Discovery of novel inhibitors of signal transducer and activator of transcription 3 (STAT3) signaling pathway by virtual screening

Article abstract of DOI:10.1016/j.ejmech.2013.01.009

Inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway has been considered a novel therapeutic strategy to treat human cancers that harbor aberrantly-active STAT3. In this study, nearly 204,000 compounds in Specs database were screened by virtual screening, and samples of top 100 compounds identified as candidate small-molecule inhibitors of STAT3 were evaluated by STAT3-dependent luciferase reporter assay as well as other cell-based assays. A benzothiazole core scaffold containing compound, 9, was identified as an inhibitor of IL-6/STAT3 signaling with an IC₅₀ of 3.567 μM. It is the first time to discover benzothiazole scaffold as a potent STAT3 signaling inhibitor. We further investigated the (structure-activity relationship) SAR of the benzothiazole analogues, and discovered compound 16w as a better small-molecule inhibitor. Both compounds inhibited the phosphorylation of STAT3 and had no obvious effect on upstream JAK2 kinase.

Full text of DOI:10.1016/j.ejmech.2013.01.009

European Journal of Medicinal Chemistry 62 (2013) 301e310
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel inhibitors of signal transducer and activator
of transcription 3 (STAT3) signaling pathway by virtual screening
,
Mingming Zhang a, Weiliang Zhu b, Yingxia Li a
*
a School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Shanghai Institute of Material Medica, Chinese Academy of Science, 555 Zuchongzhi Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the signal transducer and activator of transcription 3 (STAT3) signaling pathway has been
considered a novel therapeutic strategy to treat human cancers that harbor aberrantly-active STAT3. In
this study, nearly 204,000 compounds in Specs database were screened by virtual screening, and samples
of top 100 compounds identified as candidate small-molecule inhibitors of STAT3 were evaluated by
STAT3-dependent luciferase reporter assay as well as other cell-based assays. A benzothiazole core
scaffold containing compound, 9, was identified as an inhibitor of IL-6/STAT3 signaling with an IC50 of
Received 5 November 2012
Received in revised form
4 January 2013
Accepted 8 January 2013
Available online 17 January 2013
3.567 mM. It is the first time to discover benzothiazole scaffold as a potent STAT3 signaling inhibitor. We
Keywords:
further investigated the (structureeactivity relationship) SAR of the benzothiazole analogues, and dis-
covered compound 16w as a better small-molecule inhibitor. Both compounds inhibited the phosphor-
ylation of STAT3 and had no obvious effect on upstream JAK2 kinase.
STAT3 signaling
Small-molecule inhibitors
Virtual screening
Cell viability
Ó 2013 Elsevier Masson SAS. All rights reserved.
Antitumor activity
1. Introduction
phosphorylated, consequently leading STAT3 monomers to dimer-
ize through a reciprocal pTyr-SH2 domain interaction, translocate
The signal transducer and activator of transcription (STATs)
family proteins are a class of transcription factors that modulate
cellular responses, including cell proliferation and differentiation,
inflammation and immune responses [1,2]. STATs signaling can be
activated by upstream cytokines, growth factors, and other poly-
peptide ligands [2]. At least seven members (STAT1 e 4, STAT5a,
STAT5b and STAT6) have been identified in mammalian cells.
Among these members, STAT3 has been attached great importance
and is the most studied member because of its role in malignant
tumorigenesis and transformation. STAT3 is frequently overex-
pressed or persistently activated in most tumors, including ovarian,
cervical, breast, head and neck squamous cell carcinoma, prostate,
and leukemias [3]. STAT3-mediated tumorigenesis may be attrib-
uted to up-regulation of genes encoding antiapoptotic and
proliferation-associated proteins (like Bcl-2, Bcl-xL, Mcl-1, Cyclin
D1, and c-Myc), inducer of tumor angiogenesis (like VEGF and FGF),
and promoter of tumor cell migration and invasion (like MMP-1, -2,
-9, and -10) [4]. In addition, studies also indicated that activated
STAT3 suppressed tumor-immune surveillance [5]. During activa-
tion, tyrosine residue 705 (Tyr705) in the STAT3 SH2 domain is
into the nucleus, recognize STAT3-specific DNA binding elements,
and mediate target gene transcription [1,2]. Given its importance to
tumorigenesis, STAT3 represents an attractive target for discovery
of new anticancer drug and is currently under intense investigation.
Heretofore, several classes of STAT3 inhibitors have been iden-
tified using multiple approaches such as peptide-based rational
design, virtual screening of large chemical libraries, high through-
put screening, repositioning using multiple ligand simultaneous
docking (MLSD), and fragment-based drug design. Some of them
exhibited high potency against STAT3 signaling, including pepti-
domimetics (1), STX-0119 (2), Niclosamide (3), T3 (4), and the
natural product Cucurbitacin Q (5) (Fig. 1) [6e10]. The peptide and
peptidomimetic inhibitory approaches face several challenges in
terms of the low cell permeability, stability, and the consequential
low biological activities, whereas non-peptidic small-molecules
represent a rather more attractive approach to inhibit aberrant
STAT3 activity in cancer cells. Recently, a few STAT3-specific small-
molecule inhibitors, including OPB-31121 and OPB-51602 have
entered into clinical trials [11]. The future of research in STAT3 in-
hibition should focus on seeking potent small-molecule inhibitors
with novel scaffold. In this study, we screened nearly 204,000
compounds by virtual database screening, and reported the iden-
tification of 6-aminobenzo[d]thiazole-2-thiol analogues as novel
* Corresponding author. Tel./fax: þ86 21 51980127.
E-mail address: liyx417@fudan.edu.cn (Y. Li).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.009

302
M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
NH2
O
H
N
O
N
O
N
NO2
O
OH
Cl
O
N
H
N
H
O
O
S
O
N
O
O
HN
O
N
H
N
NH2
H
N
C15H31
O
N
Cl
OH
N
N
P
O
O
OH
1
(2)
(3)
(4)
STX-0119
Niclosamide
T3
O
H
O
O
O
H
O
H
O
S
O
S
N
H
H
OH
O
HO
HO
N
NH
(5)
16w
Cucurbitacin Q
Fig. 1. Structures of 1, STX-0119, Nicloamides, T3, Cucurbitacin Q and 16w.
potent small-molecule inhibitors of the STAT3 signaling pathway
for the first time.
was also investigated, and our results demonstrated that com-
pound 9 displayed a more than 6-fold preference over IFN- /STAT1
and did not inhibit TNF- /NF-LB signaling pathway. In the cell
g
a
viability assay, compound 9 significantly inhibited cell viability at
low micromolar concentrations in breast cell line MDA-MB-468
harboring constitutively activated STAT3, with an IC50 of
2. Results and discussion
2.1. Structure-based virtual screening
8.829 mM. Therefore, we used 9 as a potential lead structure for
further investigation.
Our structure-based virtual screening employed molecular
docking program DOCK (Version 4.0) and GLIDE (http://www.
schrödinger.com) to search for novel STAT3 inhibitors [12e14].
Twenty-five benzothiazole-based inhibitors were synthesized
(Table 2), and evaluated by STAT3-dependent luciferase reporter
assay and cell viability assay (MTT) to explore the SARs. Among
these compounds, the most potent compound 16w inhibited IL-6/
STAT3 signaling pathway with an IC50 of 1.16 mM, 3-fold increase
in potency compared with compound 9. In the cell viability assay,
16w significantly inhibited cell viability of MDA-MB-468 with an
The X-ray crystal structure of the STAT3b homodimer bound to DNA
solved at 2.25-Å resolution was retrieved from Protein Data Bank
(PDB code: 1BG1) for docking simulation. Double-stranded DNA, all
crystallographic water, and PO34ꢀ buffer molecules were removed.
The native pTyr (pY) peptide, APpYLKTKF was extracted from the
crystal structure of one of the monomers as a ligand. The residues
within a radius of 5-Å around the ligand were used to construct the
grids for docking screening. First, DOCK4.0 was used to search the
Specs chemical database (October, 2010; http://www.specs.net) for
potential binders of STAT3. Subsequently, the top 10% ranked
compounds with the highest score obtained by DOCK were selected
for the second round docking by GLIDE SP (Standard Precision
mode), then the top 500 molecules were filtered again by GLIDE XP
(Extra Precision mode). Finally, according to the scores, structural
diversity, and visual inspection, we manually selected and pur-
chased 100 molecules for inhibitory activity assay.
IC50 of 16.06 mM.
To further validate the inhibitory activity of these two com-
pounds against STAT3 pathway, western blot analysis was used. Our
results showed that treatment with compound 9 or 16w con-
sistently inhibited STAT3 phosphorylation in HepG2 cell line. As
shown in Fig. 3, curcumin dose-dependently inhibited Tyr705
phosphorylation, and compounds 9 and 16w remarkably inhibited
Table 1
Inhibitory potency of the tested molecules.
Compound IFN-
g
/STAT1 TNF-
a
/NF-LB IL-6/STAT3a
MDA-MB-468b
2.2. Biology
10
m
M
10
m
M
10
(%)
m
M
IC50
M)
19.25
10
(%)
m
M
IC50
M)
196.5
(%)
(%)
(
m
(
m
All selected compounds were tested for their inhibitory
activity using a STAT3 luciferase reporter system (Table 1). Four of
them (6e9) displayed inhibitory effect on STAT3-induced luciferase
Currumin NDc
ND
ND
ND
ꢀ4.32
ꢀ14.57
ND
30.66
24.0
32.3
38.5
34.8
21.5
49.5
3.88
NDc
NDc
NDc
6
ND
7
ND
activity in HepG2 cell at 10
m
M after 24 h incubation, indicating that
8
9
10
11
13.8
6.17
ND
3.567 66.39
8.829
these compounds strongly blocked STAT3-mediated DNA binding
and inhibited the transcriptional function of STAT3 (Fig. 2). Based
on these inhibitors, a structural similarity-based search in Specs
database was performed, leading to the discovery of another two
hits (10, 11). The most potent compound 9 showed an IC50 of
NDc
ND
ND
NDc
a
STAT3-dependent luciferase reporter gene assay in HepG2 cell. Percent inhibi-
M or IC50
Cell viability assay against MDA-MB-468 (MTT assay). Percent inhibition at
M or IC50
tion at 10
m
.
b
3.567
mM against IL-6/STAT3 signaling pathway. The selectivity
10
m
.
c
against related IFN-
g
/STAT1 and TNF- /NF-LB signaling pathway
a
Not Determined.

M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
303
N
N
N
O
O
N
S
O
O
O
N
O
O2N
NH
O
N
H
O
HN
NH
NH
O
O
O
O
O
O
6
7
8
Br
O
O
S
O
S
O
S
S
N
H
N
H
O
N
N
NH
NH
9
10
O
S
O
S
N
H
O
N
NH
11
Fig. 2. Structures of the discovered active compounds.
Tyr705 phosphorylation at 50
m
M, whereas the total STAT3 protein
another hydrogen bond with Gln635. Interestingly, this com-
pound acted as a partial peptidomimetic of phosphotyrosine
peptide (APpYLKTKF) without the segment having polar in-
teractions with the pTyr-binding pocket surrounded by Lys591,
Arg609, and Ser611.
level remained unchanged after 6 h incubation. In addition, the
influence of compounds 9 and 16w on upstream tyrosine kinase
was also determined. These two compounds had no obvious effects
on the protein levels of JAK and p-JAK after 6 h treatment.
The inhibitory activity of the synthesized compounds 16aey on
STAT3-dependent luciferase reporter gene assay is listed in Table 2.
Among the analogues, 2,6-diethylphenyl or (S)-methylbenzyl group
on R1 was optimal for the inhibitory activity against STAT3 signaling
(16n, 16sex). Aromatic ring (R2) was essential, and replacement of
this aromatic group with alkanes completely abolished the activity
(16hek). Regarding the substitution on R2 ring, 2-OMe-Ph (16f), 4-
OMe-Ph (16s), 4-pyridyl (16n), and 3-pyridyl (16o) groups were
tolerated; however, the incorporation of 2-OMe-benzyl rendered
the compound inactive (16m). In addition, the presence of long-
chain alkenes on the phenyl ring resulted in the improved activ-
ity of compounds (16tez). It might be due to the hydrophobic in-
teractions between the alkene chain and the residue Gln635,
Ile634, and Gln635.
3. Chemistry
The route adapted for the synthesis of these benzothiazole-
based derivatives 16aey is outlined in Scheme 1, using a modified
literature method [15,16]. The starting compounds 12aeg were
prepared from commercially available phenylamine or benzyl-
amine derivatives, and 2-chloroacetyl chloride. In the next step,
12aeg were condensed with 6-aminobenzothiazole-2-thiol in
refluxing acetone, affording the amines 13aeg. Then, target com-
pounds 16aey were obtained, respectively, by the reaction be-
tween 13aeg and the corresponding acyl chloride in the presence
of potassium carbonate at room temperature in dichloromethane.
The acyl chlorides are commercially available or prepared from
corresponding benzoic acids reacted with SOCl2 (15aeg). The yield
of all reactions was satisfactory. All the products were characterized
by 1H NMR, 13C NMR and MS analyses.
5. Conclusions
Here we reported the discovery of novel inhibitors of STAT3
signaling pathway by virtual screening. Among these compounds, 9
and 16w potently inhibited activation and transactivation of STAT3
4. Discussion
A docking model of compound 9 bound to the STAT3eSH2
but had no obvious effects on the closely related IFN-g/STAT1, TNF-
domain was generated on the basis of crystal structure of
a/NF-LB signaling pathway, and the upstream JAK2 kinase. Fur-
STAT3
b
homodimer (PDB code 1BG1) by GLIDE. As illustrated in
thermore, these two compounds greatly inhibited the viability of
human breast cell line MDA-MB-468 harboring constitutively
active STAT3. Therefore, they represented new potent lead com-
pounds with benzothiazole scaffold for development of STAT3
pathway inhibitors as molecular-targeted anticancer drugs. The
extensive studies of the mechanisms are currently undergoing and
will be reported in due course.
Fig. 4, 4-Br-Ph inserted into a small hydrophobic cleft in a manner
similar to Phe710 of the phosphotyrosine peptide. The amideeNH
close to R1 participated in a hydrogen bond interaction with
residue Gln644. In addition, C]O of the other amide close to R2
formed a hydrogen bond with the backbone amideeNH of
Glu638, and the oxygen atom of the methoxyl group formed

304
M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
Table 2
Structure and inhibitory activity of benzothiazole-based molecules.
Compound
Structure
R1
IL-6/STAT3 pathwaya
MDA-MB-
468b
R2
10
m
M (%)
IC50
(m
M)
10
m
M (%)
IC50
(
mM)
Currumin
16a
30.66
ꢀ23.22
19.25
3.88
2.47
196.5
16b
16c
ꢀ32.49
9.04
21.54
39.01
5.01
16d
16e
ꢀ5.89
ꢀ21.44
ꢀ0.77
16f
25.80
ꢀ5.71
2.36
23.88
6.13
16g
16h
16i
eCH2CH3
ꢀ11.98
32.36
ꢀ4.54
ꢀ17.35
ꢀ12.34
12.57
8.23
ꢀ9.23
ꢀ7.98
ꢀ8.31
ꢀ16.30
39.12
16j
16k
16l
16m
16n
16o
14.42
ꢀ13.13
5.48

M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
305
Table 2 (continued )
Compound
Structure
R1
IL-6/STAT3 pathwaya
MDA-MB-
468b
R2
10
m
M (%)
IC50
(m
M)
10
m
M (%)
IC50 (mM)
16p
16q
23.78
ꢀ20.41
ꢀ56.80
ꢀ0.31
16r
16s
42.95
18.85
ꢀ44.67
24.10
16t
25.78
34.89
16u
16v
16w
30.86
30.66
51.10
57.25
61.11
51.43
1.6
16.06
16x
22.57
11.50
44.61
3.02
16y
a
STAT3-dependent luciferase reporter gene assay in HepG2 cell. Percent inhibition at 10
Cell viability assay against MDA-MB-468 (MTT assay).
mM or IC50.
b
6. Experimental section
6.1.3. MTT assay
About 5000 cells per well were seeded into 96-well plates.
Twenty-four hours later, cells were treated with vehicle control
(DMSO) or the tested compounds for 72 h. MTT assay was per-
formed as described [17].
6.1. Biological evaluation
6.1.1. Cell lines and culture
HepG2/STAT3 cells, a gift from Prof. Xinyuan Fu (National Uni-
versity of Singapore, Singapore), were HepG2 cells stably trans-
fected with a STAT3-responsive firefly luciferase reporter plasmid.
All other cell lines were obtained from the American Type Culture
Collection (USA).
6.1.4. Western blot analysis
Whole cell lysates were prepared in 1ꢁ Laemmli sample buffer
(Sigma) to extract total proteins. Equivalent amounts of total cel-
lular protein were electrophoresed on an 8% SDS-PAGE gel and
transferred onto nitrocellulose membranes (Millipore). Membranes
were blocked with 5% nonfat milk in TBS containing 0.1% Tween-20
(TBST) for 1 h at room temperature, and then incubated with the
corresponding primary antibodies in 5% bovine serum albumin
(BSA) in TBSTat 4 ꢂC overnight. Membranes were then washed with
TBST and incubated with horseradish peroxidase-conjugated sec-
ondary antibody in 5% BSA in TBST for 1 h at room temperature.
Immune complexes were detected by enhanced chem-
iluminescence (Pierce). Antibodies used in Western blotting were
mouse anti-STAT3, mouse anti-Phospho-Stat3 (Tyr705), anti-JAK2,
anti-Phospho-JAK2 (Tyr1007/1008) and anti-GAPDH.
6.1.2. Luciferase assay
HepG2/STAT3 cells (1.5 ꢁ105 per well) were seeded into 24-well
microplates (Corning) and allowed to grow for 48 h and then
treated with test samples for 1 h followed by stimulation with
10 ng/mL interleukin (IL)-6 (BD Biosciences) for 5.5 h. Luciferase
activity was determined using a Promega luciferase kit according to
the manufacturer’s instruction. The cell number was counted at
seeding and controlled by equal seeding. All luciferase assays were
repeated at least twice to minimize the variance caused by cell
number.

306
M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
Fig. 3. Compound 9 and 16w inhibited the phosphorylation of STAT3. (A) Curcumin led to does-dependent inhibition of STAT3 phosphorylation. (B) Compounds 9 and 16w inhibited
Tyr705 phosphorylation at 50 M. (C) Compounds 9 and 16w had no obvious effect on JAK2 and p-JAK2. HepG2 cells were pretreated with compounds for 6 h before stimulation by
IL-6 (10 ng/mL) for 15 min. Whole cell lysates were processed for Western blot analysis and probed with the indicated antibodies. GAPDH served as a loading control.
m
6.2. Chemistry
solution was evaporated to give the crude product, which was
purified by Silica gel column chromatography with ethyl acetatee
petroleum ether as eluent.
6.2.1. General information
Solvents were purified by standard methods. TLCs were carried
out on Merck 60 F254 silica gel plates and visualized by UV irradi-
ation. Flash column chromatography was performed on silica gel
(200e300 mesh, Qingdao, China).1H NMR and 13C NMR spectrawere
recorded with a 400 MHz Bruker spectrometer or JEOL JNM-ECP
6.2.2.1. 2-chloro-N-(3-chloro-4-fluorophenyl)acetamide (12a). 195.5
mg as white solid, yield 88.47%. 1H NMR (400 MHz, CDCl3)
d 8.21 (s,
1H), 7.74 (dd, J ¼ 6.4, 2.7 Hz, 1H), 7.38 (ddd, J ¼ 8.9, 4.0, 2.7 Hz, 1H),
7.13 (t, J ¼ 8.7 Hz, 1H), 4.20 (s, 2H). ESI [M þ H]þ calcd for
C8H7Cl2F1N1O1: 221.99, found: 220.2.
400 MHz spectrometers. Chemical shifts (d) are expressed in ppm,
and coupling constants (J) are expressed in Hz. Mass spectra analyses
were performed on a Q-Tof Ultima Global mass spectrometer.
6.2.2.2. 2-chloro-N-(2,6-dimethylphenyl)acetamide (12b). 172.6 mg
as white solid, yield 87.59%. 1H NMR (400 MHz, DMSO-d6)
d 9.62 (s,
6.2.2. General procedure for the synthesis of 12aeg
1H), 7.07 (d, J ¼ 5.7 Hz, 3H), 4.26 (s, 2H), 2.12 (s, 6H). ESI [M þ H]þ
2-chloroacetyl chloride (1.5 mmol) was added dropwise to
a mixture of amine (1 mmol) and triethylamine (3 mmol) in 20 mL
dichloromethane originally at 0 ꢂC with stirring. Then the resulting
mixture was cooled to ambient temperature and stirred for 6 h.
After removal of dichloromethane solvents on a rotary evaporator,
water was added and extract with ethyl acetate (3 ꢁ 30 mL). The
organic layer was combined and dried with sodium sulfate. The
calcd for C10H13Cl1N1O1: 198.07, found: 198.2.
6.2.2.3. 2-chloro-N-(2,6-diethylphenyl)acetamide (12c). 186.2 mg as
white solid, yield 82.72%. 1H NMR (400 MHz, DMSO-d6)
d 9.61 (s,1H),
7.22e7.14 (m, 1H), 7.09 (d, J ¼ 7.5 Hz, 2H), 4.25 (s, 2H), 2.47
(d, J ¼ 7.7 Hz, 5H), 1.08 (t, J ¼ 7.6 Hz, 6H). ESI [M þ H]þ calcd for
C12H17Cl1N1O1: 226.1, found: 226.1.
O
R1
S
O
S
N
H
a
b
c
R1
Cl
R1 NH22
N
H
N
NH2
12a-g
13a-g
O
R1
N
S
O
S
R2
H
N
NH
16a-y
Scheme 1. Synthesis of derivatives 16aey. Reagents and conditions: (a) 2-chloroacetyl chloride, Et3N, CH2Cl2, 0 ꢂC to RT, 6 h, yield 70e82%; (b) 6-amino-2-mercaptobenzothiazole,
K2CO3, acetone, reflux, 4 h, yield 90e95%; (c) benzoyl chloride/4-methoxybenzoyl chloride/2-methoxybenzoyl chloride/4-nitrobenzoyl chloride/acetyl chloride/2-chloroacetyl
chloride/isobutyrl chloride/cyclopropanecarbonyl chloride/propionyl chloride/15aeg, K2CO3, CH2Cl2, RT, 4e12 h, yield 60e80%.

M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
307
Fig. 4. Docking mode of compound 9 with STAT3 generated by GLIDE. Visible by PyMol. Carbon atoms of compound 9 are colored cyan, and those of phosphotyrosine peptide
(APpYLKTKF) are colored yellow. Hydrogen bonding to Gln644, Glu638, and Gln635 are shown by red dashed lines. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
6.2.2.4. 2-chloro-N-(4-fluorobenzyl)acetamide (12d). 177.0 mg as
white solid, yield 88.04%. 1H NMR (400 MHz, CDCl3)
7.31e7.24
obtained respectively and could be used directly for the next step
without further purification.
d
(m, 2H), 7.09e7.00 (m, 2H), 6.86 (s, 1H), 4.47 (d, J ¼ 5.9 Hz, 2H),
4.11 (s, 2H). ESI [M þ H]þ calcd for C9H10Cl1F1N1O1: 202.04, found:
202.1.
6.2.4. General procedure for the synthesis of 14aed
4-hydrobenzoic acid (1.38 g, 10 mmol) and bromoalkane
(12.5 mmol) were dissolved in ethanol (20 mL). KOH (1.23 g,
21.83 mmol) was then added and the resulting mixture was stirred
under reflux for 24 h. Subsequently, KOH (1.23 g, 20.14 mmol) was
added and reflux was continued for a further 1 h. After cooling to
room temperature, ethanol was removed under vacuum, and the
residue was resolved in water (60 mL), acidified to pH ¼ 1 with HCl.
After that, the precipitate was collected, recrystallized twice from
ethanol, and finally dried under vacuum to give 14aed (30e44%) of
white solids.
6.2.2.5. 2-chloro-N-(4-nitrobenzyl)acetamide (12e). 174.3 mg as
yellow solid, yield 76.44%. 1H NMR (400 MHz, DMSO-d6)
d 8.89 (s,
1H), 8.18 (d, J ¼ 8.5 Hz, 2H), 7.51 (d, J ¼ 8.6 Hz, 2H), 4.42 (d,
J ¼ 6.0 Hz, 2H), 4.15 (s, 2H). ESI [M þ H]þ calcd for C9H10Cl1N2O3:
229.04, found: 229.1.
6.2.2.6. (S)-2-chloro-N-(1-phenylethyl)acetamide (12f). 165.0 mg as
white solid, yield 83.73%. 1H NMR (400 MHz, CDCl3)
d 7.42e7.27 (m,
5H), 6.78 (s, 1H), 5.22e5.06 (m, 1H), 4.16e3.96 (m, 2H), 1.54 (d,
J ¼ 6.9 Hz, 3H). ESI [M þ H]þ calcd for C10H13Cl1N1O1: 198.07, found:
198.1.
6.2.5. General procedure for the synthesis of 15aeg
Thionyl chloride (10 mL) was added to carboxylic acids
(0.381 mmol). The mixture was stirred and refluxed for 3 h. After
cooling to room temperature, the thionyl chloride was evaporated
under reduced pressure giving light yellow oil or solid. The purity of
15aeg was quite sufficient to use the product directly for the fol-
lowing synthesis.
6.2.2.7. (R)-2-chloro-N-(1-phenylethyl)acetamide (12g). 169.6 mg as
white solid, yield 86.07%. 1H NMR (400 MHz, CDCl3)
d 7.42e7.27 (m,
5H), 5.20e5.09 (m, 1H), 4.12e4.00 (m, 2H), 1.54 (d, J ¼ 6.9 Hz, 3H). ESI
[M þ H]þ calcd for C10H13Cl1N1O1: 198.07, found: 198.1.
6.2.3. General procedure for the synthesis of 13aeg
6.2.6. General procedure for the synthesis of 16aey
6-aminobenzothiazole-2-thiol (0.241 mmol) was added to
a mixture of 12aeg (0.254 mmol) and potassium carbonate
(0.688 mmol) in 10 mL of acetone originally at room temperature
with stirring. The resulting mixture was stirred for 2 h under reflux,
then cooled to ambient temperature. After removal of acetone
solvents on a rotary evaporator, the solid was dissolved in
dichloromethane (20 mL) and washed with water (3 ꢁ 10 mL).
Then, the organic phase was dried over MgSO4 and concentrated in
vacuo to give the solid product. The title compounds 13aek were
The acyl chloride (w0.381 mmol) was added to a mixture of
13aek (w0.241 mmol) and potassium carbonate (0.508 mmol) in
10 mL of dichloromethane at room temperature with stirring. The
completion of the reaction was monitored by TLC and took 4e8 h.
The precipitate was collected by filtration and washed with water
to yield the crude product. Each compound was purified by column
chromatography on silica gel using dichloromethane/ethyl acetate
as eluent or crystallized from suitable solvent to give pure target
compounds 16aey.

308
M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
6.2.6.1. N-(2-((2-((3-chloro-4-fluorophenyl)amino)-2-oxoethyl)thio)
benzo[d]thiazol-6-yl)-4-methoxybenzamide (16a). 62.78 mg as
134.00, 132.60, 130.10, 127.77, 126.45, 125.31, 121.39, 120.98, 119.65,
112.48, 112.35, 56.37, 36.91, 24.70, 15.09. ESI-HRMS [M þ H]þ calcd
for C27H28N3O3S2: 506.15666, found: 506.15757.
white solid, yield 51.2%. 1H NMR (400 MHz, DMSO-d6)
d 10.65 (s,
1H),10.30 (s,1H), 8.51 (d, J ¼ 1.9 Hz,1H), 7.95 (d, J ¼ 8.9 Hz, 2H), 7.89
(dd, J ¼ 6.9, 2.5 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.71 (dd, J ¼ 8.8,
2.1 Hz, 1H), 7.45 (dd, J ¼ 4.4, 2.5 Hz, 1H), 7.38 (t, J ¼ 9.0 Hz, 1H), 7.06
(d, J ¼ 8.9 Hz, 2H), 4.36 (s, 2H), 3.82 (s, 3H). 13C NMR (100 MHz,
6.2.6.7. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)-4-nitrobenzamide (16g). 82 mg as light yellow solid,
yield 65.43%. 1H NMR (400 MHz, DMSO-d6)
d 10.81 (s, 1H), 9.68 (s,
DMSO-d6)
d
166.12, 165.49, 164.60, 162.47, 149.19, 136.75, 136.47,
1H), 8.56 (d, J ¼ 1.9 Hz, 1H), 8.40 (d, J ¼ 8.9 Hz, 2H), 8.21 (d,
J ¼ 8.8 Hz, 2H), 7.96e7.71 (m, 2H), 7.24e7.12 (m, 1H), 7.08 (d,
J ¼ 7.5 Hz, 2H), 4.37 (s, 2H), 2.47 (d, J ¼ 7.5 Hz, 4H), 1.03 (t, J ¼ 7.5 Hz,
136.44, 135.72, 130.11, 127.21, 121.36, 121.06, 120.33, 120.04, 119.97,
117.70, 117.48, 114.12, 113.09, 55.92, 38.06. ESI-HRMS [M þ Na]þ
calcd for C23H17Cl1F1N3Na1O3S2: 524.02761, found: 524.02627.
6H). 13C NMR (100 MHz, DMSO-d6)
d 207.01, 166.34, 165.42, 149.77,
149.69, 141.95, 140.93, 135.98, 135.83, 133.99, 129.73, 127.77, 126.45,
124.08, 121.42, 120.39, 113.45, 31.16, 24.70, 15.07. ESI-HRMS
[M þ H]þ calcd for C26H25N4O4S2: 521.13117, found: 521.13185.
6.2.6.2. N-(2-((2-((3-chloro-4-fluorophenyl)amino)-2-oxoethyl)thio)
benzo[d]thiazol-6-yl)benzamide (16b). 57 mg as white solid, yield
50.21%. 1H NMR (400 MHz, DMSO-d6)
d 10.65 (s, 1H), 10.46 (s, 1H),
7.95 (d, J ¼ 7.0 Hz, 2H), 7.89 (d, J ¼ 4.4 Hz, 1H), 7.79 (d, J ¼ 8.6 Hz,
1H), 7.75e7.67 (m, 1H), 7.59 (t, J ¼ 7.2 Hz,1H), 7.53 (t, J ¼ 7.3 Hz, 2H),
7.46 (ddd, J ¼ 9.0, 4.5, 2.7 Hz,1H), 7.38 (t, J ¼ 9.1 Hz,1H), 4.37 (s, 2H).
6.2.6.8. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)propionamide (16h). 49.77 mg as white solid, yield
48.36%. 1H NMR (400 MHz, DMSO-d6)
d 10.12 (s, 1H), 9.66 (s, 1H),
13C NMR (100 MHz, DMSO-d6)
d
165.54, 165.51, 164.74, 162.45,
8.41 (d, J ¼ 1.8 Hz, 1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.53 (dd, J ¼ 8.8,
2.0 Hz, 1H), 7.26e7.12 (m, 1H), 7.07 (d, J ¼ 7.6 Hz, 2H), 4.34 (s, 2H),
2.50e2.41 (m, 4H), 2.36 (q, J ¼ 7.5 Hz, 2H), 1.10 (t, J ¼ 7.5 Hz, 3H),
149.26, 136.76, 135.69, 135.27, 130.17, 128.12, 127.21, 127.03, 121.19,
120.37, 114.10, 113.09, 55.92, 37.03, 31.16, 25.41, 18.52. ESI-HRMS
[M þ Na]þ calcd for C22H15Cl1F1N3Na1O2S2: 494.01705, found:
494.01588.
1.02 (t, J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d 172.64,
166.36, 164.30, 148.91, 141.94, 136.76, 135.92, 133.98, 127.76, 126.43,
121.42, 118.92, 111.60, 36.91, 30.00, 24.69, 15.06, 10.12. ESI-HRMS
[M þ H]þ calcd for C22H26N3O2S2: 428.14609, found: 428.14706.
6.2.6.3. N-(2-((2-((2,6-dimethylphenyl)amino)-2-oxoethyl)thio)
benzo[d]thiazol-6-yl)-2-methoxybenzamide (16c). 72.2 mg as white
solid, yield 62.78%. 1H NMR (400 MHz, DMSO-d6)
d
10.33 (s, 1H),
6.2.6.9. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
9.67 (s, 1H), 8.52 (s, 1H), 7.77 (d, J ¼ 8.9 Hz, 1H), 7.65 (dd, J ¼ 18.0,
8.1 Hz, 2H), 7.50 (t, J ¼ 7.5 Hz, 1H), 7.17 (d, J ¼ 8.5 Hz, 1H), 7.11e6.97
(m, 4H), 4.34 (s, 2H), 3.89 (s, 3H), 2.12 (s, 6H). 13C NMR (100 MHz,
[d]thiazol-6-yl)cyclopropane carboxamide (16i). 72.36 mg as white
solid, yield 68.39%. 1H NMR (400 MHz, DMSO-d6)
d 10.44 (s, 1H),
9.66 (s, 1H), 8.39 (d, J ¼ 1.9 Hz, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 7.54 (dd,
J ¼ 8.8, 2.0 Hz,1H), 7.23e7.12 (m, 1H), 7.07 (d, J ¼ 7.5 Hz, 2H), 4.34 (s,
2H), 2.50e2.40 (m, 4H), 1.02 (t, J ¼ 7.5 Hz, 6H), 0.87e0.78 (m, 4H).
DMSO-d6)
d 165.53, 165.12, 164.79, 156.97, 149.30, 136.42, 135.90,
135.69, 135.22, 132.60, 130.11, 128.14, 127.06, 125.31, 121.37, 120.99,
119.70, 112.50, 112.39, 56.38, 37.02, 18.50. ESI-HRMS [M þ H]þ calcd
for C25H24N3O3S2: 478.12536, found: 478.12633.
13C NMR (100 MHz, DMSO-d6)
d 165.54, 165.51, 164.74, 162.45,
149.26, 136.76, 135.69, 135.27, 130.17, 128.12, 127.21, 127.03, 121.19,
120.37, 114.10, 113.09, 55.92, 37.03, 31.16, 25.41, 18.52. ESI-HRMS
[M þ Na]þ calcd for C23H26N3O2S2: 440.14609, found: 440.14630.
6.2.6.4. N-(2-((2-((2,6-dimethylphenyl)amino)-2-oxoethyl)thio)
benzo[d]thiazol-6-yl)-4-methoxybenzamide (16d). 65 mg as white
solid, yield 56.52%. 1H NMR (400 MHz, DMSO-d6)
d
10.42 (s, 1H),
6.2.6.10. 2-chloro-N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)
9.81 (s, 1H), 8.52 (s, 1H), 7.99 (d, J ¼ 8.8 Hz, 2H), 7.77 (s, 2H), 7.10e
thio)benzo[d]thiazol-6-yl)propanamide (16j). 89 mg as white solid,
6.99 (m, 5H), 4.36 (s, 2H), 3.83 (s, 3H), 2.13 (s, 6H). 13C NMR
yield 80.1%.1H NMR (400 MHz, DMSO-d6)
d 10.57 (s,1H), 9.66 (s,1H),
(100 MHz, DMSO-d6)
d
165.54,165.51,164.74,162.45,149.26,136.76,
8.41 (d, J ¼ 2.0 Hz,1H), 7.81 (d, J ¼ 8.8 Hz,1H), 7.58 (dd, J ¼ 8.8, 2.1 Hz,
1H), 7.28e7.13 (m,1H), 7.07 (d, J ¼ 7.5 Hz, 2H), 4.71 (q, J ¼ 6.6 Hz,1H),
4.36 (s, 2H), 2.49e2.41 (m, 4H), 1.64 (d, J ¼ 6.6 Hz, 3H), 1.02 (t,
135.69, 135.27, 130.17, 128.12, 127.21, 127.03, 121.19, 120.37, 114.10,
113.09, 55.92, 37.03, 31.16, 25.41, 18.52. ESI-HRMS [M þ H]þ calcd
for C25H24N3O3S2: 478.12536, found: 478.12639.
J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d 167.89, 166.32,
165.23, 149.55, 141.94, 136.00, 135.75, 133.98, 127.76, 126.44, 121.56,
119.32,112.39, 55.24, 36.93, 24.69, 21.46,15.06. ESI-HRMS [M þ Na]þ
calcd for C22H24Cl1N3Na1O2S2: 484.08907, found: 484.08902.
6.2.6.5. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)-4-methoxybenzamide (16e). 69.24 mg as white
solid, yield 56.89%. 1H NMR (400 MHz, DMSO-d6)
d 10.33 (s, 1H),
9.67 (s, 1H), 8.53 (d, J ¼ 1.9 Hz, 1H), 7.99 (d, J ¼ 8.9 Hz, 2H), 7.78 (dt,
J ¼ 8.9, 5.4 Hz, 2H), 7.18 (dd, J ¼ 8.0, 7.1 Hz, 1H), 7.08 (dd, J ¼ 8.2,
3.7 Hz, 4H), 4.37 (s, 2H), 3.85 (s, 3H), 2.50e2.45 (m, 4H), 1.03 (t,
6.2.6.11. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)isobutyramide (16k). 67.14 mg as white solid, yield
63.17%. 1H NMR (400 MHz, DMSO-d6)
d 10.08 (s, 1H), 9.66 (s, 1H),
J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d
165.54, 165.51,
8.42 (d, J ¼ 2.0 Hz, 1H), 7.76 (d, J ¼ 8.8 Hz, 1H), 7.55 (dd, J ¼ 8.8,
2.1 Hz, 1H), 7.24e7.12 (m, 1H), 7.07 (d, J ¼ 7.5 Hz, 2H), 4.34 (s, 2H),
2.72e2.56 (m,1H), 2.50e2.43 (m, 4H),1.12 (d, J ¼ 6.8 Hz, 6H),1.02 (t,
164.74, 162.45, 149.26, 136.76, 135.69, 135.27, 130.17, 128.12, 127.21,
127.03, 121.19, 120.37, 114.10, 113.09, 55.92, 37.03, 31.16, 25.41, 18.52.
ESI-HRMS [M þ H]þ calcd for C27H28N3O3S2: 506.15666, found:
506.15617.
J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d 175.85, 166.36,
164.36, 161.08, 148.94, 145.14, 141.94, 136.80, 135.88, 133.98, 127.77,
126.44, 124.93, 121.39, 119.06, 111.76, 35.43, 31.17, 24.69, 19.96,
15.07. ESI-HRMS [M þ H]þ calcd for C23H28N3O2S2: 442.16174,
found: 442.16267.
6.2.6.6. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)-2-methoxybenzamide (16f). 71.14 mg as white solid,
yield 58.45%. 1H NMR (400 MHz, DMSO-d6)
d 10.37 (s, 1H), 9.67 (s,
1H), 8.55 (d, J ¼ 1.9 Hz, 1H), 7.81 (d, J ¼ 8.8 Hz, 1H), 7.71 (dd, J ¼ 8.8,
2.0 Hz,1H), 7.65 (dd, J ¼ 7.6,1.6 Hz,1H), 7.57e7.47 (m,1H), 7.26e7.13
(m, 2H), 7.09 (t, J ¼ 7.2 Hz, 3H), 4.37 (s, 2H), 3.91 (s, 3H), 2.50e2.44
(m, 4H), 1.03 (t, J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
6.2.6.12. N-(2,6-diethylphenyl)-2-((6-(2-(2-methoxyphenyl)acet-
amido)benzo[d]thiazol-2-yl)thio)acetamide (16l). 88.71 mg as white
solid, yield 70.92%. 1H NMR (400 MHz, DMSO-d6)
d 10.31 (s, 1H),
9.66 (s, 1H), 8.40 (d, J ¼ 1.9 Hz, 1H), 7.78 (d, J ¼ 8.8 Hz, 1H), 7.57 (dd,
d
166.37, 165.14, 164.76, 156.95, 149.30, 141.96, 136.42, 135.91,
J ¼ 8.8, 2.0 Hz, 1H), 7.32e7.21 (m, 2H), 7.21e7.13 (m, 1H), 7.07

M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
309
(d, J ¼ 7.5 Hz, 2H), 6.99 (d, J ¼ 8.0 Hz,1H), 6.91 (t, J ¼ 7.4 Hz,1H), 4.34
(s, 2H), 3.78 (s, 3H), 3.67 (s, 2H), 2.46 (t, J ¼ 7.5 Hz, 4H), 1.02
6.2.6.18. (S)-4-methoxy-N-(2-((2-oxo-2-((1-phenylethyl)amino)
ethyl)thio)benzo[d]thiazol-6-yl)benzamide (16r). 70.11 mg as white
(t, J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d
169.76,
solid, yield 60.99%. 1H NMR (400 MHz, DMSO-d6)
d 10.32 (s, 1H),
166.36, 164.40, 157.71, 148.99, 141.94, 136.76, 135.93, 133.98, 131.29,
128.56, 127.76, 126.44, 124.50, 121.45, 120.63, 118.98, 111.67, 111.15,
55.89, 38.20, 36.93, 24.69, 15.07. ESI-HRMS [M þ H]þ calcd for
C28H30N3O3S2: 520.17231, found: 520.17169.
8.80 (d, J ¼ 8.0 Hz, 1H), 8.51 (d, J ¼ 1.7 Hz, 1H), 7.99 (d, J ¼ 8.9 Hz,
2H), 7.77 (dt, J ¼ 8.9, 5.4 Hz, 2H), 7.27 (ddd, J ¼ 29.9,13.4, 7.0 Hz, 5H),
7.09 (d, J ¼ 8.9 Hz, 2H), 5.03e4.81 (m, 1H), 4.25e4.09 (m, 2H), 3.85
(s, 3H), 1.39 (d, J ¼ 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6)
d
165.95, 165.49, 164.94, 162.47, 149.27, 144.64, 136.67, 135.69,
6.2.6.13. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
130.11, 128.70, 127.23, 127.16, 126.41, 121.27, 120.30, 114.13,
113.04, 55.93, 48.95, 37.18, 22.88. ESI-HRMS [M þ H]þ calcd for
C25H24N3O3S2: 478.12536, found: 478.12660.
[d]thiazol-6-yl)isonicotinamide (16m). 87.1 mg as white solid, yield
75.93%.1H NMR (400 MHz, DMSO-d6)
d 10.75 (s,1H), 9.68 (s,1H), 8.81
(dd, J ¼ 4.5,1.6 Hz, 2H), 8.55 (d, J ¼ 1.9 Hz,1H), 7.89 (dd, J ¼ 4.5,1.5 Hz,
2H), 7.85 (d, J ¼ 8.8 Hz,1H), 7.77 (dd, J ¼ 8.8, 2.0 Hz,1H), 7.24e7.14 (m,
1H), 7.08 (d, J ¼ 7.5 Hz, 2H), 4.37 (s, 2H), 2.50e2.42 (m, 4H), 1.03 (t,
6.2.6.19. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)-4-(pentyloxy)benzamide (16s). 97.60 mg as white
J ¼ 7.5 Hz, 6H). 13C NMR (100 MHz, DMSO-d6)
d
166.34, 165.44,
solid, yield 72.16%. 1H NMR (400 MHz, CDCl3)
d
8.87 (d, J ¼ 7.7 Hz,
164.62, 150.79, 149.77, 142.28, 141.95, 135.86, 135.82, 133.99, 127.78,
126.45, 122.07, 121.41, 120.38, 113.47, 36.93, 24.70, 15.08. ESI-HRMS
[M þ H]þ calcd for C25H25N4O2S2: 477.14134, found: 477.14077.
1H), 8.48 (dd, J ¼ 17.5, 1.9 Hz, 1H), 8.18e7.66 (m, 4H), 7.35 (dt,
J ¼ 8.8, 3.0 Hz, 1H), 7.22e7.12 (m, 1H), 7.02 (dd, J ¼ 43.9, 8.2 Hz, 4H),
4.16 (s, 2H), 4.05 (dt, J ¼ 28.2, 6.5 Hz, 2H), 2.52 (q, J ¼ 7.5 Hz, 4H),
1.94e1.76 (m, 2H), 1.44 (ddd, J ¼ 20.7, 11.3, 5.0 Hz, 4H), 1.06 (t,
J ¼ 7.6 Hz, 6H), 0.95 (t, J ¼ 7.1 Hz, 3H). 13C NMR (100 MHz, CDCl3)
6.2.6.14. N-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
[d]thiazol-6-yl)nicotinamide (16n). 71.74 mg as white solid, yield
d 167.56, 165.77, 165.34, 162.35, 148.85, 141.63, 136.56, 135.53,
62.54%.1H NMR (400 MHz, DMSO-d6)
d
10.69 (s,1H), 9.68 (s,1H), 9.13
132.34, 128.93, 128.08, 126.45, 126.39, 121.35, 119.16, 114.54, 112.44,
68.32, 36.10, 28.82, 28.15, 24.86, 22.44, 22.39, 14.60, 14.01. ESI-
HRMS [M þ H]þ calcd for C31H36N3O3S2: 562.2193, found: 562.2199.
(d, J ¼ 1.7 Hz,1H), 8.79 (dd, J ¼ 4.8,1.5 Hz,1H), 8.55 (d, J ¼ 1.9 Hz,1H),
8.38e8.26 (m,1H), 7.85 (d, J ¼ 8.8 Hz,1H), 7.77 (dd, J ¼ 8.8, 2.0 Hz,1H),
7.60 (dd, J ¼ 7.7, 5.0 Hz,1H), 7.21e7.13 (m,1H), 7.08 (d, J ¼ 7.5 Hz, 2H),
4.37 (s, 2H), 2.50e2.43 (m, 4H), 1.03 (t, J ¼ 7.5 Hz, 6H). 13C NMR
6.2.6.20. tN-(2-((2-((2,6-diethylphenyl)amino)-2-oxoethyl)thio)benzo
(100 MHz, DMSO-d6)
d
166.35, 165.24, 164.68, 152.68, 149.64, 149.15,
[d]thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (16t). 93.51 mg as
141.95, 136.13, 135.98, 135.82, 133.99, 130.95, 127.77, 126.45, 124.03,
121.40, 120.28, 113.27, 36.93, 31.17, 24.70, 15.09. ESI-HRMS [M þ H]þ
calcd for C25H25N4O2S2: 477.14134, found: 477.14025.
white solid, yield 69.14%. 1H NMR (400 MHz, CDCl3)
d 8.87 (d,
J ¼ 8.6 Hz, 1H), 8.49 (dd, J ¼ 15.6, 2.0 Hz, 1H), 7.99 (d, J ¼ 26.4 Hz,1H),
7.93e7.71 (m, 3H), 7.34 (ddd, J ¼ 8.7, 4.2, 2.2 Hz, 1H), 7.22e6.90 (m,
5H), 4.49 (ddd, J ¼ 16.4,12.1, 6.1 Hz,1H), 4.16 (s, 2H), 2.69 (s,1H), 2.52
(q, J ¼ 7.5 Hz, 4H), 1.91e1.70 (m, 1H), 1.70e1.59 (m, 1H), 1.48 (dddd,
J ¼ 19.7, 13.7, 6.4, 3.5 Hz, 2H), 1.36 (dd, J ¼ 18.1, 6.1 Hz, 3H), 1.06 (t,
J ¼ 7.6 Hz, 6H), 0.96 (td, J ¼ 7.3, 3.4 Hz, 3H). 13C NMR (100 MHz,
6.2.6.15. N-(2-((2-((4-fluorobenzyl)amino)-2-oxoethyl)thio)benzo[d]
thiazol-6-yl)-4-methoxybenzamide (16o). 63.5 mg as white solid,
yield 51.97%. 1H NMR (400 MHz, DMSO-d6)
d 10.33 (s, 1H), 8.84
(t, J ¼ 5.9 Hz,1H), 8.52 (d, J ¼ 1.8 Hz,1H), 7.99 (d, J ¼ 8.8 Hz, 2H), 7.77
(dt, J ¼ 8.9, 5.4 Hz, 2H), 7.30 (dd, J ¼ 8.6, 5.6 Hz, 2H), 7.18e7.01 (m,
4H), 4.31 (d, J ¼ 5.9 Hz, 2H), 4.19 (s, 2H), 3.85 (s, 3H). 13C NMR
CDCl3) d 167.53, 165.77, 165.33, 161.57, 148.84, 141.63, 136.57, 135.52,
132.34, 128.97, 128.09, 127.18, 126.45, 121.37, 119.13, 115.53, 114.01,
112.43, 38.47, 38.38, 36.10, 24.86, 19.61, 18.71, 18.61, 14.61, 14.02. ESI-
HRMS [M þ H]þ calcd for C31H36N3O3S2: 562.2193, found: 562.2202.
(100 MHz, DMSO-d6) d 166.95,165.49,162.82,162.47,160.41,149.25,
136.69, 135.70, 130.11, 129.61, 129.53, 127.21, 121.32, 120.30, 115.52,
115.31, 114.13, 113.06, 110.77, 55.92, 42.31, 36.98. ESI-HRMS
[M þ H]þ calcd for C24H21F1N3O3S2: 482.10029, found: 482.10030.
6.2.6.21. (S)-4-butoxy-N-(2-((2-oxo-2-((1-phenylethyl)amino)ethyl)
thio)benzo[d]thiazol-6-yl)benzamide (16u). 93.70 mg as white solid,
yield 74.89%. 1H NMR (400 MHz, CDCl3)
d
8.44 (dd, J ¼ 11.6, 1.9 Hz,
6.2.6.16. 4-methoxy-N-(2-((2-((4-nitrobenzyl)amino)-2-oxoethyl)
1H), 8.07 (d, J ¼ 38.8 Hz, 1H), 7.97e7.76 (m, 3H), 7.67 (d, J ¼ 8.7 Hz,
1H), 7.42 (td, J ¼ 8.4, 2.1 Hz, 1H), 7.20 (d, J ¼ 10.2 Hz, 5H), 6.98 (d,
J ¼ 8.8 Hz, 2H), 5.06 (p, J ¼ 7.0 Hz, 1H), 4.08 (ddd, J ¼ 28.2, 10.1,
5.3 Hz, 2H), 3.93 (q, J ¼ 14.9 Hz, 2H), 1.94e1.74 (m, 2H), 1.54 (dt,
J ¼ 22.5, 7.6 Hz, 2H), 1.43 (d, J ¼ 6.9 Hz, 3H), 1.08e0.92 (m, 3H). 13C
thio)benzo[d]thiazol-6-yl)benzamide (16p). 68.22 mg as white solid,
yield 55.72%. 1H NMR (400 MHz, DMSO-d6)
d 10.33 (s, 1H), 8.99 (t,
J ¼ 6.0 Hz, 1H), 8.54 (d, J ¼ 1.9 Hz, 1H), 8.12 (d, J ¼ 8.7 Hz, 2H), 7.98
(dd, J ¼ 6.8, 4.9 Hz, 2H), 7.81 (d, J ¼ 8.8 Hz, 1H), 7.76 (dd, J ¼ 8.9,
2.0 Hz, 1H), 7.52 (d, J ¼ 8.7 Hz, 2H), 7.09 (d, J ¼ 8.9 Hz, 2H), 4.46 (d,
J ¼ 6.0 Hz, 2H), 4.23 (s, 2H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-
NMR (100 MHz, DMSO-d6)
d 165.54, 165.51, 164.74, 162.45, 149.26,
136.76, 135.69, 135.27, 130.17, 128.12, 127.21, 127.03, 121.19, 120.37,
114.10, 113.09, 55.92, 37.03, 31.16, 25.41, 18.52. ESI-HRMS [M þ H]þ
calcd for C28H30N3O3S2: 520.1723, found: 520.1731.
d6)
d 167.34, 165.50, 164.71, 162.46, 149.20, 147.73, 146.85, 136.77,
135.75, 130.12, 128.52, 127.24, 123.83, 121.32, 120.22, 114.13, 113.01,
55.92, 42.59, 36.88. ESI-HRMS [M þ H]þ calcd for C24H21N4O5S2:
509.09479, found: 509.09338.
6.2.6.22. (S)-N-(2-((2-oxo-2-((1-phenylethyl)amino)ethyl)thio)
benzo[d]thiazol-6-yl)-4-(pentyloxy)benzamide (16v). 85.12 mg as
6.2.6.17. (R)-4-methoxy-N-(2-((2-oxo-2-((1-phenylethyl)amino)
white solid, yield 66.24%. 1H NMR (400 MHz, CDCl3)
d 8.43 (dd,
ethyl)thio)benzo[d]thiazol-6-yl)benzamide (16q). 68.22 mg as white
J ¼ 12.4, 2.0 Hz,1H), 8.09 (d, J ¼ 42.3 Hz,1H), 7.98e7.75 (m, 3H), 7.66
(dd, J ¼ 8.7, 1.8 Hz, 1H), 7.42 (td, J ¼ 8.4, 2.1 Hz, 1H), 7.26e7.12 (m,
5H), 6.97 (d, J ¼ 8.7 Hz, 1H), 5.06 (p, J ¼ 7.0 Hz, 1H), 4.17e4.06 (m,
1H), 4.02 (t, J ¼ 6.6 Hz, 1H), 3.99e3.85 (m, 2H), 1.96e1.76 (m, 2H),
1.56e1.32 (m, 7H), 0.95 (td, J ¼ 7.1, 2.3 Hz, 3H). 13C NMR (100 MHz,
solid, yield 55.72%. 1H NMR (400 MHz, DMSO-d6)
d 10.32 (s, 1H),
8.80 (d, J ¼ 7.9 Hz, 1H), 8.51 (d, J ¼ 1.7 Hz, 1H), 7.99 (d, J ¼ 8.8 Hz,
2H), 7.77 (dt, J ¼ 8.9, 5.3 Hz, 2H), 7.31 (dt, J ¼ 14.9, 7.4 Hz, 4H), 7.22
(t, J ¼ 7.0 Hz, 1H), 7.09 (d, J ¼ 8.8 Hz, 2H), 4.93 (p, J ¼ 7.0 Hz, 1H),
4.24e4.11 (m, 2H), 3.84 (d, J ¼ 4.9 Hz, 3H),1.39 (d, J ¼ 7.0 Hz, 3H). 13C
CDCl3)
d 167.28, 165.64, 164.28, 162.35, 149.09, 142.88, 136.52,
NMR (100 MHz, DMSO-d6)
d
165.95, 165.49, 164.95, 162.46, 149.27,
135.17, 128.94, 128.60, 127.28, 126.39, 125.93, 121.26, 119.31, 114.55,
112.54, 69.41, 49.45, 36.37, 28.64, 28.15, 28.07, 22.45, 22.40, 22.00,
14.02. ESI-HRMS [M þ H]þ calcd for C29H32N3O3S2: 534.1880,
found: 534.1887.
144.64, 136.66, 135.68, 130.11, 128.70, 127.22, 127.16, 126.40, 121.27,
120.29,114.13,113.04, 55.92, 48.95, 37.17, 22.89. ESI-HRMS [M þ H]þ
calcd for C25H24N3O3S2: 478.12536, found: 478.12503.

310
M. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 301e310
6.2.6.23. N-(2-((2-oxo-2-(((S)-1-phenylethyl)amino)ethyl)thio)benzo
References
[d]thiazol-6-yl)-4-(pentan-2-yloxy)benzamide (16w). 89.34 mg as
white solid, yield 77.85%. 1H NMR (400 MHz, CDCl3)
d 8.44 (dd,
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J ¼ 11.6, 2.0 Hz, 1H), 8.05 (d, J ¼ 36.3 Hz, 1H), 7.96e7.75 (m, 3H), 7.66
(d, J ¼ 8.7 Hz,1H), 7.41 (td, J ¼ 8.6, 2.1 Hz,1H), 7.25e7.11 (m, 5H), 6.98
(dd, J ¼ 12.3, 8.8 Hz, 1H), 5.06 (p, J ¼ 7.0 Hz, 1H), 4.49 (ddd, J ¼ 15.5,
12.2, 6.1 Hz,1H), 4.00e3.84 (m, 2H),1.88e1.64 (m, 2H),1.59e1.18 (m,
8H), 1.04e0.88 (m, 3H). 13C NMR (100 MHz, CDCl3)
d 167.28, 165.66,
164.28, 161.56, 148.94, 142.88, 136.56, 135.16, 128.98, 128.60, 127.27,
126.16, 125.93, 121.23, 119.27, 115.53, 112.45, 49.44, 38.46,
38.37, 36.36, 22.00, 19.60, 18.72, 18.61, 14.02. ESI-HRMS [M þ H]þ
calcd for C29H32N3O3S2: 534.1880, found: 534.1883.
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6.2.6.24. 4-(benzyloxy)-N-(2-((2-((2,6-diethylphenyl)amino)-2-
oxoethyl)thio)benzo[d]thiazol-6-yl)benzamide (16x). 100.03 mg as
white solid, yield 69.53%. 1H NMR (400 MHz, CDCl3)
d 8.90 (s, 1H),
8.53 (d, J ¼ 1.5 Hz, 1H), 7.92 (s, 1H), 7.85 (d, J ¼ 8.6 Hz, 2H), 7.75
(d, J ¼ 8.7 Hz, 1H), 7.51e7.30 (m, 6H), 7.23e7.13 (m, 1H), 7.13e6.97
(m, 4H), 5.15 (s, 2H), 4.17 (s, 2H), 2.53 (q, J ¼ 7.5 Hz, 4H), 1.06 (t,
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d 167.50, 165.95, 165.19,
161.86, 148.91, 141.64, 136.62, 136.15, 135.38, 132.35, 128.93, 128.73,
128.29, 128.07, 127.49, 126.91, 126.45, 121.39, 119.07, 115.01,
112.44, 70.21, 36.09, 24.86, 14.61. ESI-HRMS [M þ H]þ calcd for
C33H32N3O3S2: 582.1880, found: 582.1884.
6.2.6.25. (S)-4-(benzyloxy)-N-(2-((2-oxo-2-((1-phenylethyl)amino)
ethyl)thio)benzo[d]thiazol-6-yl)benzamide (16y). 84.37 mg as white
solid, yield 63.29%. 1H NMR (400 MHz, CDCl3)
d
8.47 (d, J ¼ 1.8 Hz,
1H), 8.00e7.81 (m, 4H), 7.68 (d, J ¼ 8.7 Hz, 1H), 7.41 (ddd, J ¼ 15.4,
9.5, 7.0 Hz, 6H), 7.21 (s, 5H), 7.08 (d, J ¼ 8.8 Hz, 2H), 5.15 (s, 2H), 5.06
(p, J ¼ 7.1 Hz, 1H), 3.94 (q, J ¼ 14.9 Hz, 2H), 2.05 (s, 2H), 1.44 (d,
J ¼ 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 167.28, 165.65, 165.21,
161.86, 149.00, 142.90, 136.60, 136.17, 135.29, 128.95, 128.74, 128.60,
128.30, 127.49, 127.27, 126.94, 125.95, 121.27, 119.13, 115.01,
112.46, 70.22, 49.42, 36.36, 21.99. ESI-HRMS [M þ H]þ calcd for
C31H27N3O3S2: 554.1567, found: 554.1578.
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Acknowledgments
This work was supported by the 11th Fudan University Gradu-
ates Innovation Project and National Natural Science Foundation of
China (Grant No. 81072525).
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2013.01.009.
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