Discovery of a Novel Dual-Target Inhibitor of ERK1 and ERK5 That Induces Regulated Cell Death to Overcome Compensatory Mechanism in Specific Tumor Types

Article abstract of DOI:10.1021/acs.jmedchem.9b01896

ERK1 and ERK5 are proposed to have pivotal roles in several types of cancer. Under some circumstance, ERK5 may provide a common bypass route, which rescues proliferation upon abrogation of ERK1 signaling. Thus, we accurately classified the tumor types from The Cancer Genome Atlas (TCGA) based on the expression levels of ERK1 and ERK5. We proposed a novel therapeutic strategy to overcome the above-mentioned compensatory mechanism in specific tumor types by co-targeting both ERK1 and ERK5. On the basis of the idea of overcoming ERK5 compensation mechanism, 22ac (ADTL-EI1712) as the first selective dual-target inhibitor of ERK1 and ERK5 was discovered to have potent antitumor effects in vitro and in vivo. Interestingly, this compound was found to induce regulated cell death accompanied by autophagy in MKN-74 cells. Taken together, our results warrant the potential of this dual-target inhibitor as a new candidate drug that conquers compensatory mechanism in certain tumor types.

Full text of DOI:10.1021/acs.jmedchem.9b01896

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Article
Discovery of a novel dual-target inhibitor of ERK1 and
ERK5 that induces regulated cell death to overcome
compensatory mechanism in specific tumor types
Guan Wang, Yuqian Zhao, Yao Liu, Dejuan Sun, Yongqi
Zhen, Jie Liu, Leilei Fu, Lan Zhang, and Liang Ouyang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01896 • Publication Date (Web): 20 Feb 2020
Downloaded from pubs.acs.org on February 22, 2020
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Discovery of a novel dual-target inhibitor of ERK1 and ERK5
that induces regulated cell death to overcome compensatory
mechanism in specific tumor types
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Guan Wang,#,† Yuqian Zhao,#,† Yao Liu,† Dejuan Sun,† Yongqi Zhen,§ Jie Liu,† Leilei Fu,*,†,§ Lan
Zhang,*,†,§ Liang Ouyang,*,†
†
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, and Collaborative Innovation Center
of Biotherapy, Sichuan University, Chengdu 610041, China
§
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu, 610031, China
KEYWORDS: ERK1; ERK5; Regulated cell death; Compensatory mechanism; Dual-target inhibitor
ABSTRACT: ERK1 and ERK5 are proposed to have pivotal roles in several types of cancer. Under some circumstance,
ERK5 may provide a common bypass route, which rescues proliferation upon abrogation of ERK1 signaling. Thus, we
accurately classified the tumor types from The Cancer Genome Atlas (TCGA) based on the expression levels of ERK1 and
ERK5. We proposed a novel therapeutic strategy to overcome the above-mentioned compensatory mechanism in specific
tumor types by co-targeting both ERK1 and ERK5. Based on the idea of overcoming ERK5 compensation mechanism, 22ac
(ADTL-EI1712) as the first selective dual-target inhibitor of ERK1 and ERK5, was discovered to have potent anti-tumor
effects, in vitro and in vivo. Interestingly, this compound was found to induce regulated cell death accompanied with
autophagy in MKN-74 cells. Taken together, our results warrant the potential of this dual-target inhibitor as a new
candidate drug that conquers compensatory mechanism in certain tumor types.
INTRODUCTION
similar domains, especially ATP-binding sites, ERK1 and
ERK5 have some similar biological functions in
transduction of growth factor signals.12,13 ERK1 and ERK5
are able to phosphorylate the same substrates, such as c-
Myc and RSK, induce the same immediate early genes c-
Jun or c-Fos, and regulate cell proliferation in the G1/S
transition.14 Importantly, a recent study has demonstrated
that ERK5 may provide a common bypass route, which
rescues cell proliferation upon abrogation of ERK1
signaling, with therapeutic implications in colorectal
cancer, which would provide a possible avenue to target
the compensatory route for potential cancer therapy.15
The mitogen-activated protein kinase (MAPK) cascade
is a highly conserved module that is involved in key
cellular functions.1 Researches have shown that targeting
MAPK pathways is an effective treatment in many
tumors.2-4 The MAPK family consists of four conventional
subfamilies: extracellular signal-regulated kinase1/2
(ERK1/2), c-Jun-N-terminal kinases (JNK), p38 and ERK5.5
ERK1 and ERK2 are proteins encoded respectively by
MAPK1 and MAPK3 genes, and their sequences are 84%
identical.6 To date, numbers of studies on activation,
inhibition and substrate recognition mechanisms have
not found the difference in biological function between
ERK1 and ERK2.7,8 In fact, any specific differences in the
design and discovery of ERK1 and ERK2 inhibitors will be
important factors, but the emerging consensus is that
they are functionally redundant.9 ERK5, also known as big
More recently, there has been accumulating clinical
evidence regarding the involvement of either ERK1 or
ERK5 signaling in tumor development and progression.16
As reported, more than 30% of cancers have been proven
to have BRAF, RAS or RAF gene mutations that activate
the MAPK signaling cascade.17,18 Also, ERK was found to
be activated in more than 85% of tumors.19 So that,
targeted inhibition of ERK is expected to directly inhibit
MAP kinase
1
(BMK1), contains the adenosine
triphosphate (ATP)-competitive hinge binding domain,
which is similar with ERK1.10,11 In regarding to their highly
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the MAPK signaling pathway, and may address innately or not appropriate. 3) Except for the above two situations,
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acquired resistant tumors.20,21 Accordingly, some selective
ERK1/2 inhibitors such as BVD-523 (1), GDC0994 (2) and
SCH772984 (3) have been investigated as candidate anti-
tumor agents,22-24 whereas other selective ERK5 inhibitors
such as XMD8-92 (4) have been reported for potential
cancer therapy (Figure 1).25 However, neither ERK1 nor
ERK5 inhibitors currently meet our expectations for
preclinical and clinical trials, which may due to the
compensatory mechanism of ERK5 for ERK1. To our
knowledge, no dual-target inhibitor of ERK1/5 or their
combination therapeutic strategy has been reported so
far.
whether it is appropriated to use a dual-target inhibitor
may be further investigated. Herein, we suggest that ERK5
may provide a common bypass route rescuing cell
proliferation upon abrogation of ERK1 in such types of
cancer, not limited to colorectal cancer.10 If possible, these
findings would provide a novel therapeutic strategy to co-
targeting both ERK1 and ERK5 for overcoming such
compensatory mechanisms in specific cancer types.
Considering this hypothesis, we designed and synthesized
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a
series of candidate compounds and eventually
discovered compound 22ac (ADTL-EI1712) as the first
selective dual-target inhibitor of ERK1 and ERK5, which
could induce regulated cell death, a form of cell death
that relies on the activation of genetically encoded
machinery,26-28 to overcome compensatory mechanism in
specific cancer cells, in vitro and in vivo.
To solve this problem, we hypothesized to classify all
the tumor types into three categories: 1) When both ERK1
and ERK5 are upregulated, using a dual-target inhibitor
should be appropriate. 2) When both ERK1 and ERK5 are
downregulated, using a dual-target inhibitor should be
Cl
F
Cl
H3C
HN
N
CH3
N
N
O
O
N
OH
OH
N
NH
H
N
NH
N
Cl
CH3
1
2
(BVD-523)
(GDC0994)
N
O
H
N
N
CH3
OH
N
O
CH3
N
N
N
O
O
N
N
N
H
N
N
N
N
H
N
CH3
3
4
(XMD8-92)
(SCH772984)
Figure 1. Representative chemical structures of ERKs inhibitors.
RESULTS AND DISCUSSION
(CHOL), kidney renal clear cell carcinoma (KIRC) and
liver hepatocellular carcinoma (LIHC). While, for the case
of ERK1 or ERK5 only, a higher expression level in tumor
tissues than normal tissues only appeared in four cancer
types, stomach adenocarcinoma (STAD), glioblastoma
multiforme (GBM), head and neck squamous cell
carcinoma (HNSCC) and kidney renal papillary cell
carcinoma (KIRP). But the expression levels of ERK1 and
ERK5 were not increased in other cancer types, such as
cervical squamous cell carcinoma and endocervical
adenocarcinoma (CESC), skin cutaneous melanoma
(SKCM) and so on. Herein, three types of tumors, AML,
Design of a dual-target inhibitor of ERK1 and
ERK5. Firstly, we accurately classified all the tumor types
based upon the mRNA expression levels of ERK1 and
ERK5. Then, the data of The Cancer Genome Atlas
(TCGA) and Genotype-Tissue Expression (GTEx)
databases were downloaded, the mRNA expression levels
of ERK1 and ERK5 were analyzed by Mann-Whitney test.29
GraphPad Prism 6.0 software was used for analysis and
p<0.05 was set as the detection threshold, we found that
both ERK1 and ERK5 were expressed at high levels in
acute myeloid leukemia (AML), cholangio carcinoma
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STAD and CESC were used as the examples to map their ERK1 and ERK5 by binding site similarity analysis and
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expression levels of ERK1 and ERK5 relative to their
normal tissue comparators (Figure 2a). Based upon
above-mentioned specific classifications, we carried out
the dual-target drug design for ERK1 and ERK5.
multiple docking. The crystal structures of ERK1 and
ERK5 were extracted from protein data bank (PDB: 4QTB
and 4B99).24,32,33 Sequence alignment reveals the overall
homology of ERK1 and ERK5 is 60%, while the overall
homology is only 30-43% between ERK1 and JNK, p38
(Figure 2b and S1). It is well-known that similar receptors
may bind similar ligands; therefore, this overall sequence
homology is more likely to share a common ligand.34
Multiple docking is widely used to design a dual-target
molecule, compared with linker and pharmacophore
methods, it may reveal a novel framework candidate
compound.30,31 In regarding to the ATP-competitive hinge
binding site, we designed the first dual-target inhibitor of
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Figure 2. Design of the dual-target inhibitor of ERK1 and ERK5. (A) Differentially expressed ERK1 and ERK5 genes between
tumors (T) and match normal (N) for three cancer types in TCGA and GTEx. ERK1 and ERK5 expressions in the tumor (red) and
match normal (blue) tissues of AML, STAD, CESC. (B) Candidate binding pockets in the structure of the kinase domain of
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ERK1/2/5, JNK1/2/3, p38α/β/γ/δ. The corresponding PDB IDs were 4QTB (ERK1), 5NGU (ERK2), 4B99 (ERK5), 3PZE (JNK1), 3NPC
(JNK2), 6EKD (JNK3), 5XYX (p38α), 3GP0 (p38β), 1CM8 (p38γ), 4EYJ (p38δ). The kinase hingle, P-loop, DFG domains is shown in
yellow. The kinase αC and αD helixes is shown in green. (C) Docking poses showed the interaction of 9, 21e, 22ac with ERK1
(PDB ID: 4QTB) and ERK5 (PDB ID: 4B99). Blue markers indicate that the compound has consistent interacting amino acid
residues in ERK1 and ERK5.
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In the present study, a multiple docking strategy was
utilized to discover the lead inhibitor of ERK1 and ERK5.
First, a total of 212,255 compounds in the SPECS library
were filtered by the Lipinski's rule of five, leaving 97,360
small molecules. Second, the drug binding site was
defined around the high homology hinge domain. The
filtered SPECS library was rapidly molecular docked in the
co-crystal structures of ERK1 (PDB ID: 4QTB) using
Libdock module of Discovery Studio Software (version
3.5), and the top 1,000 hits were screened (score above
120). Next, the CDOCKER module was implemented in
ERK1 and ERK5 (PDB ID: 4B99) respectively to perform
accurate docking analysis of the 1,000 hits. ERK1 and ERK5
each obtained 231 and 208 compounds with a score
greater than 30. Finally, we performed overlapping and
docking pattern analysis of the two groups of molecules,
and screened 15 candidates (5-19). These compounds were
further filtered by bioassay, 3 and 4 were used as positive
control agents for ERK1/2 and ERK5 respectively (Figure
S2 and S3). The 2-amino-3-carboxamide-thieno[2,3-c]
pyridine scaffold (9) showed moderate potency on ERK1
and ERK5. Meanwhile, we also found that in the previous
reports, this class of ingredients has a certain anti-tumor
activity, but they have not been subjected to in-depth
target and mechanism studies.35-38 In this study,
compound 9 was identified as the leading compound
targeting ERK1/5 for further structural optimization and
structure-activity relationship analysis. According to the
molecular model of 9 with ERK1 and ERK5, the leading
molecule has a similar docking posture and pattern as the
core of the inhibitors present in the original crystal
structures. However, some hydrogen bonding interactions
with hinge region are still needed to stabilize the binding
of compound to ERKs, such as Met125 of ERK1 and Met140
of ERK5.24,33 Further analysis revealed that Ile48-Val56,
Ser170-Leu173, Cys183-Gly186 of ERK1 and Ile61-Val69,
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Ser186-Leu189, Gly199-Gly202 of ERK5 formed
a
hydrophobic channel, while 9 lacked the corresponding
group to interact with it (Figure 2c). Consequently, 9 was
designed to change the six-membered cyclic hydrocarbon
structure to piperidine ring structure, and to fill the
hydrophobic region by introducing appropriate
substituents on the N atom to increase side chains. A
series of derivatives containing different terminal groups
were designed and synthesized. The synthetic routes for
21 and 22 series compounds are shown in Scheme 1.
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Scheme 1. The synthesis of compound 21 and 22. Reaction conditions: i Ethanol, reflux, 83%; ii DIEA, acyl chloride, rt., 60-
70%; iii TFA: dichloromethane (1:1), rt., 90-95%; iv benzyl chloride, K2CO3, DMF, 80 ℃, 60-75%; v isothiocyanate, DIEA,
dichloromethane, rt., 60-75%.
Synthesis and identification of dual-target
inhibitors of ERK1 and ERK5. A highly efficient
synthesis of 21 and 22 was accomplished in four steps
(Scheme 1). The intermediate 20a was prepared by the
Gewald reaction of N-(tert-butoxycarbonyl)-4-piperidone,
sulfur and α-cyanoacetamide,39 the acylation of
intermediate 20a to intermediate 20b, and then the
deprotection of intermediate 20b with TFA, and the
Scheme 1). The results of affinity and activity suggested
that the ortho position of the benzyl ring was substituted
by electron withdrawing group chlorine (21e, 21i and 21j),
but not F, CN or electron donating group, could enhance
kinase inhibition and anti-proliferation activity. Next,
electron withdrawing groups (Cl, CF3, NO2) and electron
donating groups (CH3) were introduced at varied
positions in R1 (21l-21o, 21p-21q, 21r-21t, 21u-21v).
However, the results showed lower kinase inhibitory
activities. In this series, some compounds with mediocre
ERKs inhibition showed better cytostatic activity, such as
21e, 21t and 21o-21q. The reason may be that the core
group of 21 series compounds have other anti-tumor
mechanisms, such as rosbin (a piperidinothieno derivative
with similar structure), which has been reported to
increase reactive oxygen species accumulation to induce
cell death.33 In the modification of 21 series compounds,
reaction
of
intermediate
20c
with
benzyl
chloride/isocyanate leads to the production of targeted
compound 21/22 with a purity >95%. To establish the
structure-activity relationship of compounds, inhibitory
activities of compounds against ERK1 and ERK5 were
evaluated. In addition, inhibitory activities of all
compounds for ERK2 were also measured, due to the
redundancy of the biological functions of ERK1 and
ERK2.7-9 These results show that compounds have equal
inhibitory potential for ERK1 and ERK2, which is in
consistent with those previous reports.6,22-24
compound 21e was obtained as
a
representative
compound.
To establish the structure-activity relationship for
inhibitors, a series of 21 derivatives were synthesized and
tested in the first round of screening (Table 1). The first
set (21a-21k) comprises variations of the R2, which
retained a slight inhibition of kinase activity (Figure 3,
The docking pattern analysis of 21e and leading
compound with ERK1 and ERK5 indicated that
modifications of benzyl moiety of compounds caused a
change in displacement of the core group to the hinge
region, but still cannot produce stable interactions in the
9
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hinge region and hydrophobic channel (Figure 2c). 2-chlorophenyl group and R1 as various substituted
1
Therefore, continued studies were carried out to find
suitable groups to extend the target molecule. By
analyzing structures of the original inhibitors for ERK1
and ERK5, it was found that their chemical structures
interacting in the channel have amide groups.24,30 Instead
of amide structure, we supposed that the interaction with
this region may be further enhanced by introducing
thiourea and urea groups. On the other hand, the
introduction of thiourea or urea group also facilitates the
elongation of molecules, possibly by enhancing the
displacement of the core group and promoting interaction
with the amino acid residues in the hinge region.
Subsequently, compounds 22aa-22as (retaining R1 as
chlorophenyl group, R2 as various substituted phenyl
group, heterocyclic aromatic hydrocarbon, naphthalene
ring structure) were synthesized and evaluated. When R2
was halogen (not F) substituted phenyl structure,
compounds (22ac-22ag, 22ao-22ap in Table 2) had better
enzyme inhibitory activity, especially R2 was 2-
chlorophenyl group(22ac) (inhibition rates of ERK1/2/5 at
1 μM were 93.54%, 92.72% and 89.35%, respectively). We
next synthesized compounds 22ba-22bp that retain R2 as
phenyl group or heterocyclic aromatic hydrocarbons.
Activities screening results showed that when R1 was 2-
bromophenyl group and 2-iodophenyl group (22bd,
22be), the enzyme inhibitory activities (inhibition rates of
ERK1/2/5 were 87.69%, 89.73%, 80.46% and 83.52%,
85.80%, 80.36%) reach the activity similar to 22ac.
Moreover, we also found that the thiourea group with R3
replaced by sulfur has a favorable contribution to anti-
proliferative activities. Finally, we also comprehensively
investigated the effect of simultaneous changes of R1 and
R2 groups on the activity Then the series of cross-
products of R1 and R2 with different substituents were
synthesized (22ca-22cw), including substituted phenyl
group, heterocyclic aromatic hydrocarbons, aliphatic
hydrocarbons. The results of activity evaluations showed
that compounds in which R1 and R2 were substituted by
halogen (not F) in the ortho position of the phenyl group
(22ce, 22ch) also had a certain ERKs inhibitory activity
(Figure 3). However, compared with the 22ac, enzyme
inhibition and anti-proliferative activity were not further
improved.
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Table 1. Biochemical and Biological Evaluations of Candidate Compounds 21a-21v
O
NH2
NH
N
S
R1
R2
O
Kinase inhibitory activity (1 μM, %)[a]
Anti-proliferative activity (IC50, μM)[b]
No.
21a
21b
21c
R1
R2
ERK1
ERK2
ERK5
HL-60
MKN-74
HeLa
Cl
Cl
Cl
61.46±0.51
64.87±3.68
62.69±2.83
18.62±0.32
28.70±2.31
45.03±0.47
H
60.13±0.49
60.59±0.02
62.94±1.82
53.41±1.64
63.70±1.04
55.27±1.27
12.03±1.85
>50
14.45±1.36
>50
38.41±1.29
>50
CH3
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8
Cl
Cl
Cl
Cl
Cl
CN
Cl
F
21d
21e
21f
55.78±3.74
61.38±0.87
68.32±0.60
47.36±0.15
66.25±1.31
42.13±3.37
65.19±0.69
71.56±2.62
55.36±5.58
63.12±2.25
44.29±2.93
65.11±1.52
37.92±4.20
47.03±3.15
64.96±1.15
10.39±1.20
2.90±0.46
21.82±0.97
22.54±0.62
11.40±1.56
9.50±0.29
4.43±0.26
12.92±1.02
18.73±1.74
31.47±2.39
>50
27.48±0.31
>50
9
10
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14
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21
22
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Cl
21g
21h
>50
O2N
46.08±1.05
Cl
Cl
21i
57.82±0.51
78.13±5.85
61.85±3.06
4.85±0.73
4.74±0.08
28.83±0.72
Cl
Cl
Cl
Cl
Cl
21j
68.94±3.30
60.97±0.18
66.59±4.34
59.91±2.59
63.10±0.20
22.90±1.47
8.01±1.74
15.81±1.39
16.34±1.04
23.65±1.78
21.05±1.58
19.60±1.06
Cl
21k
Cl
F
21l
CF3
CF3
72.21±1.70
76.79±4.96
43.28±1.85
56.80±12.80
61.13±1.76
>50
>50
26.92±1.85
>50
>50
>50
Cl
21m
47.77±11.40
Cl
21n
21o
CF3
CF3
37.26±0.61
39.61±4.31
35.59±0.83
33.19±4.53
38.73±1.76
39.97±0.29
10.62±1.07
14.85±0.73
8.53±0.57
10.85±1.30
>50
>50
Cl
Cl
Cl
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Cl
2
3
4
5
6
7
8
9
CH3
CH3
21p
21q
21r
42.77±0.54
32.18±2.71
49.31±3.67
45.39±1.68
53.69±2.39
48.82±4.43
46.00±2.76
52.31±2.39
42.47±3.08
10.04±1.59
19.88±1.35
19.76±2.48
12.34±1.67
25.47±1.93
23.29±1.55
47.35±3.26
>50
Cl
Cl
Cl
Cl
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33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
49.53±1.38
Cl
F
F
21s
21t
48.36±1.75
55.71±15.40
31.63±3.26
67.17±0.44
36.88±7.62
32.38±1.10
29.31±1.08
65.34±1.54
21.11±5.57
38.05±9.61
45.23±3.73
77.90±5.24
10.27±0.94
13.06±1.59
18.35±0.16
19.83±0.65
7.43±0.46
9.61±0.17
13.49±1.12
16.52±2.06
32.97±1.18
27.46±1.78
46.11±2.07
33.02±1.78
Cl
Cl
Cl
Cl
NO2
21u
21v
Cl
NO2
Cl
Cl
aEach compound was tested in triplicate; the data are presented as the mean ± SEM (n=3).
bIC50 values obtained with cell viability assay for 24 h.
Table 2. Biochemical and Biological Evaluations of Candidate Compounds 22aa-22cw
O
NH2
H
NH
O
N
N
S
R1
R2
R3
Kinase inhibitory activity (1
Anti-proliferative activity (IC50,
μM)[b]
μM, %)[a]
No.
R1
R2
R3
ERK1
ERK2
ERK5
HL-60
MKN-74
HeLa
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7
Cl
Cl
85.42±0.1 76.54±0.5 59.52±1.5
S
46.67±0.9
6
22aa
5.94±0.74 15.59±1.34
13.39±1.84 20.65±1.27
4
2
4
82.81±5.1
7
81.26±3.6 72.45±5.2
30.76±2.6
7
22ab
S
8
9
8
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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31
32
33
34
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38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
93.54±4.9 92.72±0.2 89.35±2.4
22ac
22ad
22ae
22af
22ag
22ah
22ai
22aj
S
O
S
1.26±0.57
2.55±0.66
>50
0
2
1
Cl
89.25±0.
94
88.37+0.3 89.92±0.
49
45.43±0.0
7
12.48±1.62 16.28±1.49
3
F
88.43±2.2 82.75±2.4 70.66±6.
86
28.74±0.5
9
4.97±1.21
12.59±1.52
9
0
Br
90.86±1.0 86.94±2. 88.52±0.8
24.62±0.8
1
S
9.09±2.03 10.84±3.21
1
67
9
I
87.96±0.
89
87.35±3.3
2
80.97±2.
02
S
4.43±0.50
10.61±0.96
2.63±1.79
1.87±1.55
11.66±1.57
35.17±1.25
28.95±1.61
CF3
CH3
OCH3
83.59±3.3 85.74±2.9 76.61±5.6
26.74±2.2
7
S
8
4
9
68.41±3.3
8
72.77±3.7 59.26±4.4 22.59±3.6
S
>50
>50
2
2
2
71.30±1.4
0
70.64±0.
47
53.73±2.9 40.88±2.0
S
7
8
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SCH3
Cl
Cl
69.45±2.
60
64.58±4.1 52.62±4.6
22ak
22al
S
S
14.55±1.98 13.77±2.18 43.56±1.82
1
8
60.93±2.7 64.69±5.1
40.46±0.
72
34.37±0.91 25.83±0.71 24.88±1.18
9
4
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cl
Cl
80.54±2.4 54.38±12. 57.43±3.7
40
22am
22an
S
S
7.59±1.46
7.09±0.06
>50
3
4
F3C
78.12±6.8 68.67±13. 55.70±6.2
15.68±2.61 19.56±1.53 36.43±1.78
9
9
0
H3CO
Cl
Cl
Cl
90.66±0. 90.51±0.4
08
88.00±0.
29
26.54±0.4
22ao
22ap
S
3.95±0.82
4.02±0.21
2
5
Cl
Cl
Cl
82.74±0.8 84.95±0.
84
82.68±0.
27
24.43±0.7
4
O
17.69±2.70
6.50±0.47
27.17±0.05
3
F3C
Cl
Cl
Cl
89.32±2.3 88.84±1.7 77.44±3.1
14.96±0.2
4
48.88±1.0
9
22aq
22ar
22as
22ba
S
O
S
4
4
5
CF3
F3C
91.03±0.9 89.16±0.5
80.34±0.
63
26.68±2.5
9
38.62±0.4
6
24.33±1.05
9
8
CF3
41.94±5.5 45.42±3.7 34.87±2.8
38.62±0.9
3
33.89±1.85
>50
>50
8
7
6
N
Cl
73.84±2.9 77.46±0.3 58.90±0.7
S
13.75±1.97 11.80±0.86
4
0
4
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8
9
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
F
62.43±0.5 68.59±0. 65.24±0.8
S
19.44±0.0
8
22bb
12.53±1.04 11.07±0.58
4
62
6
64.99±0. 62.36±0.4 54.91±0.6
32.74±3.0
8
49.57±2.0
3
22bc
22bd
22be
22bf
22bg
22bh
22bi
22bj
22bk
O
S
>50
09
0
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
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38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Br
87.69±2.
90
89.73±1.3
9
80.46±0.
32
14.59±4.6
2
12.90±1.74 46.93±1.55
I
83.52±1.2 85.80±2.3 80.36±1.5
S
8.56±2.57
17.57±3.28
20.93±1.43
17.46±1.39
>50
3
7
4
F3C
78.83±5.3 80.91±1.5 56.49±1.9
21.85±0.9
9
46.94±2.0
8
S
8
2
8
H3C
70.64±3.3
7
53.04±0.5
0
10.95±0.9
5
S
65.91±1.18
35.26±3.72
>50
H3CO
H3CS
66.34±1.8
4
67.07±6. 53.49±4.8 20.84±1.8
20.47±4.8
6
S
65
6
4
67.19±3.8 63.41±5.7
30.46±3.8
2
S
49.14±1.51
6.55±5.72
16.42±1.16
43.89±1.34
34.71±5.03
2
2
75.62±0.1
9
78.96±0.
04
57.48±0.1
7
36.05±2.8
7
44.00±0.2
9
CF3
S
78.30±0.5 77.59±1.2 62.17±0.6
CF3
O
>50
>50
9
8
7
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Cl
Cl
Cl
Cl
Cl
83.16±3.6 82.80±2.4 50.35±7.2
S
26.25±2.6
CH3
22bl
22bm
22bn
22bo
22bp
8.43±1.76
11.27±1.40
1
0
1
7
70.42±0.1 71.93±0.4 53.80±0.6
32.85±0.2
8
CH3
O
S
14.62±2.50 27.55±1.43
4
9
1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NO2
64.21±0.0 58.46±0.1 70.74±0.1
40.33±0.0
7
7.04±0.09
8.85±1.16
7
6
2
NO2
71.26±0.2
4
60.40±0.
08
65.73±0.1
9
O
S
18.49±1.57 22.74±1.68
34.56±1.87 28.37±1.12
>50
>50
48.49±1.9 45.71±3.6 38.52±4.0
N
8
7
9
N
60.48±3.3 68.45±4.3 43.27±0.3
22ca
22cb
S
S
>50
>50
>50
>50
>50
>50
0
3
6
65.82±3.5 66.29±5.1 61.44±2.4
4
9
3
F3C
F
76.62±0.7 68.58±2.
60
77.32±0.8
8
28.02±2.8
9
22cc
22cd
22ce
22cf
S
S
S
S
17.90±1.25
>50
13.26±1.16
>50
6
CF3
F
Br
70.07±0.5 68.65±2.
65.69±1.4
9
>50
8
99
Br
Br
Br
80.94±4.
55
82.06±2.
68
88.42±3.2
0
24.70±1.4
8
17.56±3.97 13.68±2.37
CF3
79.33±3.0 72.09±3.4 78.58±3.7
24.28±2.9
27.41±0.34
6
39.62±3.19
2
7
6
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9
CH3
Br
65.10±2.7 60.75±1.8
46.67±0.
91
22cg
S
S
S
>50
>50
>50
>50
9
0
I
I
Br
81.98±3.4
9
80.05±4. 85.46±4.2
01
22.37±2.7
9
22ch
22ci
18.94±3.58
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CF3
OCH3
F
77.47±1.1
8
80.66±3.1 64.83±3.2
49.21±0.8
0
19.70±1.96 14.16±0.82
3
1
I
F3C
F3C
F3C
F3C
H3C
H3C
H3C
56.10±2.2 59.63±4.3 42.78±3.7
41.80±2.6
22cj
22ck
22cl
S
S
S
S
S
S
S
S
>50
>50
>50
5
0
1
6
77.23±4.6 81.62±3.3 86.49±1.4
19.62±0.4
23.51±2.62
8
2
5
6
Br
82.51±3.7
8
85.47±1.8
1
73.31±1.57 25.48±1.85 23.85±1.87
>50
CH3
OCH3
Br
64.93±3.0 62.75±2.6 48.33±0.9 25.09±0.4 47.38±0.6
22cm
22cn
22co
22cp
22cq
46.97±1.21
>50
1
5
0
6
5
77.59±1.9 74.89±2.4 61.72±0.5
>50
34.52±1.93
3
9
7
64.72±6.
87
65.19±3.8 54.86±5.3
45.19±3.6
9
>50
>50
6
3
CF3
72.01±0.9 76.93±1.2 60.07±1.5
23.17±0.16 17.43±1.88
>50
2
2
1
CH3
48.29±4. 43.00±7.0 37.13±3.9
07
>50
>50
>50
9
8
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2
3
OCH3
H3CO
44.71±0.9 48.67±2.1 32.04±1.3
S
22cr
>50
>50
>50
>50
5
6
9
4
5
6
7
8
9
82.53±0.5 79.67±0.2 57.91±8.5
CF3
22cs
22ct
22cu
22cv
S
S
S
S
5.52±0.49
12.25±1.54
5
5
5
F3C
F3C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
79.36±1.5 65.99±3.2 54.20±4.8
49.02±4.9
8
CF3
16.31±3.59 13.75±1.46
6
3
3
CF3
CF3
CF3
F3C
77.40±5.6 80.13±10. 70.76±4.8
70
CH3
11.83±1.60
14.89±1.14 17.06±1.98
8
0
F3C
NO2
80.22±2.5 74.98±3.6 82.86±4.
16.38±1.52 25.79±1.36 27.17±4.78
5
2
95
74.38±6.1
0
79.69±2. 48.63±4.5
47.41±2.9
22cw
S
-
>50
>50
>50
>50
>50
N
60
5
6
H
N
98.81±0.3
5
99.25±0.
07
3
<50
1.06±0.39
10.59±1.62
>50
-
-
98.47±0.1
2
4
-
<50
<50
>50
>50
-
-
-
-
0.88±0.83
(CI =
0.56)e
3+4c
-
NDd
NDd
NDd
aEach compound was tested in triplicate; the data are presented as the mean ± SEM (n=3).
bIC50 values obtained with cell viability assay for 24 h.
cCombined at a mole ratio of 1:1.
dNot determined.
eCombination index (CI) was calculated using the Chou-Talalay method.
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1
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7
8
9
Cl
Cl
CH3
NH
O
CF3
CH3
Cl
O
R1
NH2
NH
O
H
N
Cl
S
Cl
F
N
R1
S
R2
R2
Extended
molecules with
benzyl group
O
Cl
21 series
9 leading compound
21b
21e
21l
21q
Modifying
leading
structure
Cl
Cl
Cl
Cl
R1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
F3C
Br
Cl
NH2
R2
R3
NH
HN
S
CF3
Cl
O
Cl
O
Extended
molecules with
thiourea/urea
NH2
S
S
O
S
21a
22af
22al
22ap
22aq
H
N
NH
O
N
S
R1
R2
I
NO2
NO2
R3
Obtained
representative
inhibitor
R1
22 series
N
H
Cl
S
Cl
O
O
Br
NH2
NH
O
R2
R3
Cl
H
N
N
N
S
S
S
S
Cl
22bn
22bo
22ch
22cw
22ac
Figure 3. Structural optimization and discovery of the dual-target inhibitor of ERK1 and ERK5. Representative
inhibitors were shown in the figure.
Based on the above analysis, compounds in which R1
and R2 are phenyl substituted by halogen (not F) in ortho
position and the linker is thiourea group in which R3 is
substituted by sulfur atom has a positive effect on the
inhibition of ERKs. Moreover, activities of ERK1/2
inhibitor 3 and ERK5 inhibitor 4 were further evaluated
when used alone or in combination (Table 2). In HL-60
cells, 3 and 4 might show a certain synergistic effect, the
combination index (CI) calculated by Chou-Talalay
method was 0.56.40 In both MKN-74 and HeLa cells, 3 and
4 did not exhibit good anti-proliferative activity either
alone or in combination. Finally, with chlorophenyl at R1,
chlorophenyl at R2 and sulfur at R3, 22ac showed the
most potent kinase affinity and anti-proliferative activity
(Table 2). Based upon kinase inhibitory assays, the IC50
values of 22ac for ERK1 and ERK5 were 40.43 nM and
64.50 nM. The above results indicate that 22ac is a potent
and targeted dual-inhibitor of ERK1 and ERK5. In the
complex of 22ac-ERK1 and 22ac-ERK5, the ortho-
chlorobenzamide moiety of 22ac positioned towards the
entrance of the ATP-binding pocket. The 2-amino-3-
carboxamide-thieno scaffold occupied the purine binding
site, three hydrogen bonds were detected between 22ac
and the hinge region residues Asp123, Leu124, Lys131 of
ERK1 and one hydrogen bond was found at the hinge
region residue Met140 of ERK5. In contrast to the docking
mode of 9, the core structure of 22ac increased hydrogen
bonding interaction with Asp123, Met125 of ERK1 and
Met140 of ERK5, which was mainly attributed to the
structural modification of ortho-chlorophenyl-thiourea
group. Additionally, ortho-chlorophenyl-thiourea group
was also inserted into the hydrophobic pocket composed
of Ile48, Val56, Cys183 of ERK1 and Ile61, Val69, Ser186,
Leu189 of ERK5. Therefore, these characters suggest that
22ac could bind to ERK1 and ERK5 (Figure 2c).
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8
9
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21
22
23
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Identification of a selective dual-target inhibitor of ERK1 and ERK5. (A) Kinome tree representation of the
selectivity of 22ac. % Inhibition in kinase kinetic assays is shown as spheres as indicated in the figure capture. (B) MKN-74 cells
were transfected with specific siRNA of ERK1/2 or negative control siRNA for indicated times, followed by treatment of 22ac (0.5
μM). The expressions of p-ERK1/2 and p-ERK5 puncta were detected by immunocytochemistry. (C) The expression levels of p-
ERK1/2, ERK1/2, p-ERK5, ERK5, p-RSK, RSK, p-cMyc and c-Myc in MKN-74 cells treated with 0.5 μM 22ac for 0 h, 6 h, 12 h and 24
h were analyzed by immunoblotting. GAPDH was used as a loading control. (D) MKN-74 cells were transfected with specific
siRNA of ERK1/2 or negative control siRNA, followed by treatment of 22ac (0.5 μM ), 3 (1 μM) and 4 (1 μM), respectively. The
expression levels of ERK1/2, p-ERK1/2, ERK5 and p-ERK5 were analyzed by immunoblotting, GAPDH was used as a loading
control. (E, F) MKN-74 cells were treated for 0 h, 6 h, 12 h and 24 h with 0.5 μM 22ac, and then the expressions of Ras, Raf, p-
MEK1/2, MEK1/2, p-JNK, JNK, p-p38 and p38 were analyzed by immunoblotting. GAPDH was used as a loading control.
22ac induces regulated cell death by targeting ERK1
and ERK5. To further confirm the target selectivity of
22ac, a KINOMEscan panel of 100 kinases binding assays
with 1 μM 22ac was performed by Eurofins Pharma
Discovery Services UK Limited. The results showed that
22ac could inhibit ERK1/5 kinases specifically (ERK1/2/5
inhibition ratio was 94%, 91%, 87%, respectively) (Figure
4a). According to immunofluorescence results, we found
that 22ac could markedly reduce the fluorescence puncta
of p-ERK1/2 and p-ERK5 in MKN-74 cells (Figure 4b).
Similarly, immunoblotting assays also suggested that 22ac
inhibited the phosphorylation of ERK1/2 and ERK5 (Figure
4c). Interestingly, 22ac could not inhibit the
phosphorylation of RSK, which is directly regulated by
ERK1/2. But the phosphorylation of cMyc was blocked by
22ac, indicating that 22ac might induce cell death via
non-canonical apoptosis pathways (Figure 4c). Moreover,
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22ac and 4 rather than 3, inhibited phosphorylation of methyladenine (3-MA) to identify whether it can block
1
2
3
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ERK5 under the condition of ERK1/2 silencing (Figure 4d).
22ac-induced autophagy. We found that the upregulation
of LC3-II and degradation of p62 were significantly
reversed in the presence of 3-MA (Figure 5c). Moreover,
we knocked down an autophagy-related gene ATG5 by
specific siRNA and found that silencing of ATG5 could
block the lipidation of LC3 and degradation of p62 (Figure
5d). Compared to the negative control group, cell viability
was recovered after silencing of ATG5, indicating
activation of autophagy may be one of the essential
regulated cell death mechanisms for 22ac-induced cell
death (Figure 5e). These results suggest that 22ac induces
regulated cell death accompanied with autophagy in
MKN-74 cells.
ERK, JNK and p38 are all contained in the mammalian
MAPK family.41,42 And ERK is an important downstream
node in the Ras/Raf/MEK/ERK signaling cascade.43,44
Therefore, we examined the expressions and
phosphorylations of Ras, Raf, MEK, JNK1/2 and p38 to
illuminate the selectivity of 22ac on MAPK pathways in
cells. As expected, 22ac did not activate or inhibit these
pathways (Figure 4e, f).
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22ac induces regulated cell death accompanied
with autophagy in stomach adenocarcinoma cells.
Interestingly, we observed 22ac could induce massive
cytoplasmic vacuolization in MKN-74 cells, which is
closely associated with autophagy. Thus, we examined
whether 22ac could induce autophagy in MKN-74 cells.
Induction of autophagy was preliminarily confirmed by
the increased LC3 puncta in MKN-74 cells treated with
22ac (Figure 5a). Subsequently, we demonstrated that
treatment with 22ac resulted in elevated expression of
LC3-II and degradation of p62 in MKN-74 cells (Figure
5b). Next, we used the autophagy inhibitor 3-
Distinct from MKN-74 cells, we found that treatment
with 22ac did not induce cytoplasmic vacuolization and
autophagy in HL-60 cells. Intriguingly, 22ac exhibited
good anti-proliferative effects in both HL-60 and MKN-74
cells, which is different from the combined ERK1 and
ERK5 inhibitors (3 and 4). These above-mentioned results
suggest that 22ac-induced regulated cell death might have
an additional mechanism of action (MOA) in MKN-74
cells.
Figure 5. 22ac induces regulated cell death accompanied with autophagy in MKN-74 cells. (A) MKN-74 cells were
transfected with GFP-mRFP-LC3 plasmid, followed by treatment with 0.5 μM 22ac for 24 h. Then, the GFP-LC3 puncta were
observed by fluorescence microscope. Scale bar = 20 μm. (B) The expression levels of LC3 and p62 in MKN-74 cells treated with
0.5 μM 22ac were analyzed by immunoblotting. (C) MKN-74 cells were treated with 0.5 μM 22ac for 24 h in the presence or
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absence of 3-MA, the expression levels of LC3 and p62 were analyzed by immunoblotting. (D) MKN-74 cells were transfected
with specific siRNA of ATG5 or negative control siRNA for indicated times, followed by treatment of 0.5 μM 22ac. Then the
expression levels of ATG5, LC3 and p62 were analyzed by immunoblotting, GAPDH was used as a loading control. (E) MKN-74
cells were transfected specific siRNA of ATG5 or negative control siRNA for indicated times, followed by treatment of 0.5 μM
22ac. Then cell viabilities were detected by MTT assay. ##p<0.01, compared with si-NC administration group; ***p<0.001,
compared with si-ATG5 control group.
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Tumor inhibition in xenograft tumor models of
nude mice. Based upon the efficacy of 22ac on HL-60,
MKN-74, HeLa cells in vitro, we proceeded to evaluate its
anti-tumor activity in vivo. A total of 48 female nude mice
were divided into three groups, and xenograft mouse
models were constructed by subcutaneous injection of
HL-60 cells, MKN-74 cells and HeLa cells. Nude mice of
each tumor type were further divided into model and
treatment group (8 in each group). In vivo experiments
showed that 22ac with a per os (p.o.) dose of 50 mg/kg
once a day for 16 days significantly inhibited the tumor
volume in the xenograft mouse model of HL-60 and MKN-
74 cells, while the anti-tumor effect of HeLa cells group
was much weaker (Figure 6a). Similarly, a remarkable loss
of tumor weights was observed in groups of HL-60 and
MKN-74 cells (p<0.001) (Figure 6b). To test whether 22ac-
mediated inhibition of tumor growth in vivo was
associated with reduced cell proliferation, Ki-67 of tumor
tissues of MKN-74 group was analyzed for its
immunoreactivity. We found that 22ac treatment
obviously reduced the number of Ki-67 positive cells in
xenograft mice (Figure 6c, d). To elucidate the mechanism
for the therapeutic efficacy of 22ac in vivo, we examined
the immunoreactivity of p-ERK1/2 and p-ERK5 in tumor
tissues of MKN-74 cells xenograft mice. As a result, the
expression of p-ERK1/2 and p-ERK5 were both reduced in
22ac-treated tumor tissues (Figure 6c). Next, we carried
out western blot analysis to further clarify the mechanism
in vivo. As expected, 22ac inhibited the phosphorylation
of ERK1/2 and ERK5, which was in line with the
immunohistochemical analysis and the results obtained in
vitro (Figure 6e). Taken together, these results suggest
that 22ac can inhibit tumor growth in vivo.
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Figure 6. 22ac has a therapeutic effect in vivo. (A) Mice bearing HL-60, MKN-64 and HeLa cells (n = 8 per group) tumor
xenograft were treated with vehicle control or indicated dose of 22ac (50 mg/kg, p.o.) once a day. Tumor volume data were
presented as mean ± SEM. (B) Tumor weight change of mice in different groups. ***p<0.001, compared with control group. (C)
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Immunohistochemical analysis of proliferative marker Ki-67, p-ERK1/2 and p-ERK5. Tumor tissues were excised from the control
and 22ac (50 mg/kg) treated mice. Scale bar=200 μm. (D) Quantitative analysis of percentage of Ki-67, p-ERK1/2 and p-ERK5
positive ratio. ***p<0.001, **p<0.01, compared with control group. (E) Two individual tumor tissues (1 and 2) excised from the
xenograft mice were lysed. Then the expression levels of ERK1/2, p-ERK1/2, ERK5 and p-ERK5 were detected by western blot
analysis.
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Preliminary assessment for pharmacokinetics
profiles of 22ac. The pharmacokinetics profiles of 22ac in
rats were assessed. Two groups of rats (3 in each group)
were administered a single intravenous (i.v.) dose of 1
mg/kg and p.o. dose of 10 mg/kg to estimate
pharmacokinetic parameters. As showed in Table 3
following oral administration of 22ac, the highest
concentrations of 22ac were observed at 1.24 ± 0.20 h with
a maximum plasma concentration (Cmax) value of 1560 ±
169 ng/mL, which implied that 22ac could be absorbed
rapidly from the intestine. Meanwhile, the area under the
curve (AUC) of 22ac showed a high oral exposure with
AUC0-t = 5078 ± 282 h*ng/mL and AUC0-∞ = 5180 ± 357
h*ng/mL. In addition, 22ac also exhibited an acceptable
oral bioavailability of 27.64%. These results demonstrated
the suitability of this compound for use as oral candidate.
This study provides evidence that 22ac can be used for
future optimization.
targeting both ERK1 and ERK5 to overcome such common
bypass route/compensatory mechanism in specific cancer
types, classified by ERK1 and ERK5 expression levels. In
addition, this dual-target inhibitor, 22ac has also been
demonstrated to improve the anti-proliferative effects.
Interestingly, we found that this compound could induce
regulated cell death accompanied with autophagy in
MKN-74 cells rather than in HL-60 cells, suggesting that
additional mechanisms might be also involved in
regulated cell death in some types of cancer. Therefore,
the interrelationship of mechanisms and tumor types
remains to be further clarified in our future work.
Probably, this compound can also act as a valuable
chemical probe or lead compound for investigating the
biological function of the relationship between ERK1 and
ERK5, validating the druggable potential of two targets
and thus discovering more novel anti-tumor candidate
drugs in the future.
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EXPERIMENTAL SECTION
Table 3. Pharmacokinetics profiles of 22ac in rats.
General Methods. All chemicals and reagents were
obtained from commercial sources and used without
further purification. 1H and 13C NMR spectra were
recorded on a Bruker Analytik, DE/AVANCE Digital 400
at 400 and 100 MHz. High-resolution mass spectra
(HRMS) data were recorded on a commercial apparatus
and methanol was used to dissolve the sample. All final
compounds were purified to > 95% purity, as determined
by high-performance liquid chromatography (HPLC).
HPLC was performed on the SHIMADZU LC-20AT pump
and SPD-20A Photodiode Array detector using an Agilent
Diamonsil C18-WR, 5.0 μm, 4.6 x 250 mm (WondaSil).
Mobile phase was MeOH-H2O (85: 15), the flow rate was
1.0 mL/min. High-performance liquid chromatograms of
key representative compounds in Figure 3 were shown in
the Supporting Information.
Rats*
Parameters
i.v. (1 mg/kg)
p.o. (10 mg/kg)
T1/2 (h)
Tmax (h)
0.95±0.06
0.08±0.01
1890±94
1837±194
1841±196
1.24±0.20
0.75±0.35
1560±169
5078±282
5180±357
27.64
Cmax (ng/mL)
AUC0-t (h*ng/mL)
AUC0-∞ (h*ng/mL)
F (%)
*All data were obtained by triple testing (± standard
deviation).
CONCLUSION
In summary, we report herein for the first time that a
selective inhibitor 22ac (ADTL-EI1712) that can
simultaneously co-target ERK1 and ERK5 as a novel
therapeutic agent that induces regulated cell death to
overcome the compensatory mechanism in specific cancer
types. Since ERK5 can give a common bypass route in
certain types of cancer, which rescues cell proliferation
upon abrogation of ERK1 signaling, our work sheds light
on providing a novel drug design strategy that co-
Preparation of intermediates 20a. To a solution of tert-
butyl 4-oxopiperidine-1-carboxylate (30.0 mmol), 2-
cyanoacetamide (30.0 mmol), and sulfur (30.0 mmol) in
anhydrous ethanol (200 mL), morpholine (30.0 mmol)
was added. The mixture was refluxed for 8 h. The
suspension was cooled to ambient temperature and
filtered through a pad of Celite, and washed with absolute
ethyl alcohol (30 mL x 3). The combined filtrate was
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Journal of Medicinal Chemistry
concentrated to dryness. The crude product was diluted 2-(2-Chlorobenzamido)-4,5,6,7-tetrahydrothieno
Page 20 of 44
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7
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by 95% ethyl alcohol (30 mL), and filtered to give the
product as a white solid, yield 83%.
[2,3-c]pyridine-3-carboxamide (21a). White solid, mp
184-186, yield 73.8%. 1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 12.31 (1H, brs), 9.26 (2H, brs), 7.75 (1H, dd, J = 7.7,
1.3 Hz), 7.62 (1H, dd, J = 6.6, 1.5 Hz), 7.59 (1H, td, J = 6.6,
1.3 Hz), 7.52 (1H, td, J = 7.7, 1.5 Hz), 4.32 (2H, s), 3.39 (2H, t,
J = 5.2 Hz), 3.03 (2H, t, J = 5.2 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 166.7, 163.3, 143.5, 134.0, 133.1, 130.8,
130.7, 130.4, 128.2, 127.7, 119.4, 117.5, 41.6, 41.0, 22.6. HRMS
(ESI)+ calculated for C15H15ClN3O2S, [M+H]+: m/z 336.0599,
found 336.0574.
Preparation of intermediates 20b. To a solution of
intermediate 20a (10 mmol) and DIEA (20 mmol) in
dichloromethane (60 mL), a solution of corresponding
acyl chloride (1.1 eq) in dichloromethane (10 mL) was
added dropwise at room temperature. The mixture was
stirred for 8 h and washed with water, saturated aqueous
sodium bicarbonate and brine, and then dried over
anhydrous sodium sulfate. After removing the solvent
under reduced pressure, the crude product was purified
by flash chromatography on silica gel, eluting with
dichloromethane/methanol (5-10%), yield 60-70%.
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6-Benzyl-2-(2-chlorobenzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21b).
1
White solid, mp 168-170, yield 70.5%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.38 (1H, brs), 7.73 (1H, dd, J = 7.5, 1.4
Hz), 7.62 (1H, dd, J = 8.0, 1.3 Hz), 7.58 (1H, td, J = 8.0, 1.3
Hz), 7.50 (1H, td, J = 7.5, 1.4 Hz), 7.37 (2H, m), 7.35 (2H, m),
7.28 (1H, m), 3.69 (2H, s), 3.57 (2H, s), 2.83 (2H, t, J = 5.1
Hz), 2.71 (2H, t, J = 5.1 Hz). 13C-NMR (100 MHz, DMSO-
d6), δ(ppm): 167.6, 162.7, 142.8, 138.7, 134.2, 133.0, 130.9,
130.6, 130.3, 129.3, 129.3, 128.7, 128.7, 128.5, 128.2, 127.6, 125.2,
117.0, 61.2, 51.4, 49.7, 25.9. HRMS (ESI)+ calculated for
C22H21ClN3O2S, [M+H]+: m/z 426.1043, found 426.1045.
Preparation of intermediate 20c.
A
solution of
intermediate 20b (15 mmol) in 1:1 TFA: dichloromethane
(100 mL) was stirred at room temperature for 2 h. Upon
completion, the residue was concentrated and added 50
ml water, the mixture was basified with aqueous NaOH (1
mol/L) till pH = 10 and extracted with ethyl acetate (3 x 50
mL). The combined organic layers were washed with
saturated aqueous sodium bicarbonate and brine, and
then dried over anhydrous sodium sulfate. Then, the
solvent was removed under reduced pressure to give the
crude product. The crude product was purified by silica
sel flash chromatography (dichloromethane/methanol
10:1), as a white solid, yield 90-95%.
2-(2-Chlorobenzamido)-6-(2-methylbenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21c).
1
White solid, mp 215-217, yield 65.7%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.39 (1H, brs), 7.73 (1H, d, J = 7.5, 1.3
Hz), 7.62 (1H, td, J = 7.9, 1.4 Hz), 7.60 (1H, td, J = 8.0, 1.6
Hz), 7.57 (1H, td, J = 7.5, 1.7 Hz), 7.29 (1H, d, J = 7.6 Hz),
7.17 (1H ,m), 7.16(1H , m), 7.15 (1H, m), 3.64 (2H, s), 3.58
(2H, s), 2.82 (2H, brt, J = 4.8 Hz), 2.72 (2H, brt, J = 4.8 Hz),
2.34 (3H, s). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 167.1,
167.1, 162.2, 142.2, 137.1, 136.3, 133.7, 132.5, 130.4, 130.1, 129.8,
129.5, 128.0, 127.7, 127.1, 125.5, 124.8, 116.6, 58.8, 51.1, 49.3,
25.4, 18.8. HRMS (ESI)+ calculated for C23H23ClN3O2S,
[M+H]+: m/z 440.1183, found 440.1200.
General Procedure for the Synthesis of 21a-21v. To a
solution of intermediate 20c (10 mmol) and K2CO3 (1.5 eq)
in DMF (60 mL), benzyl chloride (15 mmol) was added
dropwise at 0℃. The resulting mixture was allowed to 80
℃for additional 8 h. After completion, the residue was
diluted with 50 ml water, extracted with ethyl acetate (3 ×
50 mL). The combined organic layers were washed with
water, saturated aqueous sodium bicarbonate and brine,
and then dried over anhydrous sodium sulfate. Then, the
solvent was removed under reduced pressure to give the
crude oil. The crude oil was purified by silica gel flash
chromatography (dichloromethane/methanol 50:1) as a
white solid.
2-(2-Chlorobenzamido)-6-(2-cyanobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21d).
1
White solid, mp 163-166, yield 69.2%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.40 (1H, brs), 7.85 (1H, m), 7.73 (2H,
m), 7.62 (2H, m), 7.60 (1H, m), 7.52 (2H, m), 3.86 (2H, s),
3.65 (2H, s), 2.85 (2H, m), 2.78 (2H, m). 13C-NMR (100
MHz, DMSO-d6), δ(ppm): 167.5, 162.7, 142.8, 142.6, 134.2,
133.6, 133.5, 133.0, 130.9, 130.6, 130.3, 129.7, 128.6, 128.4,
128.2, 124.9, 118.1, 117.1, 112.6, 59.0, 51.4, 49.8, 25.8. HRMS
(ESI)+ calculated for C23H20ClN4O2S, [M+H]+: m/z 451.1041,
found 451.0995.
General Procedure for the Synthesis of 22. To a solution
of intermediate 20c (10 mmol) in dichloromethane (70
mL), DIEA (20 mmol), isothiocyanate (11 mmol)
respectively were added dropwise at 0℃, and removed to
room temperature for additional 8 h. The mixture was
washed with water, saturated aqueous sodium
bicarbonate and brine, and then dried over anhydrous
sodium sulfate. After removing the solvent under reduced
pressure, the crude product was purified by flash
2-(2-Chlorobenzamido)-6-(2-chlorobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21e).
1
chromatography
on
silica
gel,
eluting
with
White solid, mp 217-219, yield 66.3%. H-NMR (400 MHz,
dichloromethane/methanol (2-5%).
DMSO-d6), δ(ppm): 12.38 (1H, brs), 7.73 (1H, d, J = 7.5, 1.5
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Hz), 7.62 (1H, td, J = 8.0, 1.5 Hz), 7.60 (1H, dd, J = 7.0, 1.7 brs), 9.39 (1H, brs), 7.74 (1H, dd, J = 7.4, 1.4 Hz), 7.62 (1H,
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Hz), 7.56 (1H, m), 7.51 (1H , td, J =7.6, 1.7 Hz), 7.47(1H , dd,
J =7.5, 1.4 Hz), 7.36 (1H, td, J =7.4, 1.4 Hz), 7.31 (1H, td, J
=7.5, 1.6 Hz), 3.79 (2H, s), 3.66 (2H, s), 2.86 (2H, m), 2.77
(2H, m). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 167.1,
162.2, 142.3, 133.7, 133.4, 132.5, 130.8, 130.4, 130.4, 130.1, 129.8,
129.8, 129.3, 128.8, 128.0, 127.7, 127.1, 116.6, 57.4, 50.9, 49.4,
25.4. HRMS (ESI)+ calculated for C22H20Cl2N3O2S, [M+H]+:
m/z 460.0683, found 460.0653.
m), 7.59 (1H, dd, J = 7.0, 1.4 Hz), 7.52 (3H, m), 7.38 (1H, m),
3.91 (2H, s), 3.69 (2H, s), 2.83 (2H, m), 2.83 (2H, m). 13C-
NMR (100 MHz, DMSO-d6), δ(ppm): 166.8, 163.3, 143.6,
134.2,134.0 133.0, 132.9, 130.9, 130.7, 130.6, 130.3, 129.2, 128.2,
128.1, 127.8, 119.5, 117.5, 117.1, 60.0, 50.0, 41.3, 22.6. HRMS
(ESI)+ calculated for C22H19Cl3N3O2S, [M+H]+: m/z
494.0288, found 494.0264.
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2-(2-Chlorobenzamido)-6-(2-cyanobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21j).
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2-(2-Chlorobenzamido)-6-(2-fluorobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21f).
1
Off-white solid, mp 222-224, yield 62.8%. H-NMR (400
1
White solid, mp 163-166, yield 69.5%. H-NMR (400 MHz,
MHz, DMSO-d6), δ(ppm): 13.09 (1H, brs), 7.88 (1H, m),
7.84 (1H,d, J = 7.8 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.65 (1H, t, J
= 7.8 Hz), 7.60 (1H, d, J = 2.1 Hz), 7.57 (1H, d, J = 8.4 Hz),
7.43 (1H, dd, J = 8.4, 2.1 Hz), 3.76 (2H, s), 3.70 (2H, s), 3.44
(2H, m), 2.76 (2H, m). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 168.1, 168.1, 161.5, 144.0, 135.5, 134.9, 134.7, 134.3,
132.8, 132.5, 131.6, 129.2, 128.4, 127.7, 127.4, 126.1, 124.9, 116.3,
57.4, 51.4, 49.9, 25.9. HRMS (ESI)+ calculated for
C22H19Cl3N3O2S, [M+H]+: m/z 494.0285, found 494.0264.
DMSO-d6), δ(ppm): 12.37 (1H, brs), 7.72 (1H, d, J = 6.6 Hz),
7.61 (1H, m), 7.59 (1H, m), 7.51 (1H, d, J = 7.9 Hz), 7.59 (1H ,
dd, J = 8.6, 7.9 Hz), 7.35 (1H, dd , J = 13.4, 6.1 Hz), 7.23 (1H,
d, J = 7.0, 2.4 Hz), 7.20 (1H, d, J = 13.4, 6.8 Hz), 3.75 (2H, s),
3.60 (2H, s), 2.84 (2H, brt, J =5.1 Hz), 2.74 (2H, brt, J = 5.1
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 167.5, 165.7,
162.8, 161.5 (244.0 Hz), 142.8, 134.2, 133.0, 130.9, 130.8 (3.0
Hz), 130.6, 130.5 (26.5 Hz), 130.3, 128.4, 128.2, 128.2, 124.8
(3.4 Hz), 117.1, 115.7 (21.1 Hz), 53.8, 51.0, 49.7, 25.7. HRMS
(ESI)+ calculated for C22H20ClFN3O2S, [M+H]+: m/z
444.0926, found 444.0949.
2-(2-Chlorobenzamido)-6-(3,5-dichlorobenzyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21k). White solid, mp 220-223, yield
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2-(2-Chlorobenzamido)-6-(4-chlorobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21g).
67.8%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.37 (1H,
brs), 7.73 (1H, dd, J = 7.5, 1.5 Hz), 7.62 (1H, dd, J = 8.1, 1.3
Hz), 7.58 (1H, td, J = 8.1, 1.5 Hz), 7.52 (1H, d, J = 1.8 Hz),
7.51 (1H, m), 7.43 (2H, d, J = 1.8 Hz), 3.71 (2H, s), 3.61 (2H,
1
White solid, mp 180-182, yield 64.2%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 12.40 (1H, brs), 7.72 (1H, d, J = 7.0 Hz), 7.62 (1H,
m), 7.60 (2H, m), 7.51 (1H, dd, J =7.0, 6.8 Hz), 7.40 (2H,
m), 7.39 (1H, m), 3.68 (2H, s), 3.57 (2H, s), 2.84 (2H, m),
2.71 (2H, m). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
167.6, 162.7, 142.8, 134.2, 133.0, 132.1, 131.1, 131.1, 130.9, 130.8,
130.6, 130.3, 129.4, 128.7, 128.7, 128.4, 128.2, 117.0, 60.2, 51.2,
49.7, 25.8. HRMS (ESI)+ calculated for C22H20Cl2N3O2S,
[M+H]+: m/z 460.0662, found 460.0653.
13
s), 2.84 (2H, t, J = 5.1 Hz), 2.71 (2H, t, J = 5.1 Hz). C-NMR
(100 MHz, DMSO-d6), δ(ppm): 167.6, 162.7, 143.6, 142.8,
134.4, 134.4, 134.2, 133.0, 130.9, 130.9, 130.6, 130.3, 128.4,
128.2, 127.7, 127.2, 124.9, 117.1, 59.6, 51.3, 49.6, 25.8. HRMS
(ESI)+ calculated for C22H19Cl3N3O2S, [M+H]+: m/z
495.8214, found 495.8230.
6-(2-Fluorobenzyl)-2-(4-(trifluoromethyl)
benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxamide (21l). Off-white solid, mp 297-299, yield
2-(2-Chlorobenzamido)-6-(4-nitrobenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21h).
White solid, mp 238-240, yield 64.7%. 1H-NMR (400 MHz,
DMSO-d6), δ(ppm): 13.08 (1H, brs), 8.22 (2H, d, J = 8.6
Hz), 7.88 (1H, m), 7.83 (1H, d, J = 7.7 Hz), 7.74 (1H, d, J =
7.5 Hz), 7.67 (2H, d, J = 8.6 Hz), 7.64 (1H, dd, J = 7.9, 7.7
Hz), 3.84 (2H, s), 3.61 (2H, s), 2.88 (2H, brt, J = 5.4 Hz),
1
69.7%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.17 (1H,
brs) , 8.09 (2H, d, J = 8.1 Hz), 8.00 (2H, brd, J = 8.1 Hz),
7.49 (1H, ddd, J = 7.6, 7.3, 1.8 Hz), 7.36 (1H, ddm, J = 13.4,
6.0 Hz), 7.21 (1H, d, J = 7.3 Hz), 7.19 (1H, m), 3.74 (2H, s),
3.60 (2H, s), 2.88 (2H, brt, J = 5.1 Hz), 2.74 (2H, brt, J = 5.1
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 172.1, 168.1,
161.2 (245.2 Hz), 131.9 (3.4 Hz), 129.7 (5.7 Hz), 128.6, 128.5,
128.5, 128.5, 127.8, 126.6 (3.3 Hz), 126.6 (3.3 Hz), 125.5 (30.5
Hz), 125.4, 125.2, 125.2, 124.4 (272.5 Hz), 116.7, 115.8 (21.4
Hz), 54.0, 51.4, 49.7, 25.8. HRMS (ESI)+ calculated for
C23H20F4N3O2S, [M+H]+: m/z 478.1233, found 478.1212.
13
2.75 (2H, brt, J = 5.4 Hz). C-NMR (100 MHz, DMSO-d6),
δ(ppm): 168.1, 161.7, 147.3, 147.2, 137.1, 134.3, 132.8, 132.6,
131.6, 130.2, 130.2, 128.4, 127.4, 126.1, 123.9, 123.9, 116.4, 109.4,
60.2, 51.4, 49.9, 25.9. HRMS (ESI)+ calculated for
C22H21ClN5O4S, [M+H]+: m/z 471.0918, found 471.0894.
2-(2-Chlorobenzamido)-6-(2,6-dichlorobenzyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
6-(4-Chlorobenzyl)-2-(4-(trifluoromethyl)
benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxamide (21m). Off-white solid, mp 240-243, yield
carboxamide (21i). Off-white solid, mp 182-185, yield
1
1
66.1%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.37 (1H,
69.1%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.18 (1H,
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Journal of Medicinal Chemistry
Page 22 of 44
brs), 8.08 (2H, d, J = 8.3 Hz), 7.99 (2H, d, J = 8.3 Hz), 7.41
Hz), 3.76 (2H, s), 3.63 (2H, s), 2.88 (2H, brt, J = 4.3 Hz),
2.77 (2H, brt, J = 4.3 Hz). C-NMR (100 MHz, DMSO-d6),
δ(ppm): 168.2, 167.7, 163.2, 162.8, 144.7, 143.5, 143.4, 135.5,
134.7, 132.8, 132.5, 130.1, 130.1, 129.2, 127.6, 127.6, 127.5, 124.5,
115.6, 57.4, 51.4, 49.9, 21.6. HRMS (ESI)+ calculated for
C23H22Cl2N3O2S, [M+H]+: m/z 474.0851, found 474.0810.
1
2
3
4
5
6
7
8
13
(2H, m), 7.39 (2H, m), 3.68 (2H, s), 3.58 (2H, s), 2.87 (2H,
13
m), 2.72 (2H, m). C-NMR (100 MHz, DMSO-d6), δ(ppm):
181.4, 167.8, 161.9, 144.1, 142.9, 142.9, 136.4, 132.6, 130.0,
129.4, 128.6, 128.6, 126.7 (3.0 Hz),126.7 (3.0 Hz), 125.4, 125.4,
124.4 (276.0 Hz), 124.2, 124.1 (33.9 Hz), 54.2, 47.7, 46.0,
25.8. HRMS (ESI)+ calculated for C23H20ClF3N3O2S,
[M+H]+: m/z 494.0954, found 494.0917.
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
fluorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (21r). Off-white solid, mp
265-267, yield 64.8%.1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 12.73 (1H, brs), 9.46 (1H, brs), 8.03 (1H, ddd, J =
7.7, 7.5, 1.9 Hz), 7.71 (1H, m), 7.50 (1H, d, J = 7.6 Hz), 7.46
(1H, m), 7.42 (1H, m), 7.34 (1H, m), 7.33 (1H, m), 7.29 (1H,
m), 5.14(2H, s), 4.27 (2H, s), 4.21 (2H, brt, J = 5.1 Hz), 3.02
(2H, brt, J = 5.1 Hz). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 182.0, 167.4, 160.3 (248.9 Hz), 159.6 (2.8 Hz), 143.5,
138.6, 135.4 (9.3 Hz), 132.5, 131.9, 131.8, 129.8, 129.3, 128.4,
127.7, 125.8 (3.9 Hz), 124.0, 117.2 (23.2 Hz), 116.5, 47.5, 45.7,
41.5, 25.9. HRMS (ESI)+ calculated for C22H20ClFN3O2S,
[M+H]+: m/z 444.0964, found 444.0949.
9
2-(2-Chlorobenzamido)-6-(2-methylbenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (21n).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Off-white solid, mp 278-279, yield 62.8%. H-NMR (400
MHz, DMSO-d6), δ(ppm): 13.16 (1H, brs), 8.08 (2H, d, J =
8.1 Hz), 7.98 (2H, d, J = 8.1 Hz), 7.49 (2H, d, J = 7.8 Hz),
7.37 (1H, t, J = 7.8 Hz), 3.90 (2H, s), 3.69 (2H, s), 2.84 (2H,
m), 2.84 (2H, m). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
167.6, 167.6, 161.1, 136.1, 136.1, 133.8, 132.0 (24.6 Hz), 130.0,
128.7, 128.7, 128.0, 128.0, 127.9 (4.8 Hz), 127.9 (4.8 Hz),
126.2, 126.2, 124.6, 121.3 (230.0 Hz), 116.0, 54.6, 50.6, 49.4,
25.3. HRMS (ESI)+ calculated for C23H19Cl2F3N3O2S,
[M+H]+: m/z 528.0551, found 528.0527.
6-(2,4-Dichlorobenzyl)-2-(4-(trifluoromethyl)
benzamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3-carboxamide (21o). White solid, mp 244-246, yield
6-(2,6-Dichlorobenzyl)-2-(2-fluorobenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21s). White solid, mp 234-237, yield 61.0%.
1H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.85 (1H, brs),
12.83 (1H, brs), 8.02 (1H, td, J = 7.8, 1.6 Hz), 7.70 (1H, m),
7.44 (1H, m), 7.41 (1H, m), 7.37 (1H, dd, J = 8.5, 7.6 Hz),
3.89 (2H, s), 3.68 (2H, s), 3.33 (2H, m), 2.83 (2H, m). 13C-
NMR (100 MHz, DMSO-d6), δ(ppm): 167.6, 160.4 (250.2
Hz), 159.5 (2.8 Hz), 143.3, 136.6, 136.6, 135.2 (9.1 Hz), 134.3,
131.9, 130.5, 129.1, 129.1, 128.3, 125.8 (3.2 Hz), 125.1, 120.2,
117.2 (23.1 Hz), 116.7, 55.2, 51.1, 50.0, 25.8. HRMS (ESI)+
calculated for C22H19Cl2FN3O2S, [M+H]+: m/z 478.0551,
found 478.0559.
1
67.7%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.19 (1H,
brs), 8.09 (2H, d, J = 8.2 Hz), 7.98 (2H, d, J = 8.2 Hz), 7.60
(1H, brs), 7.57 (1H, d, J = 8.2 Hz), 7.43 (1H, dd, J = 8.2, 1.5
Hz), 3.75 (2H, s), 3.65 (2H, s), 2.89 (2H, m), 2.77 (2H, m).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 168.0, 168.0, 161.7,
143.9, 136.5, 135.5, 134.7, 132.6 (32.1 Hz), 129.2, 128.5, 128.5,
127.7, 126.6 (4.2 Hz), 126.6 (4.2 Hz), 126.6, 125.1, 121.6 (272.1
Hz), 116.5, 115.5, 57.3, 51.3, 49.8, 25.9. HRMS (ESI)+
calculated for C23H19Cl2F3N3O2S, [M+H]+: m/z 528.0546,
found 528.0527.
6-(2,6-Dichlorobenzyl)-2-(4-methylbenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
6-(2,4-Dichlorobenzyl)-2-(2-fluorobenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21p). White solid, mp 246-248, yield
carboxamide (21t). White solid, mp 250-252, yield 68.8%.
1H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.16 (1H, brs),
8.79 (1H, brs), 8.48 (1H, dd, J = 8.2, 1.3 Hz), 8.39 (1H, d, J
=7.9 Hz), 7.87 (1H, dd, J = 8.2, 7.9 Hz), 7.51 (1H, brs), 7.49
(1H, brs), 7.37 (1H, dd, J = 8.2, 7.9 Hz), 3.91 (2H, s), 3.67
(2H, s), 2.93 (2H, brt, J = 5.3 Hz), 2.63 (2H, brt, J = 5.3 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.0, 167.3, 163.9,
151.4, 148.1, 148.1, 142.3, 136.6, 135.3, 134.3, 130.7, 130.6, 129.2,
129.2, 127.3, 127.2, 123.6, 122.7, 114.5, 54.6, 49.4, 23.7. HRMS
(ESI)+ calculated for C22H19Cl2FN3O2S, [M+H]+: m/z
478.0587, found 478.0559.
1
62.9%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.02 (1H,
brs), 7.79 (2H, d, J = 7.7 Hz), 7.49 (2H, d, J = 8.0 Hz), 7.41
(2H, d, J = 7.7 Hz), 7.36 (1H, t, J = 8.0 Hz), 3.88 (2H, s), 3.67
(2H, s), 2.83 (2H, m), 2.83 (2H, m). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 168.2, 167.7, 144.7, 143.5, 143.4, 134.7,
132.8, 132.5, 130.2, 130.2, 130.2, 130.2, 129.2, 127.8, 127.6,
127.6, 127.5, 127.5, 115.6, 57.3, 51.4, 49.9, 21.6. HRMS (ESI)+
calculated for C23H22Cl2N3O2S, [M+H]+: m/z 474.0848,
found 474.0810.
6-(2,4-Dichlorobenzyl)-2-(4-methylbenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
6-(2,6-Dichlorobenzyl)-2-(3-nitrobenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (21q). Off-white solid, mp 243-246, yield
1
64.1%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.03 (1H,
carboxamide (21u). Off-white solid, mp 243-246, yield
1
brs), 9.48 (1H, brs), 7.79 (2H, d, J = 7.7 Hz), 7.61 (1H, brs),
7.57 (1H, brd, J = 8.4 Hz), 7.44 (1H, m), 7.42 (2H, d, J = 7.7
66.9%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.16 (1H,
brs), 8.79 (1H, brs), 8.48 (1H, dd, J = 8.2, 1.3 Hz), 8.39 (1H,
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Journal of Medicinal Chemistry
d, J =7.9 Hz), 7.87 (1H, dd, J = 8.2, 7.9 Hz), 7.51 (1H, brs), 12.26 (1H, brs), 9.41 (1H, brs), 7.75 (1H, dd, J = 7.5, 1.5 Hz),
1
2
3
4
5
6
7
8
7.49 (1H, brs), 7.37 (1H, dd, J = 8.2, 7.9 Hz), 3.91 (2H, s),
3.67 (2H, s), 2.93 (2H, brt, J = 5.3 Hz), 2.63 (2H, brt, J = 5.3
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.0, 167.3,
163.9, 151.4, 148.1, 148.1, 142.3, 136.6, 135.3, 134.3, 130.7, 130.6,
129.2, 129.2, 127.3, 127.2, 123.6, 122.7, 114.5, 54.6, 49.4, 23.7.
HRMS (ESI)+ calculated for C22H19Cl2N4O4S, [M+H]+: m/z
505.0518, found 505.0504.
7.63 (1H, dd, J = 8.0, 1.4 Hz), 7.59 (1H, td, J = 8.0, 1.5 Hz),
7.52 (1H, td, J = 7.5, 1.4 Hz), 7.50 (1H, m), 7.35 (1H, m), 7.33
(1H, m), 7.29 (1H, m), 5.13 (2H, s), 4.20 (2H, brt, J = 5.4
Hz), 2.98 (2H, brt, J = 5.4 Hz). 13C-NMR (100 MHz, DMSO-
d6), δ(ppm): 182.0, 167.3, 162.9, 142.7, 138.6, 134.1, 133.0,
132.5, 131.8, 130.9, 130.6, 130.4, 129.8, 129.4, 128.4, 128.2,
127.7, 124.0, 117.7, 47.6, 45.7, 25.8. HRMS (ESI)+ calculated
for C22H19Cl2N4O2S2, [M+H]+: m/z 505.0332, found
505.0326.
9
6-(2,4-Dichlorobenzyl)-2-(3-nitrobenzamido)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
carboxamide (21v). White solid, mp 198-200, yield 62.3%.
1H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.16 (1H, brs),
8.79 (1H, t, J = 1.8 Hz), 8.47 (1H, d, J = 8.2 Hz), 8.40 (1H, d,
J = 8.0 Hz), 7.86 (1H, dd, J = 8.2, 8.0 Hz), 7.61 (1H, brs),
7.58 (1H, d, J = 8.2 Hz), 7.44 (1H, d, J = 8.2 Hz), 3.79 (2H,
2-(2-Chlorobenzamido)-N6-(2-chlorophenyl)-4,7-
dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxamide
(22ad). White solid, mp 208-210, yield 66.5%. H-NMR
1
(400 MHz, DMSO-d6), δ(ppm): 12.28 (1H, brs), 8.38 (1H,
brs), 7.74 (1H, dd, J = 7.4, 1.4 Hz), 7.62 (1H, dd, J = 8.0, 1.5
Hz), 7.59 (1H, td, J = 8.0, 1.6 Hz), 7.52 (2H, m), 7.47 (1H, dd,
J = 8.0, 1.4 Hz), 7.45 (1H, td, J =7.6, 1.5 Hz), .7.16 (1H, t, J
=7.9, 1.7 Hz), 4.67 (2H, s), 3.75 (2H, t, J = 5.6 Hz), 2.90 (2H,
t, J = 5.6 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
168.8, 162.4, 154.8, 142.2, 136.4, 133.7, 132.5, 130.4, 130.1,
129.9, 129.2, 128.7, 128.5, 127.7, 127.4, 127.2, 125.8, 124.0,
117.2, 42.7, 41.3, 25.4. HRMS (ESI)+ calculated for
C22H19Cl2N4O3S, [M+H]+: m/z 489.0587, found 489.0555.
13
s), 3.65 (2H, s), 2.85 (2H, m), 2.67 (2H, m). C-NMR (100
MHz, DMSO-d6), δ(ppm): 168.0, 163.9, 159.5, 148.1, 135.3,
134.7, 133.0, 132.8, 132.6, 130.7, 129.2, 127.8, 127.3, 127.0,
126.9, 125.6, 123.6, 120.9, 114.5, 50.7, 49.3, 24.1. HRMS (ESI)+
calculated for C22H19Cl2N4O4S, [M+H]+: m/z 505.0517,
found 505.0504.
2-(2-Chlorobenzamido)-6-(phenylcarbamothioyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22aa). White solid, mp 167-170, yield
2-(2-Chlorobenzamido)-6-((2-
fluorophenyl)carbamothioyl)-4,5,6,7-
1
68.4%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.94 (1H,
brs), 9.50 (1H, brs), 7.89 (1H, m), 7.84 (1H, d, J = 7.8 Hz),
7.74 (1H, d, J = 8.0 Hz), 7.64 (1H, dd, J = 8.0, 7.8 Hz), 7.32
(3H, m), 7.13 (1H, m), 5.09 (2H,s), 4.21 (2H, brt, J = 5.1 Hz),
3.01 (2H, brt, J = 5.1 Hz). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 181.7, 167.8, 161.7, 144.1, 141.3, 134.7, 134.3, 132.9,
131.6, 129.4, 128.5, 128.5, 127.5, 126.2, 126.1, 126.1, 125.1, 123.9,
116.8, 47.6, 45.8, 25.8. HRMS (ESI)+ calculated for
C22H20ClN4O2S2, [M+H]+: m/z 471.0722, found 471.0716.
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
(22ae). White solid, mp 213-215, yield 78.3%. 1H-NMR (400
MHz, DMSO-d6), δ(ppm): 12.26 (1H, brs), 9.32 (1H, brs),
7.75 (1H, dd, J = 7.4, 1.1 Hz), 7.61 (1H, m), 7.60 (1H, m), 7.52
(1H, td, J = 7.5, 1.5 Hz), 7.28 (1H, m), 7.27 (1H, m), 7.19 (1H,
m), 7.18 (1H, m), 5.11 (2H, s), , 4.20 (2H, t, J = 5.2 Hz), 2.98
(2H, t, J = 5.2 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
182.3, 167.3, 162.9, 157.9 (245.0 Hz), 142.8, 134.1, 133.0, 130.9,
130.8, 130.6, 130.4, 129.4, 129.1 (12.1 Hz), 128.3 (7.8 Hz), 128.2,
124.5 (3.1 Hz), 123.9, 117.6, 116.2 (20.1 Hz), 47.6, 45.9, 25.8.
HRMS (ESI)+ calculated for C22H19ClFN4O2S2, [M+H]+: m/z
489.0622, found 489.0613.
2-(2-Chlorobenzamido)-6-(naphthalen-1-
ylcarbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22ab). White solid, mp 151-
1
154, yield 64.3%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.91 (1H, brs), 9.71 (1H, brs), 7.95 (1H, m) 7.91 (1H, m), 7.87
(1H, m), 7.86 (2H, dd, J = 7.6, 6.4 Hz), 7.75 (1H, d, J = 8.0
Hz), 7.66 (1H, dd, J = 8.0, 7.6 Hz), 7.54 (1H, d, J = 8.0 Hz),
7.51 (1H, m), 7.50 (1H, dd, J = 6.8, 3.0 Hz), 7.36 (1H, d, J =
7.2 Hz), 5.22 (2H, s), 4.30 (2H, brt, J = 5.4 Hz), 3.09 (2H,
brt, J = 5.4 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
182.7, 167.9, 161.8, 144.1, 137.6, 134.8, 134.4, 134.3, 132.9, 131.6,
131.2, 129.5, 128.4, 127.5, 127.5, 127.1, 126.7, 126.4, 126.4, 126.2,
126.0, 124.2, 117.0, 55.4, 47.7, 45.7. HRMS (ESI)+ calculated
for C26H22ClN4O2S2, [M+H]+: m/z 521.0885, found 521.0873.
6-((2-Bromophenyl)carbamothioyl)-2-(2-
chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22af). White solid, mp 215-
1
216, yield 77.0%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.26 (1H, brs), 9.43 (1H, brs), 7.75 (1H, dd, J = 7.6, 1.5 Hz),
7.66 (1H, dd, J = 8.0, 1.2 Hz), 7.63 (1H, dd, J = 8.0, 1.2 Hz),
7.59 (1H, td, J = 8.0, 1.2 Hz), 7.52 (1H, td, J = 7.6, 1.5 Hz),
7.39 (1H, td, J = 8.0, 1.2 Hz), 7.33 (1H, dd, J = 7.9, 1.7 Hz),
7.21 (1H, td, J = 7.9, 1.7 Hz), 5.13 (2H, s), 4.20 (2H, t, J = 5.3
Hz), 2.98 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz, DMSO-
d6), δ(ppm): 181.9, 167.2, 162.9, 142.7, 140.0, 134.1, 133.0,
132.9, 132.0, 130.9, 130.6, 130.4, 129.4, 128.7, 128.3, 128.2,
124.0, 123.7, 117.7, 47.6, 45.7, 25.9. HRMS (ESI)+ calculated
for C21H19BrClN4O2S2, [M+H]+: m/z 548.9821, found
2-(2-Chlorobenzamido)-6-((2-chlorophenyl)
carbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22ac). White solid, mp 158-
1
160, yield 62.5%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
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Journal of Medicinal Chemistry
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548.9821.
td, J = 8.0, 1.5 Hz), 7.52 (1H, td, J = 7.3, 1.5 Hz), 7.23 (1H, d, J
1
2
3
4
5
6
7
8
= 7.3 Hz), 7.20 (1H, dd, J = 7.4, 1.6 Hz), 7.04 (1H, d, J = 7.4
Hz), 6.92 (1H, td, J = 7.6, 1.4 Hz), 5.09 (2H, s), 4.18 (2H, t, J
= 5.5 Hz), 3.77 (3H, s), 2.96 (2H, t, J = 5.5 Hz). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 182.3, 167.3, 162.9, 154.8,
142.8, 134.1, 133.0, 130.9, 130.6, 130.4, 130.1, 129.8, 129.4, 128.2,
127.6, 124.1, 120.3, 117.6, 112.2, 56.0, 47.4, 45.7, 25.8. HRMS
(ESI)+ calculated for C23H22ClN4O3S2, [M+H]+: m/z
501.0822, found 501.0820.
2-(2-Chlorobenzamido)-6-((2-
iodophenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22ag). White solid, mp 169-172, yield 75.6%. H-NMR
(400 MHz, DMSO-d6), δ(ppm): 12.27 (1H, brs), 9.47 (1H,
brs), 7.87 (1H, dd, J = 7.9, 1.2 Hz), 7.75 (1H, dd, J = 7.4, 1.5
Hz), 7.63 (1H, dd, J = 8.0, 1.4 Hz), 7.59 (1H, td, J = 8.0, 1.4
Hz), 7.52 (1H, td, J = 7.4, 1.5 Hz), 7.40 (1H, td, J = 7.9, 1.2
Hz), 7.28 (1H, dd, J = 8.0, 1.4 Hz), 7.03 (1H, td, J = 7.8, 1.5
Hz), 5.14 (2H, s), 4.21 (2H, t, J = 5.4 Hz), 2.99 (2H, t, J = 5.4
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.7, 167.2,
162.9, 143.2, 142.8, 139.0, 134.1, 133.0, 131.2, 130.9, 130.9, 130.6,
130.4, 129.4, 129.0, 128.8, 128.2, 117.7, 102.1, 47.6, 45.7, 26.0.
HRMS (ESI)+ calculated for C22H19ClIN4O2S2, [M+H]+: m/z
596.9683, found 596.9677.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2-(2-Chlorobenzamido)-6-((2-
(methylthio)phenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22ak). White solid, mp 202-205, yield 74.2%. H-NMR
(400 MHz, DMSO-d6), δ(ppm): 12.27 (1H, brs), 9.26 (1H,
brs), 7.75 (1H, d, J = 7.2 Hz), 7.61 (1H, m), 7.60 (1H, m), 7.51
(1H, t, J = 7.2 Hz), 7.29 (1H, m), 7.28 (1H, m), 7.16 (1H, m),
7.15 (1H, m), 5.10 (2H, s), 4.18 (2H, t, J = 5.0 Hz), 2.97 (2H, t,
J = 5.0 Hz), 2.38 (3H, s). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 182.0, 167.3, 163.0, 142.7, 138.3, 138.2, 134.1, 133.0,
130.9, 130.6, 130.4, 130.3, 129.4, 128.2, 127.7, 125.9, 125.0, 124.1,
117.7, 47.5, 45.6, 25.9, 15.0. HRMS (ESI)+ calculated for
C23H22ClN4O2S3, [M+H]+: m/z 517.0593, found 517.0588.
2-(2-Chlorobenzamido)-6-((2-
(trifluoromethyl)phenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
(22ah). White solid, mp 213-215, yield 74.3%. 1H-NMR (400
MHz, DMSO-d6), δ(ppm): 12.27 (1H, brs), 9.40 (1H, brs),
7.75 (1H, dd, J = 7.9, 1.2 Hz), 7.73 (1H, d, J = 8.0 Hz), 7.68
(1H, t, J = 7.7 Hz), 7.63 (1H, d, J = 7.9 Hz), 7.59 (1H, t, J =
8.0 Hz), 7.53 (1H, dd, J = 7.9, 1.2 Hz), 7.50 (1H, t, J = 7.7 Hz),
7.40 (1H, d, J = 7.9 Hz), 5.10 (2H, s), 4.19 (2H, t, J = 5.3 Hz),
2.97 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 182.6, 167.2, 163.0, 142.7, 139.3 (2.1 Hz), 134.1, 133.8,
133.0, 130.9, 130.6, 130.4, 129.4, 128.2, 128.0 (31.2 Hz), 127.9,
127.4 (28.3 Hz), 126.6 (4.7 Hz), 124.1 (274.7 Hz), 123.9, 117.7,
47.5, 45.7, 25.8. HRMS (ESI)+ calculated for
C23H19ClF3N4O2S2, [M+H]+: m/z 539.0590, found 539.0574.
6-([1,1'-Biphenyl]-2-ylcarbamothioyl)-2-(2-
chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22al). White solid, mp 131-
1
134, yield 75.6%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.31 (1H, brs), 9.24 (1H, brs), 7.75 (1H, dd, J = 7.4, 1.3 Hz),
7.63 (1H, dd, J = 8.0, 1.3 Hz), 7.59 (1H, td, J = 8.0, 1.5 Hz),
7.52 (1H, td, J = 7.4, 1.3 Hz), 7.44 (1H, m), 7.37 (1H, m), 7.36
(1H, m), 7.35 (1H, m), 7.30 (1H, m), 7.29 (1H, m), 7.28 (1H,
m), 7.26 (1H, m), 7.25 (1H, m), 4.94 (2H, s), 4.04 (2H, t, J =
5.3 Hz), 2.81 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 182.6, 167.3, 162.9, 142.8, 139.6, 139.6,
138.4, 134.2, 133.0, 131.5, 130.9, 130.9, 130.6, 130.4, 130.3, 129.3,
129.1, 129.1, 128.4, 128.4, 128.2, 127.9, 127.4, 124.1, 117.5, 47.4,
45.7, 25.7. HRMS (ESI)+ calculated for C28H24ClN4O2S2,
[M+H]+: m/z 547.1025, found 547.1033.
2-(2-Chlorobenzamido)-6-(o-tolylcarbamothioyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22ai). White solid, mp 213-215, yield 72.9%.
1H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.26 (1H, brs),
9.26 (1H, brs), 7.75 (1H, dd, J = 7.4, 1.5 Hz), 7.62 (1H, dd, J =
8.0, 1.3 Hz), 7.59 (1H, td, J = 8.0, 1.3 Hz), 7.52 (1H, td, J = 7.4,
1.5), 7.23 (1H, dd, J = 6.4, 2.4 Hz), 7.18 (1H, m), 7.17 (1H, m),
7.09 (1H, dd, J = 6.4, 2.4 Hz), 5.11 (2H, s), 4.18 (2H, t, J = 5.4
Hz), 2.97 (2H, t, J = 5.4 Hz), 2.18 (3H, s). 13C-NMR (100
MHz, DMSO-d6), δ(ppm): 181.8, 167.3, 162.9, 142.7, 139.9,
136.1, 134.1, 133.0, 130.9, 130.6, 130.6, 130.4, 129.4, 129.3, 128.2,
126.9, 126.5, 124.2, 117.7, 47.5, 45.6, 25.8, 18.3. HRMS (ESI)+
calculated for C23H22ClN4O2S2, [M+H]+: m/z 485.0873,
found 485.0875.
2-(2-Chlorobenzamido)-6-((4-(trifluoromethyl)
phenyl)carbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22am). White solid, mp 186-
1
188, yield 61.5%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.94 (1H, brs), 9.77 (1H, brs), 7.89 (1H, m), 7.85 (1H, brd, J
= 7.8 Hz), 7.73 (1H, d, J = 8.1 Hz), 7.67(2H, d, J = 8.6 Hz),
7.65 (1H, dd, J = 8.1, 7.8 Hz), 7.59 (2H, d, J = 8.1 Hz), 5.10
(2H, s), 4.22 (2H, brt, J = 5.1 Hz), 3.04 (2H, brt, J = 5.1 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.5, 167.8, 161.8,
145.2, 144.2, 134.7, 134.3, 132.9, 131.6, 129.4, 127.5, 126.2, 125.6
(3.9 Hz), 125.6 (3.9 Hz), 125.1, 125.1, 124.9 (271.6 Hz), 124.5
(32.4 Hz), 123.7, 116.8, 47.8, 46.2, 25.8. HRMS (ESI)+
calculated for C23H19ClF3N4O2S2, [M+H]+: m/z 539.0618,
found 539.0590.
2-(2-Chlorobenzamido)-6-((2-
methoxyphenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22aj).
White solid, mp 189-191, yield 78.4%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.27 (1H, brs), 8.99 (1H, brs), 7.75 (1H,
dd, J = 7.6, 1.4 Hz), 7.63 (1H, dd, J = 8.0, 1.5 Hz), 7.59 (1H,
1
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1
Journal of Medicinal Chemistry
2-(2-Chlorobenzamido)-6-((4-methoxyphenyl)
carbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
(3.4, 3.2 Hz), 47.9, 46.3, 25.8. HRMS (ESI)+ calculated for
C24H18ClF6N4O2S2, [M+H]+: m/z 607.0492, found 607.0464.
2
3
4
c]pyridine-3-carboxamide (22an). White solid, mp 201-
203, yield 61.7%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
N6-(3,5-Bis(trifluoromethyl)phenyl)-2-(2-
1
chlorobenzamido)-4,7-dihydrothieno[2,3-c]pyridine-
3,6(5H)-dicarboxamide (22ar). Off-white solid, mp 231-
233, yield 69.2%. 1H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.26 (1H, brs), 9.37 (1H, brs), 8.26 (2H, brs), 7.74 (1H, d, J
= 7.5, 1.3 Hz), 7.62 (1H, m), 7.61 (1H, m), 7.59 (1H, td, J =
8.0, 1.6 Hz), .7.52 (1H, td, J = 7.3, 1.5 Hz), 4.71 (2H, s), 3.79
(2H, t, J = 5.4 Hz), 2.93 (2H, t, J = 5.4 Hz). 13C-NMR (100
MHz, DMSO-d6), δ(ppm): 166.8, 162.4, 154.3, 142.4 (32.4
Hz), 142.4 (32.4 Hz), 133.6, 132.5, 130.5, 130.4, 130.2, 130.1,
129.9, 128.7, 127.7, 123.7, 123.3 (273.2 Hz), 123.3 (273.2 Hz),
118.8 (3.4 Hz), 118.8 (3.4 Hz), 117.2, 114.3 (3.2 Hz), 42.8, 41.3,
25.3. HRMS (ESI)+ calculated for C24H18ClF6N4O3S,
[M+H]+: m/z 591.0714, found 591.0692.
12.94 (1H, brs), 9.37 (1H, brs), 7.89 (1H, dd, J = 2.1, 1.6 Hz),
7.84 (1H, brd, J = 7.8 Hz), 7.73 (1H, d, J = 8.1 Hz), 7.65 (1H,
dd, J = 8.1, 7.8 Hz), 7.20 (2H, d, J = 8.9 Hz), 6.89 (2H, d, J =
8.9 Hz), 5.09 (2H, s), 4.20 (2H, brt, J = 5.4 Hz), 3.75 (3H,
5
6
7
8
9
13
s), 3.00 (2H, brt, J = 5.4 Hz). C-NMR (100 MHz, DMSO-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
d6), δ(ppm): 181.8, 167.8, 161.7, 157.1, 144.0, 134.7, 134.4, 134.1,
132.8, 131.6, 129.4, 128.1, 128.1, 127.5, 126.2, 124.1, 116.8, 113.7,
113.7, 55.7, 47.5, 45.5, 25.8. HRMS (ESI)+ calculated for
C23H22ClN4O3S2, [M+H]+: m/z 501.0845, found 501.0822.
2-(2-Chlorobenzamido)-6-((2,6-dichlorophenyl)
carbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22ao). Off-white solid, mp
264-266, yield 65.7%. 1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 12.26 (1H, brs), 9.49 (1H, brs), 7.75 (1H, dd, J = 7.4,
1.3 Hz), 7.63 (1H, dd, J = 8.1, 1.4 Hz), 7.60 (1H, td, J = 8.1, 1.4
Hz), 7.53 (1H, m), 7.52 (2H, d, J = 8.1 Hz), 7.33 (1H, t, J =7.9
Hz), 5.14 (2H, s), 4.19 (2H, t, J = 5.2 Hz), 2.98 (2H, t, J = 5.2
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.0, 166.7,
162.5, 142.2, 135.9, 135.0, 135.0, 133.6, 132.5, 130.3, 130.1, 129.9,
128.9, 128.8, 128.3, 128.3, 127.7, 123.4, 117.3, 47.2, 45.2, 25.4.
HRMS (ESI)+ calculated for C22H18Cl3N4O2S2, [M+H]+: m/z
538.9962, found 538.9937.
2-(2-Chlorobenzamido)-6-(pyridin-3-
ylcarbamothioyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22as). White solid, mp 216-
1
217, yield 78.9%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.26 (1H, brs), 9.61 (1H, brs), 8.51 (1H, d, J = 1.7 Hz), 8.33
(1H, d, J = 4.0 Hz), 7.76 (1H, m), 7.74 (1H, m), 7.63 (1H, dd,
J = 8.1, 1.4 Hz), 7.59 (1H, td, J = 8.1, 1.7 Hz), 7.52 (1H, td, J =
7.1, 1.4 Hz), 7.36 (1H, dd, J = 8.1, 4.7 Hz), 5.13 (2H, s), 4.22
(2H, t, J = 5.4 Hz), 3.00 (2H, t, J =5.4 Hz). 13C-NMR (100
MHz, DMSO-d6), δ(ppm): 181.9, 167.3, 162.9, 147.3, 145.8,
142.9, 138.1, 134.1, 133.7, 133.0, 130.9, 130.6, 130.4, 129.4, 128.2,
123.8, 123.4, 117.6, 47.6, 46.0, 25.8. HRMS (ESI)+ calculated
for C21H19ClN5O2S2, [M+H]+: m/z 472.0669, found
472.0665.
2-(2-Chlorobenzamido)-N6-(2,6-dichlorophenyl)-
4,7-dihydrothieno[2,3-c]pyridine-3,6(5H)-
dicarboxamide (22ap). White solid, mp 265-267, yield
66.3%. 1H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.29 (1H,
brs), 8.61 (1H, brs), 7.74 (1H, dd, J = 7.6, 1.5 Hz), 7.62 (1H,
d, J = 8.0, 1.5 Hz), 7.59 (1H, td, J = 7.0, 1.7 Hz), 7.52 (1H, m),
7.51 (2H, d, J = 8.0 Hz), 7.30 (1H, t, J = 8.0 Hz), 4.67 (2H, s),
3.76 (2H, t, J = 5.2 Hz), 2.90 (2H, t, J = 5.2 Hz). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 166.8, 162.4, 154.7, 142.2,
134.5, 134.5, 133.7, 132.5, 130.4, 130.1, 129.9, 129.2, 128.7, 128.4,
128.3, 128.3, 127.7, 124.0, 117.2, 42.7, 41.3, 25.5. HRMS (ESI)+
calculated for C22H18Cl3N4O3S, [M+H]+: m/z 523.0194,
found 523.0165.
2-Benzamido-6-((2-chlorophenyl)carbamothioyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22ba). White solid, mp 224-227, yield
1
75.9%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.95 (1H,
brs), 9.41 (1H, brs), 7.92 (2H, d, J = 7.1 Hz), 7.68 (1H, t, J =
7.4 Hz), 7.62 (2H, t, J = 7.1 Hz), 7.50 (1H, d, J = 7.1 Hz), 7.34
(1H, m), 7.33 (1H, m), 7.29 (1H, m), 5.12 (2H, s), 4.21 (2H, t,
J = 5.3 Hz), 3.02 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 182.1, 168.0, 163.0, 144.7, 138.6, 133.2,
132.5, 132.4, 131.8, 129.8, 129.7, 129.7, 129.2, 128.4, 127.7, 127.5,
127.5, 123.6, 116.2, 47.5, 45.7, 26.0. HRMS (ESI)+ calculated
for C22H20ClN4O2S2, [M+H]+: m/z 471.0716, found 471.0708.
6-((3,5-Bis(trifluoromethyl)phenyl)carbamothioyl)-
2-(2-Chlorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22aq). Off-white solid, mp
1
206-209, yield 69.8%. H-NMR (400 MHz, DMSO-d6),
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
δ(ppm): 12.93 (1H, brs), 10.12 (1H, brs), 8.17 (2H, brs), 7.90
(1H, m), 7.84(1H , d, J =7.7 Hz), 7.78 (1H, brs), 7.49 (1H,
brs), 7.31(1H , d, J =7.6 Hz), 7.65 (1H, dd, J = 7.7, 7.6 Hz),
5.16 (2H, s), 4.26 (2H, brt, J = 5.4 Hz), 3.07 (2H, brt, J = 5.4
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.0, 167.8,
161.8, 144.2, 143.5, 143.5, 134.7, 134.3, 132.9, 131.6, 130.2 (33.4
Hz), 130.2 (33.4 Hz), 129.4, 129.2 (284.1 Hz), 129.2 (284.1
Hz), 127.5, 126.2, 125.1 (3.2 Hz), 125.1 (3.2 Hz), 122.4, 117.3
fluorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bb). White solid, mp 245-
247, yield 69.4%. 1H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.73 (1H, brs), 9.41 (1H, brs), 8.04 (1H, td, J = 7.7, 1.6 Hz),
7.71 (1H, m), 7.49 (1H, d, J = 7.5 Hz), 7.44 (2H, m), 7.34 (1H,
m), 7.33 (1H, m), 7.29 (1H, m), 5.13 (2H, brs), 4.20 (2H, t, J
= 5.3 Hz), 3.01 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 181.5, 166.9, 159.8 (248.5 Hz), 159.1,
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143.0, 138.1, 134.9 (8.4 Hz), 131.9, 131.4, 131.3, 129.3, 128.8, (1H, d, J = 7.4 Hz), 7.35 (1H, m), 7.33 (1H, m), 7.28 (1H, m),
1
2
3
4
5
6
7
8
127.9, 127.2, 125.3 (3.2 Hz), 123.5, 119.5 (11.6 Hz), 116.8, 116.6
(3.2 Hz), 47.0, 45.2, 25.4. HRMS (ESI)+ calculated for
C22H19ClFN4O2S2, [M+H]+: m/z 489.0657, found 489.0622.
5.13 (2H, s), 4.20 (2H, t, J = 5.1 Hz), 2.97 (2H, t, J = 5.1 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.4, 166.6, 163.4,
141.8, 138.0, 133.8 (2.1 Hz), 132.9, 131.9, 131.2, 131.1 (13.3 Hz),
129.2, 128.9, 128.5, 127.8, 127.1, 126.7 (4.7 Hz), 126.1 (31.4 Hz),
123.5, 123.4 (273.2 Hz), 117.5, 47.0, 45.1, 25.2. HRMS (ESI)+
calculated for C23H19ClF3N4O2S2, [M+H]+: m/z 538.0590,
found 538.0595.
N6-(2-Chlorophenyl)-2-(2-fluorobenzamido)-4,7-
dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxamide
1
(22bc). White solid, mp 202-204, yield 73.1%. H-NMR
(400 MHz, DMSO-d6), δ(ppm): 12.74 (1H, brs), 8.37 (1H,
brs), 8.03 (1H, td, J = 7.7, 1.6 Hz), 7.70 (1H, m), 7.51 (1H, dd,
J = 8.0, 1.4 Hz), 7.47 (1H, m), 7.43 (1H, d, J = 7.2 Hz), 7.41
(1H, m), 7.30 (1H, td, J = 7.7, 1.3 Hz), 7.15 (1H, td, J = 7.7, 1.5
Hz), 4.67 (2H, brs), 3.75 (2H, t, J = 5.4 Hz), 2.93 (2H, t, J =
5.4 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):167.0,
167.0, 159.7 (250.0 Hz), 159.3, 154.8, 136.4, 134.8 (8.5 Hz),
131.4, 129.2, 128.6, 128.4, 127.4, 127.2, 125.8, 125.3 (3.4 Hz),
123.8 (17.8 Hz), 116.8, 116.6 (3.4 Hz), 116.6, 42.7, 41.2, 25.5.
HRMS (ESI)+ calculated for C22H19ClFN4O3S, [M+H]+: m/z
473.0861, found 473.0850.
9
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
methylbenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bg). White solid, mp 215-
1
218, yield 75.6%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.24 (1H, brs), 9.41 (1H, brs), 7.59 (1H, dd, J = 8.4, 1.4 Hz),
7.50 (1H, dd, J = 8.4, 1.0 Hz), 7.47 (1H, m), 7.38 (1H, m),
7.37 (1H, m), 7.34 (1H, m), 7.33 (1H, m), 7.29 (1H, m), 5.12
(2H, s), 4.20 (2H, t, J = 5.3 Hz), 2.99 (2H, t, J = 5.3 Hz), 2.47
(3H, s). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.0,
167.6, 165.6, 144.0, 138.6, 137.2, 134.2, 132.5, 132.0, 131.8, 131.6,
129.8, 129.2, 128.4, 127.7, 127.7, 126.8, 123.4, 116.6, 47.6, 45.8,
25.9, 20.3. HRMS (ESI)+ calculated for C23H22ClN4O2S2,
[M+H]+: m/z 485.0873, found 485.0872.
2-(2-Bromobenzamido)-6-((2-
chlorophenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22bd). White solid, mp 229-232, yield 77.2%. H-NMR
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
(400 MHz, DMSO-d6), δ(ppm): 12.13 (1H, brs), 9.43 (1H,
brs), 7.78 (1H, dd, J = 7.9, 0.8 Hz), 7.67 (1H, dd, J = 7.5, 1.7
Hz), 7.55 (1H, td, J = 7.5, 0.9 Hz), 7.50 (1H, m), 7.49 (1H,
m), 7.35 (1H, m), 7.33 (1H, m), 7.29 (1H, m), 5.13 (2H, s),
4.19 (2H, t, J = 5.2 Hz), 2.97 (2H, t, J = 5.2 Hz). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 182.1, 167.2, 164.0, 142.8,
138.6, 136.7, 133.9, 132.9, 132.4, 131.8, 129.9, 129.8, 129.4, 128.6,
128.4, 127.7, 124.0, 119.5, 117.7, 47.6, 45.8, 25.9. HRMS (ESI)+
calculated for C22H19BrClN4O2S2, [M+H]+: m/z 548.9821,
found 548.9819.
methoxybenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bh). White solid, mp 206-
209, yield 72.2%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
1
13.06 (1H, brs), 9.39 (1H, brs), 8.10 (1H, dd, J = 7.8, 1.7 Hz),
7.63 (1H, td, J = 7.6, 1.7 Hz), 7.50 (1H, brd, J = 7.6 Hz), 7.34
(1H, m), 7.33 (1H, m), 7.29 (1H, m), 7.26 (1H, d, J = 7.8 Hz),
7.15 (1H, t, J = 7.2 Hz), 5.11 (2H, s), 4.20 (2H, t, J = 5.3 Hz),
4.09 (3H, s), 2.99 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 181.9, 167.2, 161.6, 158.1, 143.3, 138.6,
134.9, 132.5, 132.1, 131.8, 129.8, 129.2, 128.4, 127.7, 123.6, 121.5,
119.3, 117.2, 112.9, 56.4, 47.5, 45.8, 26.0. HRMS (ESI)+
calculated for C23H22ClN4O3S2, [M+H]+: m/z 501.0822,
found 501.0815.
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
iodobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22be). White solid, mp 228-
1
231, yield 76.8%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
12.06 (1H, brs), 9.42 (1H, brs), 8.00 (1H, d, J = 7.9 Hz), 7.59
(1H, dd, J = 7.6, 1.9 Hz), 7.55 (1H, t, J = 7.5 Hz), 7.50 (1H, d,
J = 7.5 Hz), 7.34 (1H, m), 7.33 (1H, m), 7.29 (1H, m), 7.28
(1H, m), 5.13 (2H, s), 4.20 (2H, t, J = 5.4 Hz), 2.97 (2H, t, J =
5.4 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.1,
167.3, 165.7, 143.0, 140.7, 140.3, 138.6, 132.6, 132.5, 131.8, 129.8,
129.4, 129.0, 128.9, 128.4, 127.7, 124.0, 117.5, 93.9, 47.6, 45.7,
25.9. HRMS (ESI)+ calculated for C22H19ClN4O2S2, [M+H]+:
m/z 596.9683, found 596.9685.
(methylthio)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22bi).
White solid, mp 230-232, yield 76.5%. 1H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.27 (1H, brs), 9.25 (1H, brs), 7.75 (1H,
dd, J = 7.4, 1.2 Hz), 7.62 (1H, dd, J = 8.0, 1.2 Hz), 7.59 (1H,
td, J = 8.0, 1.2 Hz), 7.52 (1H, td, J = 7.4, 1.2 Hz), 7.29 (1H, m),
7.28 (1H, m), 7.16 (1H, m), 7.15 (1H, m), 5.10 (2H, s), 4.19
(2H, t, J = 5.3 Hz), 2.97 (2H, t, J = 5.3 Hz), 2.38 (3H, s). 13C-
NMR (100 MHz, DMSO-d6), δ(ppm): 182.1, 167.3, 162.9,
142.8, 138.4, 138.2, 134.1, 133.0, 130.9, 130.6, 130.4, 130.3, 129.4,
128.2, 127.7, 126.0, 125.1, 124.1, 117.7, 47.5, 45.7, 25.9, 15.0.
HRMS (ESI)+ calculated for C23H21ClN4NaO2S3, [M+Na]+:
m/z 539.0413, found 539.0419.
6-((2-Chlorophenyl)carbamothioyl)-2-(2-
(trifluoromethyl)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22bf).
1
White solid, mp 177-180, yield 71.2%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.12 (1H, brs), 9.43 (1H, brs), 7.90 (1H,
d, J = 7.8 Hz), 7.83 (1H, m), 7.82 (1H, m), 7.81 (1H, m), 7.50
((2-Chlorophenyl)carbamothioyl)-2-(4-
(trifluoromethyl)benzamido)-4,5,6,7-
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Journal of Medicinal Chemistry
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22bj).
[M+H]+: m/z 469.1123, found 469.1101.
1
2
3
4
5
6
7
8
White solid, mp 189-191, yield 67.4%. 1H-NMR (400 MHz,
DMSO-d6), δ(ppm): 13.02 (1H, brs), 9.42 (1H, brs), 8.10
(2H, d, J = 8.3 Hz), 8.00 (2H, d, J = 8.3 Hz), 7.49 (1H, d, J =
7.6 Hz), 7.34 (1H, m), 7.33 (1H, m), 7.29 (1H, m), 5.13 (2H,
s), 4.21 (2H, t, J = 5.6 Hz), 3.02 (2H, t, J = 5.6 Hz). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 181.5, 167.3, 167.3, 161.5,
143.5, 138.1, 135.9, 131.9, 131.3, 129.3, 129.1 (38.4 Hz), 128.1,
128.1, 127.9, 127.2, 126.2 (3.5 Hz), 126.2 (3.5 Hz), 123.8 (271.6
Hz), 123.6, 116.6, 47.1, 45.2, 25.4. HRMS (ESI)+ calculated
for C23H19ClF3N4O2S2, [M+H]+: m/z 539.0571, found
539.0590.
6-((2-Chlorophenyl)carbamothioyl)-2-(3-
nitrobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bn). Off-white solid, mp
164-166, yield 68.2%. 1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 13.08 (1H, brs), 9.42 (1H, brs), 8.67 (1H, s), 8.49
(1H, d, J = 7.8 Hz), 8.32 (1H, d, J = 7.6 Hz), 7.91 (1H, t, J =
7.8 Hz), 7.50 (1H, d, J = 7.6 Hz), 7.34 (1H, m), 7.33 (1H, m),
7.29 (1H, m), 5.14 (2H, s), 4.22 (2H, t, J = 5.1 Hz), 3.02 (2H, t,
9
10
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
J = 5.1 Hz). C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.5,
167.2, 160.7, 148.0, 143.2, 138.1, 133.7, 133.3, 131.9, 131.3, 130.9,
129.3, 129.0, 127.9, 127.2, 126.9, 123.7, 121.9, 116.9, 47.1, 45.2,
25.4. HRMS (ESI)+ calculated for C22H19ClN5O4S2, [M+H]+:
m/z 516.0592, found 516.0567.
N6-(2-Chlorophenyl)-2-(4-(trifluoromethyl)
benzamido)-4,7-dihydrothieno[2,3-c]pyridine-
3,6(5H)-dicarboxamide (22bk). White solid, mp 176-178,
yield 69.3%. 1H-NMR (400 MHz, DMSO-d6), δ(ppm): 13.05
(1H, brs), 8.38 (1H, brs), 8.10 (2H, d, J = 8.2 Hz), 7.99 (2H,
d, J = 8.2 Hz), 7.50 (1H, dd, J = 8.0, 1.4 Hz), 7.46 (1H, dd, J =
8.0, 1.3 Hz), 7.29 (1H, td, J = 8.0, 1.4 Hz), 7.15 (1H, td, J = 8.0,
1.5 Hz), 4.68 (2H, t, J = 5.6 Hz), 3.76 (2H, t, J = 5.6 Hz),
2.94 (2H, brs). 13C-NMR (100 MHz, DMSO-d6), δ(ppm):
167.4, 167.4, 161.4, 154.8, 136.4, 132.3, 131.1, 129.2, 128.6 (32.1
Hz), 128.4, 128.0, 128.0, 127.4, 127.2, 126.1 (3.3 Hz), 126.1 (3.3
Hz), 125.8, 124.1, 123.8 (271.5 Hz), 116.6, 42.8, 41.2, 25.4.
HRMS (ESI)+ calculated for C23H19ClF3N4O3S, [M+H]+: m/z
523.0846, found 523.0818.
N6-(2-Chlorophenyl)-2-(3-nitrobenzamido)-4,7-
dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxamide
(22bo). Off-white solid, mp 129-131, yield 70.2%. H-NMR
1
(400 MHz, DMSO-d6), δ(ppm): 13.03 (1H, brs), 8.66 (1H,
brs), 8.48 (1H, dd, J = 8.0, 1.2 Hz), 8.37 (1H, brs), 8.31 (1H, d,
J = 8.0 Hz), 7.90 (1H, t, J = 8.0 Hz), 7.51 (1H, dd, J = 8.1, 1.2
Hz), 7.47 (1H, dd, J = 7.9, 1.1 Hz), 7.30 (1H, td, J = 8.1, 1.1
Hz), 7.16 (1H, td, J = 7.9, 1.2 Hz), 4.68 (2H, s), 3.77 (2H, t, J
= 5.5 Hz), 2.94 (2H, t, J = 5.5 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 167.3, 167.3, 160.7, 154.8, 148.0, 146.4,
136.4, 133.3, 130.8, 129.2, 128.9, 128.4, 127.3, 127.2, 126.8, 125.8,
124.0, 121.9, 116.8, 42.8, 41.3, 25.5. HRMS (ESI)+ calculated
for C22H19ClN5O5S, [M+H]+: m/z 500.0813, found 500.0795.
6-((2-Chlorophenyl)carbamothioyl)-2-(4-
methylbenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bl). Off-white solid, mp
226-229, yield 67.7%. 1H-NMR (400 MHz, DMSO-d6),
δ(ppm): 12.91 (1H, brs), 9.41 (1H, brs), 7.81 (2H, d, J = 8.0
Hz), 7.50 (1H, d, J = 7.4 Hz), 7.43 (2H, d, J = 8.0 Hz), 7.35
(1H, m), 7.33 (1H, s), 7.29 (1H, m), 5.12 (2H, brs), 4.21 (2H,
6-((2-Chlorophenyl)carbamothioyl)-2-(pyrimidine-
4-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22bp). White solid, mp 235-
1
237, yield 74.7%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
13.24 (1H, brs), 9.44 (1H, brs), 9.42 (1H, brs), 9.17 (1H, d, J =
5.1 Hz), 8.16 (1H, dd, J = 5.1, 1.2 Hz), 7.50 (1H, d, J = 7.5 Hz),
7.33 (1H, m), 7.32 (1H, m), 7.29 (1H, m), 5.15 (2H, s), 4.21
(2H, t, J = 5.3 Hz), 3.03 (2H, t, J = 5.3 Hz). 13C-NMR (100
MHz, DMSO-d6), δ(ppm): 182.0, 167.1, 160.7, 160.1, 158.7,
154.9, 142.5, 138.6, 132.4, 131.8, 129.8, 129.6, 128.4, 127.7, 124.5,
119.4, 118.3, 47.6, 45.7, 25.9. HRMS (ESI)+ calculated for
C20H18N6O2S2, [M+H]+: m/z 473.0621, found 437.1110.
13
m) 3.02 (2H, m), 2.41 (3H, s). C-NMR (100 MHz, DMSO-
d6), δ(ppm): 182.0, 168.0, 162.9, 144.9, 143.5, 138.6, 138.6,
132.4, 131.8, 130.2, 130.2, 129.8, 1229.8, 128.4, 127.7, 127.5,
127.5, 123.4, 116.0, 47.5, 45.7, 26.0, 21.6. HRMS (ESI)+
calculated for C23H22ClN4O2S2, [M+H]+: m/z 485.0896,
found 485.0873.
N6-(2-Chlorophenyl)-2-(4-methylbenzamido)-4,7-
dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxamide
6-(Butylcarbamothioyl)-2-hexanamido-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22ca).
White solid, mp 228-231, yield 76.6%. H-NMR (400 MHz,
1
(22bm). White solid, mp 258-260, yield 66.5%. H-NMR
1
(400 MHz, DMSO-d6), δ(ppm): 12.93 (1H, brs), 8.37 (1H,
brs), 7.80 (2H, d, J = 8.2 Hz), 7.51 (1H, dd, J = 8.1, 1.6 Hz),
7.45 (1H, dd, J = 8.1, 1.5 Hz), 7.41 (2H, d, J = 8.2 Hz), 7.29
(1H, td, J = 8.1, 1.5 Hz), 7.15 (1H, td, J =8.1, 1.6 Hz), 4.67 (2H,
s), 3.75 (2H, t, J = 5.6 Hz), 2.94 (2H, t, J = 5.6 Hz), 2.40 (3H,
s). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 167.6, 167.6,
162.4, 154.8, 142.9, 136.4, 129.7, 129.7, 129.5, 129.2, 128.5,
128.4, 127.3, 127.2, 127.0, 127.0, 125.8, 123.3, 115.5, 42.8, 41.2,
25.5, 21.1. HRMS (ESI)+ calculated for C23H22ClN4O3S,
DMSO-d6), δ(ppm): 11.46 (1H, brs), 7.76 (1H, t, J = 5.2 Hz),
4.88 (2H, s), 4.03 (2H, t, J = 5.4 Hz), 3.50 (2H, dd, J = 12.5,
7.0 Hz), 2.82 (2H, t, J = 5.4 Hz), 2.42 (2H, t, J = 7.4 Hz),
1.59 (2H, m), 1.52 (2H, m),1.30 (2H, m), 1.29 (2H, m), 1.26
(2H, m), 0.89 (3H, t, J = 7.3 Hz), 0.87 (3H, t, J = 8.7 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.4, 170.2, 167.3,
143.2, 128.9, 123.1, 116.2, 46.6, 45.6, 45.1, 36.0, 31.3, 31.1, 25.7,
24.9, 22.3, 20.1, 14.3, 14.3. HRMS (ESI)+ calculated for
C19H31N4O2S2, [M+H]+: m/z 411.1888, found 411.1904.
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Journal of Medicinal Chemistry
Page 28 of 44
2-Benzamido-6-(phenylcarbamothioyl)-4,5,6,7- DMSO-d6), δ(ppm): 181.9, 167.2, 164.0, 142.7, 140.0, 136.7,
1
2
3
4
5
6
7
8
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
(22cb). White solid, mp 232-235, yield 74.6%. H-NMR
133.9, 132.9, 132.9, 132.0, 129.9, 129.4, 128.7, 128.6, 128.3,
124.0, 123.7, 119.5, 117.7, 47.6, 45.7, 25.9. HRMS (ESI)+
calculated for C22H19Br2N4O2S2, [M+H]+: m/z 592.9316,
found 592.9315.
1
(400 MHz, DMSO-d6), δ(ppm): 12.97 (1H, brs), 9.48 (1H,
brs), 7.92 (2H, d, J = 7.1 Hz), 7.68 (1H, t, J = 7.1 Hz), 7.62
(2H, t, J = 7.6 Hz), 7.33 (2H, m), 7.32 (2H, m), 7.14 (1H, m),
5.09 (2H, s), 4.21 (2H, t, J = 5.4 Hz), 3.02 (2H, t, J = 5.4 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.7, 168.0, 163.0,
144.8, 141.3, 133.2, 132.6, 129.7, 129.7, 129.3, 128.5, 128.5, 127.5,
127.5, 126.1, 126.1, 125.1, 123.6, 116.1, 47.6, 45.9, 25.9. HRMS
(ESI)+ calculated for C22H21N4O2S2, [M+H]+: m/z 437.1106,
found 437.1110.
2-(2-Bromobenzamido)-6-((2-
(trifluoromethyl)phenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22cf).
White solid, mp 228-230, yield 70.2%. 1H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.14 (1H, brs), 9.41 (1H, brs), 7.78 (1H,
brd, J = 7.9 Hz), 7.73 (1H, brd, J = 7.9 Hz), 7.69 (1H, m),
7.68 (1H, m), 7.55 (1H, m), 7.54 (1H, m), 7.50 (1H, m), 7.49
(1H, m), 7.40 (1H, brd, J = 7.9 Hz), 5.10 (2H, s), 4.18 (2H, t, J
= 5.2 Hz), 2.96 (2H, t, J = 5.2 Hz). 13C-NMR (100 MHz,
DMSO-d6), δ(ppm): 182.6, 167.2, 164.1, 142.8, 139.3 (2.0 Hz),
136.7, 133.9, 133.8, 133.0 (12.3 Hz), 129.9, 129.4, 128.6, 127.9,
127.4 (29.2 Hz), 126.8 (15.7 Hz), 126.7 (5.9 Hz), 124.1 (274.4
Hz), 123.9, 119.5, 117.7, 47.5, 45.7, 25.8. HRMS (ESI)+
calculated for C23H19BrF3N4O2S2, [M+H]+: m/z 583.0085,
found 583.0063.
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10
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12
13
14
15
16
17
18
19
20
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24
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26
27
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6-((3,5-Bis(trifluoromethyl)phenyl)carbamothioyl)-
2-(2-fluorobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22cc). White solid, mp 217-
1
219, yield 61.4%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.77 (1H, brs), 12.74 (1H, brs), 9.96 (1H, brs), 8.14 (2H,
brs), 8.04 (1H, td, J = 7.8, 1.8 Hz), 7.80 (1H, brs), 7.71 (1H,
m), 7.45 (1H, td, J = 8.7, 1.3 Hz), 7.42 (1H, m), 5.16 (2H, s),
4.25 (2H, m), 3.06 (2H, m). 13C-NMR (100 MHz, DMSO-
d6), δ(ppm): 181.0, 167.4, 160.4 (251.2 Hz), 159.7, 143.5 (25.3
Hz), 135.4 (9.1 Hz), 131.9, 130.1 (32.0 Hz), 130.1 (32.0 Hz),
129.3, 125.8 (3.0 Hz), 125.2, 123.8 (272.1 Hz), 123.8 (272.1
Hz), 123.7, 122.4, 120.0 (11.2 Hz), 117.3 (3.4, 3.0 Hz), 117.3,
117.0, 116.9, 47.8, 46.3, 25.8. HRMS (ESI)+ calculated for
C24H18F7N4O2S2, [M+H]+: m/z 591.0779, found 591.0759.
2-(2-Bromobenzamido)-6-(o-tolylcarbamothioyl)-
4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (22cg). White solid, mp 227-229, yield
1
73.5%. H-NMR (400 MHz, DMSO-d6), δ(ppm): 12.13 (1H,
brs), 9.26 (1H, brs), 7.78 (1H, dd, J = 7.9, 0.8 Hz), 7.67 (1H,
dd, J = 7.5, 1.7 Hz), 7.55 (1H, td, J = 7.4, 1.1 Hz), 7.49 (1H, td,
J = 7.5, 1.7 Hz), 7.23 (1H, dd, J = 6.3, 2.7 Hz), 7.17 (1H, m),
7.16 (1H, m), 7.08 (1H, dd, J = 6.6, 2.4 Hz), 5.11 (2H, s), 4.18
(2H, t, J = 5.4 Hz), 2.96 (2H, t, J = 5.4 Hz), 2.18 (3H, s). 13C-
NMR (100 MHz, DMSO-d6), δ(ppm): 181.9, 167.3, 164.0,
142.8, 139.9, 136.7, 136.1, 133.9, 132.9, 130.6, 129.9, 129.4,
129.3, 128.6, 126.9, 126.5, 124.2, 119.5, 117.7, 47.5, 45.6, 25.8,
18.3. HRMS (ESI)+ calculated for C23H22BrN4O2S2, [M+H]+:
m/z 529.0368, found 529.0370.
2-(2-Bromobenzamido)-6-((2-
fluorophenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22cd). White solid, mp 231-232, yield 78.0%. H-NMR
(400 MHz, DMSO-d6), δ(ppm): 12.13 (1H, brs), 9.33 (1H,
brs), 7.78 (1H, dd, J = 7.8, 1.0 Hz), 7.68 (1H, dd, J = 7.5, 1.8
Hz), 7.55 (1H, td, J = 7.4, 1.0 Hz), 7.50 (1H, td, J = 7.8, 1.8
Hz), 7.27 (1H, m), 7.26 (1H, m), 7.19 (1H, m), 7.18 (1H, m),
5.11 (2H, s), 4.19 (2H, t, J = 5.2 Hz), 2.97 (2H, t, J = 5.2 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.3, 167.3, 164.0,
157.9 (246.1 Hz), 142.8, 136.7, 133.9, 132.9, 130.7, 129.9, 129.4,
129.1 (12.8 Hz), 128.6, 128.3 (8.2 Hz), 124.5 (3.1 Hz) , 123.9,
119.5, 117.7, 116.2 (20.6 Hz), 47.6, 45.9, 25.8. HRMS (ESI)+
calculated for C22H19BrFN4O2S2, [M+H]+: m/z 533.0117,
found 533.0112.
6-((2-Bromophenyl)carbamothioyl)-2-(2-
iodobenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22ch). White solid, mp 230-
1
233, yield 77.4%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.13 (1H, brs), 9.43 (1H, brs), 7.77 (1H, d, J = 7.9 Hz), 7.67
(1H, m), 7.66 (1H, td, J = 7.3, 1.7 Hz), 7.55 (1H, td, J = 7.5,
0.9 Hz), 7.49 (1H, td, J = 7.5, 1.6 Hz), 7.39 (1H, td, J = 7.3, 1.2
Hz), 7.32 (1H, dd, J = 7.9, 1.6 Hz), 7.21 (1H, td, J = 7.3, 1.7
Hz), 5.13 (2H, s), 4.20 (2H, t, J = 5.3 Hz), 2.98 (2H, t, J = 5.3
Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 181.9, 167.2,
164.0, 142.8, 140.0, 136.7, 133.9, 132.9, 132.9, 132.0, 129.9,
129.4, 128.7, 128.6, 128.3, 124.9, 123.7, 119.5, 117.7, 47.6, 45.7,
25.9. HRMS (ESI)+ calculated for C22H19BrIN4O2S2, [M+H]+:
m/z 640.9177, found 640.9180.
2-(2-Bromobenzamido)-6-((2-
bromophenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22ce).
1
White solid, mp 231-233, yield 75.0%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.13 (1H, brs), 9.45 (1H, brs), 7.78 (1H,
d, J = 7.9 Hz), 7.67 (1H, td, J = 7.4, 1.7 Hz), 7.66 (1H, td, J =
7.4, 1.7 Hz), 7.55 (1H, td, J = 6.6, 1.7 Hz), 7.52 (1H, td, J = 7.4,
1.7 Hz), 7.39 (1H, td, J = 7.9, 1.0 Hz), 7.32 (1H, dd, J = 6.6, 1.7
Hz), 7.21 (1H, td, J = 7.9, 1.7 Hz), 5.13 (2H, s), 4.19 (2H, t, J =
5.2 Hz), 2.98 (2H, t, J = 5.2 Hz). 13C-NMR (100 MHz,
2-(2-Iodobenzamido)-6-((2-
(trifluoromethyl)phenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22ci).
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Journal of Medicinal Chemistry
1
White solid, mp 212-214, yield 74.3%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.07 (1H, brs), 9.40 (1H, brs), 7.99 (1H,
d, J = 7.9 Hz), 7.72 (1H, d, J = 7.9 Hz), 7.68 (1H, t, J = 7.7
Hz), 7.59 (1H, dd, J = 7.7, 1.8 Hz), 7.55 (1H, t, J = 7.7 Hz),
7.50 (1H, t, J = 7.7 Hz), 7.40 (1H, d, J = 7.9 Hz), 7.30 (1H, t, J
= 6.8 Hz), 5.11 (2H, s), 4.19 (2H, t, J = 5.0 Hz), 2.97 (1H, t, J
= 5.0 Hz). 13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.6,
167.3, 165.7, 143.0, 140.7, 140.3, 139.3 (1.7 Hz), 133.7, 133.0,
132.6, 129.4, 129.0 (9.1 Hz), 127.9, 127.4 (29.1 Hz), 126.7 (5.2
Hz), 126.6, 124.1 (274.0 Hz), 123.9, 117.5, 93.9, 47.5, 45.7,
25.8. HRMS (ESI)+ calculated for C23H19F3IN4O2S2, [M+H]+:
m/z 630.9946, found 630.9946.
129.1, 128.7, 128.3, 127.2 (4.9 Hz), 126.7 (31.7 Hz), 124.1, 123.9
(274.4 Hz), 123.7, 118.1, 47.6, 45.7, 25.8. HRMS (ESI)+
calculated for C23H19BrF3N4O2S2, [M+H]+: m/z 583.0085,
found 583.0090.
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6-(O-tolylcarbamothioyl)-2-(2-
(trifluoromethyl)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22cm). White solid, mp 215-217, yield 72.6%. H-NMR
9
(400 MHz, DMSO-d6), δ(ppm): 12.12 (1H, brs), 9.28 (1H,
brs), 7.91 (1H, d, J = 7.7 Hz), 7.82 (1H, m), 7.81 (1H, m), 7.80
(1H, m), 7.23 (1H, dd, J = 6.4, 2.2 Hz), 7.18 (1H, m), 7.17 (1H,
m), 7.08 (1H, dd, J = 7.5, 2.2 Hz), 5.12 (2H, s), 4.18 (2H, t, J
= 5.2 Hz), 2.95 (2H, t, J = 5.2 Hz), 2.18 (3H, s). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 181.8, 167.2, 167.2, 164.0,
142.4, 139.9, 136.1, 134.4 (2.1 Hz), 133.5, 131.7, 130.6, 129.5,
129.4 (13.1 Hz), 129.1, 127.2 (5.0 Hz), 126.9, 126.7 (31.6 Hz),
124.3, 123.9 (274.0 Hz), 118.0, 47.5, 45.6, 25.8, 18.3. HRMS
(ESI)+ calculated for C24H22F3N4O2S2, [M+H]+: m/z 519.1136,
found 519.1132.
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2-(2-Iodobenzamido)-6-((2-
methoxyphenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22cj).
1
White solid, mp 193-196, yield 79.1%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.14 (1H, brs), 8.97 (1H, brs), 7.78 (1H,
brt, J = 7.8 Hz), 7.67 (1H, dd, J = 7.5, 1.7 Hz), 7.55 (1H, td, J
= 7.4, 1.1 Hz), 7.49 (1H, td, J = 7.5, 1.1), 7.22 (1H, m), 7.21 (1H,
m), 7.04 (1H, dd, J = 8.2, 0.8 Hz), 6.92 (1H, td, J = 7.5, 1.1
Hz), 5.09 (2H, s), 4.18 (2H, brt, J = 5.3 Hz), 3.77 (3H, s),
2.95 (2H, t, J = 5.3 Hz). 13C-NMR (100 MHz, DMSO-d6),
δ(ppm): 182.3, 167.3, 164.0, 154.8, 142.7, 136.7, 133.9, 132.9,
130.1, 129.9, 129.8, 129.4, 128.6, 127.7, 124.1, 120.3, 119.5, 117.6,
112.2, 56.0, 47.4, 45.7, 25.8. HRMS (ESI)+ calculated for
C23H22IN4O3S2, [M+H]+: m/z 593.0178, found 593.0173.
6-((2-Methoxyphenyl)carbamothioyl)-2-(2-
(trifluoromethyl)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22cn). White solid, mp 221-223, yield 73.5%. H-NMR
(400 MHz, DMSO-d6), δ(ppm): 12.12 (1H, brs), 8.97 (1H,
brs), 7.90 (1H, d, J = 7.7 Hz), 7.84 (1H, m), 7.81 (1H, m),
7.78 (1H, m), 7.23 (1H, m), 7.21 (1H, m), 7.04 (1H, dd, J = 8.2,
0.8 Hz), 6.92 (1H, td, J = 7.6, 1.1 Hz), 5.09 (2H, s), 4.18 (2H,
6-((2-Fluorophenyl)carbamothioyl)-2-(2-
(trifluoromethyl)benzamido)-4,5,6,7-
13
brt, J = 5.4 Hz), 3.31 (3H, s), 2.94 (2H, brt, J = 5.4 Hz). C-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
(22ck). White solid, mp 212-214, yield 73.8%. 1H-NMR (400
MHz, DMSO-d6), δ(ppm): 12.11 (1H, brs), 9.32 (1H, brs),
7.91 (1H, d, J = 7.7 Hz), 7.82 (1H, m), 7.81 (1H, m), 7.80 (1H,
m), 7.28 (1H, m), 7.27 (1H, m), 7.19 (1H, m), 7.18 (1H, m),
5.11 (2H, s), 4.19 (2H, d, J = 5.3 Hz), 2.97 (2H, d, J = 5.3 Hz).
13C-NMR (100 MHz, DMSO-d6), δ(ppm): 182.3, 167.2, 164.0,
157.9 (246.7 Hz), 142.5, 134.3, 133.5, 131.7, 130.8, 129.4, 129.1
(12.1 Hz), 129.1, 128.3 (7.8 Hz), 127.2 (4.9 Hz), 126.7 (31.6
Hz), 124.5 (3.5 Hz), 124.3 (55.0 Hz), 124.0 (274.1 Hz), 118.0,
116.2 (20.1 Hz), 47.6, 45.9, 25.8. HRMS (ESI)+ calculated for
C23H19F4N4O2S2, [M+H]+: m/z 523.0886, found 523.0891.
NMR (100 MHz, DMSO-d6), δ(ppm): 182.3, 167.2, 164.0,
154.8, 142.4, 134.4, 133.5, 131.7, 130.1, 129.8, 129.5, 129.1, 127.6,
127.2 (4.8 Hz), 126.7 (31.5 Hz), 124.2, 123.9 (274.1 Hz), 120.3,
118.0, 112.2, 56.0, 47.4, 45.7, 25.8. HRMS (ESI)+ calculated
for C24H22F3N4O3S2, [M+H]+: m/z 535.1085, found 535.1092.
6-((2-Bromophenyl)carbamothioyl)-2-(2-
methylbenzamido)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (22co). White solid, mp 222-
1
223, yield 79.4%. H-NMR (400 MHz, DMSO-d6), δ(ppm):
12.25 (1H, brs), 9.45 (1H, brs), 7.66 (1H, dd, J = 8.0, 1.2 Hz),
7.59 (1H, dd, J = 7.2, 1.4 Hz), 7.48 (1H, t, J = 7.9 Hz), 7.40
(1H, m), 7.38 (1H, m), 7.37 (1H, m), 7.32 (1H, dd, J = 8.0, 1.7
Hz), 7.21 (1H, td, J = 7.9, 1.7 Hz), 5.12 (2H, s), 4.20 (2H, t, J
= 5.4 Hz), 3.00 (2H, t, J = 5.4 Hz), 2.47 (3H, s). 13C-NMR
(100 MHz, DMSO-d6), δ(ppm): 181.8, 167.6, 167.6, 165.5,
143.9, 140.0, 137.2, 134.2, 132.9, 132.0, 132.0, 131.6, 129.2, 128.7,
128.3, 127.7, 126.8, 123.7, 116.6, 47.6, 45.7, 26.0, 20.4. HRMS
(ESI)+ calculated for C23H22BrN4O2S2, [M+H]+: m/z
529.0368, found 529.0367.
6-((2-Bromophenyl)carbamothioyl)-2-(2-
(trifluoromethyl)benzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (22cl).
1
White solid, mp 220-221, yield 74.0%. H-NMR (400 MHz,
DMSO-d6), δ(ppm): 12.11 (1H, brs), 9.45 (1H, brs), 7.91 (1H,
d, J = 7.7 Hz), 7.82 (1H, m), 7.81 (1H, m), 7.80 (1H, m), 7.66
(1H, dd, J = 8.0, 1.2 Hz), 7.39 (1H, td, J = 7.9, 1.3 Hz), 7.32
(1H, dd, J = 7.9, 1.3 Hz), 7.21 (1H, td, J = 7.9, 1.8 Hz), 5.13
(2H, s), 4.19 (2H, d, J = 5.3 Hz), 2.97 (2H, d, J = 5.3 Hz). 13C-
NMR (100 MHz, DMSO-d6), δ(ppm): 181.9, 167.1, 164.0,
142.4, 140.0, 134.3 (2.2 Hz), 133.5, 132.9, 132.0, 131.7, 129.4,
2-(2-Methylbenzamido)-6-((2-
(trifluoromethyl)phenyl)carbamothioyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide
1
(22cp). White solid, mp 210-213, yield 74.6%. H-NMR
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