Design, synthesis and biological evaluation of imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as lymphoid-specific tyrosine phosphatase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104124

Lymphoid-specific tyrosine phosphatase (LYP), which exclusively exists in immune cells and down-regulates T cell receptor signaling (TCR), has becoming a potent target for various autoimmune diseases. Herein, we designed and synthesized imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as new LYP inhibitors. Among them, the cinnamic acids-based inhibitors (9p and 9r) displayed good LYP inhibitory activities (IC₅₀ = 2.85–6.95 μM). Especially, the most potent inhibitor 9r was identified as competitive inhibitor (K_i = 1.09 μM) and bind LYP reversibly. Meanwhile, 9r exhibited better selectivity over other phosphatases than known LYP inhibitor A15. Furthermore, compound 9r could regulate TCR associated signaling pathway in Jurkat T cell.

Full text of DOI:10.1016/j.bioorg.2020.104124

Journal Pre-proofs
Design, synthesis and biological evaluation of imidazolidine-2,4-dione and 2-
thioxothiazolidin-4-one derivatives as lymphoid-specific tyrosine phosphatase
inhibitors
Xiao Liang, Huansheng Fu, Peng Xiao, Hao Fang, Xuben Hou
PII:
S0045-2068(20)31421-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.104124
YBIOO 104124
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To appear in:
Bioorganic Chemistry
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25 December 2019
9 July 2020
20 July 2020
Please cite this article as: X. Liang, H. Fu, P. Xiao, H. Fang, X. Hou, Design, synthesis and biological evaluation
of imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives as lymphoid-specific tyrosine phosphatase
inhibitors, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104124
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Design, synthesis and biological evaluation of imidazolidine-
2,4-dione and 2-thioxothiazolidin-4-one derivatives as
lymphoid-specific tyrosine phosphatase inhibitors
Xiao Liang1, Huansheng Fu1,2, Peng Xiao3, Hao Fang1,* and Xuben Hou1,*
1
Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Science,
Shandong University, Ji’nan, Shandong, P.R. China
2 The Second Hospital of Shandong University, Shandong University, Ji’nan, Shandong, P.R. China
3 Department of Molecular Biology and Biochemistry, School of Medicine, Shandong University,
Ji’nan, Shandong, P.R. China
Abstract
Lymphoid-specific tyrosine phosphatase (LYP), which exclusively exists in immune
cells and down-regulates T cell receptor signaling (TCR), has becoming a potent target
for various autoimmune diseases. Herein, we designed and synthesized imidazolidine-
2,4-dione and 2-thioxothiazolidin-4-one derivatives as new LYP inhibitors. Among
them, the cinnamic acids-based inhibitors (9p and 9r) displayed good LYP inhibitory
activities (IC50 = 2.85-6.95 μM). Especially, the most potent inhibitor 9r was identified
as competitive inhibitor (Ki= 1.09 μM) and bind LYP reversibly. Meanwhile, 9r
exhibited better selectivity over other phosphatases than known LYP inhibitor A15.
Furthermore, compound 9r could regulate TCR associated signaling pathway in Jurkat
T cell.
*Corresponding authors
E-mail: hxb@sdu.edu.cn (X.H.); haofangcn@sdu.edu.cn (H.F.)
keywords: lymphoid-specific tyrosine phosphatase; inhibitor; autoimmune diseases
Introduction
Protein tyrosine phosphatases (PTPs) play important roles in various cellular
processes as they control the phosphorylation level of protein tyrosine coordinately[1].
In the past two decades, attributed to recent biomedical research findings, PTP members
have been implicated in many human diseases, including cancer, diabetes, autoimmune
as well as neurological diseases[1-3]. Nowadays, PTPs have been recognized as
sparkling target in future drug discovery[4].
As a member of classical non-receptor PTPs, lymphoid-specific phosphatase (LYP)
is encoded by PTPN22 and expressed exclusively in lymphocyte (mature B cell and T
cell) and hematopoietic cell (thymocyte)[5]. The C-terminal of LYP consist P1-P4
proline-rich motifs, and the N-terminal of LYP contains P-loop, LYP-specific insert
and WPD-loop. Specially, the conformation of WPD-loop is crucial for LYP to
recognize substrate/ligand[6]. In T cells, LYP can negatively regulate T cell receptor
1

signaling (TCR) by dephosphorylating several early TCR signal transduction mediators,
such as LCK (Y394) and ZAP70 (Y493)[7, 8]. Subsequently, through the downstream
Ras-MEK-ERK signaling pathway, LYP inhibits the activation and proliferation of T
cells[9].
Remarkably, a single-nucleotide polymorphism (SNP) (C1858T) in the PTPN22
gene is strongly associated with many autoimmune diseases, such as type 1 diabetes
(T1D)[10], rheumatoid arthritis (RA)[11], systemic lupus erythematosus (SLE)[12] and
Graves’ disease[13]. Interestingly, the PTPN22 C1858T SNP encodes LYP-R620W
mutant, which possess improved functional activity than wildtype enzyme. Therefore,
the LYP-R620W mutant has enhancive inhibition to TCR signaling, and the gain-of-
function phenotype then triggered several human autoimmune diseases[9]. Beyond that,
LYP can downregulate the MDP-induced autophagy[14], which modulates the NLRP3
activation and secretion IL1B[15].
Given LYP performs as a potential target of autoimmune diseases, various
inhibitors with different chemotypes have been developed (Figure 1.). For example,
Zhang’s group identified the salicylic acid-based LYP inhibitor I-C11[6], which
exhibited good selectivity among pest-enriched subfamily PTPs and cellular activity.
Through structure-based lead optimization, LYP inhibitor 8b was discovered in 2013
the with improved activity[16]. Notably, the breakthrough of structural biology laid the
foundation of computational strategy to rapid screen of bio-active inhibitors. Novel
LYP inhibitor A15 was identified by our group using the combination of ligand- and
structure-based virtual screening methods[17]. It was noteworthy that a selectively
allosteric LYP inhibitor (NC1) was obtained by structural modification of A15[18].
Herein, we simplified the thiazolo [3,2-a] pyrimidin core of A15[18] to
midazolidine-2,4-dione and 2-thioxothiazolidin-4-one through scaffold hopping. New
imidazolidine-2,4-dione and 2-thioxothiazolidin-4-one derivatives were designed by
introducing different substituents in the side chain. LYP inhibitory activities of these
newly designed compound were tested to explore the structure-activity relationship
(SAR). Importantly, the most active cinnamic acid-based compound 9r possessed
improved potent LYP inhibition activity (IC50 = 2.85μM) as well as better PTPs
selectivity (> 5.4-folds) compared to A15. In addition, 9r exhibited a reversible and
competitive inhibition mode against LYP. Furthermore, cellular test indicated that 9r
could efficiently improve the phosphorylation level of LCK and ERK by inhibiting
LYP, thereby regulate TCR signaling.
2

O
O
N
H
O
O
HO
HO
O
HO
HO
N
N
N
N
Cl
H
O
O
I-C11, IC50 = 4.6
PNAS, 2007
8b, IC50 = 0.26
μM
J.Med. Chem., 2013
μM
H3C
CH3
N
O
O
H3C
N
O
O
O
S
N
O
H3C
H3C
O
N
O
O
O
S
HO
H3C
N
Me
OH
O
A15, IC50 = 6.1
μM
J.Med. Chem., 2014
NC1, K = 4.3
μM
i
J. Biol. Chem., 2019
Figure 1. Structures of reported LYP inhibitors[6, 16-18].
2. Results and Discussions
2.1. Chemistry
The synthesis routes of the target compounds 5a-5e and 9a-9s are depicted in
Schemes 1-2. As exhibited in Scheme 1, compounds 3a-3e were obtained from N-
substituted reaction of hydantoin or Bucherer-Bergs reaction of aniline. Through
nucleophilic substitution reaction with methyl benzoate 4a, compounds 3a-3e were
transformed to the di-alkylated intermediates, and then immediately followed by basic
hydrolysis reaction of ester to attain the target compounds 5a-5e. As shown in Scheme
2, the N-substituted rhodanine compounds 7a-7m were synthesized by cyclization from
secondary amine 6a-6m through either carbon disulfide and sodium chloroacetate or
bis(carboxymethyl) trichiocarbonate. And the starting materials 8a-8f were
commercially free or easily prepared by Williamson reaction based on literature[19].
The intermediates 7a-7m and compounds 8a-8f got aldol condensation reaction, then
finally formed the target compounds 9a-9s.
O
HN
O
R1 Br
+
NH
a or b
O
O
2
1a-1d
R1
R1
d
R2
N
N
+
Br
N
NH
R2
O
O
NH2
c
3a-3e
4a
5a-5e
1e
Scheme 1. Synthesis of target compounds 5a-5f. Regants and conditions: (a)DBU, DMF, 60 ℃, 12h;
(b)K2CO3, DMF, RT, 1h; (c) (i) triphosgene, CH2Cl2, 0℃, 0.5 h; (ii) glycinemethylester
hydrochloride, TEA, CH2Cl2, 0 ℃, 2 h; (iii) NaH, THF, 0℃, 15 min; (d) (i)NaH, DMF, 50 ℃, 6h;
(ii)THF, H2O, NaOH, RT, 4h.
3

S
S
R1
e or f
N
g
S
R1
+
R1 NH2
N
OHC
R2
S
O
R2
O
6a-6m
7a-7m
8a-8f
9a-9s
Scheme 2. Synthesis of target compounds 9a-9s. Regants and conditions: (e) (i) CS2, Et3N, EtOH,
0℃, 30min; (ii) ClCH2COONa, H2O, RT, overnight; (iii) 6 M HCl, 85℃, 5min; (f)
SC(SCH2COOH)2, H2O, reflux, overnight; (g) NH4Ac, HAc, reflux, 30min.
2.2. LYP inhibitory activities and preliminary SARs
LYP inhibitory activities of target compounds 5a-5e and 9a-9s were summarized
in Table 1. Initially, the inhibition rates of all compounds were tested at 50μM, and
compounds with inhibition rate higher than 85% were then selected to further measure
IC50 values.
As shown in Table 1, compounds 9a-9s exhibited higher inhibition rate than 5a-
5e, indicating that 2-thioxothiazolidin-4-one scaffold is more favorable than
imidazolidine-2,4-dione scaffold in inhibiting LYP. Among 2-thioxothiazolidin-4-one
derivatives (9a-9s), substitutions in R2 position showed higher impacts on LYP
inhibitory activity than that in R1 position. In particularly, compounds with 4-
oxymethyl cinnamic acids in R2 position (9m-9s) possessed higher activities than that
with 4-oxymethyl benzoic acids (9a-9f), acetic acid (9g), acrylic acid (9h-9j), 4-
oxymethyl phenylboronic acid (9k) and benzoic acid (9l).
In terms of the substitutions in R1 position, the N-phenyl substituted compound
(9e) showed the highest inhibitory activity (IC50 = 24.21μM) among compounds with
4-oxymethyl benzoic acids in R2 position (9a-9f). For compounds with 4-oxymethyl
cinnamic acids as R2 group (9m-9s), the substituents in phenyl group of R1 substitution
also moderately affected their inhibitory activities. Compared with N-phenyl
substituted compound (9m), introduction of methoxy (9p) or chlorine (9r) groups in
the phenyl ring improve the inhibitory activity, while introduction of methyl group (9n
and 9o) played a contrary role. On the other hand, para-substituted inhibitors (9n, 9p
and 9r) performed better than corresponding ortho-substituted compounds (9o, 9q and
9s) in LYP inhibition.
Among all tested compounds, the N-phenyl-rhodanine 4-phenoxymethyl cinnamic
acids (9q and 9r) exhibited better activities (IC50= 2.85-6.95 μM) than known LYP
inhibitor A15 (IC50= 8.28 μM). Especially, the most active compound 9r is about three
time more potent than A15.
Table 1. Structure and inhibition rate of compounds 5a-5f and 9a-9s.
S
O
R1
R1
N
S
N
N
O
R2
R2
O
9a-9s
5a-5f
4

Inhibition
activitya
Inhibition
activitya
Cpd. R1
5a
R2
Cpd. R1
9h
R2
O
4.9%
53.9%
54.4%
58.9%
25.5%
22.3%
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
O2N
O
O
O
O
O
O
O
O
O
O
5b
27.3%
67.1%
12.9%
26.4%
70.7%
9i
HOOC
Me
Me
5c
9j
COOH
O
OH
B
OH
HOOC
5d
5e
9a
9b
9c
9d
9e
9k
9l
COOH
O
9m
9.72±0.62
12.75±1.02
14.01±0.86
6.95±0.43
10.43±0.35
2.85±0.19
COOH
Me
O
O
O
O
O
O
9n
21.3%
48.3%
85%
COOH
COOH
COOH
COOH
COOH
COOH
Me
9o
MeO
9p
S
OMe
24.21±1.73
64.8%
12.2%
9q
Cl
N
9f
9r
S
Cl
9g
COOH
9s
15.02±1.49
8.28±0.43
A15
a Compounds with inhibition rates higher than 85% at 50μM were selected to measure IC50 (μM)
values.
2.3. Kinetic analysis and time-dependent inhibition
Next, we measured the inhibition kinetic of compound 9r and A15. Figure 2
indicated that 9r displayed competitive inhibition (against the substrate pNPP) on LYP.
According to the Ki values, 9r (Ki = 1.1 μM) is a more potent LYP inhibitor than A15
(Ki = 4.5 μM) and recently reported LYP inhibitor NC1 (Ki = 4.3±0.3 μM)[18].
Since the structure of 9r contains α-β unsaturated ketone, which may lead to
irreversibly binding to cystine residues in LYP, we further performed time-dependent
inhibition test. As for irreversible binding inhibitor, the Kcat (inhibitor) would decrease as
preincubation time goes, whereas the reversibly binding pattern remained constant
Kcat (inhibitor). We employed reversible inhibitor A15 (20 μM) and irreversible inhibitor
A25 (50 μM) as control group[17, 20]. As results shown in Figure 3, there were no
increase in the ratio of Kcat (control) / Kcat (inhibitor) during 10-90 min preincubating of 9r
(20 μM), implying its reversible binding with LYP (Table S1 and Table S2).
5

Figure 2. Kinetic analysis of LYP inhibitors A15 (A) and 9r (B). The Lineweaver-Burk plots
displayed competitive inhibition mode.
Figure 3. The time-dependent ratios of Kcat (control)/Kcat (inhibitor) for A15, 9r and A25.
2.4 Molecular docking
To further investigate the binding mode of 9r and LYP, we performed molecular
docking using AutoDock Vina[21]. The top scored binding pose was selected as the
favorable binding mode for 9r (Figure 4). As expected, the cinnamic acid group of 9r
bind into the catalytic site of LYP, formed hydrogen bonding interactions with R233
and Q278 and mimicked the binding mode of phosphate group in substrate. This result
is in consistent with the substrate competitive and reversible binding mode of 9r
(Figure 2B and Figure 3). We also observed π-π stacking interaction between 9r and
Y60. On the other hand, the N-phenyl substituted 2-thioxothiazolidin-4-one scaffold
binding into a pocket formed by R59, L106, S107, K136 and E140. Interestingly, we
detected halogen bond interaction between chlorine atom of 9r and S107.
6

Figure 4. Predicted binding mode of 9r. (A) The interactions between 9r and residues in LYP.
Hydrogen bonding as well as halogen bonding interactions are shown in dotted line. (B) Binding
pockets of 9r in active site of LYP.
2.5. Selectivity against other phosphates
Because most classical PTPs share the conserved phospho-tyrosine binding
domain[22, 23], low isoform selectivity is a huge obstacle for PTP inhibitors accessing
clinic usage[24]. Therefore, we evaluated the selectivity of our new LYP inhibitor 9r
against a panel of PTPs, including PTPN12, PTP1B, Glepp1, STEP and SSH2 (Figure
S2-S7). According to the results in Table 2, compound 9r exhibited at least 5.4-fold of
selectivity against other PTPs, which was obviously better than A15 (mostly lower than
2.5-fold). Importantly, 9r exhibited good selectivity against PTP1B and PTPN12,
which possess high structural similarities with LYP [25].
Table 2. Selectivity of 9r and A15 against a panel of PTPs.
9r
A15
PTPs
Ratio of
selectivity
1.0
Ratio of
selectivity
1.0
IC50 (μM)
IC50 (μM)
LYP
2.85±0.19
15.46±1.67
20.72±2.46
21.91±0.92
30.56±2.52
>100
8.28±0.43
5.93±0.93
6.89±0.77
16.85±1.29
20.97±1.17
>100
PTPN12
PTP1B
Glepp1
STEP
5.4
0.7
7.3
0.8
7.7
2.0
>10
2.5
SSH2
>10
>10
2.6. Effects of 9r on TCR Signaling in lymphoid T cell
Since 9r exhibited good LYP inhibitory activity as well as PTPs selectivity in vitro,
we then evaluated its effects in T cells. Previous studies demonstrated that LYP
inhibitor could negatively modulate the dephosphorylation function of LYP, therefore
7

increasing the phosphorylation level of substrate proteins, such as LCK (Y394) and
ERK (T202 and Y204)[17]. We evaluated the effect of 9r in the Jurkat T antigen (JTAg)
human T cell line with A15 as positive control and DMSO as negative control. Similar
with known inhibitor A15, we observed increased pLCK394 and pERK levels after
treatment of 9r (Figure 5), indicating its LYP inhibitory activity in T cells.
Figure 5. Effects of A15 and 9r on phosphorylation level of LCK and ERK. The GAPDH level was
used as a control.
3. Conclusion
In current study, we designed and synthesized imidazolidine-2,4-dione and 2-
thioxothiazolidin-4-one derivatives (5a-5e and 9a-9s) based on our previously reported
LYP inhibitor A15[18]. In vitro enzymatic evaluations indicated that the most active
inhibitor 9r exhibit more potent LYP inhibitory activity than A15. Besides, the kinetic
analysis as well as time-dependent inhibition test suggested that 9r interact with LYP
under competitive and reversible pattern. Meanwhile, 9r possess more than 5.4-folds
of selectivity against other PTPs. Furthermore, cellular test indicated that 9r could
efficiently increase the phosphorylation level of LCK and ERK, thereby regulate TCR
signaling pathways. In summary, our new LYP inhibitors provide new starting point to
develop LYP-targeted clinic candidate drugs with preferable activity and selectivity.
4. Experimental section
4.1. Chemistry
Reagents and solvents used were of commercially available LR grade quality and
without further purification. All reactions were monitored by TLC on 0.25 mm silica
gel plates (60 GF-254) with UV (256nm) visualizing the spots. Melting points were
measured by the RY-1G electrothermal melting point apparatus without correction.
Brucker DRX spectrometer at 300 MHz ,400 MHz or 600 MHz were used to record the
13
1H and C NMR spectra, d in parts per million and J in hertz, choosing TMS as an
1
internal standard. Significant H NMR data are reported in the following order:
multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) number of protons. High-
resolution mass spectra (HRMS) were conducted on an Agilent G6520a Quadrupole
8

Time-of-Flight LC/MS deliver and the data were showed as m/z.
4.1.1. 3-benzylimidazolidine-2,4-dione (3a)
Hydantoin 2 (3 g, 30 mmol) and DBU (9 ml, 60 mmol) were dissolved in DMF (25 ml)
and heated at 60 ℃ for 0.5 h under N2. Benzyl bromide 1a (4.5 ml, 36 mmol) was added
to the reaction mixture and stirred for 12 h. After cooling to room temperature, the
reaction was quenched by H2O and extracted by ethyl acetate (×3). The organic layer
was washed with brine solution and then dried and reduced to crude product. The crude
product was washed by diethyl ether to obtain the light yellow solid as target compound
(2.98 g, 52%).mp:202-205℃. 1H NMR (400 MHz, DMSO) δ 8.14 (s, 1H), 7.36 – 7.30
(m, 2H), 7.27 (t, J = 5.8 Hz, 3H), 4.52 (s, 2H), 3.98 (s, 2H).
4.1.2. methyl 4-((2,5-dioxoimidazolidin-1-yl)methyl)benzoate (3b)
Using the synthetic method for 3a, the target compound was obtained as a colorless oil
1
in 66% yield. H NMR (400 MHz, DMSO) δ 8.18 (s, 1H), 7.93 (d, J = 8.2 Hz, 2H),
7.41 (d, J = 8.2 Hz, 2H), 4.61 (s, 2H), 4.01 (s, 2H), 3.85 (s, 3H).
4.1.3. 3-([1,1'-biphenyl]-4-ylmethyl)imidazolidine-2,4-dione (3c)
Using the synthetic method for 3a, the target compound was obtained as a white solid
in 46% yield. mp:132℃. 1H NMR (400 MHz, DMSO) δ 8.15 (s, 1H), 7.63 (t, J = 7.5
Hz, 4H), 7.46 (t, J = 7.6 Hz, 2H), 7.39 - 7.34 (m, 3H), 4.57 (s, 2H), 4.00 (s, 2H).
4.1.4. 2-(2,5-dioxoimidazolidin-1-yl)-N-methyl-N-phenylacetamide (3d)
Using the synthetic method for 3a, the target compound was obtained as a yellow oil in
67% yield. 1H NMR (400 MHz, DMSO) δ 8.11 (s, 1H), 7.96 (s, 1H), 7.47 (d, J = 28.7
Hz, 4H), 3.93 (t, J = 47.0 Hz, 2H), 3.20 (d, J = 16.7 Hz, 2H), 2.89 (s, 3H), 2.73 (s, 2H).
4.1.5. 3-phenylimidazolidine-2,4-dione (3e)
The solution of aniline 1f (4.6 ml, 50 mmol) in dichlormethane (100 ml) was dropwise
added to the solution of triphosgene (8.0 g, 27 mmol) in dichlormethane (70 ml) at 0
˚C and stirred for 3h. The reaction was quenched by saturated sodium bicarbonate
solution. The organic phase was dried and evaporated under vacuum and solved in
dichlormethane (100 ml) with triethylamine (8.1ml, 60mmol). The reaction mixture
was dropped into the solution of glycinemethylester hydrochloride (7.53 g, 60 mmol)
in dichlormethane (100 ml) at 0 ˚C and stirred the mixture for 2 h. The organic phase
was evaporated under vacuum to give the white solid. Then it was solved in
tetrahydrofuran (30 ml) and dropped by solution of sodium hydride (0.96 g, 40mmol)
in tetrahydrofuran (20 ml) at 0 ˚C. The reaction mixture was stirred for 15 min and
quenched by saturated ammonium chloride solution and extracted by ethyl acetate (×3).
The combined organic phase was evaporated under vacuum to give the crude product.
Then it was purified by recrystallization with solution of petroleum ether and ethyl
acetate (1:1) to give the white solid as target compound (0.3 g, 3% yield in continuous
3 steps). mp:153-154℃.1H NMR (400 MHz, DMSO) δ 8.30 (s, 1H), 7.47 (t, J = 7.6 Hz,
2H), 7.39 (d, J = 7.3 Hz, 1H), 7.36 – 7.32 (m, 2H), 4.07 (s, 2H).
4.1.6. 4-((4-((3-benzyl-2,4-dioxoimidazolidin-1-yl) methyl) phenoxy)methyl)benzoic
acid (5a)
Compound 3a (1.14 g, 6 mmol), NaH (0.5 g,12 mmol) were solved in DMF (12ml) and
heated at 50℃. Methyl 4-((4-(bromomethyl)phenoxy)methyl)benzoate 4a (2.4 g, 7.2
mmol) was added to reaction mixture and stirred for 10h. After cooling to room
9

temperature, the reaction was quenched by H2O and extracted by ethyl acetate (×3) .
The organic layer was washed with brine solution and then reduced to white oil. The
oil was solved in solution of H2O (24ml) and tetrahydrofuran (24ml), NaOH (1.2 g, 30
mmol) was added into solution then stirred for 5h at RT. The organic solvent was
evaporated under vacuum and the water phase was acidified to pH= 2 with 6 M HCl
and filtered to give the crude product. The crude product was purified by
recrystallization with ethyl alcohol to give the white solid as target compound (0.57 g,
22% ). mp:162-164℃.1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 8.2 Hz, 2H), 7.53 (d,
J = 8.1 Hz, 2H), 7.42 (d, J = 7.0 Hz, 2H), 7.32 (dd, J = 12.1, 4.7 Hz, 3H), 7.18 (d, J =
8.5 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 5.14 (s, 2H), 4.67 (s, 2H), 4.49 (s, 2H), 3.71 (s,
2H). 13C NMR (101 MHz, DMSO) δ 170.52, 167.61, 158.09, 156.75, 142.54, 137.11,
130.77, 129.94, 129.67, 129.18, 128.97, 127.94, 127.89, 127.80, 115.45, 69.09, 49.81,
45.79, 42.04. HRMS (AP-ESI) m/z Calcd for C25H22N2O5 (M+H)+431.1601, found:
431.1601.
4.1.7. 4-((4-((3-(4-carboxybenzyl)-2,4-dioxoimidazolidin-1-yl)methyl)phenoxy)
methyl)benzoic acid (5b)
Using the synthetic method for 5a, the target compound was obtained as a white solid
in 24% yield. mp: >250℃. 1H NMR (400 MHz, DMSO) δ 12.89 (s, 2H), 7.95 (d, J =
8.1 Hz, 2H), 7.85 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H),
7.18 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 5.17 (s, 2H), 4.42 (s, 2H), 4.32 (d, J
= 4.6 Hz, 2H), 3.82 (s, 2H). 13C NMR (101 MHz, DMSO) δ 171.99 (s), 167.85 (d, J =
11.2 Hz), 158.23 (s), 157.76 (s), 146.66 (s), 142.42 (s), 131.12 (s), 129.91 (s), 129.73
(s), 129.44 (d, J = 32.9 Hz), 127.76 (s), 127.22 (s), 115.18 (s), 69.13 (s), 50.06 (s), 48.30
(s), 43.93 (s). HRMS (AP-ESI) m/z Calcd for C26H22N2O7 (M-H)- 473.1354, found:
473.1358.
4.1.8. 4-((4-((3-([1,1'-biphenyl]-4-ylmethyl)-2,4dioxoimidazolidin-1yl)methyl)
phenoxy)methyl)benzoic acid (5c)
Using the synthetic method for 5a, the target compound was obtained as a white solid
in 37% yield. mp:183-186℃. 1H NMR (400 MHz, DMSO) δ 12.97 (s, 1H), 7.96 (d, J
= 8.2 Hz, 2H), 7.64 (dd, J = 7.5, 5.6 Hz, 4H), 7.55 (d, J = 8.2 Hz, 2H), 7.46 (t, J = 7.6
Hz, 2H), 7.38 (d, J = 8.3 Hz, 3H), 7.24 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.6 Hz, 2H),
5.19 (s, 2H), 4.61 (s, 2H), 4.45 (s, 2H), 3.95 (s, 2H). 13C NMR (101 MHz, DMSO) δ
170.53 (s), 167.56 (s), 158.10 (s), 156.76 (s), 142.59 (s), 140.27 (s), 139.88 (s), 136.31
(s), 130.66 (s), 129.96 (s), 129.94 (s), 129.54 (d, J = 27.2 Hz), 129.19 (s), 128.65 (s),
127.87 (d, J = 13.9 Hz), 127.23 (d, J = 19.2 Hz), 115.46 (s), 69.09 (s), 49.86 (s), 45.81
(s), 41.77 (s). HRMS (AP-ESI) m/z Calcd for C31H26N2O5 (M-H)- 505.1769, found:
505.1764.
4.1.9. 4-((4-((3-(2-(methyl(phenyl)amino)-2-oxoethyl)-2,4-dioxoimidazolidin-1-yl)
methyl)phenoxy)methyl)benzoic acid (5d)
Using the synthetic method for 5a, the target compound was obtained as a white solid
in 24% yield. mp:212-215℃. 1H NMR (400 MHz, DMSO) δ 7.95 (d, J = 8.1 Hz, 2H),
7.53 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 8.5 Hz, 2H), 5.17 (s, 2H),
4.44 (s, 2H), 3.86 (s, 2H), 3.81 (s, 2H). 13C NMR (101 MHz, DMSO) δ 170.37 (s),
169.43 (s), 168.09 (s), 158.11 (s), 156.96 (s), 141.73 (s), 129.86 (s), 129.53 (s), 129.23
(s), 127.66 (s), 115.45 (s), 69.22 (s), 49.54 (s), 45.47 (s), 41.85 (s). HRMS (AP-ESI)
m/z Calcd for C20H18N2O7 (M-H)- 397.1041, found: 397.1044.
10

4.1.10. 4-((4-((2,4-dioxo-3-phenylimidazolidin-1-yl)methyl)phenoxy)methyl)benzoic
acid (5e)
Using the synthetic method for 5a, the target compound was obtained as a white solid
in 20% yield. mp:163-166℃. 1H NMR (400 MHz, DMSO) δ 12.96 (s, 1H), 7.96 (d, J =
8.2 Hz, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.51 – 7.45 (m, 2H), 7.39 (t, J = 6.9 Hz, 3H), 7.30
(d, J = 8.6 Hz, 2H), 7.03 (d, J = 8.6 Hz, 2H), 5.21 (s, 2H), 4.50 (s, 2H), 4.01 (s, 2H).13C
NMR (101 MHz, DMSO) δ 169.64 (s), 167.57 (s), 158.12 (s), 155.88 (s), 142.62 (s),
132.77 (s), 130.67 (s), 129.87 (d, J = 17.2 Hz), 129.14 (d, J = 12.0 Hz), 128.26 (s),
127.80 (s), 127.18 (s), 115.46 (s), 69.10 (s), 49.88 (s), 45.92 (s). HRMS (AP-ESI) m/z
Calcd for C24H20N2O5 (M+H)+417.1445, found: 417.1444.
4.1.11. 3-cyclohexyl-2-thioxothiazolidin-4-one (7a)
Cyclohexanamine 6a (0.5g, 5 mmol), and triethylamine (2.35g, 25mmol) were solved
in ethanol (2ml), then carbon disulfide (0.76g, 10mmol)was dropped into mixture with
stirring for 0.5h at 0℃. The precipitate was filtered and washed by ether and solved in
solution of sodium chloroacetate (0.64 g, 5.5 mmol) at RT overnight. 6M HCl (7
ml)was added to reaction mixture and heated at 95 ℃ for 5min. The crude product was
filtered and purified by recrystallization with chloroform to give the yellow solid as
target compound (0.66g, 61% ). mp:120-122℃.1H NMR (300 MHz, CDCl3) δ 4.86 (tt,
J = 12.3 Hz,3.9 Hz, 1H), 3.82 (s, 2H), 2.31 (qd, J = 12.3 Hz, 3.0 Hz, 2H), 1.89-1.84 (m,
2H),1.70-1.32 (m, 6H).
4.1.12. 3-butyl-2-thioxothiazolidin-4-one (7b)
Using the synthetic method for 7a, the target compound was obtained as a yellow oil in
1
56% yield. H NMR (300 MHz, CDCl3) δ 3.98 (m, 4H), 1.62 (m, 2H), 1.36 (m,2H),
0.95 (t, J = 7.8 Hz, 3H).
4.1.13. 3-benzyl-2-thioxothiazolidin-4-one (7c)
The target compound was purchased from Co. J&K (041959).mp:83℃. 1H NMR (400
MHz, CDCl3) δ 7.43 (d, J = 6.9 Hz, 2H), 7.29 (t, J = 10.8 Hz, 3H), 5.18 (s, 2H), 3.97
(s, 2H).
4.1.14. 3-(thiophen-2-ylmethyl)-2-thioxothiazolidin-4-one (7d)
Thiophen-3-ylmethanamine 6d (1.13g 10mmol) and Bis(carboxymethyl)
trithiocarbonate (2.26g 10mmol) was solved in H2O (20ml) at 110℃, then potassium
carbonate (0.96g, 5mmol) was added slowly to the mixture and stirred for 12h. After
cooling to RT, the aqueous phase was extracted by ethyl acetate with 10% methanol
(×3). The organic phase was washed by H2O and dried and evaporated under vacuum
to give crude product, followed by washing with ether to give the orange solid as target
1
compound (1.19g, 51%).mp:92-94℃. H NMR (400 MHz, CDCl3) δ 7.24 – 7.19 (m,
2H), 6.94 (d, J = 3.8 Hz, 1H), 5.33 (s, 2H), 3.96 (s, 2H).
4.1.15. 3-phenyl-2-thioxothiazolidin-4-one (7e)
Using the synthetic method for 7a, the target compound was obtained as a yellow solid
in 69% yield. mp:195-197℃. 1H NMR (600 MHz, CDCl3) δ 7.54 (td, J = 7.2 Hz, 1.8
Hz, 2H), 7.50 (tt, J = 7.2 Hz, 1.8 Hz, 1H), 7.20 (dt, J = 7.2Hz, 1.8 Hz, 2H), 4.20 (s, 2H).
4.1.16. 3-(thiazol-2-yl)-2-thioxothiazolidin-4-one (7f)
Using the synthetic method for 7a, the target compound was obtained as a brown solid
in 39% yield. mp:130-131℃. 1H NMR (400 MHz, DMSO) δ 7.61 (d, J = 4.2 Hz, 1H),
11

7.16 (d, J = 4.0 Hz, 1H), 4.00 (s, 2H).
4.1.17. 3-(4-nitrophenyl)-2-thioxothiazolidin-4-one (7g)
Using the synthetic method for 7d, the target compound was obtained as a yellow solid
1
in 61% yield. mp:220 ℃. H NMR (300 MHz, DMSO-d6) δ 8.40 (m, 2H), 7.64 (m,
2H), 4.40 (s, 2H).
4.1.18. 2-thioxo-3-(o-tolyl)thiazolidin-4-one (7h)
Using the synthetic method for 7a, the target compound was obtained as a yellow solid
in 81% yield. mp:143-145℃.1H NMR (300 MHz, CDCl3) δ 7.42 (m, 1H), 7.30 (d,J =
7.8 Hz, 1H), 7.00 (m, 2H), 4.19 (s, 2H) , 2.41 (s, 3H).
4.1.19. 2-thioxo-3-(p-tolyl)thiazolidin-4-one (7i)
Using the synthetic method for 7a, the target compound was obtained as a yellow solid
in 63% yield. mp:169-171℃. 1H NMR (300 MHz, CDCl3) δ 7.34 (d, J = 8.1 Hz,
2H), 7.08 (d, J = 8.7 Hz, 2H), 4.18 (s, 2H), 2.42 (s, 3H).
4.1.20. 3-(4-methoxyphenyl)-2-thioxothiazolidin-4-one (7j)
Using the synthetic method for 7a, the target compound was obtained as a yellow solid
in 78% yield. mp:158-160℃.1H NMR (600 MHz, CDCl3) δ 7.11 (d, J = 8.4 Hz,
2H), 7.03 (d, J = 8.4 Hz, 2H), 4.16 (s, 2H), 3.84 (s, 3H).
4.1.21. 3-(2-methoxyphenyl)-2-thioxothiazolidin-4-one (7k)
Using the synthetic method for 7a, the target compound was obtained as a yellow solid
in 63% yield. mp:136-138℃. 1H NMR (600 MHz, CDCl3) δ 7.47 (td, J = 7.8 Hz,
1.8 Hz, 1H), 7.12 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 7.05 (d, J =7.8
Hz, 1H), 4.19 (d, J = 18.0 Hz, 1H), 4.13 (d, J = 18.0 Hz, 1H), 3.79 (s, 3H).
4.1.22. 3-(4-chlorophenyl)-2-thioxothiazolidin-4-one (7l)
The target compound was purchased from Co. J&K (039780).mp:128°C. 1H NMR (300
MHz, CDCl3) δ 7.51 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 4.19 (s, 2H).
4.1.23. 3-(2-chlorophenyl)-2-thioxothiazolidin-4-one (7m)
The target compound was purchased from Co. J&K (H63607). mp:113°C. 1H NMR
(300 MHz, CDCl3): δ 7.40–7.15 (m, 4H), 4.19 (s, 2H).
4.1.24. (Z)-4-((4-((3-cyclohexyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)
phenoxy)methyl) benzoic acid（9a）
Compound 7a (0.43g, 2mmol) and 4-((4-formylphenoxy)methyl)benzoic acid 8a
(0.51g, 2mmol) were added in acetic acid (8ml) and heated at 125 °C to dissolution.
Then ammonium acetate (0.62 g, 8 mmol) was added into reaction mixture and stirred
for 0.5h. The precipitate was filtered and washed by acetic acid and purified by
recrystallization with methanol to give the yellow solid as target product. (0.51g, 56%)
mp: >250 °C. 1H NMR (400 MHz, DMSO) δ 13.00 (s, 1H), 7.98 (d, J = 7.6 Hz, 2H),
7.70 (s, 1H), 7.60 (dd, J = 12.8, 8.1 Hz, 4H), 7.22 (d, J = 8.0 Hz, 2H), 5.31 (s, 2H), 4.91
(s, 1H), 2.33 (d, J = 10.6 Hz, 2H), 1.85 (d, J = 12.0 Hz, 2H), 1.68 (d, J = 9.2 Hz, 3H),
1.38 – 1.16 (m, 3H).13C NMR (101 MHz, DMSO) δ 194.32, 167.65, 167.51, 160.76,
141.92, 133.32, 132.98, 130.85, 130.00, 127.96, 126.46, 118.97, 116.42, 69.42, 57.92,
27.88, 26.06, 25.29. HRMS (AP-ESI) m/z Calcd for C24H23NO4S2 (M+H)+ 454.1141,
found: 454.1136.
12

4.1.25. (Z)-4-((4-((3-butyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)phenoxy)
methyl)benzoic acid（9b）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 40% yield. mp:238-240℃. 1H NMR (400 MHz, DMSO) δ 13.00 (s, 1H), 7.98 (d, J =
7.5 Hz, 2H), 7.79 (s, 1H), 7.61 (dd, J = 19.0, 7.8 Hz, 4H), 7.22 (d, J = 8.0 Hz, 2H), 5.31
(s, 2H), 4.03 (t, J = 7.2 Hz, 2H), 1.68 – 1.57 (m, 2H), 1.33 (dt, J = 14.7, 7.2 Hz, 2H),
0.91 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 193.65, 167.51, 167.48,
160.83, 141.88, 133.49, 133.40, 130.83, 130.00, 127.93, 126.37, 119.75, 116.40, 69.41,
44.40, 28.93, 19.98, 14.01.HRMS (AP-ESI) m/z Calcd for C22H21NO4S2 (M+H)+
428.0985, found: 428.0983.
4.1.26. (Z)-4-((4-((3-benzyl-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)phenoxy)
methyl)benzoic acid（9c）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 97% yield. mp: >250℃. 1H NMR (400 MHz, DMSO) δ 12.97 (s, 1H), 7.97 (d, J =
7.6 Hz, 2H), 7.83 (s, 1H), 7.65 (d, J = 7.9 Hz, 2H), 7.58 (d, J = 7.7 Hz, 2H), 7.36 – 7.28
(m, 5H), 7.22 (d, J = 8.0 Hz, 2H), 5.31 (s, 2H), 5.25 (s, 2H). 13C NMR (101 MHz,
DMSO) δ 193.73, 167.54, 167.51, 160.98, 141.90, 135.45, 134.09, 133.51, 130.86,
130.01, 129.04, 128.16, 128.10, 127.97, 126.36, 119.65, 116.48, 69.46, 47.58. HRMS
(AP-ESI) m/z Calcd for C25H19NO4S2 (M-H)- 460.0683, found: 460.0680.
4.1.27. (Z)-4-((4-((4-oxo-3-(thiophen-2-ylmethyl)-2-thioxothiazolidin-5-ylidene)
methyl)phenoxy)methyl)benzoic acid（9d）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 94% yield. mp: >250℃. 1H NMR (400 MHz, DMSO) δ 12.93 (s, 1H), 7.97 (d, J =
7.6 Hz, 2H), 7.83 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 7.7 Hz, 2H), 7.46 (d, J
= 5.0 Hz, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.17 (s, 1H), 6.99 (s, 1H), 5.38 (s, 2H), 5.31 (s,
2H). 13C NMR (101 MHz, DMSO) δ 192.94, 167.50, 166.98, 161.02, 141.89, 136.69,
134.27, 133.54, 130.86, 130.00, 129.10, 127.96, 127.25, 127.19, 126.30, 119.38,
116.48, 69.46, 42.33. HRMS (AP-ESI) m/z Calcd for C23H17NO4S3 (M+H)+ 468.0392,
found: 468.0394.
4.1.28. (Z)-4-((4-((4-oxo-3-phenyl-2-thioxothiazolidin-5-ylidene)methyl)phenoxy)
methyl)benzoic acid（9e）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 67% yield. mp: >250 ℃. 1H NMR (400 MHz, DMSO) δ 13.00 (s, 1H), 7.99 (d, J =
7.4 Hz, 2H), 7.83 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.63 – 7.50 (m, 5H), 7.42 (d, J = 7.5
Hz, 2H), 7.25 (d, J = 8.0 Hz, 2H), 5.33 (s, 2H).13C NMR (101 MHz, DMSO) δ 194.27,
167.53, 160.84, 141.95, 135.79, 133.40, 133.26, 130.83, 130.03, 129.90, 129.79,
129.25, 128.00, 126.48, 120.70, 116.49, 69.43. HRMS (AP-ESI) m/z Calcd for
C24H17NO4S2 (M-H)-446.0526, found: 446.0524.
4.1.29. (Z)-4-((4-((4-oxo-3-(thiazol-2-yl)-2-thioxothiazolidin-5-ylidene)methyl)
phenoxy)methyl) benzoic acid（9f）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 7% yield. mp:248-250 ℃. 1H NMR (400 MHz, DMSO) δ 12.72 (s, 1H), 8.06 (d, J =
2.4 Hz, 1H), 7.98 (d, J = 8.3 Hz, 3H), 7.86 (s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.58 (d, J
= 7.7 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 5.33 (s, 2H). 13C NMR (101 MHz, DMSO) δ
13

193.50, 166.64, 161.13, 153.99, 141.76, 134.49, 133.64, 130.02, 127.97, 126.22,
125.99, 119.68, 116.54, 69.49. HRMS (AP-ESI) m/z Calcd for C21H14N2O4S3 (M+H)+
455.0188, found: 455.0193.
4.1.30.
(Z)-4-((4-oxo-3-phenyl-2-thioxothiazolidin-5-ylidene)methyl)benzoic aci
（9g）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 76% yield. mp: >250 ℃. 1H NMR (400 MHz, DMSO) δ 13.25 (s, 1H), 8.10 (d, J =
7.7 Hz, 2H), 7.89 (s, 1H), 7.81 (d, J = 7.8 Hz, 2H), 7.55 (td, J = 14.1, 7.4 Hz, 3H), 7.44
13
(d, J = 7.4 Hz, 2H). C NMR (101 MHz, DMSO) δ 194.31, 167.33, 167.08, 137.41,
135.58, 132.55, 131.52, 131.07, 130.69, 130.02, 129.83, 129.21, 126.20. HRMS (AP-
ESI) m/z Calcd for C17H11NO3S2 (M-H)- 340.0108, found: 340.0108.
4.1.31. (E)-3-(4-((Z)-(4-oxo-3-phenyl-2-thioxothiazolidin-5-ylidene)methyl)phenyl)
acrylic acid（9h）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 51% yield. mp: >250 ℃. 1H NMR (400 MHz, DMSO) δ 13.00 (s, 1H), 7.98 (d, J =
7.6 Hz, 2H), 7.70 (s, 1H), 7.60 (dd, J = 12.8, 8.1 Hz, 4H), 7.22 (d, J = 8.0 Hz, 2H), 5.31
(s, 2H), 4.91 (s, 1H).13C NMR (101 MHz, DMSO) δ 194.20, 167.84, 167.41, 143.03,
136.89, 135.66, 134.95, 132.10, 131.52, 129.98, 129.82, 129.66, 129.23, 124.66,
121.85. HRMS (AP-ESI) m/z Calcd for C19H13NO3S2 (M-H)- 366.0264, found:
366.0267.
4.1.32. (E)-3-(4-((Z)-(3-(4-nitrophenyl)-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)
phenyl) acrylic acid（9i）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 27% yield. mp: >250 ℃. 1H NMR (400 MHz, DMSO) δ 12.54 (s, 1H), 8.44 (d, J =
8.5 Hz, 2H), 7.90 (d, J = 5.3 Hz, 3H), 7.80 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.1 Hz, 2H),
7.65 (d, J = 16.0 Hz, 1H), 6.68 (d, J = 16.0 Hz, 1H). 13C NMR (101 MHz, DMSO) δ
193.90, 167.82, 167.10, 148.38, 143.51, 143.02, 141.24, 137.02, 134.83, 132.44,
131.55, 131.12, 129.70, 129.16, 125.05, 124.63, 121.92, 120.69. HRMS (AP-ESI) m/z
Calcd for C19H12N2O5S2 (M+Cl)- 446.9882, found: 446.9884.
4.1.33. (E)-3-(4-((Z)-(4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenyl)
acrylic acid（9j）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 34% yield. mp:244-246℃.1H NMR (400 MHz, DMSO) δ 8.04 (s, 1H), 7.89 (d, J =
8.0 Hz, 2H), 7.73 (d, J = 6.7 Hz, 2H), 7.65 (d, J = 16.3 Hz, 1H), 7.47 – 7.34 (m, 4H),
6.67 (d, J = 16.0 Hz, 1H), 2.13 (s, 3H). 13C NMR (101 MHz, DMSO) δ 167.84, 166.78,
165.36, 136.75, 136.40, 134.87, 133.07, 132.62, 131.61, 131.35, 131.11, 130.31,
129.55, 129.40, 129.16, 127.49, 122.64, 121.71, 17.47. HRMS (AP-ESI) m/z Calcd for
C20H15NO3S2 (M-H)- 380.0421, found: 380.0421.
4.1.34. (Z)-(4-((4-((4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)phenoxy)
methyl)phenyl)boronic acid（9k）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 31% yield. mp:167-170 ℃. 1H NMR (400 MHz, DMSO) δ 8.06 (s, 2H), 7.85 (s, 1H),
7.82 (d, J = 7.4 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 7.44 (d, J = 4.0 Hz, 4H), 7.36 (dd, J
= 13.8, 5.9 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 5.24 (s, 2H), 2.06 (s, 3H). 13C NMR (101
14

MHz, DMSO) δ 193.43, 167.20, 161.19, 138.62, 136.34, 134.83, 134.76, 133.96,
133.50, 131.39, 130.34, 129.51, 127.67, 127.18, 126.21, 120.23, 116.52, 70.08, 17.33.
HRMS (AP-ESI) m/z Calcd for C24H20NO4S2B (M+H)+ 461.1036, found: 461.1036.
4.1.35. (Z)-4'-((4-oxo-3-phenyl-2-thioxothiazolidin-5-ylidene)methyl)-[1,1'-biph
-enyl]-4-carboxylic acid（9l）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 87% yield. mp: >250 ℃. 1H NMR (400 MHz, DMSO) δ 13.04 (s, 1H), 8.06 (d, J =
8.1 Hz, 2H), 7.98 (d, J = 8.1 Hz, 2H), 7.91 (d, J = 4.9 Hz, 3H), 7.83 (d, J = 8.3 Hz, 2H),
7.60 – 7.51 (m, 3H), 7.44 (d, J = 7.5 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 191.74,
166.71, 135.70, 131.85, 130.54, 129.82, 129.25, 128.47, 127.52, 124.12. HRMS (AP-
ESI) m/z Calcd for C23H15NO3S2 (M-H)- 416.0421, found: 416.0416.
4.1.36. (E)-3-(4-((4-((Z)-(4-oxo-3-phenyl-2-thioxothiazolidin-5-ylidene)methyl)
phenoxy)methyl)phenyl)acrylic acid（9m）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 73% yield. mp: 238-240 °C. 1H NMR (400 MHz, DMSO) δ 12.41 (s, 1H), 7.82 (s,
1H), 7.74 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.9 Hz, 2H), 7.63 (s, 1H), 7.59 – 7.51 (m,
5H), 7.42 (d, J = 7.5 Hz, 2H), 7.24 (d, J = 7.9 Hz, 2H), 6.56 (d, J = 16.0 Hz, 1H), 5.27
(s, 2H).13C NMR (101 MHz, DMSO) δ 194.25, 168.03, 167.51, 160.92, 143.93, 139.06,
135.78, 134.45, 133.39, 133.30, 129.89, 129.79, 129.24, 128.86, 128.62, 126.39,
120.61, 119.93, 116.49, 69.60. HRMS (AP-ESI) m/z Calcd for C26H19NO4S2 (M+Cl)-
508.0450, found: 508.0451.
4.1.37. (E)-3-(4-((4-((Z)-(4-oxo-2-thioxo-3-(p-tolyl)thiazolidin-5-ylidene)methyl)
phenoxy)methyl)phenyl) acrylic acid（9n）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 16% yield. mp: >250 °C.1H NMR (400 MHz, DMSO) δ 12.40 (s, 1H), 7.80 (s, 1H),
7.73 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 15.9 Hz, 1H), 7.51 (d, J
= 7.6 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 7.25 (dd, J = 16.0, 8.0 Hz, 4H), 6.55 (d, J =
15.9 Hz, 1H), 5.26 (s, 2H), 2.38 (s, 3H).13C NMR (101 MHz, DMSO) δ 194.30, 168.00,
167.51, 160.92, 143.90, 139.53, 139.06, 134.47, 133.36, 133.26, 133.13, 130.28,
128.90, 128.84, 128.60, 126.42, 120.61, 119.97, 116.50, 69.62, 21.29. HRMS (AP-ESI)
m/z Calcd for C27H21NO4S2 (M+H)+ 488.0985, found: 488.0982.
4.1.38. (E)-3-(4-((4-((Z)-(4-oxo-2-thioxo-3-(o-tolyl)thiazolidin-5-ylidene)methyl)
phenoxy)methyl)phenyl)acrylic acid（9o）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 82% yield. mp:238-240 °C. 1H NMR (400 MHz, DMSO) δ 12.41 (s, 1H), 7.86 (s,
1H), 7.72 (dd, J = 16.8, 7.9 Hz, 4H), 7.61 (d, J = 16.0 Hz, 1H), 7.52 (d, J = 7.7 Hz, 2H),
7.44 (s, 2H), 7.36 (dd, J = 14.0, 5.9 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 6.56 (d, J = 16.0
13
Hz, 1H), 5.28 (s, 2H), 2.07 (s, 3H). C NMR (101 MHz, DMSO) δ 193.41, 168.02,
167.19, 161.04, 143.92, 139.03, 136.34, 134.82, 134.47, 133.92, 133.50, 131.40,
130.33, 129.50, 128.86, 128.63, 127.67, 126.31, 120.31, 119.95, 116.51, 69.62, 17.34.
HRMS (AP-ESI) m/z Calcd for C27H21NO4S2 (M+Cl)- 522.0606, found: 522.0607.
4.1.39. (E)-3-(4-((4-((Z)-(3-(4-methoxyphenyl)-4-oxo-2-thioxothiazolidin-5-ylidene)
methyl)phenoxy)methyl)phenyl)acrylic acid（9p）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
15

in 38% yield. mp: >250 °C. 1H NMR (400 MHz, DMSO) δ 12.40 (s, 1H), 7.80 (s, 1H),
7.73 (d, J = 7.6 Hz, 2H), 7.67 (d, J = 8.3 Hz, 2H), 7.60 (d, J = 16.0 Hz, 1H), 7.51 (d, J
= 7.6 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.2 Hz,
2H), 6.55 (d, J = 15.9 Hz, 1H), 5.26 (s, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO)
δ 194.51, 168.00, 167.57, 160.90, 160.19, 143.89, 139.07, 134.47, 133.35, 133.19,
130.35, 128.85, 128.61, 128.12, 126.44, 120.61, 119.98, 116.50, 114.99, 69.61, 55.91.
HRMS (AP-ESI) m/z Calcd for C27H21NO5S2 (M+H)+ 504.0934, found: 504.0938.
4.1.40. (E)-3-(4-((4-((Z)-(3-(2-methoxyphenyl)-4-oxo-2-thioxothiazolidin-5-ylidene)
methyl)phenoxy)methyl)phenyl)acrylic acid（9q）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 68% yield. mp: >250 °C. 1H NMR (400 MHz, DMSO) δ 12.42 (s, 1H), 7.83 (s, 1H),
7.74 (d, J = 7.6 Hz, 2H), 7.68 (d, J = 7.9 Hz, 2H), 7.61 (d, J = 16.0 Hz, 1H), 7.52 (d, J
= 7.4 Hz, 3H), 7.36 (d, J = 7.7 Hz, 1H), 7.24 (d, J = 7.5 Hz, 3H), 7.12 (t, J = 7.6 Hz,
1H), 6.56 (d, J = 15.9 Hz, 1H), 5.27 (s, 2H), 3.76 (s, 3H). 13C NMR (101 MHz, DMSO)
δ 193.61, 168.01, 166.99, 161.01, 155.14, 143.92, 139.06, 134.46, 133.79, 133.45,
131.86, 130.68, 128.86, 128.63, 126.30, 123.80, 121.34, 120.20, 119.95, 116.49,
113.17, 69.61, 56.40. HRMS (AP-ESI) m/z Calcd for C27H21NO5S2 (M+Cl)- 538.0555,
found: 538.0555.
4.1.41. (E)-3-(4-((4-((Z)-(3-(4-chlorophenyl)-4-oxo-2-thioxothiazolidin-5-ylidene)
methyl)phenoxy)methyl)phenyl)acrylic acid（9r）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 47% yield. mp: >250 °C. 1H NMR (400 MHz, DMSO) δ 12.38 (s, 1H), 7.81 (s, 1H),
7.73 (d, J = 7.7 Hz, 2H), 7.70 – 7.56 (m, 5H), 7.49 (dd, J = 14.1, 7.9 Hz, 4H), 7.23 (d,
J = 8.1 Hz, 2H), 6.54 (d, J = 16.0 Hz, 1H), 5.26 (s, 2H). 13C NMR (101 MHz, DMSO)
δ 194.20, 168.02, 167.34, 160.97, 143.92, 139.05, 134.64, 134.46, 133.40, 131.27,
129.89, 128.86, 128.62, 126.35, 120.58, 119.95, 116.52, 69.60. HRMS (AP-ESI) m/z
Calcd for C26H18NO4S2Cl (M+Cl)- 542.0060, found: 542.0061.
4.1.42. (E)-3-(4-((4-((Z)-(3-(2-chlorophenyl)-4-oxo-2-thioxothiazolidin-5-ylidene)
methyl)phenoxy)methyl)phenyl)acrylic acid（9s）
Using the synthetic method for 9a, the target compound was obtained as a yellow solid
in 39% yield. mp: >250 °C. 1H NMR (400 MHz, DMSO) δ 12.38 (s, 1H), 7.88 (s, 1H),
7.76 – 7.66 (m, 5H), 7.66 – 7.55 (m, 4H), 7.51 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 8.1 Hz,
2H), 6.55 (d, J = 16.1 Hz, 1H), 5.27 (s, 2H). 13C NMR (101 MHz, DMSO) δ 193.01,
168.02, 166.67, 161.17, 143.93, 139.02, 134.47, 134.42, 133.59, 133.21, 132.22,
132.14, 131.83, 130.60, 129.04, 128.86, 128.64, 126.16, 119.95, 119.88, 116.54, 69.63.
HRMS (AP-ESI) m/z Calcd for C26H18NO4S2Cl (M+H)+ 508.0439, found: 508.0437.
4.2. Protein Expression and Purification.
The LYP protein prepared and measured for in vitro enzymic assay containing
catalytic domain (residues 1-294) and an N-terminal His tag as described previously.[26]
The expression plasmid was transformed in BL21 (DE3) cells then cultured in LB
medium with shaking at 37 °C. After the OD600 of cultures reached to 0.6-0.8, the
temperature was turned down to 18 °C and added 0.3 mM IPTG to the culture followed
by inducing for 16h. The BL21 cells were harvested by centrifugation and resuspended
in lysis buffer (20 mM Tris [pH 8.0], 300 mM NaCl, and 1 mM PMSF). Then we bound
16

the supernatant with Ni-NTA resin with vertical suspension for 2 h at 4 °C. The beads
were collected and washed with 20 mL of wash buffer (20 mM Tris [pH 8.0], 300 mM
NaCl, and 5 mM imidazole) and eluted with an imidazole gradient (20mM Tris [pH
8.0], 300 mM NaCl, and 20−200 mM imidazole). The pre-purified protein was further
refined through CM Sefinose85 with salt gradient elution. The low-salt solution
ingredients were 20 mM MES (pH 6.0), 100 mM NaCl, 1 mM EDTA, and 2 mM DTT.
The high-salt solution ingredients were 20 mM MES (pH 6.0), 1 M NaCl, 1 mM EDTA,
and 2 mM DTT. After purification using CM Sefinose, desalt with desalting gravity
column and concentration, the protein was utilized or stored at -80 °C.
4.3. Kcat and Km Measurements
The kinetic rate measurements of pNPP hydrolysis catalyzed by PTPs cited
previous work[27].The measurement process was carried in 96-well polystyrene plate
from Fisher with 50 mM 3,3-dimethylglutarate buffer(2 mM DTT and 1 mM EDTA,
with an ionic strength of 0.15 M adjusted by NaCl, pH 7.0) at 25 °C. And the substrate
concentration was set from 0.2 to 5 Km. Hydrolysis reactions were initiated by adding
appropriate amount of PTP enzymes to the mixtures and quenched by 1 M sodium
hydroxide. The absorbance at 405 nm was evaluated by plate reader. All Michaelis-
Menten parameters reported are based on non-linear curve fits of the raw data. The
steady-state kinetic parameters (kcat and Km) were calculated by GraphPad Prism 7.0
obeying the Michaelis-Menten equation (eq.1) as follows:
v = Vmax * [S] / ( Km + [S] )
(eq. 1)
4.4. In vitro Inhibition and IC50 Measurements.
The in vitro enzymatic assay assessing the negative effect of small molecular
inhibitors to the phosphatase-catalyzed pNPP hydrolysis was conducted as described
previously[17, 26]. The inhibition and IC50 of small molecule inhibitors on the pNPP
hydrolysis catalyzed by phosphatase were determined at 25 °C in 50 mM 3,3-dimethyl
glutarate buffer, and the ionic strength was adjusted to 0.15 M using NaCl (buffer A).
As for different phosphatases, the pNPP concentration was set at their respective Km
values. By adding 1 M NaOH to quench the reaction at certain time points, then we
detected product generation with monitoring the absorbance of pNPP at 405 nm. The
inhibition value and the IC50 value was obtained using GraphPad Prism 7.0
4.5. Ki Measurements.
The assay of inhibitors affecting to LYP-catalyzed hydrolysis of pNPP was
operated at 25 °C within assay buffer A. Mixed LYP and inhibitors with various fixed
concentrations, then initiated the hydrolysis reaction by adding pNPP (ranging from 0.2
to 5 times the Km) to the mixture. The reaction was quenched by 1 M NaOH when it
was finished. The inhibition constant Ki as well as the inhibition pattern were calculated
by fitting the data to the Michaelis-Menten equation for competitive inhibition (eq. 2)
using a Lineweaver-Burk plot.
17

1/v = ( Kmobs + [S] ) / Vmax[S]
Kmobs = Km + Km[I] / Ki
(eq. 2)
4.6. Cell Cultunce and Immunoblotting Assay.
Jurkat T cells were commercially available (ATCC) and cultured in RPMI 1640
medium supplemented with 10% FBS at 37 °C in a 5% CO2 humidified incubator as
described previously[17]. Then the Jurkat T cells were preincubated with small
molecule inhibitors (20 μM) or DMSO for 1h. After that, the cells were stimulated with
0.5 μg/mL anti-CD3 antibody (OKT3) or medium for 5 min and quenched at 0 °C. The
PBS-washed cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10
mM NaF, 2 mM EDTA, 10% glycerol, 1% NP-40, 0.25% sodium deoxycholate, 1 mM
Na3VO4, and a protease cocktail). The BCA protein quantitation kit (Beyotime) was
utilized to measure the concentrations of protein in lysates. Equal amounts of each cell
lysate were denatured in 2×SDS loading buffer and boiled for 10 min. The protein
specimens were subjected to western blotting.
4.7. Molecular Docking
Molecular docking was performed using AutoDock Vina[21] with default
parameters. The LYP structure (PDB: 4J51) was obtained from RCSB PDB database.
Hydrogens and missing atoms were added using biopolymer module in Sybyl-x
package. The structure of inhibitor 9r was generated using Sybyl-x, optimized using
Tripos forcefield and assigned with Gasteiger-Hückel charges. The top-scored docking
pose was selected for further analysis.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Grant
No. 81874288 and 21672127), Key Research and Development Project of Shandong
Province (Grant No. 2017CXGC1401) the Fundamental Research Funds of Shandong
University (Grant No. 2019GN045) and The Joint Research Funds for Shandong
University and Karolinska Institute (SDU-KI-2019-06).
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Graphical Abstract
22

Highlights
 Synthesis and preliminary bio-evaluation of imidazolidine-2,4-dione derivatives
and 2-thioxothiazolidin-4-one derivatives as LYP inhibitors.
 The most active compound 9r is a competitive and reversible LYP inhibitor (IC50
=2.52μM) and possess selectivity against a panel of PTPs.
 Compound 9r regulated the TCR Signaling in lymphoid T cell.
23

Declaration of interests
The authors declare that they have no known competing financial interests or personal
☒
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
24