Design, synthesis and biological evaluation of thienopyridinones as Chk1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.06.044

A series of thienopyridinone derivatives was designed and synthesized as inhibitors of checkpoint kinase 1 (Chk1). Most of them exhibited moderate to good Chk1 inhibitory activities. Among them, compounds 8q, 8t, and 8w with excellent Chk1 inhibitory activities (IC₅₀ values of 4.05, 6.23, and 2.33 nM, respectively) displayed strong synergistic effects with melphalan, a DNA-damaging agent in the cell-based assay. Further kinase profiling indicated that compound 8t was highly selective against CDK2/cyclinA, Aurora A, and PKC.

Full text of DOI:10.1016/j.bmc.2014.06.044

Bioorganic & Medicinal Chemistry 22 (2014) 4882–4892
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of thienopyridinones
as Chk1 inhibitors
b,
Pinrao Song a, Peng Peng a, Mengmeng Han b, Xianchao Cao b, Xiaodong Ma a, Tao Liu a, , Yubo Zhou
⇑
⇑
,
Yongzhou Hu a,
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thienopyridinone derivatives was designed and synthesized as inhibitors of checkpoint kinase
1 (Chk1). Most of them exhibited moderate to good Chk1 inhibitory activities. Among them, compounds
8q, 8t, and 8w with excellent Chk1 inhibitory activities (IC50 values of 4.05, 6.23, and 2.33 nM, respec-
tively) displayed strong synergistic effects with melphalan, a DNA-damaging agent in the cell-based
assay. Further kinase profiling indicated that compound 8t was highly selective against CDK2/cyclinA,
Aurora A, and PKC.
Received 14 April 2014
Revised 21 June 2014
Accepted 22 June 2014
Available online 28 June 2014
Keywords:
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Chk1 inhibitor
Thienopyridinones
Antitumor
Cell synergy
1. Introduction
Although clinical application of the first generation Chk1 inhib-
itors, exemplified by UCN-01, was hindered by its off-target effects
Checkpoint kinase 1 (Chk1), as a serine/threonine protein
kinase, plays an essential role in regulating cell cycle progression
in response to DNA damage.1 Predominantly activated by the
upstream kinase, ataxia telangiectasia and rad3 related (ATR),
Chk1 mediates the S and G2/M arrest through the proteolysis of
Cdc25A, and leads to the inactivation of Cdk2, whose function is
required in the cell cycle transition. Since most of human malig-
nancies harbor mutations or defects in the DNA-binding domain
of p53, they are more reliant on the later S or G2/M phase check-
point for cell cycle arrest.2 Activation of these checkpoints provides
an opportunity for cancer cells to repair damaged DNA and thus
reduces the effect of cancer chemotherapies and radiotherapy.
While normal cells remaining at the G1 phase via p53, are less
influenced by the abrogation of S and G2/M checkpoints. Therefore,
inhibition of Chk1 by small molecules or siRNA against Chk1, in
combination with traditional DNA damaging therapy could selec-
tively target tumor cells, and has been recognized as a promising
strategy for cancer therapy.3
and unfavorable pharmacokinetic profiles,4 recent years have wit-
nessed the development of many Chk1 inhibitors with various
chemical scaffolds (Fig. 1).5 Among these, most are single digit
nM inhibitors of Chk1 with promising prospects for further optimi-
zation of cellular potency and selectivity profiles.6 Particularly,
nine candidates have entered into clinical evaluations for the treat-
ment of cancer.7
At the beginning of our work, an initial survey of the literatures
revealed that quinolin-2-one was a favorable core for exploring
antitumor agents.8 Besides, it is also incorporated in a well-known
preclinical Chk1 inhibitor (Fig. 2, CHIR-124), which strongly inhib-
its Chk1 and potentiates the cytotoxicity of Topoisomerase I inhib-
itors.9 For the pursuit of an improved potency and selectivity
profile, we studied the structural characteristics of several Chk1
inhibitors and designed a series of novel thienopyridinone deriva-
tives based on bioisosterism strategy incorporating the following
concepts: (1) replacement of the quinolinone of CHIR-124 with
thienopyridinone; (2) various alicyclic amine and aliphatic amine
substituents were introduced at the 4-position of the thienopyrid-
inone core; (3) small substituents such as methyl, chlorine, and
bromine were introduced at the 6-position. All the synthesized
compounds were evaluated for their Chk1 inhibitory activities,
and representative compounds were performed in a combination
⇑
Corresponding authors. Tel.: +86 571 88208458; fax: +86 571 88981051 (T.L.);
tel./fax: +86 21 5080 1313 (Y.Z.); tel./fax: +86 571 8820 8460 (Y.H.).
E-mail addresses: lt601@zju.edu.cn (T. Liu), ybzhou@mail.shcnc.ac.cn (Y. Zhou),
huyz@zju.edu.cn (Y. Hu).
http://dx.doi.org/10.1016/j.bmc.2014.06.044
0968-0896/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4883
Figure 1. Structures of Chk1 inhibitors with various chemical scaffolds.
Figure 2. Design of novel thienopyridinone Chk1 inhibitors.
assay on human multiple myeloma cell line and further tested for
2.2. Chk1 kinase assay
their selectivities against other kinases.
The obtained thienopyridinone derivatives were initially evalu-
ated for their Chk1 kinase inhibitory activities by ADP-Glo lumi-
nescent assay, with AZD7762 employed as the positive control.
The results are summarized in Table 1.
2. Results and discussion
2.1. Chemistry
As shown in Table 1, most compounds exhibited Chk1
inhibitory potency in nanomolar level except for compounds
8f, 8h, 8i, and 8k–p. Incorporation of basic alicyclic amines at
the 4-amino group of the thienopyridinone core (8a–e, 8q–z,
17a, and 17b) resulted in desirable Chk1 inhibitory activity.
Among them, (S)-3-piperidine was validated as the most pre-
ferred substituent (8b, 8r, 8w, Chk1 IC50 = 5.22, 3.53, 2.33 nM,
respectively). In contrast, replacement of the alicyclic amines
with cyclohexyl (8f) or aliphatic amines (8h–j) caused dramatic
loss in potency. Compounds 8n–p, of which, the nitrogen within
piperidine or morpholine ring was directly linked to the thieno-
The synthetic routes for thienopyridinone derivatives were
summarized in Schemes 1 and 2. Methyl 2-aminothiophene-3-
carboxylates 2 and 9 were hydrolyzed with 1 N KOH aqueous solu-
tion under microwave radiation and then reacted with phosgene to
give thiaisatoic anhydrides 3 and 10, respectively.10 Treatment of 3
and 10 with p-methoxybenzyl bromide (PMB) in the presence of
K2CO3 in DMF afforded N-PMB thiaisatoic anhydrides 4a and 4d,
respectively. Compound 4b or 4c was achieved by chlorination or
bromination of 4a. The obtained 4a–d were reacted with
5-(un)substituted ethyl-2-benzimidazole-acetates in the presence
of LHMDS in THF, which furnished the key intermediates 5a–e.
Compounds 5a–e were then converted into corresponding bis-
triflates 6a–e. Treatment of 6a–e with various alkyl amines under
mild conditions provided 7a–z in good yields. Finally, removal of
the PMB and Tf groups under acidic conditions led to the target
compounds 8a–z.11 The preparation of compounds 17a and 17b
pyridinone scaffold, only displayed
activity against Chk1 with an IC50 value >10
a
negligible inhibitory
M. Besides, com-
l
pounds with small substituents, such as chlorine (8q–t), bromine
(8u, 8v), and methyl group at C-6 of thienopyridinone core (8y,
8z) and incorporation of a methoxy group at the 5-position of
benzimidazole ring (8w, 8x) exhibited
a slightly enhanced
potency. Translocation of the sulfur atom in thienopyridinone
from the 7-position to 5-position maintained potency as well,
were conducted in
a similar method starting from methyl
3-aminothiophene-2-carboxylate 11.

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P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
Scheme 1. Synthesis of thieno[2,3-b]pyridin-6(7H)-ones 8a–z. Reagents and conditions: (a) 1 N KOH, MeOH; (b) COCl2, toluene, 62–65%; (c) PMB-Br, K2CO3, DMF, 80–92%;
(d) for clorization/NCS, AcOH, 83%; for bromonization/NBS, DCM, 74%; (e) ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate, LHMDS, THF, 36–43%; (f) (TfO)2O, Py, DCM, 80–85%; (g)
R1-NH2 (for 7a–m, 7q–z), or R1 (for 7n–p), ACN, 90–95%; (h) TFA/HCl (7:1), 40–62%.
which indicated that the position of the sulfur atom had little
impact on Chk1 binding affinity.
no cytotoxicity in the absence of melphalan and thereby can be
identified as ideal Chk1 inhibitors.1,14
2.4. Kinase selectivity profiles
2.3. Cell synergy assay
Inhibition of other kinases simultaneously may antagonize
Chk1 inhibition phenotypes and even result in off-target effects,
which highlights the benefit of monitoring kinase selectivity.15
Therefore, nine compounds (8a, 8b, 8e, 8q, 8t, 8u, 8w, 8x, and
17a) were further evaluated for their selectivity profiles against
Aurora A, CDK2/cyclinA, and PKC. As shown in Table 2, all the
tested compounds demonstrated more than 20-fold selectivities
over CDK2/cyclinA, Aurora A and PKC. Compounds 8e, 8t, and 8x
sharing the common 3-aminoquinuclidine fragment at the
4-position of thienopyridinone core exhibited nearly 100-fold
selectivities over CDK2/cyclinA and PKC. Noticeably, compounds
8e and 8t showed almost no inhibition against Aurora A.
It has been widely accepted that Chk1 inhibitors could enhance
the antitumor effects of conventional DNA-damaging agents in
combination assays.12 Based on this consideration, potent Chk1
inhibitors 8q, 8t, and 8w were further evaluated for their anti-
proliferation activities against human multiple myeloma (MM) cell
line RPMI-8266 that was p53-mutant.13 As illustrated in Figure 3,
when used as single agents, all the three compounds and the posi-
tive Chk1 inhibitor AZD7762 at 100 nM have little effect on the
proliferation of RPMI-8266 cells. Whereas, they significantly
potentiated the cytotoxicity of the DNA-damaging anti-MM agent
melphalan (1–10 lM). In particular, compounds 8q and 8t showed

P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4885
Scheme 2. Synthesis of thieno[3,2-b]pyridin-5(4H)-ones 17a and 17b. Reagents and conditions: (a) 1 N KOH, MeOH; (b) COCl2, toluene, 78%; (c) PMB-Br, K2CO3, DMF, 78%;
(d) ethyl 2-(1H-benzo[d]imidazol-2-yl)acetate, LHMDS, THF, 26%; (e) (TfO)2O, Py, DCM, 78%; (f) R1-NH2, ACN, 75–78%; (g) TFA/HCl (7:1), 47–48%.
Table 1
Chk1 inhibitory profile for thienopyridinones 8a–z, 17a, and 17b
Compd
R1
R2
H
R3
H
Chk1a IC50 (nM)
15.60
Compd
R1
R2
H
R3
H
Chk1a IC50 (nM)
H
N
8a
8o
>10
>10
4.05
l
M
M
N
H
N
O
8b
8c
8d
H
H
H
H
H
H
5.22
8p
8q
8r
H
H
H
H
l
N
H
H
N
N
47.50
Cl
Cl
H
N
HN
9.94
3.53
N
H
N
8e
8f
H
H
H
H
14.02
8s
8t
Cl
Cl
H
H
40.98
6.23
N
>10 lM
H
N
H
N
8g
8h
H
H
H
H
116.10
8u
8v
Br
Br
H
H
4.83
H2N
H
N
>10
lM
15.34
(continued on next page)

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P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
Table 1 (continued)
Compd
R1
R2
H
R3
H
Chk1a IC50 (nM)
Compd
R1
R2
Cl
R3
Chk1a IC50 (nM)
2.33
H
N
H
N
8i
8j
>10
lM
8w
OMe
N
N
H
H
H
H
177.80
8x
8y
Cl
OMe
H
5.36
9.84
H
N
O
N
8k
>10
lM
Me
MeOOC
HO
H
N
8l
H
H
H
H
>10
>10
l
M
M
8z
Me
H
H
H
23.20
7.54
H
N
8m
l
17a
HO
H
N
8n
H
—
H
—
>10
lM
17b
H
H
24.73
N
AZD7762
—
7.89
a
Values are means of two experiments.
Figure 3. Synergistic effects of compounds 8q, 8t, and 8w with melphalan.
2.5. Molecular docking study
Table 2
Kinase selectivity profiles of nine Chk1 inhibitors
Based on the biological evaluation results of Chk1 kinase inhibi-
tion, two representative compounds, the highly potent inhibitor 8t,
and the weak inhibitor 8o were selected for further docking simu-
lations to analyze their binding modes with Chk1 protein using the
C-DOCKER protocol within Discovery Studio 2.1 software package.
The published X-ray crystal structure of Chk1 (PDB ID: 2GDO) was
used for the docking calculation. As shown in Figure 4, the critical
donor-acceptor-donor motif has been retained in both 8t and 8o,
which makes three hydrogen bonds with Chk1 hinge region resi-
dues Glu85 and Cys87. The presence of the 3-aminoquinuclidine
substituent in 8t fixed a basic tertiary-amine in the ribose binding
pocket, which strongly contacted to acidic residues Glu134 and
Glu91. Whereas, similar interactions cannot be observed between
compound 8o and Chk1 protein (Fig. 4B). It gave us a possible
explanation that mere hydrogen bond interactions between
IC50 (nM)
CDK2/cyclinA
Compd
Chk1
Aurora A
PKC
8a
8b
8e
8q
8t
8u
8w
8x
17a
15.60
5.22
14.02
4.05
6.23
4.83
2.33
5.36
7.54
848.82
851.83
1630.44
334.33
3187.14
246.20
393.33
4304.83
255.99
790.81
495.14
>10000
1051.51
>10000
1591.65
328.20
1400.34
201.86
695.85
522.51
1001.81
168.89
2018.88
392.71
204.48
8186.03
814.06
Compound 8w, the most potent one in the Chk1 kinase assay,
moderately inhibited other targets as well, which might partly
explain its cytotoxic effect as a single agent in the cell-based assay.

P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4887
these advantages, it can be recognized as a promising candidate
for further investigation.
4. Experimental
4.1. Chemistry
Melting points were determined with a B-540 Büchi melting-
point apparatus and are uncorrected. 1H NMR spectra were
recorded on a 500 MHz, 13C NMR were recorded on a 125 MHz
spectrometer at room temperature (chemical shifts are given in
ppm (d) relative to TMS as internal standard, coupling constants
(J) are in hertz (Hz), and signals are designated as follows: s, sin-
glet; d, doublet; t, triplet; m, multiplet; br, broad singlet, etc.).
Mass spectra (MS), ESI (positive) were recorded on an Esquire-
LC-00075 spectrometer. Thin layer chromatography was carried
out using plate silica gel F254 Merck. Reagents and solvents were
purchased from common commercial suppliers and some
unhydrous solvents were further purified before usage. All yields
are unoptimized and generally represent the result of a single
experiment.
4.1.1. Synthesis of thiaisatoic anhydrides (3, 10, 12)
2-Aminothiophene-3-carboxylate
2
(4 g, 25 mmol) was
suspended in an aqueous solution of potassium hydroxide 50 mL
(1 N, 50 mmol). The mixture was then heated under microwave
radiation (500 W) for 15 min. After cooling the solution at 0 °C,
phosgene (1.6 equiv, 21 mL of 20% COC12 solution in toluene)
was added dropwise with stirring. The mixture was allowed to
stand at room temperature overnight. The resulting precipitate
was filtered and washed successively with water and petroleum
ether.
4.1.1.1. 3-Thiaisatoic anhydride (3). White solid (65%), mp: 230–
231 °C (lit. 232 °C).
4.1.1.2. 6-Methyl-3-thiaisatoic anhydride (10). White solid
(62%), mp: 195–197 °C (lit. 193–196 °C).
Figure 4. Docking mode comparison between compound 8t and 8o bound to Chk1.
(A) Molecular docking analysis of 8t (yellow backbone) with Chk1; (B) Molecular
docking analysis of 8o (pink backbone) with Chk1. Red dashed lines indicate
hydrogen bonds, prepared using PyMOL, PDB ID: 2GDO.
4.1.1.3. 2-Thiaisatoic anhydride (12). White solid (78%), mp:
230–232 °C (lit. 230 °C).
ligands and Chk1 protein in this series were not sufficient for main-
taining Chk1 inhibitory potency, and lack of the basic amine tail
would cause dramatic drop of potency. These results predicted that
compound 8t would lead to potent biological activity, and this was
confirmed by the experimental results.
4.1.2. General procedure for the PMB protection of thiaisatoic
anhydride derivatives (4a, 4d, 13)
To a precooled (0 °C) solution of thiaisatoic anhydride deriva-
tive (3) (10 mmol) in DMF (10 mL) was added K2CO3 (12 mmol).
A solution of 4-methoxybenzylbromide (12 mmol) in DMF (3 mL)
was added dropwise. After stirring at room temperature for
40 min, the reaction mixture was poured into ice and water
(50 mL). The precipitate was filtered, washed with water and dried
in vacuo and the crude product was used directly in the next step
without further purification.
3. Conclusion
In this study, a series of thienopyridinone derivatives was
designed and synthesized as Chk1 inhibitors based on bioisoster-
ism strategy. Preliminary structure–activity relationship (SAR)
indicated that structural modification on the 4-position of thieno-
pyridinone core greatly influenced the Chk1 inhibitory activities.
Basic cyclic substituents at the 4-NH position were beneficial for
maintaining potency. The introduction of 3-piperidine at the
4-amino group contributes to the improvement in both enzymatic
and cellular synergistic efficacy, while the 3-quinuclidine fragment
was confirmed as a priority for gaining selectivity over other Ser/
Thr kinases. Based on the experimental results, it is noteworthy
that compound 8t not only demonstrated potent Chk1 inhibitory
activity but also displayed synergistic effect with melphalan
against RPMI-8266 tumor cell line. Additionally, it performed
excellent selectivity profiles against some other kinases. With all
4.1.2.1. 1-(4-Methoxybenzyl)-1H-thieno[2,3-d][1,3]oxazine-2,4-
dione (4a).
White solid (92%), mp: 112–114 °C. 1H NMR
(500 MHz, DMSO-d6) d 7.38 (d, J = 18 Hz, Ar-H, 2H), 7.28 (d,
J = 12 Hz, Ar-H, 1H), 7. 24 (d, J = 12 Hz, Ar-H, 1H), 6.94 (d,
J = 18 Hz, Ar-H, 2H), 5.06 (s, CH2, 2H), 3.74 (s, CH3, 3H). ESI-MS:
m/z = 290 [M+H]+.
4.1.2.2.
1-(4-Methoxybenzyl)-6-methyl-1H-thieno[2,3-d][1,3]
oxazine-2,4-dione (4d).
White solid (80%), mp: 130–132 °C.
1H NMR (500 MHz, DMSO-d6) d 7.37 (d, J = 18 Hz, Ar-H, 2H), 6.99
(s, Ar-H, 1H), 6.94 (d, J = 18 Hz, Ar-H, 2H), 5.01 (s, CH2, 2H), 3.74
(s, CH3, 3H), 2.37 (s, CH3, 3H). ESI-MS: m/z = 304 [M+H]+.

4888
P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4.1.2.3. 1-(4-Methoxybenzyl)-1H-thieno[3,2-d][1,3]oxazine-2,4-
dione (13). White solid (78%), mp: 179–181 °C (lit. 181–182 °C).
(m, Ar-H, 2H), 7.32 (d, J = 18 Hz, Ar-H, 2H), 6.93 (d, J = 18 Hz,
Ar-H, 2H), 5.18 (s, CH2, 2H), 3.72 (s, CH3, 3H). ESI-MS: m/z = 482
[M+H]+.
4.1.3. Synthesis of 6-chloro-1-(4-Methoxybenzyl)-1H-
thieno[2,3-d][1,3]oxazine-2,4-dione (4b)
4.1.5.4. 3-(1H-Benzimidazole-2yl)-6-methyl-4-hydroxyl-1-(4-m-
ethoxybenzyl)-thieno[2,3-b]pyridin-6(7H)-one (5d). White solid
(36%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.64 (br, NH,
1H), 7.79–7.77 (m, Ar-H, 2H), 7.33–7.31 (m, Ar-H, 2H), 7.31 (d,
J = 18 Hz, Ar-H, 2H), 7.00 (s, Ar-H, 1H), 6.91 (d, J = 18 Hz, Ar-H,
2H), 5.18 (s, CH2, 2H), 3.72 (s, CH3, 3H), 2.39 (s, CH3, 3H). ESI-MS:
m/z = 418 [M+H]+.
To a solution of compound 4a (1.95 g, 6.74 mmol) in toluene
(8 mL) and glacial acetic acid (8 mL), N-chlorosuccinimide
(1.08 g, 8.09 mmol) was added in one portion. Then the mixture
was stirred at 70 °C for 2 h. Upon cooling, the reaction mixture
was extracted with CH2Cl2 and water. And then concentrated in
vacuo, the resulting crude material was purified by column chro-
matography (petroleum ether/CH2Cl2 = 2:3, v/v) on silica gel to
afford the product as a white solid. Yield: 83%, mp: 140–142 °C.
1H NMR (500 MHz, DMSO-d6) d 7.44 (s, Ar-H, 1H), 7.40 (d,
J = 17 Hz, Ar-H, 2H), 6.95 (d, J = 17 Hz, Ar-H, 2H), 5.02 (s, CH2,
2H), 3.75 (s, CH3, 3H). ESI-MS: m/z = 324 [M+H]+.
4.1.5.5. 6-Chloro-4-hydroxy-3-(5-methoxy-1H-benzo[d]imidazo
l-2-yl)-1-(4-methoxybenzyl)thieno[2,3-b]pyridin-2-one (5e). White
solid (41%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.47
(br, NH, 1H), 7.68 (d, J = 17 Hz, Ar-H, 1H), 7.38 (s, Ar-H, 1H), 7.32
(d, J = 18 Hz, Ar-H, 2H), 7.27 (s, Ar-H, 1H), 6.97 (d, J = 17 Hz, Ar-H,
1H), 6.92 (d, J = 18 Hz, Ar-H, 2H), 5.17 (s, CH2, 2H), 3.80 (s, CH3,
3H), 3.72 (s, CH3, 3H). ESI-MS: m/z = 468 [M+H]+.
4.1.4. Synthesis of 6-bromo-1-(4-methoxybenzyl)-1H-
thieno[2,3-d][1,3]oxazine-2,4-dione (4c)
N-bromosuccinimide (1.8 g, 10.11 mmol) in CH2Cl2 (20 mL) was
added dropwise to a solution of 4a (1.95 g, 6.74 mmol) in CH2Cl2
(100 mL) under N2 protection. After the reaction was stirred at
room temperature for 2 h, water (100 mL) was added. The organic
layer was separated, washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The resulting
crude material was purified by column chromatography
(petroleum ether/ethyl acetate = 4:1, v/v) on silica gel to give a
white solid. Yield: 74%, mp: 136–138 °C. 1H NMR (500 MHz,
DMSO-d6) d 7.51 (s, Ar-H, 1H), 7.40 (d, J = 17 Hz, Ar-H, 2H), 6.95
(d, J = 17 Hz, Ar-H, 2H), 5.02 (s, CH2, 2H), 3.75 (s, CH3, 3H).
ESI-MS: m/z = 368 [M+H]+.
4.1.5.6. 6-(1H-Benzo[d]imidazol-2-yl)-7-hydroxy-4-(4-methoxy-
benzyl)thieno[3,2-b]pyridin-5(4H)-one (14). This compound
was obtained by two steps using the same method as Section 4.1.5.
The firstly generated uncyclized intermediate was separated and
then repeated the same operation again to get compound 14.
White solid (26% in two steps), mp: >250 °C. 1H NMR (500 MHz,
DMSO-d6) d 13.59 (br, NH, 1H), 7.88 (d, J = 10 Hz, Ar-H, 1H),
7.79–7.77 (m, Ar-H, 2H), 7.34–7.32 (m, Ar-H, 2H), 7.31 (d,
J = 18 Hz, Ar-H, 2H), 7.26 (d, J = 10 Hz, Ar-H, 1H), 6.88 (d,
J = 18 Hz, Ar-H, 2H), 5.34 (s, CH2, 2H), 3.69 (s, CH3, 3H). ESI-MS:
m/z = 404 [M+H]+.
4.1.5. General procedure for LHMDS cyclization
4.1.6. General procedure for the synthesis of bis-triflates 6a–e
and 15
LHMDS (1.65 mL, 1 M in THF, 1.65 mmol, 5.0 equiv) was added
dropwise to a solution of ethyl 2-(1H-benzo[d]imidazol-2-yl)ace-
tate (67.6 mg, 0.33 mmol, 1.0 equiv) and 1-(4-methoxybenzy-l)-
1H-thieno[2,3-d][1,3]oxazine-2,4-dione (0.33 mmol, 1.0 equiv) in
anhydrous THF (15 mL) at ꢀ78 °C. The resulting solution was
slowly warmed to room temperature and then stirred overnight
at 80 °C. After the reaction completed, quenched with a small
amount of NH4Cl (aq, satd). And then concentrated in vacuo, the
resulting crude material was purified by column chromatography
(petroleum ether/ethyl acetate/acetic acid = 500:100:20, v/v/v) on
silica gel to afford the product as a white solid.
To a solution of 5a (403 mg, 1 mmol) in anhydrous CH2Cl2
(80 mL) was added pyridine (20 mmol). The mixture was cooled
to ꢀ10 °C, trifluoroacetic anhydride (8 mmol) in anhydrous
CH2Cl2 (8 mL) was added dropwise. After the reaction was stirred
at ꢀ5 °C for 4 h, saturated aqueous NaHCO3 solution (20 mL) was
added. The organic layer was separated, washed with 1 N aqueous
HCl solution, 1 N aqueous NaHCO3 solution, and brine, dried over
anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the residue obtained was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 4:1, v/v) to
give 6a.
4.1.5.1.
3-(1H-Benzimidazole-2yl)-4-hydroxyl-1-(4-methoxy-
(5a). White solid
benzyl)-thieno[2,3-b]pyridin-6(7H)-one
4.1.6.1. 7-(4-Methoxybenzyl)-6-oxo-5-(1-((trifluoromethyl)sul-
fonyl)-1H-benzo[d]imidazol-2-yl)-6,7-dihydrothieno[2,3-b]py-
ridin-4-yl trifluoromethanesulfonate (6a). White solid (85%),
(43%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.64 (br,
NH, 1H), 7.81–7.77 (m, Ar-H, 2H), 7.35–7.31 (m, Ar-H, 4H), 7.30
(d, J = 11 Hz, Ar-H, 1H), 7.07 (d, J = 11 Hz, Ar-H, 1H), 6.91 (d,
J = 17 Hz, Ar-H, 2H), 5.23 (s, CH2, 2H), 3.71 (s, CH3, 3H). ESI-MS:
m/z = 404 [M+H]+.
mp: 185–187 °C. 1H NMR (500 MHz, DMSO-d6)
d 8.04 (d,
J = 15 Hz, Ar-H, 1H), 7.89 (d, J = 16 Hz, Ar-H, 1H), 7.72–7.64 (m,
Ar-H, 2H), 7.64 (d, J = 12 Hz, Ar-H, 1H), 7.35 (d, J = 18 Hz, Ar-H,
2H), 7.27 (d, J = 12 Hz, Ar-H, 1H), 6.95 (d, J = 18 Hz, Ar-H, 2H),
5.67 (d, J = 31 Hz, CH2, 1H), 5.18 (d, J = 31 Hz, CH2, 1H), 3.74 (s,
CH3, 3H). ESI-MS: m/z = 668 [M+H]+.
4.1.5.2. 3-(1H-Benzimidazole-2yl)-6-chloro-4-hydroxyl-1-(4-m-
ethoxybenzyl)-thieno[2,3-b]pyridin-6(7H)-one (5b). White solid
(42%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.57 (br, NH,
1H), 7.80–7.78 (m, Ar-H, 2H), 7.35–7.33 (m, Ar-H, 2H), 7.32 (d,
J = 18 Hz, Ar-H, 2H), 7.28 (s, Ar-H, 1H), 6.93 (d, J = 18 Hz, Ar-H,
2H), 5.17 (s, CH2, 2H), 3.72 (s, CH3, 3H). ESI-MS: m/z = 438 [M+H]+.
4.1.6.2. 2-Chloro-7-(4-methoxybenzyl)-6-oxo-5-(1-((trifluoro-
methyl)sulfonyl)-1H-benzo[d]imidazol-2-yl)-6,7-dihydrothieno
[2,3-b]pyridin-4-yl trifluoromethanesufonate (6b). White solid
(83%), mp: 157–158 °C. 1H NMR (500 MHz, DMSO-d6) d 8.04 (d,
J = 16 Hz, Ar-H, 1H), 7.89 (d, J = 17 Hz, Ar-H, 1H), 7.72–7.64 (m,
Ar-H, 2H), 7.37 (d, J = 18 Hz, Ar-H, 2H), 7.34 (s, Ar-H, 1H), 6.97 (d,
J = 18 Hz, Ar-H, 2H), 5.65 (d, J = 31 Hz, CH2, 1H), 5.10 (d, J = 31 Hz,
CH2, 1H), 3.75 (s, CH3, 3H). ESI-MS: m/z = 702 [M+H]+.
4.1.5.3. 3-(1H-Benzimidazole-2yl)-6-bromo-4-hydroxyl-1-(4-m-
ethoxybenzyl)-thieno[2,3-b]pyridin-6-one (5c). White solid
(36%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.58 (br,
NH, 1H), 7.81–7.78 (m, Ar-H, 2H), 7.39 (s, Ar-H, 1H), 7.35–7.33

P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4889
4.1.6.3. 2-Bromo-7-(4-methoxybenzyl)-6-oxo-5-(1-((trifluoro-
methyl)sulfonyl)-1H-benzo[d]imidazol-2-yl)-6,7-dihydrothieno
[2,3-b]pyridin-4-yl trifluoromethanesulfonate (6c). Yellow solid
(80%), mp: 153–155 °C. 1H NMR (500 MHz, DMSO-d6) d 8.05
(d, J = 16 Hz, Ar-H, 1H), 7.89 (d, J = 17 Hz, Ar-H, 1H), 7.72–7.64
(m, Ar-H, 2H), 7.42 (s, Ar-H, 1H), 7.37 (d, J = 18 Hz, Ar-H, 2H),
6.97 (d, J = 18 Hz, Ar-H, 2H), 5.65 (d, J = 31 Hz, CH2, 1H), 5.10
(d, J = 31 Hz, CH2, 1H), 3.75 (s, CH3, 3H). ESI-MS: m/z = 746 [M+H]+.
1H), 7.60 (d, J = 12 Hz, Ar-H, 2H), 7.28 (d, J = 12 Hz, Ar-H, 1H),
7.20–7.17 (m, Ar-H, 2H), 4.50–4.47 (m, CH, 1H), 3.57–3.54 (m, CH2,
1H), 3.32–3.30 (m, CH2, 1H), 3.09–3.02 (m, CH2, 2H), 2.29–2.27 (m,
CH2, 1H), 2.08–2.06 (m, CH2, 1H), 1.94–1.83 (m, CH2, 2H); 13C NMR
(125 MHz, DMSO-d6) d 162.01, 152.44, 151.80, 148.87, 140.43,
131.72, 122.78, 121.73, 121.31, 116.87, 116.54, 111.79, 111.05,
90.05, 48.28, 47.41, 42.69, 29.82, 20.75. ESI-MS: m/z = 366 [M+H]+.
4.1.8.2. (S)-5-(1H-Benzo[d]imidazol-2-yl)-4-(piperidin-3-ylami-
no)thieno[2,3-b]pyridin-6(7H)-one (8b). White solid (55%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.11 (s, NH, 1H),
12.33 (d, J = 15 Hz, NH, 1H), 10.05 (br, NH, 1H), 7.70 (br, Ar-H,
1H), 7.60 (d, J = 12 Hz, Ar-H, 2H), 7.28 (d, J = 12 Hz, Ar-H, 1H),
7.20–7.16 (m, Ar-H, 2H), 4.50–4.45 (m, CH, 1H), 3.55 (d, J = 24 Hz,
CH2, 1H), 3.29 (d, J = 25 Hz, CH2, 1H), 3.06–2.99 (m, CH2, 2H),
2.28 (d, J = 19 Hz, CH2, 1H), 2.06–2.03 (m, CH2, 1H), 1.93–1.81 (m,
4.1.6.4. 7-(4-Methoxybenzyl)-2-methyl-6-oxo-5-(1-((trifluoro-
methyl)sulfonyl)-1H-benzo[d]imidazol-2-yl)-6,7-dihydrothieno
[2,3-b]pyridin-4-yl trifluoromethanesulfonate (6d). White solid
(82%), mp: 161–163 °C. 1H NMR (500 MHz, DMSO-d6) d 8.04
(d, J = 16 Hz, Ar-H, 1H), 7.89 (d, J = 17 Hz, Ar-H, 1H), 7.70–7.62
(m, Ar-H, 2H), 7.37 (d, J = 18 Hz, Ar-H, 2H), 7.03 (s, Ar-H, 1H),
6.98 (d, J = 18 Hz, Ar-H, 2H), 5.64 (d, J = 31 Hz, CH2, 1H), 5.08
(d, J = 31 Hz, CH2, 1H), 3.76 (s, CH3, 3H), 2.43 (s, CH3, 3H).
ESI-MS: m/z = 682 [M+H]+.
CH2, 2H); 13C NMR (125 MHz, DMSO-d6)
d 162.03, 152.47,
151.79, 148.87, 122.82, 121.71, 121.65, 121.37, 116.86, 116.51,
111.86, 111.79, 111.04, 89.96, 48.50, 47.76, 42.88, 29.98, 21.02.
ESI-MS: m/z = 366 [M+H]+.
4.1.6.5. 2-Chloro-5-(5-methoxy-1-((trifluoromethyl)sulfonyl)-
1H-benzo[d]imidaz-ol-2-yl)-7-(4-methoxybenzyl)-6-oxo-6,7-
dihydrothieno[2,3-b]pyridin-4-yl
(6e).
trifluoromethanesulfonate
4.1.8.3. 5-(1H-Benzo[d]imidazol-2-yl)-4-(pyrrolidin-3-ylamino)
thieno[2,3-b]pyridin-6(7H)-one (8c). White solid (51%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.07 (s, NH, 1H), 12.51
(d, J = 13 Hz, NH, 1H), 10.02 (br, NH, 1H), 7.67–7.61 (m, Ar-H,
2H), 7.57 (d, J = 12 Hz, Ar-H, 1H), 7.26 (d, J = 12 Hz, Ar-H, 1H),
7.19–7.16 (m, Ar-H, 2H), 5.01–4.96 (m, CH, 1H), 3.62–3.59 (m,
CH2, 1H), 3.46–3.42 (m, CH2, 1H), 3.38–3.33 (m, CH2, 1H), 3.23–
3.19 (m, CH2, 1H), 2.46–2.39 (m, CH2, 1H), 2.22–2.16 (m, CH2,
1H). ESI-MS: m/z = 352 [M+H]+.
White solid (85%), mp: 153–155 °C. 1H NMR (500 MHz,
DMSO-d6) d 7.75 (d, J = 18 Hz, Ar-H, 1H), 7.57 (s, Ar-H, 1H), 7.37
(d, J = 18 Hz, Ar-H, 2H), 7.33 (s, Ar-H, 1H), 7.30 (d, J = 18 Hz, Ar-H,
1H), 6.97 (d, J = 18 Hz, Ar-H, 2H), 5.65 (d, J = 31 Hz, CH2, 1H), 5.09
(d, J = 31 Hz, CH2, 1H), 3.91 (s, CH3, 3H), 3.75 (s, CH3, 3H). ESI-
MS: m/z = 732 [M+H]+.
4.1.6.6. 4-(4-Methoxybenzyl)-5-oxo-6-(1-((trifluoromethyl)sul-
fonyl)-1H-benzo[d]imidazol-2-yl)-4,5-dihydrothieno[3,2-b]py-
ridin-7-yl trifluoromethanesulfonate (15). White solid (78%),
4.1.8.4. 5-(1H-Benzo[d]imidazol-2-yl)-4-(piperidin-4-ylamino)
thieno[2,3-b]pyridin-6(7H)-one (8d). White solid (56%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.12 (s, NH, 1H), 12.35
(d, J = 15 Hz, NH, 1H), 8.75 (br, NH, 1H), 7.72–7.70 (m, Ar-H, 1H),
7.58–7.56 (m, Ar-H, 2H), 7.26 (d, J = 12 Hz, Ar-H, 1H), 7.20–7.16
(m, Ar-H, 2H), 4.57–4.56 (m, CH, 1H), 3.41–3.36 (m, CH2, 2H),
3.28–3.23 (m, CH2, 2H), 2.29–2.26 (m, CH2, 2H), 1.96–1.89 (m,
CH2, 2H). ESI-MS: m/z = 366 [M+H]+.
mp: 157–159 °C. 1H NMR (500 MHz, DMSO-d6)
d 8.42 (d,
J = 11 Hz, Ar-H, 1H), 8.04 (d, J = 15 Hz, Ar-H, 1H), 7.89 (d,
J = 16 Hz, Ar-H, 1H), 7.72–7.64 (m, Ar-H, 3H), 7.35 (d, J = 16 Hz,
Ar-H, 2H), 6.92 (d, J = 17 Hz, Ar-H, 2H), 5.65 (d, J = 31 Hz, CH2,
1H), 5.35 (d, J = 31 Hz, CH2, 1H), 3.72 (s, CH3, 3H). ESI-MS:
m/z = 668 [M+H]+.
4.1.7. General procedure for the synthesis of 4-substitued
amino-3-benzimidazole-thiopyridinone intermediates (7a–z,
16a, and 16b)
To a solution of the bis-triflate (0.3 mmol) prepared above in
acetonitrile (5 mL), the corresponding amine (0.8 mmol) dissolved
in acetonitrile (3 mL) was added dropwise. The reaction mixture
was stirred at room temperature for 2 h, and then evaporated
under reduced pressure. The yellow oil obtained was used for the
next step without further purification.
4.1.8.5. 5-(1H-Benzo[d]imidazol-2-yl)-4-(quinuclidin-3-ylamino)
thieno[2,3-b]pyridin-6(7H)-one (8e). White solid (46%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.09 (s, NH, 1H), 12.62
(d, J = 14 Hz, NH, 1H), 7.72–7.69 (m, Ar-H, 1H), 7.60–7.57 (m, Ar-
H, 1H), 7.52 (d, J = 12 Hz, Ar-H, 1H), 7.25 (d, J = 12 Hz,
Ar-H, 1H), 7.19–7.16 (m, Ar-H, 2H), 4.57–4.54 (m, CH, 1H), 3.71–
3.67 (m, CH2, 1H), 3.19–3.09 (m, CH2, 2H), 3.05–3.03 (m, CH2,
2H), 3.03–3.00 (m, CH2, 1H), 2.39–2.36 (m, CH, 1H), 2.27–2.25
(m, CH2, 1H), 1.94–1.88 (m, CH2, 1H), 1.87–1.75 (m, CH2, 2H); 13C
NMR (125 MHz, DMSO-d6) d 162.12, 152.80, 152.06, 148.63,
140.63, 131.69, 122.90, 121.53, 121.20, 116.59, 115.98, 111.62,
111.22, 89.13, 57.03, 51.15, 46.85, 45.94, 26.90, 24.98, 19.79. ESI-
MS: m/z = 392 [M+H]+.
4.1.8. General procedure for the synthesis of thienopyridinones
derivatives (8a–z, 17a, and 17b)
Compound 10a (0.25 mmol) was dissolved in a mixture of tri-
fluoroacetic acid and 12 N aqueous HCl solution (7:1, v/v), and
heated at 90 °C for 18 h. The solution was cooled to room temper-
ature, and saturated aqueous NaHCO3 was added slowly to basify
the solution to pH 9. Then the reaction mixture was extracted with
ethyl acetate and brine, dried over anhydrous Na2SO4. The solvent
was evaporated under reduced pressure and the residue obtained
was purified by silica gel column chromatography (CH2Cl2/EtOH/
TEA = 500:10:10–500:15:15, v/v/v), and then suspended in water
and filtrated to get target compound 8a.
4.1.8.6. 5-(1H-Benzo[d]imidazol-2-yl)-4-(cyclohexylamino)thie-
no[2,3-b]pyridin-6(7H)-one (8f). White solid (55%), mp: >250 °C.
1H NMR (500 MHz, DMSO-d6) d 13.11 (s, NH, 1H), 12.29 (d,
J = 16 Hz, NH, 1H), 7.70–7.67 (m, Ar-H, 1H), 7.58–7.55 (m, Ar-H,
1H), 7.45 (d, J = 12 Hz, Ar-H, 1H), 7.22 (d, J = 12 Hz, Ar-H, 1H),
7.18–7.14 (m, Ar-H, 2H), 4.23–4.19 (m, CH, 1H), 2.06–2.04 (m,
CH2, 2H), 1.85–1.82 (m, CH2, 2H), 1.65–1.49 (m, CH2, 5H), 1.45–
1.39 (m, CH2, 1H). ESI-MS: m/z = 365 [M+H]+.
4.1.8.1. 5-(1H-Benzo[d]imidazol-2-yl)-4-(piperidin-3-ylamino)
thieno[2,3-b]pyridin-6(7H)-one (8a). White solid (56%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.10 (s, NH, 1H), 12.34
(d, J = 15 Hz, NH, 1H), 9.31 (br, NH, 1H), 7.72 (d, J = 9 Hz, Ar-H,
4.1.8.7. 5-(1H-Benzo[d]imidazol-2-yl)-4-((piperidin-4-ylmethyl)
amino)thieno[2,3-b]pyridin-6(7H)-one (8g). White solid (55%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.05 (s, NH, 1H),

4890
P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
12.18 (t, J = 11 Hz, NH, 1H), 7.69–7.67 (m, Ar-H, 1H), 7.64 (d,
J = 14 Hz, Ar-H, 1H), 7.56–7.54 (m, Ar-H, 1H), 7.19–7.15 (m, Ar-H,
3H), 3.76–3.73 (m, CH2, 2H), 3.26–3.23 (m, CH2, 2H), 2.86–2.80
(m, CH2, 2H), 2.08–1.95 (m, CH2, CH, 3H), 1.50–1.42 (m, CH2, 2H).
ESI-MS: m/z = 380 [M+H]+.
4.1.8.15. 5-(1H-Benzo[d]imidazol-2-yl)-4-(piperidin-1-yl)thieno
[2,3-b]pyridin-6(7H)-one (8o). White solid (45%), mp: >250 °C.
1H NMR (500 MHz, DMSO-d6) d 12.36 (br, NH, 1H), 7.57 (br, Ar-
H, 2H), 7.26 (d, J = 11 Hz, Ar-H, 1H), 7.17–7.15 (m, Ar-H, 2H), 7.14
(d, J = 12 Hz, Ar-H, 1H), 2.92 (br, CH2ꢁ 2, 4H), 1.46 (br, CH2ꢁ 3,
6H). ESI-MS: m/z = 351 [M+H]+.
4.1.8.8. 4-((2-Aminoethyl)amino)-5-(1H-benzo[d][d]imidazol-2-
yl)thieno[2,3-b]pyridin-6(7H)-one (8h). White solid (59%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.04 (br, NH, 1H),
12.15 (t, J = 10 Hz, NH, 1H), 7.64–7.62 (m, Ar-H, 3H), 7.23 (d,
J = 12 Hz, Ar-H, 1H), 7.18–7.15 (m, Ar-H, 2H), 4.01 (dd, J = 25 Hz,
13 Hz, CH2, 2H), 3.16 (t, J = 14 Hz, CH2, 2H). ESI-MS: m/z = 326
[M+H]+.
4.1.8.16. 5-(1H-Benzo[d]imidazol-2-yl)-4-morpholinothieno[2,3-
b]pyridin-6(7H)-one (8p). White solid (45%), mp: >250 °C. 1H
NMR (500 MHz, DMSO-d6) d 12.39 (br, NH, 1H), 7.58 (br, Ar-H,
2H), 7.28 (d, J = 11 Hz, Ar-H, 1H), 7.22 (d, J = 11 Hz, Ar-H, 1H),
7.20–7.16 (m, Ar-H, 2H), 3.57 (t, J = 9 Hz, CH2ꢁ 2, 4H), 2.94 (t,
J = 9 Hz, CH2ꢁ 2, 4H). ESI-MS: m/z = 353 [M+H]+.
4.1.8.9. 5-(1H-Benzo[d]imidazol-2-yl)-4-((2-(methylamino)ethyl)
amino)thieno[2,3-b]pyridin-6(7H)-one (8i). White solid (57%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.08 (s, NH, 1H),
12.21 (t, J = 11 Hz, NH, 1H), 10.01 (br, NH, 1H), 7.66 (d, J = 12 Hz,
Ar-H, 3H), 7.25 (d, J = 12 Hz, Ar-H, 1H), 7.19–7.16 (m, Ar-H, 2H),
4.16 (dd, J = 25 Hz, 13 Hz, CH2, 2H), 3.34 (t, J = 14 Hz, CH2, 2H),
2.66 (s, CH3, 3H). ESI-MS: m/z = 340 [M+H]+.
4.1.8.17. 5-(1H-Benzo[d]imidazol-2-yl)-2-chloro-4-(piperidin-3-
ylamino)thieno[2,3-b]pyridin-6(7H)-one
(8q). White
solid
(53%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.03 (br,
NH, 1H), 12.32 (d, J = 15 Hz, NH, 1H), 9.69 (br, NH, 1H), 7.63 (br,
Ar-H, 3H), 7.19–7.16 (m, Ar-H, 2H), 4.36–4.34 (m, CH, 1H), 3.47–
3.45 (m, CH2, 1H), 3.23–3.20 (m, CH2, 1H), 2.96–2.90 (m, CH2,
2H), 2.25–2.23 (m, CH2, 1H), 2.01–1.96 (m, CH2, 1H), 1.88–1.79
(m, CH2, 2H). ESI-MS: m/z = 400 [M+H]+.
4.1.8.10. 5-(1H-Benzo[d]imidazol-2-yl)-4-((2-(dimethylamino)
ethyl)amino)thieno[2,3-b]pyridin-6(7H)-one (8j). White solid
(62%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 12.99 (br,
NH, 1H), 12.23 (br, NH, 1H), 12.20 (t, J = 10 Hz, NH, 1H), 7.67 (d,
J = 15 Hz, Ar-H, 2H), 7.53 (br, Ar-H, 1H), 7.17 (d, J = 15 Hz, Ar-H,
1H), 7.16–7.12 (m, Ar-H, 2H), 3.90 (dd, J = 27 Hz, 13 Hz, CH2, 2H),
2.72 (t, J = 15 Hz, CH2, 2H), 2.35 (s, CH3ꢁ 2, 6H). ESI-MS: m/
z = 354 [M+H]+.
4.1.8.18. (S)-5-(1H-Benzo[d]imidazol-2-yl)-2-chloro-4-(piperidin-
3-ylamino)thieno[2,3-b]pyridi-n-6(7H)-one (8r). White solid
(52%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.05 (br,
NH, 1H), 12.24 (d, J = 16 Hz, NH, 1H), 7.62 (br, Ar-H, 2H), 7.53 (s,
Ar-H, 1H), 7.17–7.16 (m, Ar-H, 2H), 4.23–4.18 (m, CH, 1H), 3.35–
3.33 (m, CH2, 1H), 3.06–3.03 (m, CH2, 1H), 2.82–2.76 (m, CH2,
2H), 2.20–2.19 (m, CH2, 1H), 1.91–1.89 (m, CH2, 1H), 1.81–1.67
(m, CH2, 2H). ESI-MS: m/z = 400 [M+H]+.
4.1.8.11. 5-(1H-Benzo[d]imidazol-2-yl)-4-((2-morpholinoethyl)
amino)thieno[2,3-b]pyridin-6(7H)-one (8k). White solid (62%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.04 (br, NH, 1H),
12.27 (br, NH, 1H), 12.09 (t, J = 8 Hz, NH, 1H), 7.68 (d, J = 12 Hz,
Ar-H, 2H), 7.52–7.50 (m, Ar-H, 1H), 7.17 (d, J = 12 Hz, Ar-H, 1H),
7.16–7.14 (m, Ar-H, 2H), 3.94 (dd, J = 21 Hz, 10 Hz, CH2, 2H), 3.67
(t, J = 9 Hz, CH2ꢁ 2, 4H), 2.78 (t, J = 12 Hz, CH2, 2H), 2.58 (br,
CH2ꢁ 2, 4H). ESI-MS: m/z = 396 [M+H]+.
4.1.8.19. 5-(1H-Benzo[d]imidazol-2-yl)-2-chloro-4-(pyrrolidin-3-
ylamino)thieno[2,3-b]pyridin-6(7H)-one (8s). White solid (49%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.02 (br, NH, 1H),
12.54 (d, J = 13 Hz, NH, 1H), 10.13 (br, NH, 1H), 7.65 (br, Ar-H,
2H), 7.63 (s, Ar-H, 1H), 7.19–7.16 (m, Ar-H, 2H), 5.01–4.95 (m,
CH, 1H), 3.66–3.62 (m, CH2, 1H), 3.51–3.46 (m, CH2, 1H),
3.43–3.39 (m, CH2, 1H), 3.24 (dd, J = 24 Hz, 8 Hz, CH2, 1H),
2.49–2.39 (m, CH2, 1H), 2.23–2.17 (m, CH2, 1H). ESI-MS: m/
z = 386 [M+H]+.
4.1.8.12. Methyl-3-((5-(1H-benzo[d]imidazol-2-yl)-6-oxo-6,7-dihy-
drothieno[2,3-b]pyridin-4-yl)-amino)propanoate (8l). White solid
(62%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.01 (br, NH, 1H),
12.36 (br, NH, 1H), 12.20 (t, J = 8 Hz, NH, 1H), 7.69–7.67 (m, Ar-H, 1H),
7.62 (d, J = 12 Hz, Ar-H, 1H), 7.53–7.52 (m, Ar-H, 1H), 7.23 (d, J = 11 Hz,
Ar-H, 1H), 7.18–7.14 (m, Ar-H, 2H), 4.09 (dd, J = 12 Hz, CH2, 2H), 3.66
(s, CH3, 3H), 3.92 (t, J = 12 Hz, CH2, 2H). ESI-MS: m/z = 369 [M+H]+.
4.1.8.20. 5-(1H-Benzo[d]imidazol-2-yl)-2-chloro-4-(quinuclidin-
3-ylamino)thieno[2,3-b]pyridin-6(7H)-one (8t). White solid
(53%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.02 (br,
NH, 1H), 12.63 (d, J = 15 Hz, NH, 1H), 11.08 (br, NH, 1H), 7.71–
7.70 (m, Ar-H, 1H), 7.58–7.56 (m, Ar-H, 2H), 7.20–7.16 (m, Ar-H,
2H), 4.55 (br, CH, 1H), 3.73–3.69 (m, CH2, 1H), 3.18 (t, J = 15 Hz,
CH2, 2H), 3.10 (t, J = 16 Hz, CH2, 2H), 3.06–3.05 (m, CH2, 1H),
2.43–2.42 (m, CH, 1H), 2.28 (br, CH2, 1H), 1.99–1.93 (m, CH2, 1H),
1.89–1.80 (m, CH2, 2H); 13C NMR (125 MHz, DMSO-d6) d 161.39,
157.73, 152.05, 151.04, 146.99, 140.35, 131.75, 121.85, 121.39,
119.42, 116.73, 111.90, 109.91, 90.75, 54.52, 48.81, 45.86, 45.28,
25.91, 22.33, 17.93. ESI-MS: m/z = 426 [M+H]+.
4.1.8.13. 5-(1H-Benzo[d]imidazol-2-yl)-4-((2-hydroxyethyl)amino)
thieno[2,3-b]pyridin-6(7H)-one (8m). White solid (41%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.03 (br, NH, 1H),
12.27 (br, NH, 1H), 12.19 (t, J = 9 Hz, NH, 1H), 7.67–7.65 (m, Ar-
H, 1H), 7.62 (d, J = 12 Hz, Ar-H, 1H), 7.56–7.55 (m, Ar-H, 1H), 7.18
(d, J = 11 Hz, Ar-H, 1H), 7.16–7.12 (m, Ar-H, 2H), 3.90–3.87 (m,
CH2, 2H), 3.82–3.79 (m, CH2, 2H). ESI-MS: m/z = 327 [M+H]+.
4.1.8.21. 5-(1H-Benzo[d]imidazol-2-yl)-2-bromo-4-(piperidin-3-
ylamino)thieno[2,3-b]pyridin-6(7H)7H)-one (8u). White solid
(41%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.03 (br,
NH, 1H), 12.28 (d, J = 11 Hz, NH, 1H), 9.82 (br, NH, 1H), 7.67 (s,
Ar-H, 1H), 7.63 (br, Ar-H, 2H), 7.18–7.16 (m, Ar-H, 2H), 4.32–4.30
(m, CH, 1H), 3.40–3.39 (m, CH2, 1H), 3.17–3.15 (m, CH2, 1H),
2.89–2.85 (m, CH2, 2H), 2.23–2.21 (m, CH2, 1H), 1.99–1.95 (m,
CH2, 1H), 1.85–1.76 (m, CH2, 2H). ESI-MS: m/z = 444 [M+H]+.
4.1.8.14. 5-(1H-Benzo[d]imidazol-2-yl)-4-(4-(hydroxymethyl)pi
peridin-1-yl)thieno[2,3-b]pyridin-6(7H)-one (8n). White solid
(40%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.05 (br,
NH, 1H), 12.40 (br, NH, 1H), 7.57 (br, Ar-H, 2H), 7.24 (d, J = 11 Hz,
Ar-H, 1H), 7.17–7.15 (m, Ar-H, 2H), 7.12 (br, Ar-H, 1H), 3.28–3.25
(m, CH2, 2H), 3.21 (d, J = 12 Hz, CH2, 2H), 2.63–2.62 (m, CH2, 2H),
1.54–1.52 (m, CH2, 2H), 1.38 (br, CH, 1H), 1.22–1.16 (m, CH2,
2H). ESI-MS: m/z = 381 [M+H]+.

P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
4891
4.1.8.22. 5-(1H-Benzo[d]imidazol-2-yl)-2-bromo-4-(pyrrolidin-
3-ylamino)thieno[2,3-b]pyridin-6(7H)-one (8v). White solid
(43%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.00 (br,
NH, 1H), 12.55 (d, J = 13 Hz, NH, 1H), 10.27 (br, NH, 1H), 7.71 (s,
Ar-H, 1H), 7.66 (br, Ar-H, 2H), 7.20–7.16 (m, Ar-H, 2H), 5.03–4.98
(m, CH, 1H), 3.67–3.63 (m, CH2, 1H), 3.53–3.47 (m, CH2, 1H),
3.45–3.40 (m, CH2, 1H), 3.25 (dd, J = 24 Hz, 7 Hz, CH2, 1H), 2.48–
2.40 (m, CH2, 1H), 2.25–2.18 (m, CH2, 1H). ESI-MS: m/z = 430
[M+H]+.
121.34, 117.16, 111.85, 106.44, 89.92, 48.15, 47.52, 42.66, 30.41,
20.56. ESI-MS: m/z = 366 [M+H]+.
4.1.8.28. 6-(1H-Benzo[d]imidazol-2-yl)-7-(pyrrolidin-3-ylamino)
thieno[3,2-b]pyridin-5(4H)-one (17b). White solid (47%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.17 (s, NH, 1H), 12.33
(d, J = 16 Hz, NH, 1H), 12.09 (br, NH, 1H), 8.97 (br, NH, 1H), 8.13
(d, J = 11 Hz, Ar-H, 1H), 7.73–7.71 (m, Ar-H, 1H), 7.59–7.58 (m,
Ar-H, 1H), 7.19–7.18 (m, Ar-H, 2H), 7.10 (d, J = 10 Hz, Ar-H, 1H),
4.54–4.52 (m, CH, 1H), 3.64 (dd, J = 24 Hz, 5 Hz, CH2, 1H), 3.28 (d,
J = 25 Hz, CH2, 1H), 3.18–3.14 (m, CH2, 1H), 3.08–3.03 (m, CH2,
1H), 2.32 (d, J = 19 Hz, CH2, 1H), 2.09–2.07 (m, CH2, 1H). ESI-MS:
m/z = 352 [M+H]+.
4.1.8.23. (S)-2-Chloro-5-(5-methoxy-1H-benzo[d]imidazol-2-yl)-4-
(piperidin-3-ylamino)thieno[2,3-b]pyridin-6(7H)-one (8w). White
solid (58%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 12.92 (br,
NH, 1H), 12.24 (br, NH, 1H), 9.99 (br, NH, 1H), 7.61 (s, Ar-H, 1H),
7.51 (br, Ar-H, 1H), 7.27 (d, J = 18 Hz, Ar-H, 1H), 6.82 (dd, J = 18 Hz,
5 Hz, Ar-H, 1H), 4.36–4.35 (m, CH, 1H), 3.79 (s, CH3, 3H), 3.50 (d,
J = 22 Hz, CH2, 1H), 3.28 (d, J = 25 Hz, CH2, 1H), 3.00–2.93 (m, CH2,
2H), 2.26–2.24 (m, CH2, 1H), 2.02–2.00 (m, CH2, 1H), 1.87–1.81 (m,
CH2, 2H); 13C NMR (125 MHz, DMSO-d6) d 161.38, 155.49, 146.84,
138.40, 130.99, 128.76, 127.71, 125.58, 122.05, 119.42, 118.47,
111.61, 109.78, 91.01, 55.39, 48.38, 47.95, 42.93, 30.03, 21.21. ESI-
MS: m/z = 430 [M+H]+.
4.2. Biological evaluation
4.2.1. In vitro Chk1 kinase assay
The Chk1 inhibitory activities of compounds 8a–z, 17a, and 17b
were determined using the luminescent ADP-Glo assay kit (Pro-
mega Corporation), according to the manufacturer’s instructions.
White low-volume 384-well polystyrene plates (ProxiPlate-384
Plus, PerkinElmer, Waltham, MA) were used, in which containing
1
lL of each compound dilution and 2
lL of 4 ng/mL solution of
4.1.8.24. 2-Chloro-5-(5-methoxy-1H-benzo[d]imidazol-2-yl)-4-
(quinuclidin-3-ylamino)thieno-[2,3-b]pyridin-6(7H)-one
(8x).
Chk1 kinase. The reactions were started via the addition of 2 lL
substrates to a final assay volume of 5 L per well. After incubation
l
White solid (61%), mp: >250 °C. 1H NMR (500 MHz,
for 60 min at room temperature, 5 lL ADP-Glo™ Reagent was
DMSO-d6) d 12.99 (br, NH, 1H), 12.39 (d, J = 14 Hz, NH, 1H), 7.51
(s, Ar-H, 1H), 7.43 (d, J = 18 Hz, Ar-H, 1H), 7.28 (s, Ar-H, 1H), 6.81
(d, J = 18 Hz, Ar-H, 1H), 4.30 (br, CH, 1H), 3.78 (s, CH3, 3H), 3.44–
3.39 (m, CH2, 1H), 2.89–2.68 (m, CH2, 5H), 2.12–2.10 (m, CH, 1H),
2.04 (br, CH2, 1H), 1.77–1.75 (m, CH2, 1H), 1.69–1.65 (m, CH2,
1H), 1.52 (br, CH2, 1H); 13C NMR (125 MHz, DMSO-d6) d 161.74,
155.53, 152.64, 150.71, 141.42, 132.44, 122.18, 118.71, 117.07,
110.38, 109.88, 99.54, 95.41, 90.32, 55.44, 50.68, 46.83, 45.92,
45.68, 26.83, 24.84, 19.74. ESI-MS: m/z = 456 [M+H]+.
added to terminate the kinase reaction and deplete the uncon-
sumed ATP. After an additional incubation for 40 min at room tem-
perature, 10 lL kinase detection reagent was added to convert ADP
to ATP and luciferase or luciferin was introduced to detect ATP.
After incubation for 40 min at room temperature, luminescence
was measured using a plate-reading luminometer. IC50 values were
calculated using GraphPad Prism 4 software. AZD7762 was used as
the positive control. Assays were performed in duplicate and
repeated on separate days.
4.1.8.25. 5-(1H-Benzo[d][d]imidazol-2-yl)-2-methyl-4-(piperidin-
3-ylamino)thieno[2,3-b]pyridin-6(7H)-one (8y). White solid
(54%), mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.12 (br,
NH, 1H), 12.21 (d, J = 16 Hz, NH, 1H), 9.75 (br, NH, 1H), 7.67–7.58
(m, Ar-H, 2H), 7.28 (s, Ar-H, 1H), 7.18–7.15 (m, Ar-H, 2H), 4.38–
4.36 (m, CH, 1H), 3.46–3.42 (m, CH2, 2H), 3.20–3.17 (m, CH2, 1H),
2.94–2.89 (m, CH2, 2H), 2.49 (s, CH3, 3H), 2.25–2.23 (m, CH2, 1H),
1.87–1.77 (m, CH2, 2H). ESI-MS: m/z = 380 [M+H]+.
4.2.2. Cell synergy assay
The inhibitory effect of Chk1 inhibitors on multiple myeloma
cell growth was assessed with MTS assay. Briefly, in a volume of
100 lL, human myeloma RPMI 8226 cells were seeded in 96-well
plates at a density of 5 ꢁ 103 cells per well. Cells were added with
100 nM Chk1 inhibitor or with melphalan (1, 3 and 10 lM) or both
of them, cultured for 72 h, and incubated with MTS at a final con-
centration of 0.5 mg/mL for 2–4 h. Optical density was determined
at 490 nm (background subtraction at 690 nm) with a SpectraMax
340 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
4.1.8.26. 5-(1H-Benzo[d]imidazol-2-yl)-2-methyl-4-(pyrrolidin-3-
ylamino)thieno[2,3-b]pyridin-6(7H)-one (8z). White solid(50%),
mp: >250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.08 (br, NH, 1H),
12.40 (d, J = 14 Hz, NH, 1H), 9.96 (br, NH, 1H), 7.64 (br, Ar-H, 2H),
7.27 (s, Ar-H, 1H), 7.18–7.15 (m, Ar-H, 2H), 4.97–4.92 (m, CH, 1H),
3.62–3.59 (m, CH2, 1H), 3.47–3.42 (m, CH2, 1H), 3.38–3.34 (m,
CH2, 1H), 3.20 (dd, J = 23 Hz, 7 Hz, CH2, 1H), 2.48 (s, CH3, 3H),
2.44–2.38 (m, CH2, 1H), 2.20–2.14 (m, CH2, 1H). ESI-MS: m/z = 366
[M+H]+.
4.2.3. Kinase selectivity assay
For the kinase selectivity assays, the recombinant Aurora A pro-
tein was expressed in Escherichia coli cells, CDK2/cyclin A was pur-
chased from Carna company. Aurora A and PKC kinase assays were
carried out by using the Invitrogen Z0-LYTETM Kinase Assay kits.
CDK2/cyclin A kinase assay was carried out using the HTRF Kinase
Assay Kit (Cisbio). Compounds were initially tested at a final con-
centration of 1
lg/mL. Compounds displaying more than 40% inhi-
4.1.8.27. 6-(1H-Benzo[d]imidazol-2-yl)-7-(piperidin-3-ylamino)
thieno[3,2-b]pyridin-5(4H)-one (17a). White solid (48%), mp:
>250 °C. 1H NMR (500 MHz, DMSO-d6) d 13.18 (br, NH, 1H),
12.33 (d, J = 16 Hz, NH, 1H), 12.10 (br, NH, 1H), 9.15 (br, NH, 1H),
8.13 (d, J = 11 Hz, Ar-H, 1H), 7.73–7.71 (m, Ar-H, 1H), 7.60–7.59
(m, Ar-H, 1H), 7.20–7.18 (m, Ar-H, 2H), 7.11 (d, J = 11 Hz, Ar-H,
1H), 4.58–4.52 (m, CH, 1H), 3.65 (dd, J = 24 Hz, 6 Hz, CH2, 1H),
3.29 (d, J = 25 Hz, CH2, 1H), 3.18–3.14 (m, CH2, 1H), 3.09–3.04 (m,
CH2, 1H), 2.33–2.31 (m, CH2, 1H), 2.09–2.06 (m, CH2, 1H), 1.93–
1.81 (m, CH2, 2H); 13C NMR (125 MHz, DMSO-d6) d 162.30,
158.39, 152.39, 151.30, 143.70, 140.46, 132.80, 131.74, 121.76,
bition at 1 g/mL were further tested for dose-response IC50 values.
And results represented an average of at least two experiments.
l
4.3. Molecular docking
Docking analysis were carried out by using C-DOCKER module
(Discovery Studio, version 2.1; Accelrys, San Diego, CA, USA, 2008)
to compare the binding modes between compound 8t bound to
Chk1 and 8o bound to Chk1. The X-ray crystal structure of Chk1
(PDB ID: 2GDO)was used for the dockingcalculation. After removing
the ligand and water molecules, the CHARMm-force field was

4892
P. Song et al. / Bioorg. Med. Chem. 22 (2014) 4882–4892
Fischmann, T. O.; Madison, V. Bioorg. Med. Chem. Lett. 2011, 21, 471; (c)
Lainchbury, M.; Matthews, T. P.; McHardy, T.; Boxall, K. J.; Walton, M. I.; Eve, P.
D.; Hayes, A.; Valenti, M. R.; de Haven Brandon, A. K.; Box, G. J. Med. Chem.
2012, 55, 10229.
applied to the protein. And the ATP binding pocket was chosen as the
active site with a radius set as 9 Å. The ligands were generated ran-
dom conformations using CHARMm-based molecular dynamics
(1000 steps), and then docked into the defined Chk1 binding site.
The other parameters were set as default. The final binding confor-
mation of 8t and 8o was determined based on the calculated CDOCK-
ING ENERAGE. The most stable binding modes among the top 10
docking poses of 8t and 8o were presented in Figure 4, respectively.
6. (a) Walton, M. I.; Eve, P. D.; Hayes, A.; Valenti, M.; Brandon, A. D. H.; Box, G.;
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Acknowledgments
The authors are grateful to the support of the National Natural
Science Foundation of China (81172929) and the support of the
Program for Zhejiang Leading Team of S&T Innovation
(2011R50014). The authors also thank Jianyang Pan (Institute of
Pharmaceutical Informatics, Zhejiang University, China) for per-
forming NMR spectrometry and Mass spectrometry for structure
elucidation.
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