Design, synthesis and biological evaluation of novel tetrahydrothieno [2,3-c]pyridine substitued benzoyl thiourea derivatives as PAK1 inhibitors in triple negative breast cancer

Article abstract of DOI:10.1080/14756366.2020.1797710

The overexpression of P21-activated kinase 1 (PAK1) is associated with poor prognosis in several cancers, which has emerged as a promising drug targets. Based on high-throughput virtual screening strategy, tetrahydrothieno [2,3-c]pyridine scaffold was identified as an initial lead for targeting PAK1. Herein we reported our structure-based optimisation strategy to discover a potent PAK1 inhibitor (7j) which displayed potent PAK1 inhibition and antiproliferatory activity in MDA-MB-231 cells. 7j induced obviously G2/M cell cycle arrest via PAK1-cdc25c-cdc2 pathway, and also inhibited MAPK-ERK and MAPK-JNK cascade to induce MDA-MB-231 cell death. Together, these results provided a novel chemical scaffold as PAK1 inhibitor for breast cancer treatment.

Full text of DOI:10.1080/14756366.2020.1797710

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation of
novel tetrahydrothieno [2,3-c]pyridine substitued
benzoyl thiourea derivatives as PAK1 inhibitors in
triple negative breast cancer
Dahong Yao , Jian Huang , Jinhui Wang , Zhendan He & Jin Zhang
To cite this article: Dahong Yao , Jian Huang , Jinhui Wang , Zhendan He & Jin Zhang (2020)
Design, synthesis and biological evaluation of novel tetrahydrothieno [2,3-c]pyridine substitued
benzoyl thiourea derivatives as PAK1 inhibitors in triple negative breast cancer, Journal of Enzyme
Inhibition and Medicinal Chemistry, 35:1, 1524-1538, DOI: 10.1080/14756366.2020.1797710
To link to this article: https://doi.org/10.1080/14756366.2020.1797710
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
020, VOL. 35, NO. 01, 1524–1538
2
https://doi.org/10.1080/14756366.2020.1797710
ARTICLE
Design, synthesis and biological evaluation of novel tetrahydrothieno
[
2,3-c]pyridine substitued benzoyl thiourea derivatives as PAK1 inhibitors
in triple negative breast cancer
a,b
c
c
a,b
a,d
Dahong Yao , Jian Huang , Jinhui Wang , Zhendan He and Jin Zhang
a
b
School of Pharmaceutical Sciences, Shenzhen Technology University, Shenzhen, China; School of Pharmaceutical Sciences, Guangdong Key
Laboratory for Genome Stability and Human Disease Prevention, Shenzhen University Health Science Center, Shenzhen University, Shenzhen,
c
d
China; Drug Development Department, Shenzhen Honghui Bio-Pharmaceutical Co. Ltd., Shenzhen, China; Collaborative Innovation Center for
Biotherapy, West China Hospital, Sichuan University, Chengdu, China
ABSTRACT
ARTICLE HISTORY
Received 2 March 2020
Revised 7 July 2020
Accepted 10 July 2020
The overexpression of P21-activated kinase 1 (PAK1) is associated with poor prognosis in several cancers,
which has emerged as a promising drug targets. Based on high-throughput virtual screening strategy, tet-
rahydrothieno [2,3-c]pyridine scaffold was identified as an initial lead for targeting PAK1. Herein we
reported our structure-based optimisation strategy to discover a potent PAK1 inhibitor (7j) which
displayed potent PAK1 inhibition and antiproliferatory activity in MDA-MB-231 cells. 7j induced obviously
G2/M cell cycle arrest via PAK1-cdc25c-cdc2 pathway, and also inhibited MAPK-ERK and MAPK-JNK cas-
cade to induce MDA-MB-231 cell death. Together, these results provided a novel chemical scaffold as
PAK1 inhibitor for breast cancer treatment.
KEYWORDS
High-throughput virtual
screening; PAK1 inhibitor;
anti-proliferation; cell cycle
arrests; breast cancer
1
. Introduction
the similarity between the ATP-binding pockets of kinases. Recently, to
achieve kinase selectivity, allosteric PAK1 inhibitors were designed and
P21-activated kinases (PAKs) belong to the STE20 family of serine/
threonine kinases which is comprised of group I (PAK1, PAK2, and
PAK3) and group II (PAK4, PAK5, and PAK6) based on sequence and
structural homology . PAKs functions as GTPase effector that links
the Rho-related GTPases CDC42 and RAC1 to the JNK MAP kinase
pathway that widely involve in cancer cell migration, proliferation,
15
16
synthesised by targeting the specific site, such as AL3 and IPA-3 .
Unfortunately, to date only pan-PAK inhibitor PF-35783099 progressed
into clinical trials but is now stopped because of its poor potency
in vivo. Consequently, there has been significant interest in the identifi-
cation of potent PAK1 inhibitors with novel scaffold that are capability
of clinical development for the breast cancers treatment.
In this study, we aimed to discover novel scaffolds with potential to
optimise as potent PAK1 inhibitors. To achieve this, we identified the tet-
rahydrothieno[2,3-c]pyridine scaffold as a promising lead for targeting
1
2–5
cell cycle and survival mechanisms . Targeting p21-activated Kinase
Inhibits Growth and Metastasis via Raf1/MEK1/ERK Signalling in
1
6
Oesophageal Squamous Cell Carcinoma Cells . In particular, it was
reported that PAK1 gene amplification and protein overexpression
were associated with poor prognosis in a variety of human cancers,
including breast cancer , Non-Small Cell Lung Cancer , renal cell car-
cinoma and so forth. Furthermore, it was shown recently that com-
bination of PAK1 inhibitor (FRAX1036) with taxane treatment could
induce microtubule disorganisation, cell cycle arrests and cellular
apoptosis in the luminal subtype of breast cancer . PAK1 have significant cell cycle arrest and cell death in MDA-MB-231 cells.
PAK1 by using
a high-throughput virtual screening strategy.
Subsequently, a series of tetrahydrothieno [2,3-c]pyridine substitued
benzoyl thiourea derivatives was designed and synthesised based on
structure-based strategy. A potent PAK1 inhibitor (7j) was discovered,
which presented an IC50 value of 209nM and inhibited MDA-MB-231
cell proliferation with an IC50 value of 4.67 lM. In vitro, 7j could induce
7
8
9
1
0
emerged as a promising oncology targets and attracted a lot of Together, these results demonstrated that 7j is a novel potent PAK1
11
pharmacologist interest due to its critical roles in cancers .
inhibitor, which may provide a candidate drug for future cancer therapy.
Several PAK1 inhibitors have been described over the past few
years (Figure 1). The ATP-competing PAK1 inhibitors have been exten-
sively studied, but few chemical scaffolds, mainly including Oxindole/
2
. Results and discussion
12
2.1. The discovery of tetrahydrothieno [2,3-c]pyridine scaffold
by virtual screening
Maleimide-based inhibitors, such as Staurosporine , Aminopyrazole-
13
based inhibitors, such as PF-3758309 , and Aminopyrimidine-based
14
inhibitors, such as FRAX597 . These ATP-competing inhibitors dis- In order to discover potential lead with novel skeleton, we adopt
played high affinity and poor selectivity of PAK isoforms because of a comprehensive screening strategy combining virtual screening
CONTACT Jin Zhang
zhangjinjin8916@163.com
School of Pharmaceutical Sciences, Shenzhen Technology University, Shenzhen, 518060, China; Collaborative
hezhendan@sztu.edu.cn School of
Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China; Zhendan He
Pharmaceutical Sciences, Shenzhen Technology University, 3002 Lantian Road, Pingshan District, Shenzhen, 518060, Guangdong Province, China; School of
Pharmaceutical Sciences, Guangdong Key Laboratory for Genome Stability and Human Disease Prevention, Shenzhen University Health Science Center, Shenzhen
University, Shenzhen, 518060, China
Supplemental data for this article is available online at https://doi.org/10.1080/14756366.2020.1797710.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1525
Figure 1. Some known PAK1 inhibitors.
with enzymic analyses (Figure 2(A)). Firstly, we eliminated com- 2.2. Chemistry
pounds with unfavourable druglike descriptors and physicochemi-
The synthesis of compounds 5a–q was carried out by using com-
mercially available, piperidin-4-one (1) as the starting materials
cal properties, the Chemdiv and Specs chemical library.
Subsequently, these compounds were applied in structure-based
pharmacophore (SBP) virtual screening through the Libdocking
protocol in Discovery Studio 3.5 (DS). Only compounds ranked
(Scheme 1). Piperidin-4-one was protected by t-butyloxycarboryl
to yielded intermediate 3. The Gewald reaction of intermediate 3
with S8 and morpholine gave intermediate 4. Treatment of inter-
mediate 4 with KSCN and Acyl chloride derivatives afforded com-
pounds 5a–q. Furthermore, the compounds 7a–n were prepared
from intermediate 5m (Scheme 2). The synthesis of intermediate 6
was obtained by the deprotection of intermediate 5m with TFA
1
0000 according to the fit scores were retained to access to next
docking. The top 200 hits were obtained by a semi-rigid docking
protocol. Additionally, molecular dynamics based on AMBER 10
were performed to further screen to product 13 hits carrying
diverse scaffolds (Table S1). Subsequently, we conducted the PAK1
inhibitory activity assay to investigate the potency of these hits
purchased commercially. The results showed that hit 2169–1087
with tetrahydrothieno[2,3-c]pyridine scaffold presented 78% inhib-
inCH
2 2
Cl . Treatment of intermediate 6 with acyl chloride deriva-
tives yielded compounds 7a–n.
ition rate against PAK1 at 30 lM (Figure 2(B)). Furthermore, we 2.3. Analysis of the structure-activity relationship
detected its half inhibitory concentration against PAK1, the result
revealed that 2169–1087 showed a weak activity with IC50 value
of 23.5 lM. In cell viability assay, 2169–1087 showed no antiproli-
feratory activity at 50 lM in MDA-MB-231 cells. In spite of this, we
are not absolutely discouraged these poor results. In addition, we
pounds showed significant improvement of PAK1 inhibitory activ-
heavily analysed the binding poses of 2169–1087 and PAK1. As
shown in Figure 2(C), 2169–1087 bound the ATP-binding site, and
the formamide group at 3-site of thiophene was initiated a hydro-
To optimise the lead (2169–1087) based on the inhibitory activity
against PAK1, we preferentially incorporated substituted-benzoyl
thiourea to serve as the suitable linker, yielding compounds 5a–q.
The enzymic inhibitory results revealed that most of these com-
ity compared to the lead (Table 1). Among 5a–e, the methyl
substitution showed no contribution to inhibitory activity, and 3-
methyl (5d) displayed relative benefit. 5e and 5f with 4-site sub-
gen bond with residue GLU345 located kinase hinge of ATP bind-
ing site. The benzoyl moiety at 2-site positioned the hydrophobic
pocket II near the active loop of kinase, and the Boc-group
located the entrance of kinase hinge by hydrophobic interaction.
Collectively, 2169–1087 possessed the basic profiles serving as
stitution presented an activity decrease, suggesting that 4-site
substitution has a negative effect on affinity, which was further
confirmed by 5g, 5j and 5n. Next, some halogen substitution
derivatives (5g–l and 5o) were synthesised, but these compounds
had no significant improvement in activity. Trifluoromethyl group
the frame to optimise PAK1 inhibitor. Structurally, a longer linker was incorporated into 3- and 4 site respectively to obtain com-
should be incorporated to make the hydrophobic group at 2-site pounds 5m and 5n. Intriguingly, 5m showed higher inhibitory
of thiophene occupy the hydrophobic pocket II, and initiated potency with IC50 value of 2.02 lM.
hydrogen bond interactions with the gate control residues of the
According to the above SAR analysis, we hypothesised the sub-
active loop. So benzoyl thiourea group was designed to serves as stitution groups at 6-site of tetrahydrothieno [2,3-c]pyridine might
the linker and some hydrophobic groups were introduced to dis- have a huge influence on affinity. To confirm this suppose, we
cuss the chemical space.
synthesised compounds 7a–n keeping the 3-trifluoromethylphenyl

1
526
DAHONG YAO ET AL.
Figure 2. Virtual screening schematic model for the discovery process of novel PAK1 inhibitors.
Table 1. The PAK1 inhibitory activity of compounds 5a–q
substitution (Table 2). The results revealed that the introduction of
aromatic substituents (7a–c) has an adverse effect on affinity.
Subsequently, some simple alkyl substituents (7d–i) were also dis-
cussed. Among them, only compound 7d showed a slight
improvement in activity compared to 5m. Lastly, we further
designed some cycloalkane substitution derivatives (7j–n).
Unexpectedly, the cyclopropyl substitution (7j) showed a signifi-
cant elevation in activity with IC50 value of 0.21 lM. As the group
size increases, the activity decreased rapidly.
a
Kinase inhibitory activity (IC50, lM)
Compound
R
1
PAK1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
phenyl
7.64 ± 0.72
7.28 ± 0.78
6.21 ± 0.69
4.32 ± 0.51
11.22 ± 1.01
16.38 ± 1.78
9.35 ± 0.97
5.33 ± 0.65
7.82 ± 0.71
9.12 ± 0.95
8.65 ± 0.93
5.15 ± 0.54
2.02 ± 0.27
12.15 ± 1.35
7.43 ± 0.81
19.33 ± 1.85
15.56 ± 1.64
2
.4. Docking and molecular dynamic (MD) simulation
4-methylphenyl
2-methylphenyl
3-methylphenyl
2,4,6-trimethylphenyl
4-methoxyphenyl
4-bromophenyl
4-fluorophenyl
2-fluorophenyl
4-chlorophenyl
2-chlorophenyl
To explore the binding pose of 7j and PAK1, a computational
study including molecular docking, molecular dynamics simulation
and binding free energy calculation were performed. The root-
mean-square deviation (RMSD) of the heavy ligand atoms and the
backbone atom of protein around 8 Å ligand were assessed in
1
00 ns MD simulation, the RMSD fluctuated between 0.4 and 1.0
indicated the system was a well-behaved setup (Figure 3(B)). The
3-chlorophenyl
binding free energy was ꢀ35.20 kcal/mol, the nonpolar term
3-trifluoromethylphenyl
4-trifluoromethylphenyl
2,3-dichlorophenyl
cyclohexyl
(
ꢀ54.16 kcal/mol) played a primary role in 7j binding to PAK1
(Figure 3(C)). Furthermore, a detailed view of the interactions was
displayed in Figure 3(D–F). As presented, the tetrahydrothieno[2,3-
c]pyridine scaffold of 7j located the ATP-binding site and the for-
2-thiophenemethyl
Each compound was tested in triplicate; the data are presented as mamide moiety at 3-site initiated two conserved hydrogen bonds
the mean ± SD.
with residues Glu345 and LEU347 at the kinase hinge.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1527
2 2 8
Scheme 1. General synthesis of compounds 5a–q. Reagents and conditions: (i) triethylamine, CH Cl , r.t., 16 h; (ii) S , morpholine, EtOH, rt, 12 h; (iii) a. KSCN, Acyl chlor-
ide derivatives, acetonitrile, reflux, 6 h.
2 2 2 2 1
Scheme 2. General synthesis of compounds 7a–n. Reagents and conditions: (iv) TFA, CH Cl r.t; (v) CH Cl , R COCl, TEA, r.t.
Additionally, a key hydrogen bond was observed between Thr406 time, 7j inhibited the phosphorylation of PAK1 at Ser199, which
and imide of linker, and the 3-trifluoromethylphenyl occupied the confirmed the inhibition of PAK1 activity after 7j treatment. In
hydrophobic pocket II by hydrophobic interactions. An additional addition, we performed CETSA assay to investigate whether 7j
halogen bond was formed by the trifluoromethyl and residue could directly bind to PAK1. The results demonstrated that as the
Val328 which is a gate control residue at the activity loop. The temperature increasing, 7j could stabilise PAK1, which proved that
cyclopropanecarbonyl positioned the hydrophobic entrance of kin- 7j could directly bind to PAK1 (Figure 4(B)). Taken together, 7j
ase hinge. Collectively, 7j is potent ATP-competing binding could inhibit PAK1 activity in MDA-MB-231 cells.
PAK1 inhibitor.
2.6 7j inhibits MDA-MB-231 cell proliferation and induces G2/M
2.5 7j inhibits PAK1 activity in MDA-MB-231 cells
cell cycle arrest
Next, to confirm whether 7j could inhibit PAK1 activity in MDA- To further explore the in vitro activity of 7j, we firstly detected the
MB-231 cells, we firstly detected the expression and phosphoryl- effect of 7j on MDA-MB-231 cells proliferation. As show in Figure
ation of PAK1 after 7j treatment. As shown in Figure 4(A), 7j did 5(A,B), 7j obviously inhibited the proliferation and colony forma-
not affect PAK1 expression in smaller dose (2.5 lM and 5 lM), but tion ability of MDA-MB-231 cells. Considering the effect of PAK1
suppressed PAK1 expression in a larger dose (10 lM). At the same on cell cycle progression, we next evaluated the cell cycle

1
528
DAHONG YAO ET AL.
Table 2. The PAK1 inhibitory activity of compounds 7a–n.
phosphorylation of p-c-Raf, p-MEK1/2 and p-ERK1/2 and the
results also demonstrated that 7j suppressed MAPK-ERK pathway
(Figure 7(B)). In addition, we investigated the expression of JNK
and c-Jun as well as their phosphorylation. The results illuminated
that 7j inhibited the activation of MAPK-JNK pathway with the
decrease of JNK and c-Jun phosphorylation (Figure 7(B)). In short,
7j could inhibit MAPK-ERK and MAPK-JNK cascade.
a
Kinase inhibitory activity (IC50, lM)
Compound
R
1
PAK1
3
. Conclusion
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
j
k
l
m
n
2-methylbenzoyl
4-chlorobenzoyl
2-chlorobenzoyl
acetyl
10.25 ± 1.13
13.18 ± 1.39
9.37 ± 1.03
1.15 ± 0.97
3.08 ± 0.29
3.87 ± 0.35
5.75 ± 0.63
4.14 ± 0.51
3.42 ± 0.33
0.21 ± 0.05
0.68 ± 0.07
1.09 ± 0.09
4.55 ± 0.51
4.87 ± 0.65
0.023 ± 0.006
In summary, using a high-throughput virtual screening strategy,
we found the lead (2169–1087) with tetrahydrothieno [2,3-c]pyri-
dine scaffold. Based on structure-based optimisation, a series of
novel tetrahydrothieno [2,3-c]pyridine substitued benzoyl thiourea
derivatives was designed, synthesised and screened for inhibitory
activity as novel PAK1 inhibitors. The results revealed 7j displayed
favourable effect in the PAK1 kinase assay and antiproliferative
assay. Although 7j is over 10-fold less PAK1 inhibitory activity in
comparison to the standard FRAX597, the scaffold was first
reported as PAK1 inhibitor, which enriched the few PAK1 inhibitor
skeleton types. In addition, the molecular docking and MD simula-
tions experiments have revealed that the binding pose of 7j with
PAK1 is typical. In vitro, 7j could bind and inhibit PAK1 activity,
and induced PAK1 inhibition regulated cell death. Of note, 7j
induced G2/M cell cycle arrest via PAK1-cdc25c-cdc2 pathway, and
isopropionyl
3-methylbutanoyl
3-chloropropanoyl
(E)-but-2-enoyl
methacryloyl
cyclopropanecarbonyl
cyclobutanecarbonyl
cyclopropanecarbonyl
cyclohexanecarbonyl
tetrahydro-2H-pyran-4-carbonyl
–
FRAX597
a
Each compound was tested in triplicate; the data are presented as
the mean ± SD.
distribution after 7j treatment. The results demonstrated that 7j
induced obviously G2/M cell cycle arrest (Figure 5(C)). Taken
7j also inhibited PAK1 regulated MAPK-ERK and MAPK-JNK path-
way which contribute to cell death (Figure 8). Together, these
together, 7j suppressed MDA-MB-231 cell proliferation and indu- results demonstrated that 7j is a novel potent PAK1 inhibitor,
ces G2/M cell cycle arrest.
which may provide a candidate drug for future cancer therapy.
2
.7. 7j induced G2/M cell cycle arrest via PAK1 regulated
4. Experimental
cdc25c/cdc2 pathway
4.1. Materials and measurements
Subsequently, to detect the mechanism of 7j-induced cell cycle
All commercially available reagents and solvents were used as
received. 1H-NMR spectra were recorded at 400 MHz and 13 C-
NMR data were collected at 100 MHz with complete proton
decoupling. ESI-HRMS spectra of all compounds were recorded by
Synapt G2-SiTM (Q-TOF-MS) equipped with a high-pressure liquid
chromatography (Waters Acquity I-ClassTM). Flash column chroma-
tography was carried out on silica gel (300–400 mesh, Qingdao
Marine Chemical Ltd, Qingdao, China). Thin layer chromatography
arrest in MDA-MB-231 cells, we firstly measured the expression of
Tyr15
p-cdc2
which always be inhibited when cells entry into G2/M
cell cycle. As shown in Figure 6(A), 7j obviously increased p-
Tyr15
cdc2
expression which demonstrated the inhibition of cdc2.
Since cdc25c could active cdc2 by inducing cdc2 dephosphoryla-
tion. We next investigated the expression level of cdc25c and
cyclinB which is the regulatory subunit of cdc2. And we also
detected the expression of Pin1 and NEDD8 which also involved
(TLC) was performed on TLC silica gel 60 F254 plates. Melting
17,18
in cell cycle regulation
decrease the expression of cdc25c, cyclinB1, Pin1 and NEDD8
Figure 6(B)). Next, the knockdown of PAK1 was performed to
. The results revealed that 7j could
points were uncorrected and determined on Shenguang melting
point apparatus (SGW X-4). The purities of all final compounds
were determined by HPLC to be above 95%. HPLC instrument:
Waters 2695/2996 HPLC (Column: YMC-Pack ODS-A C18
(
detect whether 7j induced G2/M cell cycle arrest via PAK1. After
PAK1 knockdown, 7j almost did not affect the phosphorylation of
(
4.6 ꢁ 150 mm, 5 lm), S/N：109DA80059. Elution: MeOH in water;
p-cdc2 at Tyr15, and this confirmed that the increase of p-
Flow rate: 1.0 ml/min.
Tyr15
cdc2
after 7j treatment was mainly induced by PAK1 inhib-
ition (Figure 6(C)). Collectively, these results demonstrated that 7j
induced G2/M cell cycle arrest via PAK1 regulated cdc25-
cdc2 inhibition.
4
.2. Synthesis
4.2.1. Synthesis of intermediate 3
To a solution of 4-piperidone hydrochloride (1.54 g, 10 mmol) in
dichloromethane (30 ml), triethylamine (2.02 g, 20 mmol) was added
at room temperature, after 30 min, Di-tert-butyl dicarbonate (3.27 g,
2
.8. 7j inhibited MAPK-ERK and MAPK-JNK pathways
Considering that MAPK-ERK and MAPK-JNK are two classical path-
15 mmol) was added, and stirred for additional 16 h. The residue was
1
9
ways that contribute to PAK1 regulated cell proliferation , we washed with water (3ꢁ 30 ml), the combined organic layers were
next evaluated whether 7j could affect these pathways. Firstly, we washed with saturated aqueous sodium bicarbonate and brine, and
Thr202/Tyr204
examined the expression and distribution of p-ERK1/2
then dried over anhydrous sodium sulphate. After removing the solv-
in MDA-MB-231 after 7j treatment. As shown in Figure 7(A), 7j ent under reduced pressure to give the crude product. The purity
inhibited its expression and nucleus distribution, which confirmed was prepared by re-crystallization from ethyl alcohol, as a white solid,
1
7j
impede MAPK-ERK activation. Next, we assessed the yield 79%. tert-butyl 4-oxopiperidine-1-carboxylate (3) H-NMR

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1529
Figure 3. 7j is a novel potent PAK1 inhibitor. (A) The structure of 7j; (B) The time evolution of RMSD of backbone atoms for the residues around 5 Å of 7j and heavy
atoms of 7j; (C) The binding free energy calculated by MM/GBSA methods (kcal/mol) of 7j and PAK1. (D) The p interaction surface; (E, F). The detailed interactions
between 7j and PAK1.
(
400 MHz, CDCl ), d(ppm): 3.72 (4H, t, J¼ 6.1 Hz), 2.44 (4H, t,
3
13 6
(30.0 mmol) in anhydrous ethanol (200 ml) was added morpholine
30.0 mmol). The resulting was allowed to reflux for 8 h. The sus-
pension was cooled to ambient temperature and filtered through
a pad of Celite, and washed with absolute ethyl alcohol (30 ml x
J¼ 6.1 Hz), 1.49 (9H, s); C-NMR (100 MHz, DMSO-d ), d(ppm): 207.7,
54.5, 80.4, 42.9, 42.9, 41.1, 41.1, 28.4, 28.4, 28.4
(
1
3
). The combined filtrate was concentrated to dryness. The crude
4
To
.2.2. Synthesis of intermediate 4
product was diluted by 95% ethyl alcohol (30 ml), and filtered to
1
a
solution of tert-butyl 4-oxopiperidine-1-carboxylate give the product as a white solid. H-NMR (400 MHz, DMSO-d6),
(30.0 mmol), 2-cyanoacetamide (30.0 mmol), and sulphur d(ppm): 6.98 (2H, s), 6.59 (2H, brs), 4.27 (2H, s), 3.50 (2H, t,

1
530
DAHONG YAO ET AL.
Ser199
Figure 4. 7j inhibited PAK1 activity in MDA-MB-231 cells. (A) Western blot analysis of PAK1, p-PAK1
ꢂꢂ
in MDA-MB-231 cells treated with 2.5, 5, 10 mM of 7j for
Ser199
ꢂꢂꢂ
p < .001 and
ꢂꢂꢂꢂ
4
8 h. Relative PAK1 and p-PAK1
expression levels were quantified by normalisation to b-actin. p < .01,
treated Control. (B) CETSA assay of PAK1 after 7j treatment. Relative PAK1 expression levels were quantified by normalisation to b-actin. p < .05, compared to DMSO
p < .0001, compared to DMSO
ꢂ
ꢃ
##
ꢃ
treated Control at 48 C. p < .01, compared to DMSO treated Control at 52 C.
13
13
J ¼ 5.4 Hz), 2.67 (2H, t, J ¼ 5.4 Hz), 1.42 (9H, s); C-NMR (100 MHz, J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 2.39 (3H, s), 1.44 (9H, s); C-
DMSO-d ), d(ppm): 168.0, 160.1, 154.2, 129.8, 112.6, 107.7, 79.6, NMR (100 MHz, DMSO-d ), d(ppm): 174.3, 170.7, 166.3, 154.2,
141.3, 135.5, 128.9, 127.8, 127.3, 126.1, 125.2, 122.7, 79.7, 43.0,
6
6
4
2.5, 41.9, 28.5, 28.5, 28.5, 26.5;
4
C
2.3, 36.9, 28.5, 28.5, 28.5, 25.4; HRMS (ESI)þ calculated for
þ
22
27
H N
4 4
O S
2
, [M þ H] : m/z 475.1474, found 475.1477.
4
.2.3. General procedure synthesis of compounds 5a–q
Tert-butyl-3-carbamoyl-2–(3-(2-methylbenzoyl)thioureido)-4,7-
To a solution of KSCN (117.0 mg, 1.2 mmol) in acetonitrile (10 ml),
a solution of R COCl (1.2 eq) in dichloromethane (2 ml) was added
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5c)
1
1
Yellow solid, yield 87%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
dropwise at room temperature. The mixture was allowed to heat
reflux for 10 min. And a solution of intermediate 4 in acetonitrile
1
7
4.38 (1H, s), 11.84 (1H, s), 7.69 (1H, br. s), 7.50 (1H, d, J ¼ 7.4 Hz),
.44 (1H, t, J ¼ 7.4 Hz), 7.33 (1H, br. s), 7.31 (1H, d, J ¼ 8.4 Hz), 7.29
(
10 ml) was added, refluxed for additional 6 h. The suspension was (1H, t, J ¼ 8.4 Hz), 4.51 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t,
1
3
cooled to ambient temperature and filtered through a pad of cel- J ¼ 5.0 Hz), 2.39 (3H, s), 1.44 (9H, s); C-NMR (100 MHz, DMSO-d ),
6
ite, and washed with absolute ethyl alcohol to afford the product d(ppm): 174.5, 169.5, 166.4, 154.2, 141.6, 136.5, 134.4, 131.4, 131.1,
as a yellow solid.
Tert-butyl-2–(3-benzoylthioureido)-3-carbamoyl-4,7-dihydro-
thieno[2,3-c]pyridine-6(5H)-carboxylate (5a)
128.9, 128.6, 125.9, 125.1, 122.6, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5,
þ
25.5, 19.9; HRMS (ESI)þ calculated for C H N O S , [M þ H] : m/z
2
2 27 4 4 2
475.1474, found 475.1476.
1
Yellow solid, yield 83%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
Tert-butyl-3-carbamoyl-2–(3-(3-methylbenzoyl)thioureido)-4,7-
1
7
4.46 (1H, s), 11.73 (1H, s), 7.97 (2H, d, J ¼ 6.6 Hz), 7.71 (1H, br. s), dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5d)
1
.66 (2H, d, J ¼ 6.6 Hz), 7.53 (1H, t, J ¼ 6.7 Hz), 7.35 (1H, br. s), 4.52
6
Yellow, yield 86%. H-NMR (400 MHz, DMSO-d ), d(ppm): 14.46
(
2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.78 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); (1H, s), 11.66 (1H, s), 7.83 (1H, s), 7.76 (1H, d, J ¼ 7.5 Hz), 7.67 (1H,
1
3
C-NMR (100 MHz, DMSO-d
6
), d(ppm): 174.6, 167.5, 166.4, 154.2, br. s), 7.47 (1H, d, J ¼ 7.5 Hz), 7.42 (1H, t, J ¼ 7.5 Hz), 7.37 (1H, br.
1
7
41.5, 133.6, 132.5, 129.2, 129.2, 129.0, 128.9, 128.9, 125.2, 122.8, s), 4.51 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 2.39
1
3
9.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.5; HRMS (ESI)þ calculated for (3H, s), 1.44 (9H, s); C-NMR (100 MHz, DMSO-d ), d(ppm): 174.7,
6
þ
C H N O S , [M þ H] : m/z 461.1317, found 461.1319.
167.5, 166.3, 154.2, 141.4, 138.3, 134.2, 132.3, 129.6, 129.0, 128.8,
126.3, 125.2, 122.7, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.4, 21.3;
2
1 25 4 4 2
Tert-butyl-3-carbamoyl-2–(3-(4-methylbenzoyl)thioureido)-4,7-
þ
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5b)
HRMS (ESI)þ calculated for C H N O S , [M þ H] : m/z 475.1474,
2
2 27 4 4 2
1
Yellow solid, yield 74%. H-NMR (400 MHz, DMSO-d
6
), d(ppm): found 475.1474.
1
4.47 (1H, s), 11.63 (1H, s), 7.89 (2H, d, J ¼ 8.0 Hz), 7.66 (1H, br. s),
Tert-butyl-3-carbamoyl-2–(3-(2,4,6-trimethylbenzoyl)thioureido)-
7
.37 (1H, br. s), 7.34 (2H, d, J ¼ 8.0 Hz), 4.51 (2H, s), 3.57 (2H, t, 4,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate(5e)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1531
Figure 5. 7j inhibited MDA-MB-231 cells proliferation and induced G2/M cell cycle arrest. (A) Cell viability were measured by MTT assay after 7j treated for 24 h and
8 h. (B) Colony formation assay of DMSO or 7j treated MDA-MB-231 cells. (C) MDA-MB-231 cells treated with 2.5, 5, 10 lM 7j for 48 h and subjected to cell cycle ana-
4
lysis following treatment with propidium iodide.
1
Yellow solid, yield 88%. H-NMR (400 MHz, DMSO-d
6
), d(ppm): 56.1, 43.0, 42.3, 28.5, 28.5, 28.5, 25.5; HRMS (ESI)þ calculated for
þ
1
7
2
4.41 (1H, s), 11.92 (1H, s), 7.68 (1H, br. s), 7.63 (1H, d, J ¼ 7.5 Hz),
C
22
27
H N
4 5
O S
2
, [M þ H] : m/z 491.1423, found 491.1425.
.33 (1H, br. s), 6.92 (2H, s), 4.51 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz),
Tert-butyl-2–(3-(4-bromobenzoyl)thioureido)-3-carbamoyl-4,7-
.79 (2H, t, J ¼ 5.0 Hz), 2.26 (3H, s), 2.22 (6H, s), 1.44 (9H, s); C- dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5g)
1
3
1
NMR (100 MHz, DMSO-d
6
), d(ppm): 174.3, 170.3, 166.4, 154.2,
6
Yellow solid, yield 71%. H-NMR (400 MHz, DMSO-d ), d(ppm):
1
7
41.7, 139.2, 134.4, 134.4, 133.5, 128.9, 128.4, 128.4, 125.2, 122.6, 14.43 (1H, s), 11.84 (1H, s), 7.90 (2H, d, J ¼ 8.6 Hz), 7.75 (2H, d,
9.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.4, 21.2, 19.4, 19.4; HRMS (ESI)þ J ¼ 8.6 Hz), 7.67 (1H, br. s), 7.35 (1H, br. s), 4.51 (2H, s), 3.57 (2H, t,
þ
13
calculated
found 503.1788.
for
C
24
H
31
N
O S
4 4 2
,
[M þ H] :
m/z
503.1787, J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); C-NMR (100 MHz,
6
DMSO-d ), d(ppm): 174.5, 166.7, 166.4, 154.2, 141.5, 131.9, 131.9,
Tert-butyl-3-carbamoyl-2–(3-(4-methoxybenzoyl)thioureido)-4,7-
131.7, 131.3, 131.3, 129.0, 127.5, 125.2, 122.7, 79.7, 43.0, 42.3, 28.5,
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5f)
28.5, 28.5, 25.5; HRMS (ESI)þ calculated for C21
4 4 2
H24BrN O S ,
1
þ
Yellow solid, yield 81%. H-NMR (400 MHz, DMSO-d
6
), d(ppm): [M þ H] : m/z 539.0422, found 539.0425.
1
7
3
4.48 (1H, s), 11.84 (1H, s), 8.00 (2H, d, J ¼ 8.8 Hz), 7.66 (1H, br. s),
Tert-butyl-3-carbamoyl-2–(3-(4-fluorobenzoyl)thioureido)-4,7-
.37 (1H, br. s), 7.06 (2H, d, J ¼ 8.8 Hz), 4.51 (2H, s), 3.86 (3H, s), dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5h)
1
3
1
.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); C-NMR
), d(ppm): 174.8, 166.7, 166.3, 163.7, 154.2, 14.32 (1H, s), 12.11 (1H, s), 7.69 (1H, br. s), 7.63 (1H, d, J ¼ 7.5 Hz),
41.4, 131.5, 131.5, 128.9, 125.1, 124.1, 122.8, 114.2, 114.2, 79.7, 7.55 (2H, d, J ¼ 7.6 Hz), 7.45 (2H, d, J ¼ 7.6 Hz), 7.32 (1H, br. s), 4.52
6
Yellow solid, yield 77%. H-NMR (400 MHz, DMSO-d ), d(ppm):
(100 MHz, DMSO-d
6
1

1
532
DAHONG YAO ET AL.
Figure 6. 7j induces G2/M cell cycle arrest via cdc25c/cdc2 pathway. (A) Representative immunofluorescence images of p-cdc2 in MDA-MB-231 cells treated with
ꢂꢂ
DMSO (control) or 5 mM of 7j for 48h. The nuclei was labelled with DAPI (blue). Relative p-cdc2 intensity was quantified by Image J software, p < .01. Scale bar ¼
Tyr15
5
0 lm. (B) Western blot analysis of cdc25c, p-cdc2
, CyclinB1, Pin1 and NEDD8 in MDA-MB-231 cells treated with 2.5, 5, 10 mM of 7j for 48 h. Relative cdc25c, p-
Tyr15
ꢂꢂ
expression levels were quantified by normalisation to b-actin. p < .01,
ꢂꢂꢂ
p < 0.001 and
ꢂꢂꢂꢂ
p < .0001, compared to DMSO treated Control. (C) MDA-MB-
cdc2
2
31 cells transfected with si-NC or si-PAK1 and then treated with 5 lM 7j for 48 h, respectively. The expression of PAK1 and p-cdc2Tyr15 were detected by western
Tyr15
ꢂꢂꢂꢂ
expression levels were quantified by normalisation to b-actin. p < .0001, compared to si-NC treated Control. ns, not significance, com-
blot. Relative p-cdc2
pared to si-PAK1 treated Control.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1533
Figure 7. 7j inhibits MAPK-ERK and MAPK-JNK activation. (A) Representative immunofluorescence images of p-ERK1/2 in MDA-MB-231 cells treated with DMSO (con-
ꢂ
trol) or 5 mM of 7j for 48 h. The nuclei was labelled with DAPI (blue). Relative p-ERK1/2 intensity was quantified by Image J software, p < .05. Scale bar ¼ 20 lm. (B)
Ser259
Ser217/221
Thr202/Tyr204
Western blot analysis of p-c-Raf
, p-MEK1/2
, and p-ERK1/2
in MDA-MB-231 cells treated with 2.5, 5, 10 mM of 7j for 48h. Relative p-MEK1/
Ser217/221
Thr202/Tyr204
ꢂ ꢂꢂꢂ
expression levels were quantified by normalisation to b-actin. p < .05, and p < .001, compared to DMSO treated Control. (C)
2
, and p-ERK1/2
Western blot analysis of JNK, p-JNK, c-Jun and p-c-Jun in MDA-MB-231 cells treated with 2.5, 5, 10 mM of 7j for 48 h. Relative p-JNK, and p-c-Jun expression levels
ꢂ
ꢂꢂ ꢂꢂꢂ
were quantified by normalisation to b-actin. p < .05, p < .01 and p < .001, compared to DMSO treated Control.
(
2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); (1H, d, J ¼ 7.0 Hz), 4.51 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t,
1
3
1
3
C-NMR (100 MHz, DMSO-d
6
), d(ppm): 174.1, 166.5, 166.7, 166.4, J ¼ 5.0 Hz), 1.44 (9H, s);
6
C-NMR (100 MHz, DMSO-d ), d(ppm):
1
54.2, 141.7, 134.8, 132.5, 130.5, 130.0, 129.8, 128.9, 127.6, 125.2, 173.9, 166.4, 164.5, 160.6, 158.9, 154.2, 141.6, 134.6, 130.9, 129.0,
1
22.6, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.4; HRMS (ESI)þ calcu- 125.0, 122.6, 122.5, 116.7, 116.5, 79.7, 43.0, 42.3, 36.9, 28.5, 28.5,
þ
þ
lated for C21
4
H24FN O
4 2
S
, [M þ H] : m/z 479.1223, found 479.1226.
28.5, 25.5; HRMS (ESI)þ calculated for C21
H24FN
4
O
4 2
S
, [M þ H] : m/
Tert-butyl-3-carbamoyl-2–(3-(2-fluorobenzoyl)thioureido)-4,7-
z 479.1223, found 479.1227.
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5i)
Tert-butyl-3-carbamoyl-2–(3-(4-chlorobenzoyl)thioureido)-4,7-
6
), d(ppm): dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5j)
1
Yellow solid, yield 81%. H-NMR (400 MHz, DMSO-d
1
1
4.34 (1H, s), 11.86 (1H, s), 7.71 (1H, d, J ¼ 7.2 Hz), 7.69 (1H, br. s),
6
Yellow solid, yield 77%. H-NMR (400 MHz, DMSO-d ), d(ppm):
7
.65 (1H, t, J ¼ 7.2 Hz), 7.37 (1H, br. s), 7.36 (1H, t, J ¼ 7.0 Hz), 7.34 14.43 (1H, s), 11.84 (1H, s), 7.98 (2H, d, J ¼ 8.6 Hz), 7.67 (1H, br. s),

1
534
DAHONG YAO ET AL.
1
3
1
1
1
2
5
6
.44 (9H, s); C-NMR (100 MHz, DMSO-d ), d(ppm): 174.4, 166.4,
66.2, 154.2, 141.5, 133.6, 133.4, 130.1, 129.9, 129.6, 129.2, 129.0,
28.4, 126.1, 126.0, 125.6, 125.2, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5,
þ
5.4; HRMS (ESI)þ calculated for C22
24
H F
3 4
N O
S
4 2
, [M þ H] : m/z
29.1191, found 529.1195.
Tert-butyl-3-carbamoyl-2–(3-(4-(trifluoromethyl)benzoyl)thiour-
eido)-4,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5n)
1
Yellow solid, yield 73%. H-NMR (400 MHz, DMSO-d
4.44 (1H, s), 12.00 (1H, s), 8.14 (2H, d, J ¼ 7.6 Hz), 7.91 (2H, d,
J ¼ 7.6 Hz), 7.68 (1H, br. s), 7.35 (1H, br. s), 4.52 (2H, s), 3.58 (2H, t,
6
), d(ppm):
1
1
3
J ¼ 5.0 Hz), 2.80 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); C-NMR (100 MHz,
DMSO-d ), d(ppm): 174.4, 166.5, 166.4, 154.2, 141.6, 136.6, 133.3,
32.9, 132.6, 130.2, 130.2, 129.1, 125.7, 125.7, 125.6, 122.9, 122.7,
6
1
7
9.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.6, 25.5; HRMS (ESI)þ calculated
þ
for C H F N O S , [M þ H] : m/z 529.1191, found 529.1193.
2
2 24 3 4 4 2
Tert-butyl-3-carbamoyl-2–(3-(2,3-dichlorobenzoyl)thioureido)-
4
,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5o)
Yellow solid, yield 85%. H-NMR (400 MHz, DMSO-d
1
6
), d(ppm):
1
4.31 (1H, s), 12.18 (1H, s), 7.79 (2H, dd, J ¼ 8.0 1.2 Hz), 7.71 (1H,
br. s), 7.63 (2H, dd, J ¼ 8.0 1.2 Hz), 7.48 (1H, t, J ¼ 7.9 Hz), 7.32 (1H,
br. s), 4.52 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.81 (2H, t, J ¼ 5.0 Hz),
1
3
1
1
1
6
.44 (9H, s); C-NMR (100 MHz, DMSO-d ), d(ppm): 173.8, 166.5,
65.8, 154.2, 141.7, 137.1, 132.6, 132.3, 129.1, 129.0, 128.6, 128.2,
25.4, 122.5, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.6; HRMS (ESI)þ
þ
calculated for C H Cl N O S , [M þ H] : m/z 529.0538,
2
1
23
2 4 4 2
found 529.0543.
Figure 8. The schematic model of 7j-induced cell death pathways, associated
with cell cycle arrest and proliferation inhibition.
Tert-butyl-3-carbamoyl-2–(3-(cyclohexanecarbonyl)thioureido)-
4,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5p)
1
Yellow solid, yield 66%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
1
4.20 (1H, s), 11.47 (1H, s), 7.59 (1H, br. s), 7.34 (1H, br. s), 4.48
(
2H, s), 3.54 (2H, t, J ¼ 5.0 Hz), 2.75 (2H, t, J ¼ 5.0 Hz), 1.79 (2H, d,
7
.61 (1H, d, J ¼ 8.6 Hz), 7.36 (1H, br. s), 4.51 (2H, s), 3.57 (2H, t,
13
J ¼ 12.0 Hz), 1.74 (2H, d, J ¼ 12.0 Hz), 1.63 (1H, m), 1.43 (9H, s),
J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); C-NMR (100 MHz,
1
3
1
1
2
.37 ꢀ 1.16 (6H, m); C-NMR (100 MHz, DMSO-d
6
), d(ppm): 177.1,
DMSO-d ), d(ppm): 174.5, 166.5, 166.4, 154.2, 141.5, 138.4, 131.3,
6
74.8, 166.2, 154.2, 141.2, 128.9, 125.1, 122.8, 79.7, 44.1, 43.0, 42.3,
1
4
31.2, 131.2, 129.0, 128.9, 128.9, 122.7, 114.2, 114.2, 79.7, 43.0,
9.0, 29.0, 29.0, 28.5, 28.5, 28.5, 25.6, 25.4; HRMS (ESI)þ calculated
2.3, 28.5, 28.5, 28.5, 25.4; HRMS (ESI)þ calculated for
þ
þ
for C H N O S , [M þ H] : m/z 467.1787, found 467.1788.
C H ClN O S , [M þ H] : m/z 495.0927, found 495.0931.
21 31 4 4 2
2
1
24
4 4 2
Tert-butyl-3-carbamoyl-2–(3-(2-(thiophen-2-yl)acetyl)thioureido)-
Tert-butyl-3-carbamoyl-2–(3-(2-chlorobenzoyl)thioureido)-4,7-
4
,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5q)
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5k)
1
1
Yellow solid, yield 89%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
Yellow, yield 78%. H-NMR (400 MHz, DMSO-d ), d(ppm): 14.47
6
1
7
4.44 (1H, s), 11.78 (1H, s), 8.06 (2H, t, J ¼ 8.6 Hz), 7.67 (1H, br. s),
(
(
1H, s), 11.63 (1H, s), 7.90 (2H, d, J ¼ 8.0 Hz), 7.67 (1H, br. s), 7.37
.37 (1H, br. s), 7.35 (2H, t, J ¼ 8.6 Hz), 4.51 (2H, s), 3.57 (2H, t,
1H, br. s), 7.34 (2H, d, J ¼ 8.0 Hz), 4.51 (2H, s), 3.57 (2H, t,
13
J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 2.26 (3H, s), 2.22 (6H, s), 1.44
J ¼ 5.0 Hz), 2.79 (2H, t, J ¼ 5.0 Hz), 1.44 (9H, s); C-NMR (100 MHz,
1
3
6
(9H, s); C-NMR (100 MHz, DMSO-d ), d(ppm): 174.3, 170.3, 166.4,
DMSO-d ), d(ppm): 174.7, 167.3, 166.3, 154.2, 144.1, 141.4, 129.5,
6
1
2
29.3, 129.0, 125.1, 122.8, 127.5, 125.2, 122.7, 79.7, 43.0, 42.3, 28.5, 154.2, 141.7, 139.2, 134.4, 134.4, 133.5, 128.9, 128.4, 128.4, 125.2,
8.5, 28.5, 21.6; HRMS (ESI)þ calculated for C H ClN O S , 122.6, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.4, 21.2, 19.4, 19.4; HRMS
21
24
4
4
2
þ
þ
[M þ H] : m/z 495.0927, found 495.0931.
(ESI)þ calculated for
C H N
20 25
O S
4 4
,
3
[M þ H] : m/z 481.1038,
Tert-butyl-3-carbamoyl-2–(3-(3-chlorobenzoyl)thioureido)-4,7-
found 481.1043.
dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5l)
1
Yellow solid, yield 65%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
4
.2.5. Synthesis of compound 6
1
4.42 (1H, s), 11.88 (1H, s), 8.03 (1H, s), 7.90 (1H, d, J ¼ 7.7 Hz), 7.72
To a solution of 5m (1.0 mmol) in dichloromethane (10 ml), tri-
fluoroacetic acid (10 ml) was added. Upon completion removing
the solvent under reduced pressure, the resulting was added to
water (30 ml), and basified with 1 N NaOH till pH ¼ 9, and filtered
(
1H, d, J ¼ 7.8 Hz), 7.69 (1H, br. s), 7.56 (1H, dd, J ¼ 7.8 7.7 Hz), 7.37
(
1H, br. s), 4.51 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.79 (2H, t,
13
J ¼ 5.0 Hz), 1.44 (9H, s);
6
C-NMR (100 MHz, DMSO-d ), d(ppm):
1
1
74.4, 166.4, 166.2, 154.2, 141.7, 134.5, 133.5, 133.2, 130.8, 129.0,
1
give the product as a grey solid, yield 87%. H-NMR (400 MHz,
28.9, 127.9, 125.2, 122.7, 79.7, 43.0, 42.3, 28.5, 28.5, 28.5, 25.4;
þ
HRMS (ESI)þ calculated for
95.0927, found 495.0928.
Tert-butyl-3-carbamoyl-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
eido)-4,7-dihydrothieno[2,3-c]pyridine-6(5H)-carboxylate (5m)
C
21
H24ClN
4
O
4 2
S ,
[M þ H] : m/z DMSO-d
6
), d(ppm): 10.15 (1H, s), 9.97 (1H, s), 8.94 (2H, s), 8.16 (1H,
4
s), 8.15 (1H, d, J ¼ 7.5 Hz), 7.92 (1H, d, J ¼ 7.5 Hz), 7.71 (1H, t,
J ¼ 7.5 Hz), 6.65 (1H, s), 4.22 (2H, s), 3.32 (2H, t, J ¼ 5.4 Hz), 3.17
1
3
(2H, t, J ¼ 5.4 Hz); C-NMR (100 MHz, DMSO-d6), d(ppm): 174.6,
1
Yellow solid, yield 68%. H-NMR (400 MHz, DMSO-d
6
), d(ppm): 166.4, 166.2, 141.8, 141.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6,
1
4.45 (1H, s), 12.07 (1H, s), 8.33 (1H, s), 8.24 (1H, d, J ¼ 7.8 Hz), 8.02 129.4, 129.4, 129.2, 128.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8,
(
1H, d, J ¼ 7.8 Hz), 7.78 (2H, t, J ¼ 7.8 Hz), 7.68 (1H, br. s), 7.36 (1H, 122.5, 121.6, 43.1, 42.1, 26.4; HRMS (ESI)þ calculated for
þ
br. s), 4.52 (2H, s), 3.57 (2H, t, J ¼ 5.0 Hz), 2.80 (2H, t, J ¼ 5.0 Hz),
C
17
16
H F
3 4
N O
S
2 2
, [M þ H] : m/z 429.0667, found 429.0669.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1
1535
4
.2.6. Synthesis of compound 7a–n
Yellow solid, yield 76%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
To a solution of
6
(214.2 mg, 0.5 mmol) and triethylamine 14.45 (1H, s), 12.04 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
(
(
101.1 mg, 1 mmol) in THF (20 ml), a solution of dichloromethane (1H, d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.64 (1H, br. s), 7.36 (1H,
ꢃ
3 ml) was added dropwise at 0 C. The mixture was stirred for 4 h br. s), 4.63 (2H, s), 3.75 (2H, t, J ¼ 5.0 Hz), 2.98 (1H, m), 2.88 (2H, t,
1
3
at room temperature. After completion, the residue was evapo- J ¼ 5.0 Hz), 1.03 (6H, d, J ¼ 6.2 Hz); C-NMR (100 MHz, DMSO-d
),
6
rated and added 50 ml water, extracted with ethyl acetate d(ppm): 175.3, 174.5, 166.4, 166.2, 141.8, 141.4, 133.6, 133.3, 130.1,
(
2 ꢁ 30 ml). The combined organic layers were washed with satu- 129.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9, 127.0, 126.0, 125.6,
rated aqueous sodium bicarbonate and brine, and then dried over 125.2, 123.4, 122.8, 122.5, 121.6, 43.1, 42.1, 30.8, 26.4, 19.7, 19.7;
þ
HRMS (ESI)þ calculated for
99.1085, found 499.1087.
–(3-methylbutanoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7f)
C
21
H
22
F
3
N
4
O
3
S
2
,
[M þ H] : m/z
anhydrous sodium sulphate. After removing the solvent under
reduced pressure to give the crude product. The crude product
was purified by silica sel flash chromatography (dichloromethane/
methanol 10:1) as a yellow solid.
4
6
1
6
Yellow, yield 72%. H-NMR (400 MHz, DMSO-d ), d(ppm): 14.41
6–(2-methylbenzoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
(
1H, s), 12.05 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01 (1H,
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7a)
1
d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.64 (1H, br. s), 7.34 (1H, br.
s), 4.62 (2H, s), 3.70 (2H, t, J ¼ 5.0 Hz), 2.86 (2H, t, J ¼ 5.0 Hz), 2.31
Yellow solid, yield 67%. H-NMR (400 MHz, DMSO-d
4.50 (1H, s), 12.12 (1H, s), 8.38 (1H, s), 8.33 (1H, d, J ¼ 7.8 Hz), 8.03
1H, d, J ¼ 7.8 Hz), 7.61 (2H, t, J ¼ 7.8 Hz), 7.71 (1H, br. s), 7.68 (2H,
d, J ¼ 7.7 Hz), 7.56 (2H, d, J ¼ 7.7 Hz), 7.40 (1H, br. s), 4.83 (2H, s),
6
), d(ppm):
1
1
3
(
2H, d, J ¼ 6.9 Hz), 2.04 (1H, m), 0.91 (6H, d, J ¼ 6.4 Hz); C-NMR
(
(
100 MHz, DMSO-d ), d(ppm): 174.5, 170.9, 166.4, 166.2, 141.8,
6
1
3
141.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2,
28.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.1,
2.1, 41.7, 26.4, 23.0, 22.9, 22.6; HRMS (ESI)þ calculated for
3
.60 (2H, t, J ¼ 5.0 Hz), 2.95 (2H, t, J ¼ 5.0 Hz), 2.43 (3H, s); C-NMR
1
4
C
(
100 MHz, DMSO-d6), d(ppm): 174.6, 166.4, 166.2, 141.8, 141.4,
1
1
1
36.9, 136.4, 133.6, 133.3, 131.3, 130.1, 129.8, 129.8, 129.6, 129.4,
29.4, 129.2, 128.9, 128.4, 127.1, 127.0, 126.0, 125.6, 125.2, 123.4,
þ
22
H
6
24
F
3
N4O
3
S
2
, [M þ H] : m/z 513.1242, found 513.1244.
–(3-chloropropanoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7 g)
22.8, 122.5, 121.6, 43.1, 42.1, 26.4, 19.2; HRMS (ESI)þ calculated
þ
for C H F N O S , [M þ H] : m/z 547.1085, found 547.1091.
2
5
21
3
4
3
2
1
6
Yellow solid, yield 78%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6–(4-chlorobenzoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
1
4.40 (1H, s), 12.06 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7 b)
1
(1H, d, J ¼ 7.8 Hz), 7.66 (2H, t, J ¼ 7.8 Hz), 7.35 (1H, br. s), 7.26 (1H,
Yellow solid, yield 72%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
br. s), 4.64 (2H, s), 3.83 (2H, t, J ¼ 8.0 Hz), 3.72 (2H, t, J ¼ 5.0 Hz),
1
4.50 (1H, s), 12.12 (1H, s), 8.38 (1H, s), 8.33 (1H, d, J ¼ 7.8 Hz), 8.03
1H, d, J ¼ 7.8 Hz), 7.61 (2H, t, J ¼ 7.8 Hz), 7.71 (1H, br. s), 7.68 (2H,
d, J ¼ 7.7 Hz), 7.56 (2H, d, J ¼ 7.7 Hz), 7.40 (1H, br. s), 4.83 (2H, s),
1
3
2
.96 (2H, t, J ¼ 8.0 Hz), 2.87 (2H, t, J ¼ 5.0 Hz); C-NMR (100 MHz,
(
DMSO-d
6
), d(ppm): 174.5, 172.5, 166.4, 166.2, 141.8, 141.4, 133.6,
1
3
133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9, 127.0,
3
.60 (2H, t, J ¼ 5.0 Hz), 2.95 (2H, t, J ¼ 5.0 Hz); C-NMR (100 MHz,
1
2
5
26.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.1, 42.1, 41.1, 37.1,
DMSO-d ), d(ppm): 174.5, 171.1, 166.4, 166.2, 141.8, 141.4, 135.3,
þ
6
6.4; HRMS (ESI)þ calculated for C20
H
19ClF
3 4
N O
S
3 2
, [M þ H] : m/z
1
1
2
5
33.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9,
27.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.1, 42.1,
19.0539, found 519.0540.
(
E)-6-(but-2-enoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thioureido)-
þ
6.4; HRMS (ESI)þ calculated for C H ClF N O S , [M þ H] : m/z
24
19
3 4 3 2
4
,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7 h)
67.0539, found 567.0539.
1
Yellow solid, yield 65%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
6–(2-chlorobenzoyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
1
(
4.47 (1H, s), 12.11 (1H, s), 8.38 (1H, s), 8.28 (1H, d, J ¼ 7.8 Hz), 8.07
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7c)
1H, d, J ¼ 7.8 Hz), 7.70 (1H, br. s), 7.41 (1H, br. s), 6.73 (1H, dd,
ꢃ
1
White solid, mp 166–169 C, yield 76%. H-NMR (400 MHz,
DMSO-d ), d(ppm): 14.50 (1H, s), 12.12 (1H, s), 8.38 (1H, s), 8.33
J ¼ 14.4, 7.5 Hz), 6.67 (1H, d, J ¼ 14.4 Hz), 4.73 (2H, s), 3.87 (2H, t,
6
13
J ¼ 5 Hz), 2.91 (2H, d, J ¼ 5.0 Hz), 1.93 (3H, d, J ¼ 7.5 Hz); C-NMR
(
7
1H, d, J ¼ 7.8 Hz), 8.03 (1H, d, J ¼ 7.8 Hz), 7.61 (2H, t, J ¼ 7.8 Hz),
(
6
100 MHz, DMSO-d ), d(ppm): 174.5, 166.4, 166.2, 165.2, 141.8,
.71 (1H, br. s), 7.68 (2H, d, J ¼ 7.7 Hz), 7.56 (2H, d, J ¼ 7.7 Hz), 7.40
1
1
4
41.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2,
28.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.1,
(
1H, br. s), 4.83 (2H, s), 3.60 (2H, t, J ¼ 5.0 Hz), 2.95 (2H, t,
13
J ¼ 5.0 Hz);
C-NMR (100 MHz, DMSO-d ), d(ppm): 174.5, 171.1,
6
2.1, 26.4, 18.2; HRMS (ESI)þ calculated for C H F N O S ,
2
1 20 3 4 3 2
1
1
1
66.4, 166.2, 141.8, 141.4, 135.3, 133.6, 133.3, 130.1, 129.8, 129.8,
29.6, 129.4, 129.4, 129.2, 128.9, 127.0, 126.0, 125.6, 125.2, 123.4,
þ
[M þ H] : m/z 497.0929, found 497.0933.
6
-methacryloyl-2–(3-(3-(trifluoromethyl)benzoyl)thioureido)-
22.8, 122.5, 121.6, 43.1, 42.1, 26.4; HRMS (ESI)þ calculated for
4
,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7i)
þ
1
C
24
H
6
19ClF
3 4
N O
3 2
S
, [M þ H] : m/z 567.0539, found 567.0547.
Yellow solid, yield 81%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
-acetyl-2–(3-(3-(trifluoromethyl)benzoyl)thioureido)-4,5,6,7-tet-
1
(
4.51 (1H, s), 12.12 (1H, s), 8.38 (1H, s), 8.29 (1H, d, J ¼ 7.8 Hz), 8.07
rahydrothieno[2,3-c]pyridine-3-carboxamide (7d)
1H, d, J ¼ 7.8 Hz), 7.84 (2H, t, J ¼ 7.8 Hz), 7.72 (1H, br. s), 7.40 (1H,
ꢃ
1
Yellow solid, mp 166–169 C, yield 76%. H-NMR (400 MHz,
br. s), 5.32 (1H, br. s), 5.15 (1H, br. s), 4.72 (2H, s), 3.77 (2H, t,
1
3
DMSO-d ), d(ppm): 14.33 (1H, s), 11.97 (1H, s), 8.26 (1H, s), 8.16
6
J ¼ 5.0 Hz), 2.92 (2H, t, J ¼ 5.0 Hz), 1.97 (3H, s); C-NMR (100 MHz,
DMSO-d ), d(ppm): 174.5, 170.7, 166.4, 166.2, 141.8, 141.4, 133.6,
33.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9, 127.0,
(
7
2
1H, d, J ¼ 7.8 Hz), 7.95 (1H, d, J ¼ 7.8 Hz), 7.71 (2H, t, J ¼ 7.8 Hz),
6
.58 (1H, br. s), 7.26 (1H, br. s), 4.54 (2H, s), 3.62 (2H, t, J ¼ 5.0 Hz),
1
13
.81 (2H, t, J ¼ 5.0 Hz), 2.04 (3H, s); C-NMR (100 MHz, DMSO-d ), 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 115.7, 43.1, 42.1,
6
þ
d(ppm): 174.5, 169.2, 166.4, 166.2, 141.8, 141.4, 133.6, 133.3, 130.1, 26.4, 20.6; HRMS (ESI)þ calculated for C H F N O S , [M þ H] :
2
1 20 3 4 3 2
1
1
29.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9, 127.0, 126.0, 125.6, m/z 497.0929, found 497.0931.
25.2, 123.4, 122.8, 122.5, 121.6, 43.1, 42.1, 26.4, 21.6; HRMS (ESI)þ
6-(cyclopropanecarbonyl)-2–(3-(3-(trifluoromethyl)benzoyl)th-
[M þ H] : m/z 471.0772, ioureido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7j)
þ
calculated for
found 471.0774.
C
19
H
18 3
F N
4 3
O S
2
,
1
Yellow solid, yield 61%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
6
-isobutyryl-2–(3-(3-(trifluoromethyl)benzoyl)thioureido)-4,5,6,7-
14.41 (1H, s), 12.03 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
(1H, d, J ¼ 7.8 Hz), 7.78 (2H, t, J ¼ 7.8 Hz), 7.65 (1H, br. s), 7.32 (1H,
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7e)

1
536
DAHONG YAO ET AL.
br. s), 4.64 (2H, s), 3.94 (4H, t, J ¼ 3.9 Hz), 2.92 (2H, d, J ¼ 5.0 Hz),
parameters were set as default. For each docking study, a total of 10
docking poses were retained. The root-mean square deviation (RMSD)
between docking poses were calculated.
13
2
.11 (1H, m), 0.77 (4H, br. s);
6
C-NMR (100 MHz, DMSO-d ),
d(ppm): 174.5, 171.9, 166.4, 166.2, 141.8, 141.4, 133.6, 133.3, 130.1,
1
1
29.8, 129.8, 129.6, 129.4, 129.4, 129.2, 128.9, 127.0, 126.0, 125.6,
25.2, 123.4, 122.8, 122.5, 121.6, 43.1, 42.1, 26.4, 14.4, 7.47, 7.47;
þ
4
.4. Molecular dynamics (MD) simulations
HRMS (ESI)þ calculated for C H F N O S , [M þ H] : m/z
21 20 3 4 3 2
4
97.0929, found 497.0932.
-(cyclobutanecarbonyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
22
The MD simulation was performed by Amber 10 package . The
first restraining energy minimisation was carried out by the steep-
est descent method with 0.1 kcal/molꢄÅ2 restraints for all atoms
of the complexes for 5000 steps. And then, we removed the
restraints of ligand (only restraining the protein) to perform the
second energy minimisation, and another energy minimisation
was made under releasing all the restraints. 5000 steps were set
for each energy minimisation. To handle the long-range
Coulombic interactions, the particle mesh Ewald (PME) summation
was used. The SHAKE algorithm was employed on all atoms cova-
lently bonded to a hydrogen atom, allowing for an integration
time step of 2 fs in the equilibration and subsequent production
runs. The annealed programme was from 0 to 310 K for 50 ps.
Under releasing all the restraints, the system was again equili-
brated for 500 ps. The production phase of the simulations was
run without any restraints for a total of 100 ns.
6
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7k)
1
Yellow solid, yield 68%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
1
4.41 (1H, s), 12.05 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
(
1H, d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.64 (1H, br. s), 7.34 (1H,
br. s), 4.61 (2H, s), 3.70 (2H, t, J ¼ 5.0 Hz), 3.43 (1H, m), 2.82 (2H, t,
J ¼ 5.0 Hz), 2.22 (1H, m), 2.19–2.12 (3H, m), 1.92 (1H, m), 1.76 (1H,
1
3
m);
C-NMR (100 MHz, DMSO-d ), d(ppm): 174.5, 172.9, 166.4,
6
1
1
1
66.2, 141.8, 141.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4,
29.4, 129.2, 128.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5,
21.6, 43.5, 42.3, 37.1, 36.8, 36.8, 26.4, 17.9; HRMS (ESI)þ calcu-
þ
lated for C H F N O S , [M þ H] : m/z 511.1085, found 511.1086.
2
2 22 3 4 3 2
6
-(cyclopentanecarbonyl)-2–(3-(3-(trifluoromethyl)benzoyl)th-
ioureido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxa-
mide (7 l)
1
Yellow solid, yield 78%. H-NMR (400 MHz, DMSO-d
6
), d(ppm):
1
4.44 (1H, s), 12.05 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
1H, d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.64 (1H, br. s), 7.34 (1H,
br. s), 4.61 (2H, s), 3.74 (2H, t, J ¼ 5.0 Hz), 2.88 (2H, t, J ¼ 5.0 Hz),
(
4
.5. Cell culture, antibodies and reagents
1
3
MDA-MB-231 were purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA). Cells were cultured in
DMEM with 10% foetal bovine serum and incubated with 5% CO2.
Antibodies used in this study were as follows: cdc25c (4688, CST),
pcdc2
2
.68 (1H, m), 1.71–1.64 (4H, m), 1.37–1.33 (4H, m);
C-NMR
(
100 MHz, DMSO-d ), d(ppm): 174.5, 170.0, 166.4, 166.2, 141.8,
6
1
1
4
41.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2,
28.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.5,
Tyr15
(4539, CST), cyclinB1 (4135, CST), PAK1 (ab223849, CST),
2.3, 40.8, 29.7, 29.7, 26.4, 26.0, 21.8; HRMS (ESI)þ calculated for
Ser199
þ
p-PAK1
(ab192814, abcam), JNK (9252, CST), p-JNK (9255,
C
23
H
6
23
F
3 3
N4O S
2
, [M þ H] : m/z 525.1242, found 539.1268.
Ser259
CST), c-Jun (9165, CST), p-c-Jun (3270, CST), p-c-Raf
CST), p-MEK
b-actin (3700, CST). MTT (M2128) was purchased from Sigma-
Aldrich (St. Louis, MO, USA).
(9421,
-(cyclohexanecarbonyl)-2–(3-(3-(trifluoromethyl)benzoyl)thiour-
Ser217/221
Thr202/Tyr204
(9154, CST), p-ERK
(9101, CST),
eido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (7 m)
1
Yellow solid, yield 73%. H-NMR (400 MHz, DMSO-d
4.44 (1H, s), 12.05 (1H, s), 8.33 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
1H, d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.64 (1H, br. s), 7.34 (1H,
br. s), 4.61 (2H, s), 3.74 (2H, t, J ¼ 5.0 Hz), 2.88 (2H, t, J ¼ 5.0 Hz),
6
), d(ppm):
1
(
1
3
4.6. Cell viability assay
2
.68 (1H, m), 1.71–1.64 (5H, m), 1.37–1.33 (5H, m);
C-NMR
4
(
100 MHz, DMSO-d ), d(ppm): 174.5, 170.0, 166.4, 166.2, 141.8, Cells were dispensed in 96-well plates at a density of 5 ꢁ 10
6
1
1
4
41.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4, 129.2, cells/ml. After 24 h or 48 h incubation, cells were treated with dif-
28.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6, 43.5, ferent concentrations of compounds for the indicated time peri-
2.3, 40.8, 29.7, 29.7, 26.4, 26.0, 26.0, 21.8; HRMS (ESI)þ calculated ods. Cell viability was measured by the MTT assay.
þ
for C H F N O S , [M þ H] : m/z 539.1398, found 539.1398.
2
4 26 3 4 3 2
6
-(tetrahydro-2H-pyran-4-carbonyl)-2–(3-(3-(trifluoromethyl)ben-
zoyl)thioureido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxa-
4.7. Immunofluorescence analysis
mide (7n)
For immunofluorescence staining, non-specific antibody binding
was blocked by incubating with PBS containing 1.5% goat serum.
The MDA-MB-231 cells were sequentially incubated, starting with
1
Yellow solid, yield 71%. H-NMR (400 MHz, DMSO-d ), d(ppm):
6
1
4.46 (1H, s), 12.03 (1H, s), 8.32 (1H, s), 8.23 (1H, d, J ¼ 7.8 Hz), 8.01
1H, d, J ¼ 7.8 Hz), 7.77 (2H, t, J ¼ 7.8 Hz), 7.63 (1H, br. s), 7.32 (1H,
br. s), 4.39 (2H, s), 3.61 (4H, t, J ¼ 3.9 Hz), 3.43 (2H, d, J ¼ 5.0 Hz),
(
Tyr15
Thr202/Tyr204
p-cdc2
antibody (1:200), p-ERK1/2
antibody (1:200)
ꢃ
diluted in PBS containing 1% BSA incubated overnight at 4 C, fol-
lowed by addition of fluorescent-labelled secondary antibodies for
1
3
3
.17 (4H, t, J ¼ 3.9 Hz), 2.86 (2H, t, J ¼ 5.0 Hz), 2.50 (1H, m); C-
NMR (100 MHz, DMSO-d ), d(ppm): 174.5, 166.4, 166.2, 163.2,
41.8, 141.4, 133.6, 133.3, 130.1, 129.8, 129.8, 129.6, 129.4, 129.4,
6
1
h at room temperature.
1
1
6
29.2, 128.9, 127.0, 126.0, 125.6, 125.2, 123.4, 122.8, 122.5, 121.6,
6.4, 66.4, 45.4, 44.3, 30.8, 30.8, 25.4; HRMS (ESI)þ calculated for
4.8. Western blot
þ
C
23
H
24 3
F N
O S
4 4 2
, [M þ H] : m/z 541.1191, found 541.1193.
Cells were treated with 7j for indicated times. Both adherent and
floating cells were collected. Western blot analysis was carried
out. Briefly, the cell pellets were resuspended with lysis buffer
consisting of Hepes 50 mM pH 7.4, Triton-X-100 1%, sodium ortho-
vanada 2 mM, sodium fluoride 100 mM, edetic acid 1 mM, PMSF
4.3. Molecular docking
2
0
The discovery Studio 3.5 docking program was adopted here . The
2
1
preparation of protein structure (PDB code 4ZY5) , including adding
hydrogen atoms, removing water molecules, and assigning Charmm
forcefield. Goldscore was selected as the score function, and the other
1 mM, aprotinin (Sigma, MO, USA) 10 mg/L and leupeptin (Sigma)
ꢃ
1
0 mg/L and lysed at 4 C for 1 h. After 12,000 rpm centrifugation

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1537
for 15 min, the protein content of supernatant was determined by Statistical comparisons were made by One-way ANOVA and
the Bio- Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, Student’s t-test. p < .05 was considered statistically significant.
USA). Equal amounts of the total protein were separated by
1
0–15% SDS-PAGE and transferred to PVDF membranes, the mem-
Disclosure statement
branes were soaked in blocking buffer (5% skimmed milk or BSA).
Proteins were detected using primary antibodies, followed by
HRP-conjugated secondary antibody and visualised by using ECL
as the HRP substrate. Quantification of immunoblot was per-
formed by Quantity One 4.4.
The authors declare no conflict of interest.
Author contributions
Jin Zhang and Zhendan He conceived and designed the experi-
ments; Dahong Yao, performed the experiments. Dahong Yao,
Jian Huang and Jinhui Wang wrote and revised the paper.
4
.9. Cell cycle assay
For cell cycle detection, MDA-MB-231 cells were treated with 2.5,
ꢃ
5
, 10 lM 7j for 48 h and then ethyl alcohol-fixed at 4 C for 24 h.
Then the cell cycle distribution were determined by flow cytome- Funding
try analysis of PI staining.
This work was supported by National Natural Science Foundation
of China [Grant No. 81803365]; Post-Doctor Research Project
2018M643510, 2019M650213]. The National Key R & D Program
[
4
.10. Transfection assays
Cells were transfected with PAK1 (6361, CST) and negative control
6568, CST) siRNAs at 100 nM final concentration using
of China [2018ZX09735005, 2017YFA0503900]; Foundation
Committee of basic and applied basic research of Guangdong
Province (2019B1515120029, 2019A1515110482] and Shenzhen
Natural Science Foundation [JCYJ20190808171803553]. Post-
Doctor Research Project of West China Hospital, Sichuan
University [Grant No. 2018HXBH065]. Figure 8 are created with
BioRender.com, we thank this professional website and we appre-
ciate their generosity and kindness.
(
Lipofectamine RNAiMAX/Lip3000 reagent (Invitrogen) according to
the manufacturer’s instructions. The transfected cells were used
for subsequent experiments 48 h later.
4.11. The enzymatic assay
19
These assays were carried out as described previously . All of the
ꢃ
enzymatic reactions were conducted at 30 C for 40 min. The 50 ml
reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, References
0
.1 mg/ml BSA, 1 mM DTT, 50 mM ATP, 0.2 lg/ml PAK1 and 100 uM
lipid substrate. The compounds were diluted in 10% DMSO and
ml of the dilution was added to a 50 ml reaction so that the final
1
.
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2
.
concentration of DMSO is 1% in all of the reactions. The assay
was performed using the Kinase-Glo Plus luminescence kinase
assay kit and ADP-Glo Plus luminescence kinase assay kit. It meas-
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.12. Cellular thermal shift assay (CETSA)
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23
intact cells, was analysed as described before . Briefly, cells cul-
tured in 100 ꢁ 20 mm tissue culture dishes at 90% confluence
were treated with media containing DMSO or 7j (10 lM) for 24 h.
After that cells were detached with trypsin and re-suspended in
PBS. The cell suspension was heated for 3 min to 48, 52, and
7
.
.
ꢃ
5
6 C. Subsequently, cells were lysed using liquid nitrogen and
two repeated cycles of freeze-thaw. Precipitated proteins were
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