): δ
3
1
(
(
7
2
2-18). White solid. Yield: 51%. mp 209.1−212.5 °C. H NMR
7.68−7.51 (m, 2H), 7.41 (m, 4H), 7.07 (dd, J = 16.9, 10.3 Hz, 1H),
6.93 (t, J = 8.6 Hz, 2H), 6.52 (dd, J = 16.9, 1.5 Hz, 1H), 5.90 (dd, J =
CDCl ): δ 7.83 (s, 1H), 7.65−7.52 (m, 1H), 7.42−7.30 (m, 1H),
3
1
3
.21−7.15 (m, 1H), 7.14−7.04 (m, 1H), 3.92−3.81 (m, 1H), 2.72−
10.3, 1.5 Hz, 1H), 4.86−4.50 (m, 1H), 3.06−2.60 (m, 2H). C NMR
.58 (m, 1H), 2.40−2.26 (m,1H), 1.48−1.39 (d, J = 6.6 Hz, 3H). ESI-
(151 MHz, DMSO-d ): δ 171.02, 168.55, 142.05, 136.22, 132.06,
6
+
MS (positive), 194.1 (M + 1) .
1
31.37, 130.61, 129.09, 129.03, 128.52, 128.30, 48.79, 41.51. ESI-MS
+
General Procedure for the Synthesis of 5-Acryloyl-2,3-dihydro-2-
substituted-1,5-benzothiazepin-4(5H)-one 4. To a solution of 3 (1
mmol) in dichloromethane (4 mL) was added DIEA (3 mmol), and
the reaction mixture was cooled to 0 °C and acryloyl chloride (2
mmol) was added dropwise. After addition, the reaction mixture was
allowed to warm to room temperature and stirred overnight. The
reaction mixture was diluted with dichloromethane and washed with
brine, and the organic layer was dried over anhydrous MgSO and
concentrated. The crude product was purified by chromatography on
silica to afford pure product 4.
(positive), 387.8, 389.8 (M, M + 2) .
5
-Acryloyl-2,3-dihydro-2-(4-methylphenyl)-1,5-benzothiazepin-
4
2
8
(5H)-one (4-8). Prepared using the general procedure starting from
,3-dihydro-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one (3-
1
). Yield: 51%. White solid. mp 152.3−154.5 °C. H NMR
(
CDCl ): δ 7.69−7.06 (m, 9H), 6.51 (dd, J = 16.9, 1.5 Hz, 1H),
3
5
7
1
1
1
.89 (dd, J = 10.3, 1.5 Hz, 1H), 4.70 (d, J = 7.7 Hz, 1H), 2.88 (d, J =
13
4
.1 Hz, 2H), 2.34 (s, 3H). C NMR (151 MHz, DMSO-d ): δ
6
71.15, 168.51, 141.98, 136.78, 132.03, 130.40, 128.96, 128.89,
28.38, 125.86, 49.42, 41.91, 20.46. ESI-MS (positive), 324.0 (M +
5
-Acryloyl-2,3-dihydro-2-(2-thienyl)-1,5-benzothiazepin-4(5H)-
+
) .
one (4-1). Prepared using the general procedure starting from 2,3-
5
-Acryloyl-2,3-dihydro-2-(4-methoxyphenyl)-1,5-benzothiaze-
dihydro-2-(2-thienyl)-1,5-benzothiazepin-4(5H)-one (3-1). Yield:
0%. White solid. mp 118.2−121.5 °C. H NMR (CDCl ): δ 7.46−
.64 (m, 8H), 6.49 (dd, J = 16.9, 1.5 Hz, 1H), 5.87 (dd, J = 10.3, 1.5
Hz, 1H), 4.98 (dd, J = 12.0, 5.6 Hz, 1H), 2.90 (m, 2H). C NMR
100 MHz, CDCl ): δ 170.9, 168.5, 146.3, 142.2, 137.5, 131.5, 130.6,
30.2, 129.1, 128.1, 126.8, 126.6, 124.9, 123.9, 77.3, 77.0, 76.7, 50.9,
1
pin-4(5H)-one (4-9). Prepared using the general procedure starting
from 2,3-dihydro-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-
6
6
3
1
1
3
one (3-9). Yield: 66%. White solid. mp 121.6−124.0 °C. H NMR
(
1
CDCl ): δ 7.83−7.32 (m, 4H), 7.19−6.66 (m, 5H), 6.51 (dd, J =
3
(
1
4
3
6.9, 1.5 Hz, 1H), 5.89 (dd, J = 10.3, 1.5 Hz, 1H), 4.70 (d, J = 8.0 Hz,
1
3
+
1H), 3.79 (d, J = 12.6 Hz, 3H), 3.75 (q, J = 7.0 Hz, 2H). C NMR
151 MHz, DMSO-d ): δ 171.20, 168.57, 158.55, 142.01, 132.08,
6.3, 44.1. ESI-MS (positive), 316.0 (M + 1) .
(
6
5
-Acryloyl-2,3-dihydro-2-phenyl-1,5-benzothiazepin-4(5H)-one
4-2). Prepared using the general procedure starting from 2,3-dihydro-
-phenyl-1,5-benzothiazepin-4(5H)-one (3-2). Yield: 65%. White
1
5
30.41, 129.01, 128.93, 128.55, 128.42, 127.21, 113.80, 113.54, 54.99,
4.90, 49.31, 47.92, 42.20. ESI-MS (positive), 340.0 (M + 1) .
(
2
+
1
5
-Acryloyl-2,3-dihydro-2-(4-trifluoromethylphenyl)-1,5-benzo-
solid. mp 131.2−133.3 °C. H NMR (CDCl ): δ 7.73−7.26 (m,
H), 7.06 (dd, J = 16.8, 10.4 Hz, 3H), 6.49 (dd, J = 16.9, 1.6 Hz, 1H),
3
thiazepin-4(5H)-one (4-10). Prepared using the general procedure
starting from 2,3-dihydro-2-(4-trifluoromethylphenyl)-1,5-benzothia-
7
5
8
1
4
.88 (dd, J = 10.3, 1.5 Hz, 1H), 4.69 (t, J = 8.8 Hz, 1H), 2.88 (d, J =
.7 Hz, 2H). 13C NMR (100 MHz, CDCl ): δ 171.5, 168.6, 131.6,
zepin-4(5H)-one (3-10). Yield: 62%. White solid. mp 114.6−115.1
3
1
°
C. H NMR (CDCl ) δ 7.83−7.08 (m, 9H), 6.51 (d, J = 16.7 Hz,
30.3, 130.1, 129.2, 128.9, 128.2, 126.1, 77.3, 77.0, 76.7, 50.9, 50.6,
3.0. ESI-MS (positive), 310.1 (M + 1) .
3
+
13
1H), 5.89 (d, J = 10.2 Hz, 1H), 4.72 (m, 1H), 2.86 (m, 2H).
C
5
-Acryloyl-2,3-dihydro-2-(4-fluorophenyl)-1,5-benzothiazepin-
NMR (151 MHz, DMSO-d ): δ 171.17, 168.57, 142.07, 141.76,
6
4
2
(5H)-one (4-3). Prepared using the general procedure starting from
139.42, 136.27, 132.09, 130.53, 129.07, 129.02, 128.82, 128.51,
127.41, 126.76, 126.66, 126.50, 49.33, 41.86. ESI-MS (positive),
,3-dihydro-2-(4-fluorophenyl)-1,5-benzothiazepin-4(5H)-one (3-3).
1
+
Yield: 55%. White solid. mp 119.3−122.2 °C. H NMR (CDCl ): δ
378.0 (M + 1) .
3
7
=
.73−7.31 (m, 4H), 7.03 (s, 5H), 6.53 (d, J = 1.4 Hz, 1H), 5.90 (dd, J
5-Acryloyl-2,3-dihydro-2-(2-trifluoromethylphenyl)-1,5-Benzo-
thiazepin-4(5H)-one (4-11). Prepared using the general procedure
starting from 2,3-dihydro-2-(2-trifluoromethylphenyl)-1,5-benzothia-
1
3
10.3, 1.5 Hz, 1H), 4.71 (d, J = 7.6 Hz, 1H), 3.04−2.69 (m, 2H). C
NMR (151 MHz, DMSO-d ): δ 171.08, 168.56, 142.05, 136.21,
6
1
4
32.07, 130.56, 129.07, 129.01, 128.51, 128.10, 115.33, 115.19, 48.79,
1.98. ESI-MS (positive), 328.0 (M + 1) .
zepin-4(5H)-one (3-11). Yield: 57%. White solid. mp 107.9−109.6
+
1
°C. H NMR (CDCl ): δ 7.73−6.96 (m, 8H), 6.86 (d, J = 6.0 Hz,
3
5
-Acryloyl-2,3-dihydro-2-(4-chlorophenyl)-1,5-benzothiazepin-
1H), 6.52 (dd, J = 16.9, 1.5 Hz, 1H), 5.90 (dd, J = 10.3, 1.4 Hz, 1H),
.40−5.13 (m, 1H), 2.86 (d, J = 7.6 Hz, 2H). C NMR (151 MHz,
DMSO-d ): δ 170.73, 168.44, 141.95, 136.32, 133.12, 131.91, 130.98,
4
2
(5H)-one (4-4). Prepared using the general procedure starting from
13
5
,3-dihydro-2-(4-chlorophenyl)-1,5-benzothiazepin-4(5H)-one (3-4).
6
1
Yield: 68%. White solid. mp 96.8−100.7 °C. H NMR (CDCl ): δ
3
129.28, 129.25, 128.72, 128.23, 127.11, 125.82, 44.92, 41.63. ESI-MS
7
1
(
1
1
.71−7.29 (m, 5H), 7.28−6.87 (m, 4H), 6.52 (dd, J = 16.9, 1.5 Hz,
H), 5.91 (dd, J = 10.3, 1.5 Hz, 1H), 4.85−4.49 (m, 1H), 3.10−2.59
+
(
positive), 378.0 (M + 1) .
5
-Acryloyl-2,3-dihydro-2-(4-methoxycarbonylphenyl)-1,5-benzo-
m, 2H). 13C NMR (151 MHz, DMSO-d ): δ 171.04, 168.55, 142.05,
6
thiazepin-4(5H)-one (4-12). Prepared using the general procedure
32.06, 130.61, 129.27, 129.09, 129.03, 128.52, 128.09, 127.97,
27.07, 48.73, 47.67, 41.59. ESI-MS (positive), 344.0 (M + 1) .
starting from 2,3-dihydro-2-(4-methoxycarbonylphenyl)-1,5-benzo-
+
thiazepin-4(5H)-one (3-12). Yield: 72%. White solid. mp 105.8−
5
-Acryloyl-2,3-dihydro-2-(3-chlorophenyl)-1,5-benzothiazepin-
1
1
(
07.9 °C. H NMR (CDCl ): δ 7.96 (d, J = 7.4 Hz, 2H), 7.73−7.49
3
4
2
(5H)-one (4-5). Prepared using the general procedure starting from
m, 2H), 7.47−7.29 (m, 2H), 7.22−6.97 (m, 3H), 6.50 (dd, J = 16.9,
1.5 Hz, 1H), 5.89 (dd, J = 10.3, 1.5 Hz, 1H), 4.73 (d, J = 9.2 Hz, 1H),
.91 (s, 3H), 2.88 (d, J = 9.2 Hz, 2H). C NMR (100 MHz, CDCl ):
,3-dihydro-2-(3-chlorophenyl)-1,5-benzothiazepin-4(5H)-one (3-5).
1
Yield: 51%. White solid. mp 92.3−95.0 °C. H NMR (CDCl3): δ
1
3
3
3
7
1
(
.81−6.78 (m, 9H), 6.50 (dd, J = 16.9, 1.1 Hz, 1H), 5.89 (dd, J =
δ 171.2, 168.5, 166.5, 147.4, 142.3, 136.9, 131.5, 130.6, 130.2, 129.3,
0.3, 1.2 Hz, 1H), 4.81−4.48 (m, 1H), 3.03−2.64 (m, 2H). ESI-MS
+
128.5, 126.2, 77.3, 77.2, 77.0, 76.7, 58.5, 52.2, 50.1, 42.6. ESI-MS
positive), 344.0 (M + 1) .
+
(
positive), 368.0 (M + 1) .
5
-Acryloyl-2,3-dihydro-2-(2-chlorophenyl)-1,5-benzothiazepin-
5
-Acryloyl-2,3-dihydro-2-(4-nitrophenyl)-1,5-benzothiazepin-
4
2
(5H)-one (4-6). Prepared using the general procedure starting from
4
(5H)-one (4-13). Prepared using the general procedure starting from
,3-dihydro-2-(2-chlorophenyl)-1,5-benzothiazepin-4(5H)-one (3-6).
1
2,3-dihydro-2-(4-nitrophenyl)-1,5-benzothiazepin-4(5H)-one (3-13).
Yield: 56%. White solid. mp 90.1−92.4 °C. H NMR (CDCl ): δ
3
1
Yield: 59%. White solid. mp 137.2−144.3 °C. H NMR (CDCl ): δ
7
5
.77−7.28 (m, 7H), 7.19−6.79 (m, 2H), 6.50 (d, J = 17.0 Hz, 1H),
3
1
3
8.33−7.97 (m, 2H), 7.87−7.31 (m, 5H), 7.23 (dd, J = 15.4, 8.5 Hz,
H), 6.53 (dd, J = 15.6, 1.5 Hz, 1H), 5.93 (dd, J = 10.3, 1.5 Hz, 1H),
.96−4.60 (m, 1H), 3.09−2.68 (m, 2H). C NMR (151 MHz,
DMSO-d ): δ 170.84, 168.52, 146.54, 142.03, 136.18, 131.97, 130.78,
.89 (d, J = 10.3 Hz, 1H), 5.20−4.92 (m, 1H), 2.91 (m, 2H).
C
2
4
NMR (151 MHz, DMSO-d ): δ 171.16, 168.45, 142.13, 138.41,
6
1
3
1
1
36.26, 131.96, 131.48, 130.72, 129.57, 129.17, 128.98, 128.53,
27.43, 126.44, 125.38, 46.20. ESI-MS (positive), 344.0 (M + 1) .
+
6
5
-Acryloyl-2,3-dihydro-2-(4-bromophenyl)-1,5-benzothiazepin-
129.15, 129.11, 128.56, 127.43, 123.65, 48.47, 40.94. ESI-MS
+
4
(5H)-one (4-7). Prepared using the general procedure starting from
(positive), 355.0 (M + 1) .
7
353
https://doi.org/10.1021/acs.jmedchem.0c02254
J. Med. Chem. 2021, 64, 7341−7358

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
5
-Acryloyl-2,3-dihydro-2-(4-cynophenyl)-1,5-benzothiazepin-
fragmentor of 175 V, a capillary voltage of 3000 V, and a m/z 600−
3200 mass window. Mass deconvolution was later performed using
Agilent MassHunter Qualitative Analysis B.06.00 software.
4
2
(5H)-one (4-14). Prepared using the general procedure starting from
,3-dihydro-2-(4-cynophenyl)-1,5-benzothiazepin-4(5H)-one (3-14).
1
Yield: 66%. White solid. mp 143.5−146.8 °C. H NMR (CDCl ): δ
Identification of Protein Modification Site to 4-3. Protein
Precipitation and Digestion. The incubated protein was precipitated
with acetone. The protein pellet was dried by using a Speedvac for 1−
2 min and subsequently dissolved in 8 M urea and 100 mM Tris−HCl
(pH 8.5). TCEP (Thermo Scientific, final concentration is 5 mM)
and iodoacetamide (Sigma, final concentration is 10 mM) were added
to the solution for reduction and alkylation and incubated at room
temperature for 20 and 15 min, respectively. The protein mixture was
diluted four times and digested overnight with trypsin at 1:50 (w/w)
(Promega, http://www.promega.com/).
LC/Tandem MS (MS/MS) Analysis of Peptide. The peptide
mixture was analyzed on an ACQUITY UPLC M-Class system
(Waters Corporation, Milford, MA, USA) equipped with a Waters
BEH C18 analytical column (75 μm*250 mm, 1.7 μm) at the
temperature of 60 °C during the experiments. The mobile phase and
elution gradient used for peptide separation were as follows: 0.1%
formic acid in water as buffer A and 0.1% formic acid in acetonitrile as
buffer B, 0−1 min, 2−5% B; 1−200 min, 5−35% B; 200−210 min,
35−100% B; 210−220 min, 100% B; 220−230 min, 100−2% B; and
230−240 min, 2% B. The flow rate was set as 300 ml/min.
3
8
1
.24−6.97 (m, 9H), 6.51 (d, J = 16.8 Hz, 1H), 5.90 (d, J = 10.4 Hz,
13
H), 4.75 (s, 1H), 2.92 (d, J = 9.0 Hz, 2H). C NMR (151 MHz,
DMSO-d ): δ 173.61, 167.20, 153.29, 142.81, 142.51, 136.85, 130.55,
30.17, 128.96, 128.78, 127.43, 126.65, 126.52, 126.40, 125.62,
25.23, 122.85, 122.54, 122.27, 122.08, 120.78, 44.44, 42.32. ESI-MS
positive), 335.0 (M + 1) .
6
1
1
(
+
5
-Acryloyl-2,3-dihydro-2-([1,1′-biphenyl]-4-yl)-1,5-benzothiaze-
pin-4(5H)-one (4-15). Prepared using the general procedure starting
from 2,3-dihydro-2-([1,1′-biphenyl]-4-yl)-1,5-benzothiazepin-4(5H)-
1
one (3-15). Yield: 51%. White solid. mp 152.8−154.8 °C. H NMR
(
1
1
CDCl ): δ 7.82−7.32 (m, 11H), 7.23−6.99 (m, 3H), 6.53 (dd, J =
3
6.9, 1.5 Hz, 1H), 5.91 (dd, J = 10.3, 1.5 Hz, 1H), 4.76 (t, J = 8.8 Hz,
+
H), 2.94 (d, J = 8.4 Hz, 2H). ESI-MS (positive), 386.0 (M + 1) .
5
-Acryloyl-2,3-dihydro-2-(4-morpholinophenyl)-1,5-Benzothia-
zepin-4(5H)-one (4-16). Prepared using the general procedure
starting from 2,3-dihydro-2-(4-morpholinophenyl)-1,5-benzothiaze-
pin-4(5H)-one (3-16). Yield: 48%. White solid. mp 167.4−169.2
1
°
1
6
C. H NMR (CDCl ): δ 7.23−6.80 (m, 9H), 6.48 (d, J = 16.9 Hz,
3
H), 5.85 (d, J = 10.3 Hz, 1H), 4.65 (s, 1H), 3.78 (m, 5H), 2.99 (m,
+
H). ESI-MS (positive), 395.2 (M + 1) .
Mass Spectrometry. Data-dependent tandem MS (MS/MS)
analysis was performed with a Q Exactive Orbitrap mass spectrometer
(Thermo Scientific, San Jose, CA) equipped with a nanoelectrospray
ionization source using a distal 2 kV spray voltage. A cycle of one full-
scan MS spectrum (m/z 300−1800) was acquired followed by top 20
MS/MS events, sequentially generated on the first to the twentieth
most intense ions selected from the full MS spectrum at a 30%
normalized collision energy. The number of microscans was one for
both MS and MS/MS scans and the maximum ion injection time was
50 and 100 ms, respectively. The dynamic exclusion settings used
were as follows: charge exclusion, 1 and >8; exclude isotopes, on; and
exclusion duration, 30 s. MS scan functions and LC solvent gradients
were controlled by the Xcalibur data system (Thermo Scientific).
Data Analysis. The acquired MS/MS data were analyzed against a
Spodoptera frugiperda database (including all target peptides and
proteins) using Peaks8.5. Mass tolerances for precursor ions were set
at 20 ppm and for MS/MS were set at 0.02 Da. Trypsin was defined
as a cleavage enzyme. Cysteine alkylation by iodoacetamide was
specified as a fixed modification with mass shift 57.02146 and
methionine oxidation, protein C H FNO S modification as a
5
-Acryloyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (4-17).
Prepared using the general procedure starting from 2,3-dihydro-1,5-
benzothiazepin-4(5H)-one (3-17). Yield: 85%. White solid. mp 95.9
1
°
C. H NMR (DMSO-d ): δ 7.65 (dd, J = 7.6, 1.6 Hz, 1H), 7.54−
6
7
.47 (m, 1H), 7.44−7.37 (m, 1H), 7.30 (dd, J = 8.0, 1.4 Hz, 1H),
.99−6.89 (m, 1H), 6.26−6.19 (m, 1H), 5.88−5.82 (m, 1H), 3.22 (d,
6
J = 7.6 Hz, 2H), 2.58−2.48 (m, 2H). ESI-MS (positive), 234.1 (M +
+
1
) .
5
-Acryloyl-2,3-dihydro-2-methyl-1,5-benzothiazepin-4(5H)-one
(
4-18). Prepared using the general procedure starting from 2,3-
dihydro-2-methyl-1,5-benzothiazepin-4(5H)-one (3-18). Yield: 91%.
White solid. mp 179.9 °C. H NMR (DMSO-d ): δ 7.63 (d, J = 7.7
1
6
Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.34−7.26
(
1
1
m, 1H), 6.97−6.87 (m, 1H), 6.28−6.17 (m, 1H), 5.87−5.82 (m,
H), 3.69 (s, 1H), 2.64 (dd, J = 12.8, 6.4 Hz, 1H), 2.06 (s, 1H),
+
.32−1.18 (m, 3H). ESI-MS (positive), 248.0 (M + 1) .
N-(2-(4-Oxo-2-phenyl-2,3-dihydro-1,5-benzothiazepin-5(2H)-yl)-
1
ethyl)acrylamide (7). H NMR (DMSO-d ): δ 9.56 (s, 1H), 8.37 (s,
6
1
9
1
9
H), 7.77 (s, 1H), 7.66 (s, 2H), 7.60 (d, J = 15.4 Hz, 2H), 7.45 (d, J =
.5 Hz, 3H), 7.30 (s, 1H), 7.26−7.16 (m, 1H), 7.09 (d, J = 15.8 Hz,
H), 6.19 (d, J = 11.3 Hz, 1H), 6.10 (d, J = 17.0 Hz, 1H), 5.60 (d, J =
.8 Hz, 1H), 3.33 (s, 2H), 3.00 (d, J = 7.3 Hz, 2H). C NMR (151
MHz, DMSO-d ): δ 164.71, 163.77, 140.36, 137.35, 134.59, 131.37,
1
8
14
2
variable with mass shift 327.0729. A decoy database containing the
reversed sequences of all the proteins was appended to the target
database to accurately estimate peptide probabilities and false
discovery rate (FDR), and FDR was set at 0.01.
1
3
6
1
1
30.77, 129.65, 128.81, 127.65, 127.00, 125.54, 125.27, 124.56,
21.92, 38.05, 32.77.
MS for the Covalent Binding and Label Site of 4-3 to GSK-
β. The measurements were performed by National Center for
Biology. In Vitro Biological Evaluation Conditions. Human
recombinant GSK-3β (catalog number 14−306) was purchased from
Millipore Corporation (Dundee, UK). The prephosphorylated
polypeptide substrate GS-2 was synthesized by GL Biochem Ltd.
(Shanghai, China). The Kinase-Glo Luminescent Kinase Assay
(catalog number V6713) was obtained from Promega Corporation
(Madison, WI). ATP·2Na was purchased from Roche. TDZD-8
(catalog number 098K4602V) was supplied by Sigma-Aldrich (St.
Louis, MO). Assay buffer contained 50 mM HEPES (pH 7.5), 1 mM
EDTA, 1 mM EGTA, and 15 mM magnesium acetate. Glow-type
luminescence was recorded by Fluoroskan Ascent Fl (Thermo
Electron, US).
LiCl was of molecular biology grade and purchased by Sangon
Biotech Co., Ltd. (Shanghai, China), which was dissolved in sterile
phosphate-buffered saline (PBS) and stock solution (10 mM) was
stored at −20 °C. For experimental use, the drugs were freshly
prepared from the stock solution. Benzyloxycarbonyl-Val-Ala-Asp
(OMe) fluoromethylketone (Z-VAD-FMK) was acquired from Enzo
Life Sciences, Inc (Lausen, Switzerland). Corn oil, Cat no.1148806,
Merck, Darmstadt, Germany. NB4 is an ATRA (all-trans retinoic
acid)-sensitive human APL cell line and NB4-R1 is an ATRA-resistant
human APL cell line, both of which were kindly supplied from the
Institute of Hematology, the Affiliated Hospital of Ruijin, Jiaotong
3
Protein Science Shanghai (NCPSS), China. Human recombinant
GSK-3β was purchased from Millipore (Millipore, Billerica, USA).
Preparation of Protein Sample. A stock solution of 10 μg of the
enzyme in a buffer (50 mM HEPES, 1 mM EDTA, 2 mM EGTA, and
15 mM Mg(OAc) ·4 H O) was prepared. Compound 4-3 (500 μM)
2 2
was dissolved in dimethyl sulfoxide (DMSO), then 1.6 μL of the
compound solution was added to 14.4 μL of the enzyme stock
solution (10 μM) after incubating at 30 °C under constant shaking
(500 rpm) for 30 min.
LC−MS Analyses for the Covalent Mode. The measurements
were performed using liquid chromatography−MS (LC−MS) in the
positive-ion mode with an Agilent 6550 quadrupole-time-of-flight
mass spectrometer (Santa Clara, CA) coupled with an Agilent 1260
HPLC (Santa Clara, CA). The incubated protein sample was eluted
from a Phenomenex Jupiter C4 300 Å LC Column (2 × 150 mm, 5
μm) over a 20 min gradient from 5 to 100% acetonitrile containing
0.1% formic acid at a flow rate of 0.5 mL/min. The acquisition
method in the positive-ion mode with Dual Agilent Jet Stream
electrospray voltage used a capillary temperature of 250 °C, a
7
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Article
University (Shanghai, China). HL-60 (human APL) cells and Raji
the manufacturer. Briefly, cells were seeded in 96-well plates at 3 ×
10 cells/well and treated either with these different inhibitors at serial
4
(
Burkitt lymphoma) cell lines were purchased from the Cell Bank of
the Chinese Academy of Sciences (Shanghai, China). HUVEC and
concentrations or for various times (0, 24, or 48 h). After treatment,
CCK-8 solution (10 μL) was added to each well, followed by 3 h of
incubation at 37 °C. The absorbance wavelength at 450 nm was
recorded for each well in a FlexStation 3 microplate reader (Molecular
Devices, Sunnyvale, California, USA), and cell viability was then
calculated according to the manufacturer’s instruction.
Detection of Apoptosis with Annexin V-FITC/PI Staining.
Cell apoptosis was determined by the Annexin V-fluorescein
isothiocyanate (FITC)/PI Apoptosis Detection Kit (BD Pharmingen,
LO (human normal liver cells) cell lines were kindly provided by the
2
Liver Cancer Research Institute of Zhongshan Hospital, Fudan
University (Shanghai, China), and maintained according to the
ATCC guidelines at our center. All cells were cultured in RPMI-1640
or Dulbecco’s modified Eagle’s medium (high-glucose medium)
(
Thermo Scientific HyClone, Logan, Utah, USA) containing 10%
fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% (v/v)
penicillin−streptomycin (Gibco Invitrogen, Grand Island, NY, USA)
at 37 °C. in a humidified atmosphere with 5% CO .
2
California, USA). Cells were cultured in a 24-well plate at a density of
Inhibition of GSK-3β. The measurement of GSK-3β inhibition
was performed in assay buffer using blank 96-well plates according to
4
6
× 10 cells/mL and treated with GSK-3β inhibitors for 24 h. The
cells were harvested and resuspended in binding buffer, then stained
with 5 μL Annexin V-FITC and 5 μL PI, and incubated for 20 min at
room temperature in the dark. At last, the stained cells were detected
within 30 min with the BD FACS Aria II flow cytometer (BD
Biosciences, San Jose, California, USA).
36
the Kinase-Glo assay method of Baki. In a typical assay, 4 μL of
interest compound with different concentrations (dissolved in
DMSO) was diluted by 14 μL of assay buffer, and 2 μL (20 ng) of
enzyme solution was added to each well, followed by 20 μL of assay
buffer containing 12.5 μM substrate and 4 μM ATP. After 30 min of
incubation at 30 °C, the enzymatic reaction was stopped with 40 μL
of Kinase-Glo reagent. Glow-type luminescence was recorded after 10
min. The activity is proportional to the difference of the total and
consumed ATP. The inhibitory activities were calculated on the basis
of maximal activities measured in the absence of inhibitor. The IC50
value was defined as the concentration of each compound that
reduces 50% of the enzymatic activity to that without inhibitors.
Kinetic Analysis on GSK-3β. The protocol of kinetic experiments
was much similar to the GSK-3β inhibition tests. The activities of
compound 4-2 were measured separately at 2 and 4 μM. In the
experiments for testing the relationship between 4-2 and ATP, the
concentration of substrate GS-2 was kept unchanged at 6.25 μM,
while the concentration of ATP was set at 0.5, 1, 2, 4, and 8 μM
separately. Then, in the following experiments for testing the
relationship between 4-2 and GS-2, the concentration of ATP was
kept unchanged at 2 μM, while GS-2 concentration was set at 0.78,
Western Blotting. Cells were harvested and washed twice with
ice-cold PBS. Total protein was extracted using the RIPA kit
(Beyotime Biotechnology Inc., Shanghai, China), and equal amounts
of protein were separated on polyacrylamide gels and transferred to
PVDF membranes. The membranes were blocked and incubated with
anti-Phospho-Glycogen Synthase (Ser641) (Cell Signaling Technol-
ogy, #47043, MA, USA), Glycogen Synthase (15B1) (Cell Signaling
Technology, #3886, MA, USA), and anti-β-actin, AF0003 (Beyotime
Biotechnology Inc., Shanghai, China) at 4 °C overnight and were then
incubated with horseradish peroxidase-conjugated goat anti-rabbit or
anti-mouse IgG secondary antibodies at room temperature for 1 h.
The proteins were visualized using an enhanced chemiluminescence
kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) and
analyzed by the Bio-Rad ChemiDoc MP Imaging System (California,
USA).
PK Studies. The PK analysis was performed in male ICR mice (30
g body weight) by i.p. bolus dose (15 mg/kg) of compound 4-3
dissolved in corn oil. Blood samples (∼0.2 mL) were obtained by
orbital blood collection into tri-potassium ethylenediaminetetraacetic
acid (K3EDTA)-containing tubes at 0.08, 0.17, 0.5, 1, 2, 4, and 8 h
after administration. Within each group, blood samples of three
animals were collected, resulting in a composite PK profile. The blood
samples were centrifuged at 8000 rpm for 5 min to obtain plasma, and
40 μL of plasma was pipetted into a clean tube, 160 μL of ACN was
added and thoroughly vortexed by a vortex mixer for 1 min. The
mixture was centrifuged at 14000 rpm for 10 min, and then 100 μL of
supernatant was for the analysis by AB 4000 QTRAP LC−MS/MS.
The PK parameters were calculated using the non-compartmental
analysis tool of WinNonlin Pro (version 6.3).
1.56, 3.13, 6.25, and 12.5 μM separately. The irreversibility of 4-2 was
determined by evaluating its activity to the enzyme at different
incubation times. The incubation time was set at 0, 5, and 10 min,
while the concentration of 4-2 was kept at 6.25 and 12.5 μM
separately.
Selectivity Studies of Tyrosine Kinase Inhibition. The
experimental procedures for the inhibition of tyrosine kinases were
46
conducted using the ELISA method reported by Geng et al. Briefly,
0 μg/mL of poly (Glu, Tyr) 4:1 (Sigma) was pre-coated as a
2
substrate in 96-well plates. About 50 μL of 10 μM ATP solution
diluted with kinase reaction buffer (50 mM HEPES pH 7.4, 50 mM
MgCl , 0.5 mM MnCl , 0.2 mM Na VO , and 1 mM DTT) was
2
2
3
4
added to each well. Various concentrations of compounds diluted in
0 μL of 1% DMSO (v/v) were added to each reaction well, with 1%
1
Efficacy of GSK-3β Inhibition on the Leukemia Xenograft
Model In Vivo. Balb/c female nude mice were obtained from
Shanghai SLAC Laboratory Animal Ltd. Co. The animals were fed a
sterile diet and water and kept in individually ventilated cages to avoid
infection under a 12 h light/dark cycle at constant temperature and
DMSO (v/v) used as the negative control. The kinase reaction was
initiated by the addition of purified tyrosine kinase proteins diluted
with 40 μL of kinase reaction buffer solution. After incubation for 60
min at 37 °C, the plate was washed three times with PBS containing
0
.1% Tween 20 (T-PBS). Next, 100 μL of anti-phospho-tyrosine
6
humidity. 2 × 10 leukemia cells were injected subcutaneously into
(PY99) antibody (1:500 diluted in 5 mg/mL BSA T-PBS) was added.
the right flanks of 4 week-old nude mice. Tumor-bearing mice about
After 30 min of incubation at 37 °C, the plate was washed three times.
A solution of 100 μL of horseradish peroxidase-conjugated goat anti-
mouse IgG (1:2000 diluted in 5 mg/mL BSA T-PBS) was added. The
plate was reincubated at 37 °C for 30 min and washed as mentioned
before. Finally, 100 μL of a solution containing 0.03% H O and 2
0
.5 cm in diameter were randomly divided into vehicle control (corn
oil) and 4-3 (dissolved in corn oil) treatment group (n = 5).
Intraperitoneal administration of these animals with 4-3 (15 mg/kg/
d) and vehicle control was carried out once a day for 2 weeks. Tumors
were measured by caliper, and volumes were calculated by (length ×
width )/2 once every other day. The animal experimental protocols
were performed and approved by the Animal Ethics Committee of
Zhongshan Hospital, Fudan University.
2
2
mg/mL o-phenylenediamine in 0.1 mM citrate buffer, pH 5.5, was
added, and samples were incubated at room temperature until color
emerged. The reaction was terminated by the addition of 50 μL of 2
M H SO , and the plate was read using a multi-well spectropho-
2
2
4
tometer (VERSA max, Molecular Devices, Sunnyvale, CA, USA) at
90 nm. The inhibition rate (%) was calculated using the following
equation: [1-(A490/A490 control)]× 100%.
Cell Proliferation Assay. Cell proliferation of target compounds
and LiCl was performed using the Cell Counting Kit-8 (CCK-8) assay
4
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
(Dojindo Laboratories, Kumamoto, Japan) as per the instruction of
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02254.
7
355
https://doi.org/10.1021/acs.jmedchem.0c02254
J. Med. Chem. 2021, 64, 7341−7358

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
HPLC traces for key target compounds and figures for
the candidate of 4-3 including hERG inhibition and
exposition curve in mouse plasma (PDF)
Pharmacology, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica) for assistance with the
biological evaluation.
Molecular formula strings and biological data (CSV)
ABBREVIATIONS
■
AUTHOR INFORMATION
Corresponding Authors
■
EGFR, epidermal growth factor receptor; BTK, Bruton’s
tyrosine kinase; NSCLC, non-small cell lung carcinoma;
CK2, casein kinase 2; PKA, protein kinase A; Flt-1, Fms-
related tyrosine kinase 1; KDR, kinase insert domain receptor;
FGFR, fibroblast growth factor receptor; PDGFR, platelet-
derived growth factor receptor; EPH, ephrin; RET, rearranged
during transfection; Src, sarcoma; ABL, Abelson murine
leukemia viral oncogene homolog; HUVEC, human umbilical
vein endothelial cells
Yunfeng Cheng − Institute of Clinical Science, Zhongshan
Hospital, Fudan University, Shanghai 200032, China;
Phone: +86-21-60267312; Email: yfcheng@fudan.edu.cn;
Fax: +86-21-60267312
Yong Chu − Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, China;
Phone: +86-21-51980125; Email: cy110@fudan.edu.cn;
Fax: +86-21-51980125
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Natural Science Foundation of China (grant no. 81373275 to
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