Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition

Article abstract of DOI:10.1016/j.bmcl.2019.05.021

Structure based virtual screening attempts to discover DUSP1 inhibitors have yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its analogues were synthesized to explore structure activity relationship (SAR) of DUSP1 inhi

Full text of DOI:10.1016/j.bmcl.2019.05.021

Accepted Manuscript
Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition
Bo Yeon Kim, Ji Hee Yoon, Myeongbin Kim, Jae Nyoung Kim, Hwangseo Park,
Seong Eon Ryu, Sangku Lee
PII:
S0960-894X(19)30318-X
https://doi.org/10.1016/j.bmcl.2019.05.021
BMCL 26441
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 April 2019
1 May 2019
13 May 2019
Please cite this article as: Yeon Kim, B., Hee Yoon, J., Kim, M., Nyoung Kim, J., Park, H., Eon Ryu, S., Lee, S.,
Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.021
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Synthesis and biological evaluation of acylthiourea against DUSP1
inhibition
Bo Yeon Kim,a Ji Hee Yoon,a Myeongbin Kim,b Jae Nyoung Kim,c Hwangseo Park,d
Seong Eon Ryub,* and Sangku Leea,*
aAnticancer Agent Research Center, KRIBB, Cheongju 28116, Republic of Korea
bDepartment of Bioengineering, Hanyang University, Seoul 04763, Republic of Korea
cDepartment of Chemistry, Chonnam National University, Gwangju 61186, Republic of
Korea
dDepartment of Bioscience and Biotechnology, Sejong University, Seoul 05006, Republic of
Korea
*Corresponding Author. E-mail: sangku@kribb.re.kr (SL); ryuse@hanyang.ac.kr (SER)
Key Words: Dual specificity phosphatase, Mitogen-activated protein kinase, Enzyme
inhibition, Structure activity relationship, Cancer
Abstract: Structure based virtual screening attempts to discover DUSP1 inhibitors have
yielded a scaffold featuring benzoxazole and acylthiourea pharmacophore. A series of its
analogues were synthesized to explore structure activity relationship (SAR) of DUSP1
inhibition.
Dual specificity phosphatases (DUSPs) represent a large family of enzymes that are able to
dephosphorylate both phospho-tyrosine and phospho-threonine residues on mitogen-activated
1

protein kinases (MAPKs).1,2 Because the MAPK family play a critical role in the cellular
signalling pathways leading to the regulation of cell growth and inflammation,3 regulating
MAPK-mediated pathways is a therapeutically attractive strategy. DUSP1, known as
mitogen-activated protein kinase phosphatase 1 (MKP1), is frequently activated in a variety
of human diseases including cancers,4 depression,5 arthritis,6 Alzheimer’s disease7 and
asthma.8 In this regard, DUSP1 represents attractive potential drug targets.
Three-dimensional structure of DUSP1 was reported in complex with a substrate analogue
(PDB entry: 6APX).9 The presence of structural information on the interaction of a small-
molecule ligand in the active site would make it possible to design the competitive inhibitors
that can develop into a drug candidate. The discovery of druggable inhibitors can be
accelerated to a great extent when assisted with the effective computer-aided molecular
design.
scaffold 1
C
W
A
B
HN
O
HN
S
X
Y
O
N
X, Y, W = hydrophobic groups
Z = polar moieties
Z
Figure 1. Structure of scaffold 1 (1).
Structure based virtual screening with the automated AutoDock program10 yielded a few
weak DUSP1 inhibitors sharing the molecular core shown in Figure 1. The structure of
scaffold 1 (1) features benzoxazole and acylthiourea pharmacophore. Herein, we designed
and synthesized its analogues to explore structure activity relationship (SAR) of DUSP1
inhibition for a potential small molecule lead in the development of new therapeutics against
cancers and neurological diseases.
2

NH2
NO2
NO2
TCICA
PPh3
X
Y
X
Y
OH
X
Y
O
N
H2
O
N
+
HO2C
Pd/C
NH2
1,4-dioxane
MW
(98%)
Z
Z
Z
2
4
5
3
(79%)
1
X
Z
Y
W
W
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
Cl
H
Cl
H
Cl
H
CH3
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
CH3
C2H5
t-Bu
Br
HN
W
COCl
O
HN
6
S
X
Y
H
O
CH3
C2H5
t-Bu
Br
H
2-F
Br
NH4SCN
(79%)
N
1
Z
Br
CH3
CH3
OCH3
OCH3
O
O
S
Br
S
CH3
NH
NH
N
S
N
NH
NH
S
7
8
O
O
S
H3C
S
CH3
NH
NH
Cl
NH
10
NH
N
9
O
O
S
Br
S
CH3
NH
NH
H3C
NH
H3C
NH
N
12
N
11
Scheme 1. Synthesis of scaffold 1 analogues.
Our synthetic approach to the scaffold 1 was outlined in Scheme 1. Commercially available
2-aminophenol 2 was reacted with 3-nitrobenzoic acid 3 in the presence of
trichloroisocyanuric acid (TCICA) and PPh3 in 1,4-dioxane under microwave irradiation to
yield benzoxazole 4. When the reaction mixture was refluxed under classical heating, the
intermediate amide prior to the formation of benzoxazole was mainly formed even at longer
reaction times. However, heating by microwave irradiation enhanced the benzoxazole
formation in terms of yield and short reaction times. Reduction of the nitro group in
compound 4 with Pd/C afforded amine 5, which was reacted in refluxing acetone with the
3

intermediate aroyl thiocyanate formed by treatment of acyl chloride 6 with ammonium
thiocyanate, to afford acylthiourea 1 in 60-95% yield.
Table 1. Inhibitory activity of synthesized compounds against DUSP1
Compd
MKP1(20μM) VHR(20μM) MKP1(IC50)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
7
23
26
47
91
10
34
33
52
88
14
21
41
8
34
16
50
80
20
44
19
63
82
15
61
37
11
5
74
59
48
13
102
103
99
103
101
99
16
6.6
21
-
7.9
16
8.3
27
-
8.7
25
17
4.8
2.7
35
17
19
11
-
3
42
52
53
19
99
103
98
103
98
95
8
9
10
11
12
-
-
-
-
-
To explore structure-activity relationship (SAR) of scaffold 1 (1) and DUSP1 inhibition, we
synthesized a series of 18 analogues with modifications in three functional groups of the 1
pharmacophore, and additionally 6 analogues lacking benzoxazole moiety. Inhibitory activity
of the synthesized compounds against DUSP1 (mitogen-activated protein kinase phosphatase
1 (MKP1)) was listed in table 1. Chemical complementation assays11 showed that compound
7-12, which lacked benzoxazole moiety did not inhibit MKP1, although the others 1a-r
inhibited the dual specificity phosphatases MKP1 and VHR. Inhibitory activity of compounds
was selective towards either MKP1 or VHR depending on substituents. For example, 1m and
1n were specific to MKP1, whereas 1b and 1r were specific to VHR, consistent with
structural differences in the active site of the two enzymes.12 The substituents in A moiety
(benzoxazole) of scaffold 1 influence the activity, but lacked the correlation between the
substituent and biological activity. Electron donating substituent could increase the inhibitory
activity (1a vs 1k, 1b vs 1p) or decrease the activity (1e vs 1m vs 1n). The substituent in B
4

moiety did not influence the activity (1a vs 1f, 1b vs 1g) and there was no difference (1a vs
1f, 1b vs 1g) in inhibitory activity whether the substituent is electron donating or electron
withdrawing group. The presence of electron withdrawing group in C moiety increased
inhibitory activity against MKP1 (1e, 1m, 1n). Among the tested compounds, compound 1n
exhibited the strongest inhibitory activity. The SAR studies revealed a strong correlation
between the substituent of C moiety and inhibition of cellular DUSP1. The existence of
electron withdrawing substituent in C moiety of scaffold 1 was crucial to inhibitory activity
against mitogen-activated protein kinase phosphatase 1 (MKP1).
In conclusion, a series of novel acylthiourea derivatives were synthesized through efficient
synthetic routes and evaluated for inhibitory activity against mitogen-activated protein kinase
phosphatase 1 (MKP1). The SAR studies revealed that the existence of electron withdrawing
substituent in C moiety of scaffold 1 was crucial to the inhibitory activity. Among all the
compounds designed, compound 1n possessing a bromine functional group showed the
strongest inhibitory activity.
Acknowledgements
This research was supported by a grant from KRIBB Research Initiative Program.
References and Notes
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Friess, H. Gastroenterology 2003, 124, 1830-1845; (b) Wang, H. Y.; Cheng, Z.;
Malbon, C. C. Cancer Lett. 2003, 191, 229-237.
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11. The gene for catalytic domain of DUSP1 corresponding to residues 152-323 was
cloned into NdeI and XhoI restriction sites of the expression vector pET21b. Cells
containing the vector were induced with 0.1mM IPTG and further grown at 18 C for
16 hours. Cell pellets were resuspended in the lysis buffer containing 50 mM Tris-
HCl (pH 7.5), 0.5 M NaCl, 1mM PMSF, and 0.05% -mercaptoethanol. The
resuspension was then lysed by sonication on ice. The His-tagged DUSP1 catalytic
domain was purified by a TALON crude column and dialyzed against 20 mM Tris-
HCl (pH 8.0), 0.5 mM EDTA, 200 mM NaCl and 10 mM -mercaptoethanol. The
enzyme assay was performed by monitoring the extent of hydrolysis of 6,8-difluoro-
4-methyl-umbelliferyl phosphate (DiFMUP) with spectrofluorometric analysis. The
purified DUSP1 (20 nM), DiFMUP (2.5 M), and candidate inhibitors were
incubated in the reaction mixture containing 20 mM Tris-HCl (pH 8.0), 0.01%
Trition X-100, and 5 mM DTT for 30 minutes. The resulting fluorescence was
measured by using the Perkin Elmer 2030 multiplate reader with excitation and
emission wavelengths of 355 nm and 460 nm, respectively. IC50 values of the potent
inhibitors were determined from direct regression curve analysis in duplicate.
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Hwang, H. S.; Ryu, S. Y.; Park, H.; Kim, D. S.; Kim, S. J.; Ryu, S. E. Acta
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T.; Raran-Kurussi, S.; Tropea, J. E.; Cherry, S.; Waugh, D. S. 2008, Protein Sci. 27,
561-567.
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Graphical Abstract
Synthesis and biological evaluation of acylthiourea against DUSP1 inhibition
Bo Yeon Kim, Ji Hee Yoon, Myeongbin Kim, Jae Nyoung Kim, Hwangseo Park, Seong Eon
Ryu and Sangku Lee
W
HN
O
HN
S
X
Y
O
N
Z
7