Discovery and anti-inflammatory evaluation of benzothiazepinones (BTZs) as novel non-ATP competitive inhibitors of glycogen synthase kinase-3β (GSK-3β)

Article abstract of DOI:10.1016/j.bmc.2018.09.027

Glycogen synthase kinase-3β (GSK-3β) has been identified to promote inflammation and its inhibitors have also been proven to treat some inflammatory mediated diseases in animal models. Non-ATP competitive inhibitors inherently have better therapeutical value due to their higher specificity than ATP competitive ones. In this paper, we designed and synthesized a series of new BTZ derivatives as non-ATP competitive GSK-3β inhibitors. Kinetic analysis revealed two typical compounds 6j and 3j showed the different non-ATP competitive mechanism of substrate competition or allosteric modulation to GSK-3β, respectively. As expected, the two compounds showed good specificity in a panel test of 16 protein kinases, even to the closest enzymes, like CDK-1/cyclin B and CK-II. The in vivo results proved that both compounds can greatly attenuate the LPS-induced acute lung injury (ALI) and diminish inflammation response in mice by inhibiting the mRNA expression of IL-1β and IL-6. Western blot analysis demonstrated that they negatively regulated GSK-3β, and the mechanism of the observed beneficial effects of the inhibitors may involve both the increased phosphorylation of the Ser9 residue on GSK-3β and protein expression of Sirtuin 1 (SIRT1). The results support that such novel BTZ compounds have a protective role in LPS-induced ALI, and might be attractive candidates for further development of inflammation pharmacotherapy, which greatly thanks to their inherently high selectivities by the non-ATP competitive mode of action. Finally, we proposed suggesting binding modes by Docking study to well explain the impacts of compounds on the target site.

Full text of DOI:10.1016/j.bmc.2018.09.027

Accepted Manuscript
Discovery and Anti-inflammatory Evaluation of Benzothiazepinones (BTZs) as
Novel Non-ATP Competitive Inhibitors of Glycogen Synthase Kinase-3β
(GSK-3β)
Yang Gao, Peng Zhang, Anfeng Cui, De-Yong Ye, Meng Xiang, Yong Chu
PII:
S0968-0896(18)30697-7
https://doi.org/10.1016/j.bmc.2018.09.027
BMC 14552
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 April 2018
12 September 2018
22 September 2018
Please cite this article as: Gao, Y., Zhang, P., Cui, A., Ye, D-Y., Xiang, M., Chu, Y., Discovery and Anti-
inflammatory Evaluation of Benzothiazepinones (BTZs) as Novel Non-ATP Competitive Inhibitors of Glycogen
Synthase Kinase-3β (GSK-3β), Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2
018.09.027
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Discovery and Anti-inflammatory Evaluation of Benzothiazepinones (BTZs) as
Novel Non-ATP Competitive Inhibitors of Glycogen Synthase Kinase-3β (GSK-
3
β)
1
,3
1,3
2
1
2,
Yang Gao †, Peng Zhang †, Anfeng Cui †, De-Yong Ye , Meng Xiang *, Yong
1
,
Chu *
1Department of Medicinal Chemistry, School of Pharmacy, Fudan University,
Shanghai 201203, China
2Department of Physiology and Pathophysiology, School of Basic Medical Sciences,
Fudan University, Shanghai 200032, China
3State Key Lab of New Drug &Pharmaceutical Process, Shanghai Key Lab of Anti-
Infectives, State Institute of Pharmaceutical Industry, Shanghai, 201203, China
†
Contributed equally
*
Corresponding author. E-mail addresses: xmeng@shmu.edu.cn (Meng Xiang),
cy110@fudan.edu.cn (Yong Chu).
Abstract
Glycogen synthase kinase-3β (GSK-3β) has been identified to promote inflammation
and its inhibitors have also been proven to treat some inflammatory mediated diseases
in animal models. Non-ATP competitive inhibitors inherently have better
therapeutical value due to their higher specificity than ATP competitive ones. In this
paper, we designed and synthesized a series of new BTZ derivatives as non-ATP
competitive GSK-3β inhibitors. Kinetic analysis revealed two typical compounds 6j
and 3j showed the different non-ATP competitive mechanism of substrate

competition or allosteric modulation to GSK-3β, respectively. As expected, the two
compounds showed good specificity in a panel test of 16 protein kinases, even to the
closest enzymes, like CDK-1/cyclin B and CK-II. The in vivo results proved that both
compounds can greatly attenuate the LPS-induced acute lung injury (ALI) and
diminish inflammation response in mice by inhibiting the mRNA expression of IL-1β
and IL-6. Western blot analysis demonstrated that they negatively regulated GSK-3β,
and the mechanism of the observed beneficial effects of the inhibitors may involve
both the increased phosphorylation of the Ser9 residue on GSK-3β and protein
expression of Sirtuin 1 (SIRT1). The results support that such novel BTZ compounds
have a protective role in LPS-induced ALI, and might be attractive candidates for
further development of inflammation pharmacotherapy, which greatly thanks to their
inherently high selectivities by the non-ATP competitive mode of action. Finally, we
proposed suggesting binding modes by Docking study to well explain the impacts of
compounds on the target site.
Keywords: Glycogen synthase kinase 3β; Sirtuin 1; non-ATP competitive inhibitors;
benzothiazepinones; inflammation.
Abbreviations: GSK-3β, Glycogen synthase kinase-3β; SIRT1, Sirtuin 1; NF-κB,
nuclear factor kappaB; BTZ, benzothiazepinone; ALI, acute lung injury; LPS,
lipopolysaccharide; HOAt, 1-hydroxy-7-azabenzotriazole; PBS, phatephosphate
buffered
Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
hexafluorophosphate.
saline;
HATU,
O-(7-azabenzotriazol-1-yl)-1-
[
3-oxid

1
. Introduction
Glycogen synthase kinase-3β (GSK-3β) is a constitutively active kinase that
plays a key role in the regulation of multiple signaling proteins and transcription
factors. It is well known that phosphorylation of GSK-3β modulates a lot of cellular
processes, including gene transcription, metabolic pathways, cell growth, and
differentiation, as well as apoptosis.1-4 Recent years, the inflammatory effects of
GSK-3β have also been identified. GSK-3β negatively regulates anti-inflammatory
5
interleukin-10 for lipopolysaccharide (LPS) - induced inflammation, and plays a
fundamental role in the regulation of the activity of nuclear factor kappaB (NF-κB)
within the phosphatidylinositol 3-kinase (PI3K) pathway.6 GSK-3β inhibition
prevented LPS-induced pulmonary pathology in guinea pigs, and that locally reduced
7
LPS-induced beta-catenin activation. According to that line, the GSK-3β inhibitor
had been proven to reduce infarct volume and to suppress insult-induced neuro-
8
inflammation in rat cortex after ischemic brain injury. Thus, they might provide a
potential therapeutic option for some inflammatory-mediated diseases, such as
depression, bipolar disorders, and cancer.9,10
However, most of the known inhibitors bound to the ATP-binding pocket of
GSK-3β, which might lead widespread effects due to the high homology between
human kinases. Recently, the study of non-ATP competitive inhibitors of GSK-3β had
been attractive to medicinal chemists because such compounds are inherently more
specific and generally better efficacy in vivo assays, therefore might be less adverse
effects and potential therapeutical effects.11-13 These compounds include allosteric

modulators and substrate competitive inhibitors, but there are still challenges to find
these types of candidates. The main reasons for it are due to their binding sites
generally have not been well defined and their interaction is also relatively weak
because of binding to outside ATP-binding pocket.
To our best knowledge, some chemically different non-ATP competitive GSK-
3
β inhibitor have been already reported (Fig. 1), including substrate competitive
1
4
inhibitors as small peptide L803-mts, small molecules of 5-imino-1,2,4-thiadiazoles
(
ITDZs),15 and marine natural product of alkaloid manzamine A, and allosteric
1
6
17
inhibitors as VP0.7, palinurin, as well as irreversible inhibitors thiadiazolidinones
1
8
19,20
(
TDZDs), and halomethyl ketones (HMKs).
Among them, one compound called
tideglusib (NP-12), belonging to TDZDs, is currently undergoing Phase IIb clinical
trials.21

Substrate competitive inhibitors
R2
O
O
H
N
S
S
N
N
R1
N
10
H
N
N
O
N
H
HN
R3
X
OH
ITDZs
N
IC50 = 0.3-7 M
Manzamine A
IC50 = 10.2 M
N
BTO-5s
IC50 = 10.0 M
O
HO
O
HO P O
O
O
H
O
O
O
O
H
N
H
N
N
N
N
N
N
N
H
N
N
N
N
H
H
H
O
O
O
O
O
O
OH
O
O
NH2
L803-mts
IC50 = 40 M
NH2
Allosteric inhibitors
OH
O
O
O
Palinurin
IC50 = 1.9 M
S
N
O
O
OH
N
O
O
H
H
S
N
N
N
10
N
H
10
H
O
O
O
N
O N
2
BTO-5h
IC50 = 8.0 M
BTZ-6V
IC50 = 23.0 M
VP0.7
IC50 = 3.0

M
Other non-ATP competitive inhibitors
R
R
N
O
O
X
S
N
S
O
R'
TDZDs
IC50 = 2-10 M
HMKs
IC50 = 1-5 M
Fig. 1. Examples of reported non-ATP competitive GSK-3β inhibitors.
Recently, we have reported some novel non-ATP competitive GSK-3β
inhibitors, such as benzothiazepinones (BTZs) and benzothiazinones (BTOs).22,23
Among them, BTO-5s acts in substrate-competitive mode while both of BTO-5h and
BTZ-6v are allosteric inhibitors (Fig. 1). In this paper, we synthesized a series of new

BTZ derivatives as GSK-3β non-ATP competitive inhibitors with low micromolar
activities. Among of them, two typical compounds 3j and 6j will be discussed for
their in vitro efficacy, including enzyme inhibitory activity, mode of inhibition,
selectivity for kinases profile, activation mechanism, and in vivo anti-inflammatory
activity. We also reported our preliminary efforts on evaluating the mechanism of
selected compounds for their properties in the GSK-3β-SIRT1 pathway. Although it is
well known that SIRT1 plays a pivotal role in regulating inflammation as an
+
important NAD -dependent protein deacetylate, which can directly interact with and
deacetylate the RelA/p65 component of the NF-κB complex,24 there still have few
reports on the relationship between GSK-3β and SIRT1. Finally, a suggesting binding
mode for the selected inhibitor is also discussed based on a docking study.
O
Cl
O
N
R1
H
S
N
N
H
S
N
S
O
S
N
O
O
O N
R2
N
H
2
O
O N
2
BTZ-6v
6j
3j
Fig. 2. The design of benzothiazepinones (BTZs) and the structure of 3j and 6j.
2
. Results and discussion
2
.1. Chemistry
Compounds 3j and 4a-4h were prepared following the general route described in
Scheme 1. The synthesis of 3-substituted-acrylic acids 2 was performed by the
Knoevenagel condensation of various aromatic formaldehyde 1 with propanedioic
acid in the presence of pyridine and piperidine under reflux condition. Followed by

the cyclization of resulting acids 2 with 2-aminothiophenol gave the key intermediates
of 2,3-dihydro-1,5-benzothiazepin-4(5H)-one analogs 3. We successfully expanded
the structure scope of 3 analogs with an efficient one-pot procedure which involved
the construction of 1,5-benzothiozepinone frame by using tetrabutylammonium
fluoride (TBAF) as the catalyst.25 Alkylation of 3 through the electrophilic
substitution with substituted benzyl bromide obtained the analogs 4a-4h. Compound
3
f was hydrolyzed by LiOH to afford intermediate acid 3i, which was then condensed
with 3-Cl-benzylamine by using HATU/HOAt as condensation reagent to give
compound 3j.
R1
R1
S
CHO
COOH
a
b
S
R1
c
R1
N
N
H
O
O
1a-1h
2a-2h
3
a-3h
R1
R1
O N
2
3
f
a 4-F
b 4-C
c 4-Ph
d 4-Me
e 4-CN
f 4-COOMe
d
4
a-4h
3i
g 3-COOMe
h 2-COOMe
i 4-COOH
e
3
j
j 4-CONH(3'-Cl-Bn)
Scheme 1. (a) Propanedioic acid, Pyridine, Piperidine, reflux, 4 h, 75~95%; (b) 2-
aminothiophenol, TBAF·3H O, 80 °C, 24 h, 25~71%; (c) 2-NO -BnBr, K CO ,
2
2
2
3
CH CN, reflux, overnight, 81~94%; (d) LiOH, THF, MeOH, H O, 0.5 h, reflux, 75%;
3
2
(e) HATU, HOAt, DIPEA, 3-Cl-benzylamine, DCM, overnight, RT, 80%.
Compounds 6 were prepared following the general route described in Scheme 2.
Compound 3f was alkylated with different substituted-benzyl bromide to afford
compounds 4f, 4i-4j. The above intermediates then converted to the corresponding
free acids 5a-5c by saponification of the methyl ester, following to obtain amide 6a-

6
v by amidation of the free acid with different amines. Compound 6v was hydrolyzed
by catalysis of H SO to give amide 6w.
2
4
Amide 6x was prepared following a different way. Compound 3f was arylated in
N5 position under Buchwald-Hartwig condition to obtain compound 4k, which was
then under hydrolysis to afford acid 5d. Compound 5d was condensed with 3-chloro-
benzyl amine to afford target 6x.
O
O
O
R2
OH
OMe
N
H
S
N
S
N
S
N
a
b
c
O
O
O
O
R1
R1
R1
R1
a-6u
OMe
R1
5a NO2
b CN
5c MeSO NH
6
4f
4i
4j
NO2
CN
MeSO NH
1
6v R =CN
5
S
d
1
2
6w R =CONH
2
2
N
H
O
O
O
O
R2
OMe
f
OH
N
H
3f
e
g
S
S
S
N
N
O
N
O
O
R1
R1
5d R =MeSO
R1
6x R =MeSO
2
1
1
1
4k R =MeSO
2
2
1
Scheme 2. (a) 2-R -BnBr, K CO , CH CN, reflux, overnight, 87%; (b) LiOH, THF,
2
3
3
2
MeOH, H O, 0.5 h, reflux, 95%; (c) HATU, HOAt, DIPEA, R NH , DCM, overnight,
2
2
RT, 60~94%; (d) Conc. H SO , H O, reflux, 8 h, 84%; (e) 1-bromo-3-
2
4
2
o
(
methylsulfonyl)benzene, Pd (dba) , Xantphos, Cs CO , Dioxane, microwave, 110 C,
2 3 2 3
4
h, 75%; (f) LiOH, THF, MeOH, H O, 0.5 h, reflux, 82%; (g) HATU, HOAt, DIPEA,
2
(3-chlorophenyl)methanamine, DCM, overnight, RT, 91%.
Compound 7 was prepared following the general route described in Scheme 3.
Compound 4f was treated with 1.0 M BH -THF solution and reacted under reflux
3

condition for 2 h to afford compound 7.
O
O
OMe
OMe
S
S
N
a
N
O
O2N
2
O N
4f
7
Scheme 3. (a) 1.0 M BH -THF, reflux, 2 h, 88%.
3
The structures of all the new compounds were elucidated from their
1
13
spectroscopic data ( H, C NMR, and MS) which were collected in the Experimental
Section.
2
2
.2. In vitro assays
.2.1. GSK-3β kinase inhibitory activity
Synthesized compounds were tested for their inhibitory activities in vitro against
GSK-3β using Kinase-Glo™ luminescent kinase Assay (see Experimental section).22
Briefly, the GSK-3β enzyme was incubated with ATP and GS-2 as substrates in the
presence/absence of the corresponding test compound. The assay quantified the
amount of ATP remaining in solution following a kinase reaction. Both 6j and 3j
showed good inhibitory activities with IC values of 28 μM and 25 μM, respectively
5
0
(Table 1).
Based on our previous SAR results published before, the group of 2-nitrobenzyl
attached to the N-5 of the BTZ ring could dramatically contribute to the inhibitory
1
potency, so we first made the structure modification on the R group. In these
1
compounds, the ones with the R group as methyl or 4-methoxycarbonyl showed

better inhibitory activity (Table 1. 4d and 4f).
However, it is well known that the ester group is generally not stable enough in
the metabolic process, the following modification mainly focused on the replacing of
ester group with a stable amide group. Most resulted amide compounds showed
moderate to good inhibitory activities to GSK-3β when they had larger and more
hydrophobic groups at 4-position in the phenyl ring. Among them, 6a, 6j-6n, 6q-6s
showed the acceptable activities with the IC values lower than 30 μM.
5
0
Finally, we tried to replace the nitro group with some isosteres and the results
were listed in Table 1. It was observed that methylsulfonyl group (6t) was the best
substituent contributing for inhibitory activity among these modifications, and amide
group could dramatically reduce activity (6w), which indicated that nitro group may
play a hydrogen bond acceptor role in this case. To our surprise, compound 3j, just
2
having a hydrogen atom as an R group, could keep good inhibitory activity, which
indicated that it might interact with GSK-3β in a different mechanism from the others.
Besides, to evaluate the effect of carbonyl group at 4-position in seven member ring,
the carbonyl group of compound 4f was reduced to give compound 7. Biological test
of both showed the IC50 values greatly increased from 35 μM of 4f to 100 μM of
compound 7, which indicated that the carbonyl group played an important role in
keeping the inhibitory activity.
Table 1
Target compounds and their GSK-3β inhibitory activities.

R1
S
N
O
R2
R1
R2
IC (μM)
50
a
Compd.
3
4
4
4
4
4
4
4
4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
j
(3-Cl-Bn)NHCO
4-F
H
25
a
b
c
d
e
f
2-NO -benzyl
82
71
51
20
45
35
66
80
22
48
90
2
4-Cl
2-NO -benzyl
2
4- Ph
2-NO -benzyl
2
4-Me
2-NO -benzyl
2
4- CN
2-NO -benzyl
2
4-COOMe
3-COOMe
2-COOMe
nBuNHCO
tBuNHCO
2-NO -benzyl
2
g
h
a
b
c
d
e
f
2-NO -benzyl
2
2-NO -benzyl
2
2-NO -benzyl
2
2-NO -benzyl
2
Et NHCO
2-NO -benzyl
2
2
cyclopropylNHCO
CyclohexthylNHCO
1-PiperidinylCO
2-NO -benzyl
>100
32
2
2-NO -benzyl
2
2-NO -benzyl
49
2
g
h
i
4-Me-1-PiperazinylCO
2-NO -benzyl
>100
75
2
2-PyridylCH NHCO
2-NO -benzyl
2
2
2-Pyridyl-NHCO
BnNHCO
2-NO -benzyl
35
2
j
2-NO -benzyl
28
2
k
l
Ph(CH ) NHCO
2-NO -benzyl
21
2
2
2
2-F-BnNHCO
3-F-BnNHCO
4-F-BnNHCO
3-Cl-BnNHCO
2-NO -benzyl
20
2
m
n
o
2-NO -benzyl
19
2
2-NO -benzyl
18
2
2-NO -benzyl
35
2

6
6
6
6
6
6
6
p
q
r
4-Cl-BnNHCO
4-Br-BnNHCO
2-NO -benzyl
33
25
18
27
17
27
100
18
2
2-NO -benzyl
2
4-CH -BnNHCO
2-NO -benzyl
3
2
s
4-CH O-BnNHCO
2-NO -benzyl
3
2
t
(3-Cl-Bn)NHCO
2-MeSO NH-benzyl
2
u
w
x
2-FurylCH NHCO
2-NO -benzyl
2
2
BnNHCO
2-CONH -benzyl
2
6
(3-Cl-Bn)NHCO
3-MeSO -phenyl
2
a
IC50, the mean of triplicate experiments.
2
.2.2. Mechanism of GSK-3β inhibition
To further investigate the mechanism of GSK-3β inhibition, kinetic analyses
were performed to two typical compounds 6j and 3j by the kinase assay method
2
2
described before. Briefly, the enzyme inhibitory activities of both compounds were
measured at different concentrations separately meanwhile keeping the concentration
of one of two substrates (ATP and GS-2) unchanged and varying the concentrations
of the other. Double reciprocal plotting of the data confirmed that both compounds
acted as a non-ATP competitive inhibitor (Fig. 3A and 3C). Furthermore, 3j acted no
competition to GS-2 (Fig. 3D), properly being allosteric modulator while 6j might be
the substrate competitive inhibitor because of acting as a competitive inhibitor of GS-
2
(Fig. 3B).

6j
(
A)
(B)
0
0
0
.0004
.0003
.0002
0.0025
0.0020
0.0015
0.0010
0.0005
2
5 μM
2
5 μM
15 μM
Control
1
5 μM
Control
0.0001
-1
1
2
3
-2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0
1/[GS-2]
-0.0005
3j
1/[ATP]
(
C)
(D)
0.0020
0.0015
0.0010
0.0005
0.0008
3
0 μM
0 μM
30 μM
0.0006
0.0004
0.0002
2
control
2
0 μM
control
-1.5
-1.0
-0.5
0.5
1.0
1.5
-0.5
0.5
1.0
1.5
2.0
2.5
1/[GS-2]
-0.0005
1/[ATP]
Fig. 3. Kinetic data determined for compounds 6j and 3j.
.2.3. Kinase selectivity studies
2
High selectivity for protein kinase inhibitors is critical to avoid off-target effects
in a potential therapy. In order to check the kinase selectivity of these new
compounds, both of 6j and 3j were evaluated against a panel of related 16
serine/threonine and tyrosine kinases, which are closely related to GSK-3β and/or
play important roles in several signaling pathways. The inhibitory assays were carried
out at an inhibitor concentration of 100 μM. As expected, the results showed that both
compounds displayed little or even no inhibitory effect against most of 16 kinases
(Table 2).
Table 2
Inhibitory activities (% inhibition) of 6j and 3j (100 μM) against kinases.

O
N
Cl
H
O
N
H
S
N
S
O
N
H
O
O2N
6j
3j
%
inhibitiona
% inhibitiona
% inhibitiona
kinase
kinase
kinase
6
j
3j
6j
3j
6j
3j
GSK-3β
PDGFR-αb 7.2
PDGFR-βb 5.5
PKCα
EPH-A2d
FGFR1e
28 μM 25 μM
CDK1 16
c-Kitb 28
22
-
-6
-7
0
Flt-1b
KDRe
ErbB4c 12.2
EGFRc 12
RETb
6.8
-41
8.9
8.7
-
11.2
18.5
-
41.2
2
CK2
PKA
20
0
12
17.4
13.5
13.8
5.4
c-Srcd 7.2
ABLd
68
0
46.6
a
b
The mean value of two independent experiments. Su11248 as reference inhibitor
for Flt-1 (74.8% inhibition), c-Kit (49.4% inhibition), PDGFR-α (71.4% inhibition),
c
PDGFR-β (70.6% inhibition) and RET (97% inhibition), respectively. BIBW2992 as
reference inhibitor for EGFR (89% inhibition) and ErbB4 (88.1% inhibition),
d
respectively. Dasatinib as reference inhibitor for EPH-A2 (94.8% inhibition), c-Src
e
(
94% inhibition) and ABL (93.2 % inhibition). AP24534 as reference inhibitor for
KDR (IC = 1.7 nM) and FGFR1 (IC = 0.7 nM), respectively.
5
0
50
2
2
.3. In vivo biology
.3.1. In vivo LPS-induced lung injury model
GSK-3β plays a vital factor in inflammation process by modulating many
transcription factors including NF-κB, Wnt/β-catenin pathways. Activation of NF-κB
is involved in initiating an inflammatory cascade and releases a variety of pro-
2
6
inflammatory cytokines. Thus, we hypothesized that 6j and 3j as GSK-3β inhibitors
serve as an important anti-inflammatory protection in LPS-induced lung injury.

Many studies have demonstrated that mice exposed LPS by intratracheal
instillation is an accepted method to determine the ALI. Therefore, this model was
established to evaluate our new GSK-3β inhibitors. Briefly, The C57BL/6 mice were
intraperitoneally injected with compounds 6j (80 μM), 3j (80 μM), or phosphate
buffered saline (PBS), respectively. After 12 hours, LPS (10 mg/kg) was
intraperitoneally administered to mice. The lung tissues were collected at 24 hours
after LPS administration for pathological analysis, western blotting, and RT-PCR.
2
.3.2. Histological damage assessment
Tissue damage was evaluated by morphological analysis. The lung lesions were
scored based on the following: lung hemorrhage, lung edema, interstitial
inflammatory cell infiltration, hyaline membrane, and atelectasis. The degree of each
variable was graded from 0 to 4. No injury = 0; less than 25% injury of field = 1;
2
5%-50% injury of field = 2; 50%-70% injury of field = 3; more than 75% injury of
2
7
field = 4. The score for each parameter was calculated. Six random fields of each
slice were photoed by light microscopic fields (× 200) (Leica SCN400, Leica
Microsystems, Wetzlar, Germany).
As shown in Fig. 4, 24 h after LPS challenge, the lung had moderated congestion
and inflammatory cell infiltration, the injury score exhibited at 9.25-fold compared
with PBS group. 6j and 3j treated in mice that subjected to LPS were associated with
a marked reduction in the pulmonary tissue pathological compared with LPS group,
the injury score was decreased 65% and 49%, respectively. These results suggested
that 6j and 3j as a GSK-3β intervention may block inflammation progress.

Fig. 4. Lungs were obtained from PBS or GSK-3β compounds (6j, 3j) treated mice
after LPS administration. Lung tissue was fixed, embedded, sectioned, and stained
with H&E. The pathological lung injury scores were shown as mean ± SEM (n = 5
mice).
2
.3.3. mRNA expression of IL-1β and IL-6
It is important to note that GSK-3β activity is involved in the development and
progression of LPS-induce ALI by up-regulation pro-inflammatory cytokines. To
assess the potential role of 6j and 3j as GSK-3β inhibitor in LPS-induced lung injury,
we assessed the RNA level of IL-6, IL-1β in lung tissue.
Total RNA from the lung tissue was isolated and the relative mRNA levels in the
experimental groups were standardized by the control group, to obtain fold increases
in mRNA expression. The Fig. 5 showed that 6j and 3j resulted in a significant
decrease in the expression of IL-1β and IL-6 mRNA, which were 43.18%, 28.84%
and 38.16%, 24.35% of those in LPS group, respectively. The results suggested that
6
j and 3j protect lung tissue by inhibiting the release of inflammatory factors, which

was consistent with the change of lung pathological.
Fig. 5. The lungs were harvested from the mice at 24 h and detected for IL-1β and IL6
expression by RT-PCR. Data are expressed as means ± SE of 4 independent
experiments.
2
.3.4. Western blot analysis
There are several mechanisms to down-regulate the activity of GSK-3β. The
most well-defined inactivation mechanism of GSK-3β is phosphorylation of Ser9.28
Several studies also demonstrated that SIRT1 as a NAD(+)-regulated deacetylase,29
3
0
directly interact with NF-κB, and inhibit NF-κB dependent gene expression, such as
3
1
IL-1β, IL-6. However, whether the newly BTZ inhibitors directly enhance GSK-3β
phosphorylation or promote the expression of SIRT1 to negatively regulate NF-κB-
dependent proinflammatory cytokines is still unknown.
In the current study, we utilized western blot to estimate the expression of SIRT1
and the phosphorylation of GSK-3β in the acute injury lung tissue. The Fig. 6 showed
that 6j up-regulated the expression of p-GSK-3β and SIRT1, which were 1.42 and

1
.93-fold as those in LPS group, respectively. When treated with 3j, the SIRT1
expression was 1.34-fold as that in LPS group, but without effect on the
phosphorylation of GSK-3β. These changes render that 6j has a greater ability than 3j
to inactivate GSK-3β through phosphorylation of the Ser9 residue and up-regulation
of SIRT1.
Fig. 6. The lungs were harvested from the mice at 24 h and detected for p-GSK-3β
and SIRT1 expression by Western blot. Data are expressed as means ± SE of 4
independent experiments.
2
.4. Molecular docking
Molecular docking is a very helpful tool to rationalize the structure-activity
relationship of compounds, but it is not meaningful for 3j because of the unclear
binding site of it. The binding mode of the representative compound 6j was modeled
using docking simulation. To validate the docking result, the activity of each pure
enantiomer is necessary. The pure enantiomers were separated by chromatography of
racemic 6j on a chiral stationary phase using a Superchiral S-OD column. Both of the
(R)- and (S)-enantiomers were obtained with high enantiomeric purity (> 98.1%). To

assign the absolute configuration of the enantiomers, we successfully got crystals
from one of the enantiomers and it was confirmed by X-ray crystallography to be of
(
S)-isomer (Fig. 7). The other isomer evidently was (R)- configuration. The activity of
R)- and (S)-6j against GSK-3β was 23.7 μM and 20.0 μM, respectively. From that
(
result, it seemed that the configuration of the α-carbon of the benzothiazepinone
moiety did not play an important role in the action.
Fig. 7. X-ray crystal structure of (S)-6j.
The docking studies for (R)-6j and (S)-6j were performed by utilizing the
Schrodinger software. The GSK-3β crystal structure (PDB ID: 1H8F) was chosen as
the model of the receptor since it was the only published crystal structure for substrate
binding. The ligands were prepared by minimizing the energy of both compounds
using Sybyl 6.931 with MMFF94 force, and the binding site was defined as a sphere
of 10 Å radius around Lys205, which is suggested to be the substrate recognition
binding site for the GSK-3β binding process. The suggested binding mode was shown
in Fig. 8 and several key interactions were observed in both configurations. To (R)-6j,
its ortho-nitro substituted benzyl group attached to the N5 of the BTZ ring extended to
the positively charged pocket composed of Arg96, Arg180 and Lys205, which
allowed nitro to interact with side chains of these residues by means of hydrogen

interaction. In addition, the C2-substituted group of the inhibitor extended to the
hydrophobic region consisting of three amino residues (Ser66, Phe67, and Val70), and
its benzene ring bound with Phe67 by π-π interaction (Fig. 8A). As for (S)-6j, the
ortho-nitro substituted benzyl group, like that in (R)-6j, also extended to the positively
charged pocket composed of Arg96, Arg180 and Lys205 allowed nitro to interact
with side chains of these residues by means of hydrogen interaction. One hydrogen
bond was attributed to the oxygen of carbonyl on BTZ ring with Arg180. Although
the C2-substituted group in (S)-6j extended to the outside of the target enzyme instead
of extending to the hydrophobic region consisting of three amino residues (Ser66,
Phe67, and Val170) as (R)-isomer, the similar π-π interaction between its directly
attached benzene ring with Try216 was also observed (Fig. 8B). The docking
experiments partly made clear that why the activities of the isomers were similar.
Fig. 8. Suggested binding mode for compounds (R)-6j (A) and (S)-6j (B) bound with
GSK-3β (PDB ID: 1H8F).
3
. Conclusions
GSK-3β has recently been proven to regulate some critical transcription factors,
such as NF-κB, NFAT, and STATs, and so promotes the process of inflammation.
These discoveries led to the rapid application of its inhibitors to some of the animal

models to demonstrate their potential for therapeutic intervention. However, the low
specificity of kinase inhibitors should be the great obstacle for their clinical
application. Recently, the development of non-ATP competitive inhibitors might
point out a new way to achieve high selectivity and prove their clinical values. In this
paper, we found two BTZ compounds 6j and 3j showing inhibitory activity to GSK-
3
β in a low micromolar level which are identified with the reported non-ATP
competitive inhibitors. Furthermore, they were proved to act in a non-ATP
competitive inhibition mode, and as expected they really showed good specificity in a
panel test of other 16 protein kinases, even to the closest CDK-1/Cyclin B and CK-II.
The results of in vivo assay proved both compounds can attenuate the LPS-induced
ALI and diminish inflammation response in mice by inhibiting the mRNA expression
of IL-1β and IL-6. Western blot analysis showed that 6j and 3j negatively regulated
GSK-3β, and demonstrated that the mechanism of the observed beneficial effects of
the inhibitors may involve the increased phosphorylation of the Ser9 residue on GSK-
3
β and the expression of SIRT1 (Fig. 9). The in vitro and in vivo results support these
BTZ compounds of GSK-3β inhibitors have a protective role in LPS-induced ALI and
might be potential candidates for further development of inflammation
pharmacotherapy, greatly thanks to their inherently high selectivities from the non-
competitive mechanism. Finally, docking studies were performed to give suggesting
binding modes that can well explain the impacts of candidates on the enzyme.

Fig. 9. Model of inhibitors inactivating GSK-3β through phosphorylation of the Ser9
and up-regulation SIRT1 to inhibit NF-κB signaling pathway.
4
4
4
. Experimental section
.1. Chemistry
.1.1. General
All chemicals used were of reagent grade. Yields refer to purified products and
are not optimized. Reagents were purchased from commercial sources and then used
without further purification except for special case. Flash column chromatography
was carried out at medium pressure using silica gel (200-300 mesh) purchased from
Qingdao Haiyang Chemical Co. Ltd. All the reactions were monitored by thin layer
chromatography (TLC) on silica gel. H and 13C NMR spectra were recorded on a
1
1
13
Mercury Plus 400 spectrometers ( H, 400 MHz, C, 100 MHz). Chemical shifts (δ)
are reported in parts per million (ppm) relative to internal tetramethylsilane (TMS)
and J values are reported in Hertz. Melting points were determined by an SGW X-4
thermometer and were uncorrected (slide method). MS spectra were recorded on an
Agilent LC-MS 1100 instrument with an ESI mass selective detector. High resolution
mass spectra were taken with AB 5600+ Q TOF mass spectrometer using electrospray

ionization (ESI) technique. All final compounds possessed purity > 95% as
determined by high-performance liquid chromatography (HPLC). The HPLC methods
used the following conditions: Cosmosil packed column 5C18-MS-II, 4.6× 150 mm at
3
5 °C with a 1.0 mL/min flow rate, UV purity detected at 254 nm. The mobile phase
was constituted of methanol and water. A gradient from 20% to 95% methanol over 7
min was used at a flow rate of 1.0 mL/min and the elapsed time was 15 min.
4
.1.2. General procedure for the synthesis of 3-substituted acrylic acid (2a-2h)
A solution of suitably substituted carbaldehyde (200 mmol), propenedioic acid
(20.8 g, 200 mmol) in 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
cooled to room temperature. The solid was filtered, washed with water and
recrystallized in ethanol/water.
4
4
4
4
4
4
2
4
2
4
.1.3 (E)-3-(4-Florophenyl)acrylic acid (2a). Yield: 88%; mp. 205.8-207.4 °C.
.1.4. (E)-3-(4-Chlorophenyl)acrylic acid (2b). Yield: 95%; mp 247.1-250.5 °C.
.1.5. (E)-3-(4-(1,1'-Biphenyl))acrylic acid (2c). Yield: 75%; mp 224.6-227.8 °C.
.1.6. (E)-3-(4-Methylphenyl)acrylic acid (2d). Yield: 91%; mp 208.2-211.3 °C.
.1.7. (E)-3-(4-Cyanophenyl)acrylic acid (2e). Yield: 87%; mp 253.3-255.8 °C.
.1.8. (E)-3-(4-(Methoxycarbonyl)phenyl)acrylic acid (2f). Yield: 80%; mp 230.6-
33.0 °C.
.1.9. (E)-3-(3-(Methoxycarbonyl)phenyl)acrylic acid (2g). Yield: 75%; mp 235.4-
37.1 °C.
.1.10. (E)-3-(2-(Methoxycarbonyl)phenyl)acrylic acid (2h). Yield: 75%; mp 245.8-

2
4
47.6 °C.
.1.11. General procedure for the synthesis of 2-substituted-2,3-dihydrobenzo-1,5-
thiazepin-4(5H)-one (3a-3h)
A mixture of 2-aminothiophenol (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 (DCM) was added. The mixture was washed with
aq HCl (2 N), aq Na CO (saturated), and brine. Then, the organic layer was dried
2
3
over MgSO and then concentrated. The crude product residue was purified by
4
chromatography on silica to give pure product.
4
5
7
5
1
1
.1.12. 2-(4-Florophenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one (3a). Yield:
1
5%; White solid; mp 182.9-184.0 °C; H NMR (400 MHz, CDCl ) δ 9.99 (s, 1H),
3
.54 (d, J = 8.0 Hz, 1H), 7.47-7.43 (m, 1H), 7.39-7.35 (m, 2H), 7.21-7.11 (m, 4H),
1
3
.01 (dd, J = 8.0, 4.0 Hz, 1H), 2.69-2.48 (m, 2H); C NMR (100 MHz, CDCl ) δ
3
71.5, 162.4, 160.8, 140.7, 138.5, 135.2, 129.7, 127.6, 126.2, 125.8, 122.7, 115.1,
+
14.9, 51.8, 40.9. ESI-MS m/z 273.9 [M + H] ; HRMS m/z calcd for C H FNOS
1
5
12
+
[
M + H] 274.0696, found 274.0696.
4
7
7
.1.13. 2-(4-Chlorophenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one (3b). Yield:
1
1%; White solid; mp 205.4-207.9 °C; H NMR (400 MHz, CDCl ) δ 10.00 (s, 1H),
3
.55-7.52 (m, 1H), 7.47-7.43 (m, 1H), 7.38-7.33 (m, 4H), 7.21-7.14 (m, 2H), 5.01 (dd,
1
3
J = 8.0, 4.0 Hz, 1H), 2.70-2.48 (m, 2H); C NMR (100 MHz, CDCl ) δ 171.5, 141.1,
3
1
2
40.7, 135.2, 133.0, 129.8, 128.3, 127.2, 126.2, 125.6, 122.7, 51.9, 40.7. ESI-MS m/z
+
+
89.9 [M + H] ; HRMS m/z calcd for C H ClNOS [M + H] 290.0401, found
1
5
12

2
4
90.0403.
.1.14. 2-(4-(1,1'-Biphenyl)phenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one (3c).
1
Yield: 57%; mp 189.6-191.0 °C; H NMR (400 MHz, CDCl ) δ 8.30 (s, 1H), 7.69 (d,
3
J = 8.0 Hz, 21), 7.54 (t, J = 8.0 Hz, 4H), 7.44-7.32 (m, 6H), 7.27-7.19 (m, 2H), 4.94
1
3
(
dd, J = 8.0, 4.0 Hz, 1H), 2.97-2.83 (m, 2H); C NMR (100 MHz, CDCl ) δ 171.5,
3
1
5
4
6
1
7
2
1
41.7, 140.7, 140.2, 139.9, 135.3, 129.6, 126.9, 126.8, 126.4, 126.2, 126.1, 122.7,
2.3, 40.8. ESI-MS m/z 332.1 [M + H]+.
.1.15. 2-(4-Methylphenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one (3d). Yield:
1
5%; Off-white solid; mp 175.6-179.1 °C; H NMR (400 MHz, CDCl ) δ 8.06 (s,
3
H), 7.65 (d, J = 7.7 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.26-7.20 (m, 3H), 7.17 (d, J =
.9 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 4.86 (dd, J = 10.6, 5.8 Hz, 1H), 3.79 (s, 3H),
1
3
.90-2.75 (m, 2H); C NMR (100 MHz, CDCl ) δ 171.5, 158.5, 140.6, 135.3, 134.9,
3
29.5, 126.9, 126.2, 122.6, 113.5, 54.7, 52.1, 41.0, 29.1. ESI-MS m/z 270.1 [M + H]+;
+
HRMS (EI) m/z calcd for C H NOS [M + H] 270.0947, found 270.0949.
1
6
15
4
2
1
2
.1.16. 2-(4-Cyanophenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one (3e). Yield:
1
5%; White solid; mp 195.5-197.1°C; H NMR (400 MHz, DMSO-d ) δ 10.03 (s,
6
H), 8.13 (d, J = 8.0 Hz, 1H), 8.05-8.02 (m, 3H), 7.53-7.45 (m, 6H), 7.24-7.17 (m,
1
3
H), 5.09 (dd, J = 8.0, 4.0 Hz, 1H), 2.79-2.48 (m, 2H); C NMR (100 MHz, DMSO-
d6) δ 170.1, 165.9, 152.5, 146.4, 141.4, 135.0, 134.2, 131.8, 129.7, 127.2, 126.4,
1
25.7, 125.3, 124.5, 123.0, 122.6, 122.2, 51.7, 40.6. ESI-MS m/z 280.1 [M + H]+;
+
HRMS m/z calcd for C H N OS [M + H] 281.0743, found 281.0746.
1
6
12
2
4
.1.17.
2-(4-(Methoxycarbonyl)phenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one

1
(
3f). Yield: 57%; White solid; mp 184.2-186.7 °C; H NMR (400 MHz, CDCl ) δ
3
8
7
3
1
4
3
4
.83 (brs, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.46-7.42 (m, 1H),
.37 (d, J = 8.0 Hz, 2H), 7.26-7.21 (m, 2H), 4.92 (dd, J = 8.0, 4.0 Hz, 1H), 3.90 (s,
1
3
H), 2.90-2.78 (m, 2H); C NMR (100 MHz, CDCl ) δ 171.9, 166.0, 147.6, 140.9,
3
35.2, 129.8, 129.6, 129.0, 126.1, 125.8, 125.3, 122.8, 76.7, 76.4, 76.2, 52.3, 51.5,
+
+
0.5. ESI-MS m/z 314.0 [M + H] ; HRMS m/z calcd for C H NO S [M + H]
1
7
15
3
14.0845, found 314.0847.
.1.18.
2-(3-(Methoxycarbonyl)phenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
3g). Yield: 51%; mp 175.5-178.3 °C; H NMR (400 MHz, CDCl ) δ 7.95 (d, J = 8.3
3
Hz, 2H), 7.59 (d, J = 7.5 Hz, 1H), 7.55-7.47 (m, 1H), 7.41-7.31 (m, 2H), 7.26 (s, 2H),
7
2
4
.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,
H). ESI-MS m/z 314.0 [M + H]+.
.1.18.
2-(2-(Methoxycarbonyl)phenyl)-2,3-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
3h). Yield: 43%; mp 179.0-182.3 °C; H NMR (400 MHz, CDCl ) δ 8.50 (s, 1H),
3
7
8
.97 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.36 (d, J =
.0 Hz, 2H), 7.26-7.19 (m, 2H), 4.91 (dd, J = 8.0, 4.0 Hz, 1H), 3.90 (s, 3H), 2.92-2.78
(
m, 2H); 13C NMR (100 MHz, CDCl ) δ 171.4, 166.0, 147.5, 140.8, 135.3, 129.8,
3
1
29.6, 129.0, 126.2, 125.8, 125.5, 122.7, 52.1, 51.5, 40.4. ESI-MS m/z 314.0 [M +
H]+.
.1.19. 4-(4-Oxo-2,3,4,5-tetrahydrobenzo[b][1,4]thiazepin-2-yl)benzoic acid (3i)
A solution of 3f (3.13 g, 10 mmol) in THF (30 mL), MeOH (10 mL) and H O
4
2
(10 mL) was added LiOH (1.2 g, 30 mmol), and the mixture was stirred under reflux

for 0.5 h. Conc. hydrochloric acid was added to adjust the pH 2-3, and the present
solid was collected by filtration, washed with water and recrystallized from
ethanol/water to give compound 3i as off-white solid (2.24 g, 75% yield), which was
used for next step without further purification.
4
.1.20.
N-(3-Chlorobenzyl)-4-oxo-2,3,4,5-tetrahydrobenzo-1,5-thiazepin-2-
yl)benzamide (3j)
To a mixture of 3i (299 mg, 1 mmol), HATU (760 mg, 2 mmol), HOAt (272 mg,
2
mmol), substituted amine (1.2 mmol) and DCM (10 mL) at 0 °C was added DIPEA
(0.52 mL, 3 mmol), and the reaction mixture was then slowly warm to room
temperature and stirred overnight. The reaction was quenched by water and the
mixture was extracted by ethyl acetate, washed with water and brine, dried over
Na SO and then subjected to flash chromatograph to give compound 3j as off-white
2
4
1
solid. Yield: 80%; H NMR (400 MHz, DMSO-d ) δ 10.7 (s, 1H), 9.05 (t, J = 5.6 Hz,
6
1
2
H), 7.80 (d, J = 7.4 Hz, 2H), 7.39-7.24 (m, 7H), 7.19 (t, J = 7.6 Hz, 1H), 6.97 (m,
H), 4.46 (d, J = 5.7 Hz, 2H), 3.88 (dd, J = 9.5, 5.7 Hz, 1H), 3.23 (dd, J = 14.1, 5.6
1
3
Hz, 1H), 2.76 (dd, J = 14.0, 9.9 Hz, 1H); C NMR (100 MHz, DMSO-d ) δ 167.0,
6
1
1
66.8, 143.0, 141.7, 137.6, 133.6, 133.2, 130.9, 129.8, 128.5, 127.9, 127.8, 127.7,
27.4, 126.5, 123.8, 118.3, 117.6, 43.0, 42.8, 35.2. ESI-MS m/z 423.1 [M + H]+;
+
HRMS m/z calcd for C H ClN O S [M + H] 423.0929, found 423.0928. HPLC
2
3
19
2
2
purity: 95.4%.
4
1
.1.21. General procedure for the synthesis of 2,5-disubstituted-2,3-dihydrobenzo-
,5-thiazepin-4(5H)-one (4a-4h)

A solution of 2-substituted-2,3-dihydrobenzo-1, 5-thiazepin-4(5H)-one (2
mmol), K CO (3 mmol) in anhydrous CH CN (10 mL) were stirred at -10 °C for 10
2
3
3
min. Then 2-nitrobenzyl bromide (1.2 mmol) was added dropwise and the mixture
was heated to reflux and then stirred for 10 h. The reaction was cooled to room
temperature and water was added. The mixture was extracted by ethyl acetate, washed
with water and brine, dried over Na SO and then subjected to flash chromatograph.
2
4
4
.1.22. 5-(2-Nitrobenzyl)-2-(4-Florophenyl)-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
4a). Yield: 91%; mp 166.5-168.8 °C; H NMR (400 MHz, CDCl ) δ 8.37-6.55 (m,
3
1
2H), 5.53 (q, J = 17.3 Hz, 2H), 4.88 (dd, J = 12.7, 5.4 Hz, 1H), 3.13-2.46 (m, 2H);
1
3
C NMR (100 MHz, CDCl ) δ 170.7, 148.1, 145.8, 139.5, 136.8, 133.6, 132.9, 130.8,
3
1
30.0, 129.1, 128.8, 128.1, 127.7, 126.8, 125.0, 124.6, 123.5, 115.6, 52.2, 49.4, 42.2.
+
+
ESI-MS m/z 409.1 [M + H] ; HRMS m/z calcd for C H FN O S [M + H]
2
2
17
2
3
4
4
09.1017, found 409.1025. HPLC purity: 96.6%.
.1.23. 5-(2-Nitrobenzyl)-2-(4-chlorophenyl)-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
4b). Yield: 88%; mp 164.8-167.4 °C; H NMR (400 MHz, CDCl ) δ 8.29-6.77 (m,
3
1
2H), 5.53 (q, J = 17.2 Hz, 2H), 4.85 (dd, J = 12.7, 5.4 Hz, 1H), 3.15-2.69 (m, 2H);
1
3
C NMR (100 MHz, CDCl ) δ 170.6, 148.1, 145.8, 142.1, 136.8, 133.6, 132.6, 130.9,
3
1
4
4
4
29.6, 129.0, 128.1, 127.5, 127.4, 126.7, 125.0, 123.5, 52.2, 49.4, 41.9. ESI-MS m/z
+
+
25.0 [M + H] ; HRMS m/z calcd for C H ClN O S [M + H] 425.0721, found
2
2
17
2
3
25.0725. HPLC purity: 97.3%.
.1.24. 5-(2-Nitrobenzyl)-2-((1,1'-biphenyl)-4-yl)-dihydrobenzo-1,5-thiazepin-4(5H)-
1
one (4c). Yield: 89%; H NMR (400 MHz, CDCl ) δ 8.05 (dd, J = 8.2, 1.2 Hz, 1H),
3

7
1
1
.93 (d, J = 7.8 Hz, 1H), 7.77-7.21 (m, 15H), 5.58 (m, 2H), 4.96 (dd, J = 12.8, 5.3 Hz,
1
3
H), 3.20-2.73 (m, 2H); C NMR (100 MHz, CDCl ) δ 169.3, 145.0, 141.9, 140.2,
3
39.7, 136.1, 132.9, 132.0, 130.0, 128.9, 128.2, 126.9, 124.4, 122.4, 53.2, 49.2, 40.7.
+
+
ESI-MS m/z 467.1 [M + H] ; HRMS m/z calcd for C H N O S [M + Na] 489.1243,
2
8
22
2
3
found 489.1239. HPLC purity: 96.1%.
4
.1.25. 5-(2-Nitrobenzyl)-2-(4-methylphenyl)-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
4d). Yield: 92%; mp 194.0-194.7 °C; H NMR (400 MHz, CDCl ) δ 8.04 (dd, J = 8.2,
3
1
3
3
1
1
.1 Hz, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.63 (dd, J = 11.5, 7.5 Hz, 2H), 7.52-7.31 (m,
H), 7.28-6.99 (m, 5H), 5.56 (q, J =17.3 Hz, 2H), 4.89 (dd, J = 12.7, 5.2 Hz, 1H),
1
3
.11-2.73 (m, 2H), 2.35 (s, 3H); C NMR (100 MHz, CDCl ) δ 170.4, 147.4, 145.1,
3
40.2, 137.0, 136.2, 133.1, 132.1, 130.0, 128.9, 128.8, 127.4, 126.8, 126.5, 125.3,
+
24.4, 122.8, 52.1, 48.9, 41.4, 20.5. ESI-MS m/z 405.1 [M + H] ; HRMS m/z calcd
+
for C H N O S [M + H] 405.1267, found 405.1289. HPLC purity: 99.6%.
2
3
20
2
3
4
.1.26. 5-(2-Nitrobenzyl)-2-(4-cyanophenyl)-dihydrobenzo-1,5-thiazepin-4(5H)-one
1
(
4e). Yield: 94%; mp 182.1-183.5 °C; H NMR (400 MHz, CDCl ) δ 8.17 (d, J = 8.0
3
Hz, 2H), 8.02 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.63-7.59 (m, 2H), 7.5-
7
2
1
4
4
4
.26 (m, 6H), 5.54 (q, J = 17.1 Hz, 2H), 4.95 (dd, J = 12.6, 5.4 Hz, 1H), 3.04-2.74 (m,
1
3
H); C NMR (100 MHz, CDCl ) δ 170.2, 150.3, 148.2, 147.4, 145.8, 136.7, 133.6,
3
32.4, 131.3, 129.5, 128.2, 127.8, 127.1, 126.1, 125.1, 124.2, 123.8, 58.5, 51.9, 49.5,
+
+
1.4, 29.7. ESI-MS m/z 416.0 [M + H] ; HRMS m/z calcd for C H N O S [M + H]
2
3
17
3
3
16.1063, found 416.1061. HPLC purity: 98.5%.
.1.27. 5-(2-Nitrobenzyl)-2-((4-methoxycarbonyl)phenyl)-dihydrobenzo-1,5-