Synthesis and Pin1 inhibitory activity of thiazole derivatives

Article abstract of DOI:10.1016/j.bmc.2016.09.049

Pin1 (Protein interacting with NIMA1) is a peptidyl prolyl cis–trans isomerase (PPIase) which specifically catalyze the conformational conversion of the amide bond of pSer/Thr-Pro motifs in its substrate proteins and is a novel promising anticancer target. A series of new thiazole derivatives were designed and synthesized, and their inhibitory activities were measured against human Pin1 using a protease-coupled enzyme assay. Of all the tested compounds, a number of thiazole derivatives bearing an oxalic acid group at 4-position were found to be potent Pin1 inhibitors with IC₅₀values at low micromolar level. The detailed structure–activity relationships were analyzed and the binding features of compound 10b (IC₅₀5.38 μM) was predicted using CDOCKER program. The results of this research would provide informative guidance for further optimizing thiazole derivatives as potent Pin1 inhibitors.

Full text of DOI:10.1016/j.bmc.2016.09.049

Accepted Manuscript
Synthesis and Pin1 inhibitory activity of thiazole derivatives
Hailong Zhao, Guonan Cui, Jing Jin, Xiaoguang Chen, Bailing Xu
PII:
S0968-0896(16)30766-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.049
BMC 13302
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2016
19 September 2016
20 September 2016
Please cite this article as: Zhao, H., Cui, G., Jin, J., Chen, X., Xu, B., Synthesis and Pin1 inhibitory activity of
thiazole derivatives, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.049
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and Pin1 inhibitory activity of thiazole derivatives
Hailong Zhaoa,#, Guonan Cuia,#, Jing Jina, Xiaoguang Chena,*, Bailing Xua,*
aBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences＆Peking Union Medical College,
Beijing, 100050, China
Abstract: Pin1 (Protein interacting with NIMA1) is a peptidyl prolyl cis-trans
isomerase (PPIase) which specifically catalyze the conformational conversion of the
amide bond of pSer/Thr-Pro motifs in its subtrate proteins and is a novel promising
anticancer target. A series of new thiazole derivatives were designed and synthesized,
and their inhibitory activites were measured agaist human Pin1 using a
protease-coupled enzyme assay. Of all the tested compounds, a number of thiazole
derivatives bearing an oxalic acid group at 4-position were found to be potent Pin1
inhibitors with IC50 values at low micromolar level. The detailed structure–activity
relationships were analyzed and the binding features of compound 10b (IC50 5.38 µM)
was predicted using CDOCKER program. The results of this research would provide
informative guidance for further optimizing thiazole derivatives as potent Pin1
inhibitors.
Keywords: Thiazole derivatives; Pin1; Pin1 inhibitor; PPIase; Anti-cancer agents
1. Introduction
Pin1 (Protein interacting with NIMA1) is a member of the peptidyl prolyl
cis-trans isomerase (PPIase) family which catalyze the isomerization of the amide
bond prior to the proline in its target proteins1-3. Pin1 is structurally and fuctionally
distinct from other PPIase members such as cyclophilins and FKBPs which were
identified as the targets of immunosuppressive drugs cyclosporine A and FK506,
respectively4-11. Pin1 has its own unique substrate specificity and it isomerizes
specific phosphoSer/Thr-Pro motifs and induces conformational changes of its
substrate proteins1, 12-14. Proline-directed phosphorylation on Ser or Thr is a common
regulation mechanism in cells, thus Pin1 plays critical roles in many cellular process
including cell cycle progression, cell signalling, cell proliferation and immune
responses15-18
.
In comparision with pathological roles of Pin1 in other diseases, such as
Alzheimer’s disease and cardiovascular disease19-21, its functions in tumorgenesis and
tumor invasion have been investigated more extensively22-25. Pin1 is overexpressed in
# The first two authors contributed equally.
* Corresponding author. Bailing Xu, Tel: +86 10 63166764; Xiaoguang Chen, Tel: +86 10 63166302
E-mail address: xubl@imm.ac.cn (B. Xu); chxg@imm.ac.cn (X. Chen).

various types of cancer cells, including breast, prostate, colon, cervical, liver and
esophagus cancers, and overexpression of Pin1 is associated with aggressive tumor
progression and poor prognosis in cancer2, 22-26. Furthermore, it has been demonstrated
that Pin1 as a molecular switch simultaneously impacts multiple oncogenic signaling
pathways in tumor cells3, 27; therefore, inhibiting Pin1 is expected to be a more
effective way for fighting against tumors, especially the aggressive and drug-resistant
tumors1, 28, 29
.
Up to now, a number of structurally distinct small-molecule inhibitors of Pin1
have been reported26, 30-39. Among them, the most potent Pin1 inhibitor containing an
aminophenylpropanol scaffold (A, Fig. 1 Ki = 0.006 µM) was disclosed by Pfizer33.
However, it lacked activity versus cells most likely because the phosphate group
confers poor permeability32-34. In an effort to develop Pin1 inhibitors with improved
physicochemical properties, several unnatural amino acid derivatives were found with
enzymatic activity at submicromolar level, and their antiproliferative activity in cells
at micromolar level (e.g. B, Fig. 1 Ki = 0.138 µM)14, 32. Thus, it is challenging to
discover druggable Pin1 inhibitors as anti-cancer agents. Until recently, it was
reported that all-trans retinoic acid (ATRA), a therapeutic agent for acute
promyelocytic leukemia, was a potent Pin1 inhibitor and its inhibitory effect on Pin1
was responsible for the anti-cancer activity40. These results gave more expectations on
the development of Pin1 inhibitors for the treatment of cancers.
The crystal structures of some small molecule inhibitors complexed with Pin1
were reported32-36. The binding site of catalytic domain features three subpockets,
including a prolyl pocket formed with His59, His157, Met130 and Phe13, a slightly
shallow hydrophobic shelf lined with Ala118 and Leu22, and a unique phosphate
binding pocket embracing Lys63, Arg68 and Arg69. In our previous work, we found
that substituted pyrimidine derivatives possessed Pin1 inhibitory activity with IC50
values in the double digit micromolar level (e.g. C, Fig. 1 IC50 = 26.7 µM)39. Based
on the binding features of compound C and the known structure-activity
relationships32, 39, we envisioned that the pyrimidine scaffold could be replaced with a
thiazole ring, and the thiazole scaffold may be preferable in terms of directing the
pharmacophore group into the subpockets. Therefore, we designed a series of novel
thiazole derivatives, in which an oxalic acid or acetic acid group was attached onto
the 4-position, a small hydrophobic moiety was placed at the 2-position and a bulky
aromatic substituent was incorporated into the 5-position. Herein, we present the
synthesis and biochemical evaluation of a series of thiazol derivatives as Pin1
inhibitors.
Figure 1 The chemical structures of some known Pin1 inhibitors and the general structure of

designed compounds
2. Chemistry
Scheme 1: Reagents and conditions (a) ethyl bromoacetate, triethylamine, ethanol, 93%; (b) NaH,
(Boc)2O, THF, 90%; (c) NaOH, THF, H2O 45°C, 90%; (d) EDC, HOAt, DMAP, DIEA, DCM,
DMF, 50%-72%; (e) mCPBA, CHCl3, reflux, 50%-78%; (f) R1OH, NaH, DMF, 26%-86%; (g)
TFA, DCM, 61%-94%; (h) tert-butyl 2-chloro-2-oxoacetate, toluene, 58%-93%; (i) TFA, DCM,
59%-92%.
The synthesis of target compounds (10a–10x and 19a–19e) was depicted in
Schemes 1–3, and their chemical structures were shown in Tables 1-2. Initially, we
took the synthetic route as presented in scheme 1 to construct the target compounds.
A mixture of potassium methyl cyanimidodithiocarbonate 1, ethyl bromoacetate
and triethylamine in ethanol was heated to give rise to the key thiazole scaffold 2 in
93% yield. Upon subsequent Boc protection, hydrolysis of the ethyl ester,
condensation with an array of primary amines, and oxidation of the methylthio group,
compound 2 was converted into compounds 6a-6f smoothly. In the presence of NaH
and various substituted phenols in DMF, compounds 7a-7l were prepared by the
nucleophilic substitution reaction, which were further transformed into compounds
8a-8l by removal of the Boc group with trifluoroacetic acid. The acylation of amines
8a-8k with tert-butyl 2-chloro-2-oxoacetate in toluene afforded compounds 9a-9k,
which were subsequently transformed into target compounds 10a-10k by treatment of

with TFA.
Scheme 2: Reagents and conditions (a) ArNH2, Py, DCM, 41%; (b) 0°C, KOH, CS2, ethanol,
72%; (c) i) 0-25°C, acetone, water, ii) ethanol, MeI, 73%; (d) tert-butyl 2-chloro-2-oxoacetate,
tolueme, 92%; (e) m-CPBA, CHCl3, 44%; (f) R1OH, NaH, DMF, 41%-72%; (g) TFA, DCM,
69%-97%; (h) TFA, DCM, 69%.
During the course of synthesis using the first synthetic route, we found that
methylsulfonyl compounds 6a-6f, particularly 6a had poor solubility that was
troublesome for purification. Thus we tried an alternative synthetic route as shown in
Scheme 2. In this approach, naphthylamine was first coupled with 2-bromoacetyl
bromide to give rise to the intermediate 12 in 41% yield, and then 2-bromo-acetamide
41
12 was reacted with potassium cyanocarbonimidodithioate 14
forming the key
intermediate 15 in 72% yield. The acylation of amine 15 with tert-butyl
2-chloro-2-oxoacetate in toluene proceeded smoothly to afford compound 16, which
was subsequently transformed into target compounds 10l-10x in three steps by
oxidation, substitution and deprotection. In fact, compared with the first synthetic
method, this approach was more concise and efficient.
In addition, with an aim to explore SAR on 2-position of thiazole ring,
compound 18 was prepared by removing the t-butyl group from compound 16 using
TFA.

COOH
F
F
NH
NH
2
a
N
N
H
N
H
N
Ar
O
Ar
O
S
S
O
O
8c, 8i-8l
19a-19e
8c, 19a:
8i, 19b:
8j, 19c:
8k, 19d:
8l, 19e:
Ar = Naphthalen-2-yl
Ar = 3,5-Dichlorobenzyl
)
Ar = 3,5-Bis(trifluoromethyl benzyl
Ar = 2,6-Dichlorophenethyl
Ar = 4-PhPh
Scheme 3: Reagents and conditions (a) glyoxylic acid, NaBH3CN, HOAc, MeOH/THF,
38%-88%.
The acetic acid substituted target compounds 19a-19e were synthesized starting
from intermediates 8c and 8i-8l (Scheme 3). In the presence of sodium
cyanoborohydride and acetic acid using MeOH and THF as solvents, compounds 8c
and 8i-8l reacted with glyoxylic acid by reductive amination giving compounds
19a-19e (38%-88% yield).
3. Biological results and discussion
All target compounds were screened against Pin1 by a protease-coupled enzyme
assay with Suc-Ala-Glu-Pro-Phe-pNA as the substrate42, 43. The reported Pin1
inhibitors compound A and compound B were used as reference molecules. The
inhibitory activities were expressed as IC50 values and presented in Tables 1-2.
Table 1
The chemical structures and inhibitory activities against hPin1 of compounds 10a-10x
and 18
IC50ƏSD(µM) a
4.57Ə1.14
IC50ƏSD(µM) a
6.04Ə3.35
Cpd.
R1
Ar
Cpd.
R1
Ar
10a
10n
10b
10c
5.38Ə0.35
5.50Ə4.23
10o
10p
5.02Ə0.90
5.42Ə1.15

Cl
10d
10e
10f
4.68Ə0.41
9.44Ə2.96
5.18Ə2.32
3.15Ə0.65
2.93Ə0.39
10q
10r
10s
10t
10u
13.4Ə2.74
4.67Ə0.99
9.35Ə1.23
8.11Ə0.46
11.1Ə0.70
10g
10h
10i
10j
10k
25.8Ə27.2%b
51.1Ə6.43% b
53.3Ə3.56% b
10v
23.4Ə4.98
14.8Ə0.29
10w
10x
18
21.1Ə3.15
10l
48.9Ə2.26% b
-11.6Ə8.38%b
10m
3.22Ə1.20
a. The measured IC50 for compound A was 0.05 µM, the reported Ki for compound A was 0.006
µM; the measured IC50 for compound B was 2.96 µM, the reported Ki for compound B was 0.138
µM; SD, standard deviation of two independent assays.
b. The percentage inhibition at the concentration of 100 µM of tested compounds.
As shown in Table 1, we initially investigated the SARs on the substituents at
2-position of thiazole ring by taking the naphthyl fragment as the bulky aromatic

group attached on the 5-position. When phenyl or mono-substituted phenyl group was
introduced as R1 group, the corresponding compounds (10a-10d and 10l-10t)
exhibited varied inhibitory activities against Pin1. Among them, most
mono-substituted compounds showed similar potency with unsubstituted compound
10a, they had IC50 values in the single digit micromolar level (IC50 3.22-9.35 µM). It
was noted that 2-chloro substituted compound 10q had somewhat lower activity (IC50
13.4 µM), and 2-methyl substituted compound 10l displayed the weakest inhibition on
Pin1 (48.9% @ 100 µM) in this series. Collectively, these results suggest that
placement of a substituent on the 3-position of the benzene ring was allowed and
incorporation of a fragment at the 2-position of the benzene ring is unfavorable for
potency.
In addition, three 2, 5-disubstituted compounds (10u-10w) were also tested and
their IC50s varied from 11.1 µM to 23.4 µM. It further suggested that introducing a
substituent on 2-position on the benzene ring would lead to reduction in potency in
comparison with the corresponding mono-substituted compounds 10b and 10c.
Interestingly, when an isobutyl group substituted for the phenyl moiety as R1
group, compound 10x possessed inhibitory activity as well, although its potency (IC50
21.1 µM) was lower than that of compound 10a. This result gave an indication that
alkyl moieties could be used as R1 group to snugly fit into the prolyl pocket. This
might offer a chance to further improve both potency and physicochemical properties.
With an aim to expanding the SARs on 2-substitutents, we also tested the enzymatic
activity of compound 18. It had no inhibition on Pin1, presumably due to the
methylthio group that cannot form a hydrogen bond with Pin1, compared with
compound 10x.
The preliminary SAR of 5-substituents on thiazole ring was also explored. The
incorporation of bulky aromatic groups, such as 4-phenoxyphenyl (10e and 10f) and
4-biphenyl moieties (10g and 10h) through an amide linker resulted in comparable
potency with naphthyl substituted compounds 10a and 10b. However, the
replacement of naphthyl group of compound 10c with 3, 5-dichlorobenzyl (10i), 3,
5-bis(trifluoromethyl)benzyl (10j) or 2, 6-dichlorophenethyl (10k) group led to
reduction in inhibition markedly.
Table 2
The chemical structures and inhibitory activities against hPin1 of compounds 19a-19e
IC50ƏSD (µM) a
27.7Ə10.8%b
IC50ƏSD (µM) a
5.41Ə7.64%b
Cpd.
Ar
Cpd.
Ar
19a
19d

19b
19c
14.
0
5.52
19e
9.4
0
2.43% b
18.1 3.54
a. The measured IC50 for compound A was 0.05 µM, the reported Ki for compound A was 0.006
µM; the measured IC50 for compound B was 2.96 µM, the reported Ki for compound B was 0.138
µM; SD, standard deviation of two independent assays.
b. The percentage inhibition at the concentration of 100 µM of tested compounds.
We substituted the acetic acid group for an oxalic acid moiety producing
compounds 19a-19e (Table 2). Among them, the potency of compounds 19a, 19d and
19e were noticeably lost. Interestingly, compounds 19b and 19c had inhibitory
activity and their IC50s were 14.0 µM and 18.1 µM, respectively. From these data, it
seemed that an oxalic acid group was more favorable than the acetic acid counterpart
to interact with Pin1.
A
Figure 2. CDOCKER-modeled binding mode of compound 10b (carbon atoms colored blue) in
comparison with the co-crystal structure (3JYJ in PDB)34 of a carboxylate Pin1 inhibitor (carbon
atoms colored brown). (A) The binding pose of compound 10b within the binding site of Pin1; (B)
The interactions of compound 10b with key amino acids within the binding stie. H-Bonding
interactions were presented with blue lines. Molecular image was generated with UCSF
Chimera44.
With an aim to probe the binding features of the thiazole-based Pin1 inhibitors,
molecular docking was performed using CDOCER protocol integrated in Accelrys
Discovery Studio 2.545. The coordinates of X-ray co-crystal structure of a carboxylate
inhibitor (reference molecule) with Pin1 (PDB code: 3JYJ) reported by Pfizer in
201034 was employed for docking the thiazole derivatives. The binding modes of the
oxalic acid substituted thiazoles were somewhat similar and exemplified by the
representative compound 10b as shown in Figure 2. Compound 10b could nicely
situate in the binding pocket and took a very similar binding pose with the reference
molecule.
The carboxylate of oxalic acid interacted with the positive charged side chain of

Lys63 via the key charge-charge interaction. The 2-carbonyl oxygen of oxalic acid
formed a hydrogen bond with Arg 69 and a hydrogen bond with Ser154 mediated by a
water molecule. Therefore, the oxalic acid moiety played a critical role in the binding
and could be a choice for replacement of the phosphorus acid group to improve the
potency and physicochemical properties of Pin1 inhibitors. It was noticed that the
oxygen atom as an ether linker could interact with Ser154 via a hydrogen bond as we
intended, since the cocrystal structures of known Pin1 inhibitors have demonstrated
that Ser154 had a key role in the binding32, 34. In fact, compound 18 bearing a
methylthio group on the 2-poistion of the thiazole scaffold was inactive, a result
consistent with the oxygen atom on the 2-position benefical for binding. In addition,
the nitrogen atom on the thiazole ring also formed an H-bond with Ser154 via a water
molecule. This may further contribute to the favorable binding affinity.
The hydrophobic 3-methylbenzene ring occupied the prolyl pocket consisting of
Leu122, Phe134 and Met130 residues, the bulky naphthalene ring extended to the
hydrophobic shelf including the side chains of Leu122 and Cys113. These
hydrophobic interactions had positive contributions to the binding affinity as we and
other groups have demonstrated34, 39
.
4. Conclusion
In summary, based on the pyrimidine Pin1 inhibitors we found previously, a
series of novel thiazole derivatives containing an oxalic acid or an acetic acid group
was designed and synthesized. The enzymatic assay was performed against Pin1 and
some of compounds displayed potent inhibitory activity with IC50 values at the
micromolar level. The preliminary SAR and the binding features of the thiazole
derivatives were analyzed. These results suggest that an oxalic acid moiety could be
useful in developing potent Pin1 inhibitors, and the thiazole ring could serve as an
appropriate scaffold for discovering structurally novel and drug-like Pin1 inhibitors.
These results will provide guidance for further modifications to achieve more potent
thiazole derivatives as Pin1 inhibitors.
5. Experimental section
5.1. General
Melting points were measured on a Yanaco micro melting point apparatus and
are uncorrected.1H NMR (300 MHz or 400 MHz) on a Varian Mercury 300 or 400
spectrometer was recorded in DMSO-d6, acetone-d6 or CDCl3. Chemical shifts are
reported in δ (ppm) units relative to the internal standard tetramethylsilane (TMS).
High resolution mass spectra (HRMS) were obtained on an Agilent Technologies
LC/MSD TOF spectrometer. All chemicals and solvents used were of reagent grade
without purified or dried before use. All the reactions were monitored by thin-layer

chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp.
Column chromatography separations were performed with silica gel (200–300 mesh).
5.2. Synthesis of compounds 10a-10k
5.2.1
2-((5-(Naphthalen-2-ylcarbamoyl)-2-phenoxythiazol-4-yl)amino)-2-oxoacetic acid
(10a)
To a stirred solution of 9a (80 mg, 0.16 mmol) in DCM (15 mL) was added TFA
(1.5 mL) dropwise, the reaction mixture was then allowed to stir at room temperature
overnight. DCM and excess TFA were removed under reduced pressure,
dichloromethane (2 mL) and petroleum ether (5 mL)
were then added to the
residue. After filtration and drying under high vacuum, compound 10a was obtained
as a yellow solid without further purification (50 mg, 74%); mp 155-157 °C; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 11.94 (brs, 1H), 10.13 (s, 1H), 8.23 (s, 1H), 7.85 (t, J
= 7.2 Hz, 3H), 7.67 (d, J = 8.8 Hz, 1H), 7.53-7.61 (m, 4H), 7.40-7.49 (m, 3H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 173.81, 161.18, 160.52, 154.22, 147.01, 135.71,
133.10, 130.71, 130.14, 128.12, 127.56, 127.42, 126.41, 125.05, 121.19, 120.92,
117.55; HRMS (ESI) Calcd. for C22H16N3O5S [M+H]+: 434.0805; Found: 434.0808.
5.2.2
2-((5-(Naphthalen-2-ylcarbamoyl)-2-(m-tolyloxy)thiazol-4-yl)amino)-2-
oxoacetic acid (10b)
Following the preparation protocol of compound 10a, starting from compound
9b (90 mg, 0.18 mmol), the title compound 10b was obtained as a yellow solid (70
°
mg, 88%); mp 123-125 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.96 (brs, 1H),
10.13 (s, 1H), 8.24 (s, 1H), 7.84-7.89 (m, 3H), 7.68 (dd, J1 = 8.7 Hz, J2 = 1.5 Hz,1H),
7.41-7.52 (m, 3H), 7.31-7.39 (m, 2H), 7.28 (d, J = 7.2 Hz, 1H), 2.39 (s, 3H); HRMS
(ESI) Calcd. for C23H18N3O5S [M+H]+: 448.0962; Found: 448.0963.
5.2.3
2-((2-(3-Fluorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxo
acetic acid (10c)
Following the preparation protocol of compound 10a, starting from compound
9c (70 mg, 0.14 mmol), the title compound 10c was obtained as a yellow solid (54
°
mg, 87%); mp 98-100 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.92 (s, 1H),
10.18 (s, 1H), 8.24 (s, 1H), 7.84-7.89 (m, 3H), 7.68 (d, J = 9.9 Hz, 1H); 7.58-7.63 (m,

2H), 7.40-7.50 (m, 3H), 7.30 (t, J = 8.7 Hz, 1H); HRMS (ESI): m/z, calcd. for
C22H15N3O5FS [M+H]+: 452.0711, Found: 452.0713.
5.2.4
2-((2-(3-Methoxyphenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-o
xoacetic acid (10d)
Following the preparation protocol of compound 10a, starting from compound
9d (60 mg, 0.12 mmol), the title compound 10d was obtained as a yellow solid (47
°
mg, 89%); mp 106-108 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.96 (brs, 1H),
10.13 (s, 1H), 8.24 (s, 1H), 7.83-7.88 (m, 3H), 7.68 (d, J = 7.2 Hz, 1H), 7.40-7.51 (m,
3H), 7.19 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.01 (d, J = 6.6 Hz, 1H), 3.81 (s, 3H);
HRMS (ESI) Calcd. for C23H18N3O6S [M+H]+: 464.0911; Found: 464.0900.
5.2.5
2-((2-Phenoxy-5-((4-phenoxyphenyl)carbamoyl)thiazol-4-yl)amino)-
2-oxoacetic acid (10e)
Following the preparation protocol of compound 10a, starting from compound
9e (100 mg, 0.19 mmol), the title compound 10e was obtained as a light yellow solid
°
(67 mg, 75%); mp 123-125 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.95 (brs,
1H), 9.96 (s, 1H), 7.34-7.61 (m, 9H), 7.11 (t, J = 6.9 Hz, 1H), 6.97-6.99 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 173.71, 161.14, 160.23, 157.00, 154.20, 152.70,
146.90, 133.67, 130.70, 129.96, 127.54, 123.15, 122.92, 120.90, 119.00, 118.11;
HRMS (ESI): m/z, calcd. for C24H18N3O6S [M+H]+: 476.0911, Found: 476.0904.
5.2.6
2-((5-((4-Phenoxyphenyl)carbamoyl)-2-(m-tolyloxy)thiazol-4-yl)amino)-2-oxoacet
ic acid (10f)
Following the preparation protocol of compound 10a, starting from compound 9f
(45 mg, 0.08mmol), the title compound 10f was obtained as a yellow solid (37 mg,
92%); mp 124-127 °C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.94 (s, 1H), 9.94 (s,
1H), 7.60 (d, J = 8.7 Hz, 2H), 7.24-7.48 (m, 6H), 7.10 (t, J = 6.9 Hz, 1H), 6.98 (d, J =
8.7 Hz, 4H), 2.37 (s, 3H); HRMS (ESI): m/z, calcd. for C25H20N3O6S [M+H]+:
490.1067, Found: 490.1070.

5.2.7
2-((5-([1,1'-Biphenyl]-4-ylcarbamoyl)-2-phenoxythiazol-4-yl)amino)-2-oxoacetic
acid (10g)
Following the preparation protocol of compound 10a, starting from compound
9g (70 mg, 0.14 mmol), the title compound 10g was obtained as a yellow solid (50
°
mg, 81%); mp 139-141 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.96 (s, 1H),
10.02 (s, 1H), 7.52-7.78 (m, 10H), 7.42-7.45 (m, 3H), 7.32 (d, J = 6.8 Hz, 1H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 173.80, 160.35, 154.22, 147.08, 139.55, 137.58,
135.79, 130.72, 128.88, 127.56, 127.14, 126.75, 126.29, 121.33, 120.93; HRMS (ESI):
m/z, calcd. for C24H18N3O5S [M+H]+: 460.0962, Found: 460.0961.
5.2.8 2-((5-([1,1'-Biphenyl]-4-ylcarbamoyl)-2-(m-tolyloxy)thiazol-4-yl)amino)-2-
-oxoacetic acid (10h)
Following the preparation protocol of compound 10a, starting from compound
9h (60 mg, 0.16 mmol), the title compound 10h was obtained as a yellow solid (40
°
mg, 76%); mp 112-114 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.96 (brs, 1H),
10.01 (s, 1H), 7.63-7.72 (m, 6H), 7.41-7.49 (m, 3H), 7.22-7.35 (m, 4H), 2.38 (s, 3H);
HRMS (ESI) Calcd. for C25H20N3O5S [M+H]+: 474.1118; Found: 474.1109.
5.2.9
2-((5-((3,5-Dichlorobenzyl)carbamoyl)-2-(3-fluorophenoxy)thiazol-4-yl)amino)-2-
oxoacetic acid (10i)
Following the preparation protocol of compound 10a, starting from compound 9i
(120 mg, 0.22 mmol), the title compound 10i was obtained as a grey white solid
°
(90 mg, 84%); mp 102-104 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.90 (brs,
1H), 8.84 (t, J = 5.6 Hz, 1H), 7.55-7.63 (m, 2H), 7.49 (s, 1H), 7.38 (dd, J1 = 8.4 Hz, J2
= 2.4 Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.28 (td, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 4.39
(d, J = 6.0 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.29, 162.38 (d, J =
259.1 Hz), 161.60, 161.23, 154.72 (d, J = 11.1 Hz), 146.05, 143.35 , 133.91, 131.86
(d, J = 9.2 Hz), 126.55 , 126.04 , 116.97 (d, J = 3.0 Hz), 114.21 (d, J = 20.7 Hz),
108.91 (d, J = 27.5 Hz), 41.64; HRMS (ESI) Calcd. for C19H13N3O5Cl2FS [M+H]+:
483.9932; Found: 483.9923.

5.2.10
2-((5-((3,5-Bis(trifluoromethyl)benzyl)carbamoyl)-2-(3-fluorophenoxy)thiazol-4-y
l)amino)-2-oxoacetic acid (10j)
Following the preparation protocol of compound 10a, starting from compound 9j
(90 mg, 0.171 mmol), the title compound 10j was obtained as a light yellow solid
°
(50 mg, 59%); mp 101-103 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.85 (brs,
1H), 8.91 (t, J = 6.0 Hz, 1H), 8.00 (s, 3H), 7.56-7.63 (m, 2H), 7.39 (d, J = 8.0 Hz, 1H),
7.28 (t, J = 8.4 Hz, 1H), 4.57 (d, J = 5.6 Hz, 2H); HRMS (ESI) Calcd. for
C21H13N3O5Cl2F7S [M+H]+: 552.0459; Found: 552.0450.
5.2.11 2-((5-((2,6-Dichlorophenethyl)carbamoyl)-2-(3-fluorophenoxy)thiazol-4-yl)
-amino)-2-oxoacetic acid (10k)
Following the preparation protocol of compound 10a, starting from compound
9k (95 mg, 0.171mmol), the title compound 10k was obtained as a pale yellow solid
°
(50 mg, 59%); mp 104-106 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (brs,
1H), 8.48 (brs, 1H), 7.54-7.64 (m, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.38 (d, J = 8.0 Hz,
1H), 7.28 (t, J = 8.0 Hz, 2H), 3.40-3.42 (m, 2H), 3.10 (t, J = 6.0 Hz, 2H); HRMS (ESI)
Calcd. for C20H15N3O5Cl2FS [M+H]+: 498.0088; Found: 498.0088.
5.3. Synthesis of compounds 10l-10x and 18
5.3.1
2-((5-(Naphthalen-2-ylcarbamoyl)-2-(o-tolyloxy)thiazol-4-yl)amino)-2-oxoacetic
acid (10l)
Following the preparation protocol of compound 10a, starting from compound 9l
(120 mg, 0.238 mmol), the title compound 10l was obtained as a yellow solid (75 mg,
°
71.4%); mp 169-171 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.98 (brs, 1H),
10.11 (s, 1H), 8.24 (s, 1H), 7.84-7.88 (m, 3H), 7.67 (d, J = 8.8 Hz, 1H), 7.36-7.51 (m,
6H), 2.28 (s, 3H); HRMS (ESI) Calcd. for C23H18N3O5S [M+H]+: 448.0962; Found:
448.0948.

5.3.2
2-((5-(Naphthalen-2-ylcarbamoyl)-2-(p-tolyloxy)thiazol-4-yl)amino)-2-oxoacetic
acid (10m)
Following the preparation protocol of compound 10a, starting from compound
9m (70 mg, 0.14 mmol), the title compound 10m was obtained as a yellow solid (51
mg, 82%); mp 124-126 °C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.95 (brs, 1H),
10.09 (s, 1H), 8.23 (s, 1H), 7.83-7.87 (m, 3H), 7.65-7.68 (m, 1H), 7.36-7.50 (m, 6H),
2.38 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.40, 161.24, 160.59,
152.20, 147.34, 137.21, 135.76, 133.10, 131.02, 130.81, 130.12, 128.10, 127.41,
126.40, 125.02, 121.17, 120.77, 117.50, 20.44; HRMS (ESI) Calcd. for C23H18N3O5S
[M+H]+: 448.0962; Found: 448.0952.
5.3.3
2-((2-(2-Methoxyphenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-o
xoacetic acid (10n)
Following the preparation protocol of compound 10a, starting from compound
9n (70 mg, 0.14 mmol), the title compound 10n was obtained as a light yellow solid
°
(45 mg, 94%); mp 139-141 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (brs,
1H), 10.09 (s, 1H), 8.24 (s, 1H), 7.83-7.87 (m, 3H), 7.68 (d, J = 8.4 Hz, 1H),
7.42-7.54 (m, 4H), 7.34 (d, J = 8.0 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 3.85 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 174.60, 161.23, 160.53, 150.79, 146.98, 142.48,
135.75, 133.10, 130.12, 129.07, 128.10, 127.41, 126.40, 125.02, 122.69, 121.41,
121.18, 117.48, 114.20, 109.29, 56.06; HRMS (ESI) Calcd. for C23H18N3O6S [M+H]+:
464.0911; Found: 464.0902.
5.3.4
2-((2-(2-Fluorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxo
acetic acid (10o)
Following the preparation protocol of compound 10a, starting from compound
9o (80 mg, 0.16 mmol), the title compound 10o was obtained as a yellow solid (65 mg,
°
92%); mp 124-126 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.85 (brs, 1H),
10.20 (s, 1H), 8.25 (s, 1H), 7.85-7.90 (m, 3H), 7.67-7.76 (m, 2H), 7.37-7.60 (m, 6H);
HRMS (ESI) Calcd. for C22H15N3O5FS [M+H]+: 452.0711; Found: 452.0700.

5.3.5
2-((2-(3-Chlorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxo
acetic acid (10p)
Following the preparation protocol of compound 10a, starting from compound
9p (80 mg, 0.15 mmol), the title compound 10p was obtained as a yellow solid (69
mg, 97%); mp 177-180 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (brs, 1H),
10.19 (s, 1H), 8.26 (s, 1H), 7.86-7.90 (m, 3H), 7.80 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H),
7.62 (t, J = 8.0 Hz, 1H), 7.42-7.56 (m, 5H); HRMS (ESI) Calcd. for C22H15N3O5ClS
[M+H]+: 468.0416; Found: 468.0407.
5.3.6
2-((2-(2-Chlorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxo
acetic acid (10q)
Following the preparation protocol of compound 10a, starting from compound
9q (70 mg, 0.13 mmol), the title compound 10q was obtained as a light yellow solid
°
(58 mg, 94%); mp 132-134 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.89 (brs,
1H), 10.18 (s, 1H), 8.25 (s, 1H), 7.85-7.89 (m, 3H), 7.78 (dd, J1 = 3.0 Hz, J2 = 1.8 Hz,
1H), 7.75 (dd, J1 = 3.3 Hz, J2 = 1.8 Hz, 1H), 7.68 (dd, J1 = 9.0 Hz, J2 = 1.8 Hz, 1H),
7.58 (dt, J1 = 7.8 Hz, J2 = 1.8 Hz, 1H), 7.42-7.53 (m, 3H); HRMS (ESI) Calcd. for
C22H15N3O5ClS [M+H]+: 468.0416; Found: 468.0407.
5.3.7
2-((2-(3-Cyanophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxo
acetic acid (10r)
Following the preparation protocol of compound 10a, starting from compound
9r (40 mg, 0.09 mmol), the title compound 10r was obtained as a pale yellow solid
°
(32 mg, 89%); mp 180-183 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.92 (brs,
1H), 10.23 (s, 1H), 8.26 (s, 1H), 8.21 (s, 1H), 7.86-7.95 (m, 5H), 7.78 (t, J = 8.1 Hz,
1H), 7.70 (d, J = 7.5 Hz, 1H), 7.37-7.53 (m, 3H); HRMS (ESI) Calcd. for
C23H15N4O5S [M+H]+: 459.0758; Found: 459.0748.
5.3.8 2-((5-(Naphthalen-2-ylcarbamoyl)-2-(3-(trifluoromethyl) phenoxy)thiazol-4-
-yl)amino)-2-oxoacetic acid (10s)
Following the preparation protocol of compound 10a, starting from compound 9s
(95 mg, 0.17 mmol), the title compound 10s was obtained as a yellow solid (70 mg,

°
83%); mp 178-181 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (brs, 1H),
10.20 (s, 1H), 8.25 (s, 1H), 8.06 (s, 1H), 7.81-7.90 (m, 7H), 7.70 (d, J = 8.8 Hz, 1H),
7.42-7.51 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.48, 161.15, 160.37,
154.12, 146.46, 135.67, 133.11, 131.88, 131.05 (d, J = 32.1 Hz), 130.19, 128.16,
127.44, 126.43, 125.27, 125.09, 123.93, (d, J = 2.9 Hz), 121.23, 118.17 (d, J = 4.4
Hz), 117.63; HRMS (ESI) Calcd. for C23H15N3O5F3S [M+H]+: 502.0679; Found:
502.0669.
5.3.9 2-((5-(Naphthalen-2-ylcarbamoyl)-2-(2-(trifluoromethyl) phenoxy)thiazol-4-
-yl)amino)-2-oxoacetic acid (10t)
Following the preparation protocol of compound 10a, starting from compound 9t
(130 mg, 0.23 mmol), the title compound 10t was obtained as a light yellow solid (80
mg, 69%); mp 138-140 °C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.87 (brs, 1H),
10.22 (s, 1H), 8.26 (s, 1H), 7.85-7.98 (m, 6H), 7.67 (d, J = 8.1 Hz, 2H), 7.41-7.52 (m,
2H); HRMS (ESI) Calcd. for C23H15F3N3O5S [M+H]+: 502.0679; Found: 502.0680.
5.3.10 2-((2-(2-Fluoro-5-methylphenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-
-4-yl)amino)-2-oxoacetic acid (10u)
Following the preparation protocol of compound 10a, starting from compound
9u (100 mg, 0.19 mmol), the title compound 10u was obtained as a pale yellow solid
°
(72 mg, 81%); mp 124-126 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.88 (brs,
1H), 10.19 (s, 1H), 8.26 (s, 1H), 7.84-7.90 (m, 3H), 7.67 (d, J = 8.8 Hz, 1H), 7.54 (d,
J = 7.2 Hz, 1H), 7.41-7.52 (m, 4H), 7.29 (brs, 1H), 2.35 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 172.77, 161.13, 160.21, 149.90, 146.16, 140.42 (d, J = 12.9 Hz),
135.78 (d, J = 3.4 Hz), 134.40 (d, J = 260.3 Hz), 130.16, 129.39 (d, J = 6.7 Hz),
128.15, 127.43, 126.43, 125.06, 123.67, 121.15, 117.50, 117.30 (d, J = 17.5 Hz),
20.15; HRMS (ESI) Calcd. for C23H17N3O5FS [M+H]+: 466.0868; Found: 466.0861.
5.3.11 2-((2-(5-Fluoro-2-methylphenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-
-4-yl)amino)-2-oxoacetic acid (10v)
Following the preparation protocol of compound 10a, starting from compound
9v (80 mg, 0.15 mmol), the title compound 10v was obtained as a yellow solid (65 mg,
°
92%); mp 124-126 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.94 (s, 1H), 10.15
(s, 1H), 8.25 (s, 1H), 7.84-7.90 (m, 3H), 7.68 (dd, J1 = 8.7 Hz, J2 = 1.8 Hz, 1H), 7.57
(dd, J1 = 9.0 Hz, J2 = 2.4 Hz, 1H), 7.41-7.52 (m, 3H), 7.25 (td, J1 = 8.4 Hz, J2 = 2.7
Hz, 1H), 2.24 (s, 3H); HRMS (ESI) Calcd. for C23H17N3O5FS [M+H]+: 466.0868;
Found: 466.0860.

5.3.12
2-((2-(2,5-Difluorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)
amino)-2-oxoacetic acid (10w)
Following the preparation protocol of compound 10a, starting from compound
9w (60 mg, 0.11 mmol), the title compound 10w was obtained as a yellow solid (50
°
mg, 93%); mp 118-120 C; 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.83 (s, 1H),
10.25 (s, 1H), 8.26 (s, 1H), 7.84-7.91 (m, 4H), 7.59-7.72 (m, 3H), 7.37-7.53 (m, 3H);
HRMS (ESI) Calcd. for C22H14N3O5F2S [M+H]+: 470.0617; Found: 470.0609.
5.3.13
2-((2-Isobutoxy-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)-2-oxoacetic
acid (10x)
Following the preparation protocol of compound 10a, starting from compound
9x (140 mg, 0.30 mmol), the title compound 10x was obtained as a light yellow solid
°
(85 mg, 70%); mp 170-172 C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.89 (brs,
1H), 10.14 (s, 1H), 8.26 (s, 1H), 7.87-7.91 (m, 3H), 7.72 (d, J = 8.4 Hz, 1H),
7.43-7.52 (m, 2H), 4.27 (d, J = 6.4 Hz, 2H), 2.16 (hept, J = 6.4 Hz, 1H), 0.99 (d, J =
6.8 Hz, 6H); HRMS (ESI) Calcd. for C20H20N3O5S [M+H]+: 414.1118; Found:
414.1107.
5.3.14. Synthesis of 2-((2-(Methylthio)-5-(naphthalen-2-ylcarbamoyl) thiazol-4-yl)
amino)-2-oxoacetic acid (18)
Following the preparation protocol of compound 10a, starting from compound
16a (100 mg, 0.22 mmol), the title compound 18 was obtained as a yellow solid (60
mg, 69%); mp 146-148 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.78 (brs, 1H),
10.30 (s, 1H), 8.28 (s, 1H), 7.89 (t, J = 9.6 Hz, 3H), 7.73 (d, J = 8.8 Hz, 1H), 7.50 (t, J
= 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 2.78 (s, 3H); HRMS (ESI) Calcd. for
C17H14N3O4S2 [M+H]+: 388.0420; Found: 388.0407.
5.4. Synthesis of compounds 19a-19e
5.4.1. 2-((2-(3-Fluorophenoxy)-5-(naphthalen-2-ylcarbamoyl)thiazol-4-yl)amino)
acetic acid (19a)
To a stirred solution of compound 8c (80 mg, 0.211 mmol) in MeOH (5.0 mL)
and THF (3.0 mL) was added glyoxylic acid (130 mg, 0.843 mmol) and acetic acid
°
(30 mg, 0.43 mmol). The mixture was stirred at 45 C for 1 h, then sodium

cyanoborohydride (78 mg, 1.27 mmol) was added, and the reaction mixture was
continuously stirred at room temperature for 1 day. The solvent was evaporated under
reduced pressure. The crude was purified by silica gel column chromatography
(dichloromethane/isopropanol/HOAc 120:2:1) to afford the title compound 19a (80
°
mg, 55.6%). Light yellow solid; mp 199-201 C; 1H NMR (400 MHz, DMSO-d6) δ
(ppm): 12.66 (s, 1H), 9.46 (s, 1H), 8.27 (s, 1H), 8.09 (t, J = 5.6 Hz, 1H), 7.81 (d, J =
8.8 Hz, 2H), 7.78 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 (dd, J1 = 15.6 Hz,
J2 = 8.0 Hz, 1H), 7.54 (d, J = 9.6 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.2 Hz,
2H), 7.30 (t, J = 8.4 Hz, 1H), 4.11 (d, J = 6.0 Hz, 2H); HRMS (ESI) Calcd. for
C22H17N3O4FS [M+H]+: 438.0918; Found: 438.0902.
5.4.2.
2-((5-((3,5-Dichlorobenzyl)carbamoyl)-2-(3-fluorophenoxy)thiazol-4-yl)amino)ac
etic acid (19b)
Following the preparation protocol of compound 19a, starting from compound 8i
(150 mg, 0.364 mmol), the title compound 19b was obtained as a white solid (65 mg,
38%); mp 150-152 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.60 (brs, 1H), 8.09
(t, J = 6.0 Hz, 1H), 7.79 (t, J = 6.0 Hz, 1H), 7.57 (dd, J1 = 15.6 Hz, J2 = 8.0 Hz, 1H),
7.46-7.50 (m, 2H), 7.24-7.343 (m, 4H), 4.29 (d, J = 5.6 Hz, 2H), 4.01 (d, J = 6.0 Hz,
2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.58, 171.85, 163.48, 162.39 (d, J =
244.9 Hz), 158.38, 154.66 (d, J = 11.3 Hz), 144.53, 133.79, 131.73 (d, J = 9.6 Hz),
126.26, 126.01, 117.10 (d, J = 3.4 Hz), 114.14 (d, J = 20.9 Hz), 109.00 (d, J = 24.7
Hz), 44.75, 41.38; HR-MS (ESI): m/z, calcd. for C19H15O4N3Cl2FS [M+H]+ 470.0139,
Found: 470.0133.
5.4.3.
2-((5-((3,5-Bis(trifluoromethyl)benzyl)carbamoyl)-2-(3-fluorophenoxy)thiazol-4-y
l)amino)acetic acid (19c)
Following the preparation protocol of compound 19a, starting from compound 8j
(170 mg, 0.36 mmol), the title compound 19c was obtained as a white solid (90 mg,
48%); mp 115-117 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.55 (brs, 1H), 8.17
(t, J = 5.6 Hz, 1H), 7.93-7.99 (m, 3H), 7.77 (t, J = 5.6 Hz, 1H), 7.58 (dd, J1 = 15.2 Hz,
J2 = 8.0 Hz, 1H), 7.48 (d, J = 9.6 Hz, 1H), 7.31-7.36 (m, 1H), 7.26 (t, J = 8.4 Hz, 1H),
4.47 (d, J = 5.6 Hz, 2H), 4.01 (d, J = 5.6 Hz, 2H); HR-MS (ESI): m/z, calcd. for
C21H15O4N3F7S [M+H]+ 538.0666, Found: 538.0657.

5.4.4. 2-((5-((2,6-Dichlorophenethyl)carbamoyl)-2-(3-fluorophenoxy) thiazol-4-yl)
amino)acetic acid (19d)
Following the preparation protocol of compound 19a, starting from compound
8k (200 mg, 0.47 mmol), the title compound 19d was obtained as a yellow solid (120
mg, 53%); mp 167-169 °C; 1H NMR (400 MHz, Acetone-d6) δ (ppm): 10.98 (brs, 1H),
7.91 (brs, 1H), 7.54 (dd, J1 = 14.4 Hz, J2 = 7.6 Hz, 1H), 7.38-7.40 (m, 2H), 7.27-7.30
(m, 3H), 7.16-7.18 (m, 1H), 6.91 (brs, 1H), 4.19 (d, J = 5.2 Hz, 2H), 3.54 (q, J = 6.4
Hz, 2H), 3.19-3.23 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 172.18, 171.95,
163.38, 162.38 (d, J = 245.6 Hz), 157.92, 154.70 (d, J = 11.1 Hz), 134.86, 134.78 ,
131.68 (d, J = 9.5 Hz), 128.95, 128.41, 117.04 (d, J = 2.1 Hz), 114.02 (d, J = 21.2 Hz),
108.94 (d, J = 25 Hz), 44.70, 36.94, 31.45; HR-MS (ESI): m/z, calcd. for
C20H17O4N3Cl2FS [M+H]+ 484.0295, Found: 484.0300.
5.4.5.
2-((5-([1,1'-Biphenyl]-4-ylcarbamoyl)-2-(3-fluorophenoxy)thiazol-4-yl)amino)ace
tic acid (19e)
Following the preparation protocol of compound 19a, starting from compound 8l
(80 mg, 0.20 mmol), the title compound 19e was obtained as a light yellow solid (80
mg, 88%); mp 215-217 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.62 (brs, 1H),
9.34 (s, 1H), 8.05 (t, J = 5.6 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.58-7.65 (m, 5H),
7.53 (d, J = 9.6 Hz, 1H), 7.43 (t, J = 7.2 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.32 (t, J =
7.2 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 4.10 (d, J = 6.0 Hz, 2H); HR-MS (ESI): m/z,
calcd. for C24H19O4N3FS [M+H]+ 464.1075, Found: 464.1066.
Note: Except for target compounds, the synthesis and characterization of all other
compounds mentioned in the Schemes 1-3 were described in the supporting
information.
5.5. Biological evaluation
5.5.1. Protein expression and purification
The Pet28a-Pin1 plasmid was a gift from Professor Joseph P. Noel (The Salk
Institute for Biological Studies, La Jolla, California). The N-terminally His6-tagged
o
Pin1 was expressed at 22 C in E. coli strain BL21 following induction at an optical
density of 0.6 (600 nm) with 0.5 mM IPTG for 20 h in terrific broth. Cells were
resuspended in 25 mM Tris-Cl, 500 mM NaCl, 10 mM imidazole, 100 g/mL cocktail,
o
0.5 mg/mL lysozyme. Following sonication at 4 C, the soluble supernatant was

loaded onto an Ni-NTA (Qiagen) column and washed with 10 bed volumes of
washing buffer (50 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl). His6-Pin1 was
eluted with 400 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl and condensed by
ultrafiltration (Millipore 5 kDa) with 20 mM Tris-Cl, 100 mM NaCl, 5 mM DTT.
5.5.2. Pin1 PPIase assay and IC50 measurements of Pin1 inhibitors
PPIase activities were measured at 6 °C JASCO V-650 spectrophotometer using
protease-coupled
assay
according
to
Wang
et
al[42,43]
.
Suc-Ala-Glu-Pro-Phe-4-nitroanilide in 0.47 M LiCl/trifluoroethanol was used as the
substrate. In brief, the assay buffer (855 μL of 35 mM HEPES at pH 7.8), Pin1 (0.5
μL of 850 μg/mL stock solution), and inhibitors (5 μL of varying concentrations in
DMSO) were pre-equilibrated in the 1.0 mL quartz cuvette in spectrophotometer for
10 min. Then, 100 μL of ice-cooled chymotrypsin (60 mg/mL in 0.001 M HCl) was
added and mixed immediately. Additional 40 μL of substrate (2.5 mM in 0.47 M
LiCl/trifluoroethanol) was added to the cuvette and the reaction was monitored by
absorbance at 390 nm for 90 s. For each compound, three concentrations (100 μM, 10
μM, 1μM) were chosen, and the assay was performed in duplicate. The data was
analyzed by Graphpad Prism 5.01. The inhibition at each concentration was
calculated according the following equation: Inhibition ratio (%)
=
[1-(kX-k1)/(kD-k1)]х100, where kX represents the reaction rate in the presence of tested
compound; kD is the reaction rate of DMSO control without the tested compound; k1
means the reaction rate of blank control without Pin1 protein, it represents the
reaction rate of thermal isomerization without any catalysis. The IC50 was measured
according to the inhibition ratio at each concentration of tested compound.
5.6. Computational studies
All molecular computation studies were performed using CDOCER protocol
integrated in Accelrys Discovery Studio Client 2.545 (Accelrys Software Inc., San
Diego, CA). The co-crystal structure of Pin1 complexed with a non-phosphate small
molecule inhibitor (PDB ID: 3JYJ) was chosen for molecular modeling. Using Clean
Protein tool of DS, the water molecules in protein were removed and the protein was
added hydrogen and corrected the incomplete residues, then the protein was refined
with CHARMm force field. The active site was defined as all residues within 10 Å
radius of the co-crystallized reference molecule. Compound 10Ab was minimized
using Prepare Ligands tool of DS and refined with CHARMm force field. Then it was
docked into the prepared Pin1 protein with CDOCKER using the default parameters
except that the Random Conformation was defined as 20. The 20 final docked
conformations were ranked according to their binding free energy. The docking mode
was chosen on the basis of binding rationality.
6. Acknowledgement

This work is supported by National Natural Science Foundation of China (No.
81273380) and “863” Program of China (No. 2012AA020302).
References:
1. Lu, K. P.; Hanes, S. D.; Hunter, T. Nature 1996, 380, 544.
2. Yeh, E. S.; Means, A. R. Nat. Rev. Cancer 2007, 7, 381.
3. Lu, K. P.; Finn, G.; Lee, T. H.; Nicholson, L. K. Nat. Chem. Biol. 2007, 3, 619.
4. Yaffe, M. B.; Schutkowski, M.; Shen, M.; Zhou, X. Z.; Stukenberg, P. T.; Rahfeld, J. U.; Xu, J.;
Kuang, J.; Kirschner, M. W.; Fischer, G.; Cantley, L. C.; Lu, K. P. Science 1997, 278, 1957.
5. Ranganathan, R.; Lu, K. P.; Hunter, T.; Noel, J. P. Cell 1997, 89, 875.
6. Moore, J. D.; Potter, A. Bioorg. Med. Chem. Lett. 2013, 23, 4283.
7. Litchfield, D. W.; Shilton, B. H.; Brandl, C. J.; Gyenis, L. BBA-Gen. Subjects 2015, 1850, 2077.
8. Liu, J.; Farmer, J. J.; Lane, W. S.; Friedman, J.; Weissman, I.; Schreiber, S. L. Cell 1991, 66, 807.
9. Gothel, S. F.; Schmid, R.; Wipat, A.; Carter, N. M.; Emmerson, P. T.; Harwood, C. R.; Marahiel,
M. A. Eur J Biochem 1997, 244, 59.
10. Compton, L. A.; Davis, J. M.; Macdonald, J. R.; Bachinger, H. P. Eur J Biochem 1992, 206, 927.
11. Rahfeld, J. U.; Rucknagel, K. P.; Schelbert, B.; Ludwig, B.; Hacker, J.; Mann, K.; Fischer, G.
FEBS Lett. 1994, 352, 180.
12. Verdecia, M. A.; Bowman, M. E.; Lu, K. P.; Hunter, T.; Noel, J. P. Nat Struct Biol 2000, 7, 639.
13. Lu, P. J.; Zhou, X. Z.; Shen, M.; Lu, K. P. Science 1999, 283, 1325.
14. Jiang, B.; Pei, D. J. Med. Chem. 2015, 58, 6306.
15. Chen, L.; Liu, J.; Tao, X.; Wang, G.; Wang, Q.; Liu, X. Biochem. Bioph. Res. Co. 2015, 464, 487.
16. Tong, Y.; Ying, H.; Liu, R.; Li, L.; Bergholz, J.; Xiao, Z. Cell Death and Disease 2015, 6, e1640.
17. Lee, Y. M.; Shin, S. Y.; Jue, S. S.; Kwon, I. K.; Cho, E. H.; Cho, E. S.; Park, S. H.; Kim, E. C.
Stem Cells Dev. 2014, 23, 618.
18. Barberi, T. J.; Dunkle, A.; He, Y. W.; Racioppi, L.; Means, A. R. PLoS One 2012, 7, e29808.
19. Chen, C.; Li, W.; Sultana, R.; You, M.; Kondo, A.; Shahpasand, K.; Kim, B. M.; Luo, M.;
Nechama, M.; Lin, Y.; Yao, Y.; Lee, T. H.; Zhou, X. Z.; Swomley, A. M.; Allan Butterfield, D.; Zhang,
Y.; Lu, K. P. Neurobiol. Dis. 2015, 76, 13.
20. Blair, L. J.; Baker, J. D.; Sabbagh, J. J.; Dickey, C. A. J. Neurochem. 2015, 133, 1.
21. Costantino, S.; Paneni, F.; Lüscher, T. F.; Cosentino, F. Int. J. Cardiol. 2016, 203, 702.
22. Wang, T.; Liu, Z.; Shi, F.; Wang, J. Mol. Cell. Biochem. 2016, 413, 179.
23. Ayala, G.; Wang, D.; Wulf, G.; Frolov, A.; Li, R.; Sowadski, J.; Wheeler, T. M.; Lu, K. P.; Bao,
L. Cancer Res. 2003, 63, 6244.
24. Tan, X.; Zhou, F.; Wan, J.; Hang, J.; Chen, Z.; Li, B.; Zhang, C.; Shao, K.; Jiang, P.; Shi, S.; Feng,
X.; Lv, N.; Wang, Z.; Ling, Y.; Zhao, X.; Ding, D.; Sun, J.; Xiong, M.; He, J. Cancer Biol. Ther. 2010,
9, 111.
25. Leung, K. W.; Tsai, C. H.; Hsiao, M.; Tseng, C. J.; Ger, L. P.; Lee, K. H.; Lu, P. J. Oncol. Rep.
2009, 21, 1097.
26. Nakagawa, H.; Seike, S.; Sugimoto, M.; Ieda, N.; Kawaguchi, M.; Suzuki, T.; Miyata, N. Bioorg.
Med. Chem. Lett. 2015, 25, 5619.
27. Ryo, A.; Uemura, H.; Ishiguro, H.; Saitoh, T.; Yamaguchi, A.; Perrem, K.; Kubota, Y.; Lu, K. P.;
Aoki, I. Clin. Cancer Res. 2005, 11, 7523.
28. Winkler, K. E.; Swenson, K. I.; Kornbluth, S.; Means, A. R. Science 2000, 287, 1644.

29. Xu, G. G.; Etzkorn, F. A. Drug News Perspect 2009, 22, 399.
30. Hennig, L.; Christner, C.; Kipping, M.; Schelbert, B.; Rucknagel, K. P.; Grabley, S.; Kullertz, G.;
Fischer, G. Biochemistry 1998, 37, 5953.
31. Zhang, C. J.; Zhang, Z. H.; Xu, B. L.; Wang, Y. L. Acta Pharmaceutica Sinica 2008, 43, 9.
32. Guo, C.; Hou, X.; Dong, L.; Marakovits, J.; Greasley, S.; Dagostino, E.; Ferre, R.; Catherine
Johnson, M.; Humphries, P. S.; Li, H.; Paderes, G. D.; Piraino, J.; Kraynov, E.; Murray, B. W. Bioorg.
Med. Chem. Lett. 2014, 24, 4187.
33. Guo, C.; Hou, X.; Dong, L.; Dagostino, E.; Greasley, S.; Ferre, R.; Marakovits, J.; Johnson, M. C.;
Matthews, D.; Mroczkowski, B.; Parge, H.; VanArsdale, T.; Popoff, I.; Piraino, J.; Margosiak, S.;
Thomson, J.; Los, G.; Murray, B. W. Bioorg. Med. Chem. Lett. 2009, 19, 5613.
34. Dong, L.; Marakovits, J.; Hou, X.; Guo, C.; Greasley, S.; Dagostino, E.; Ferre, R.; Johnson, M. C.;
Kraynov, E.; Thomson, J.; Pathak, V.; Murray, B. W. Bioorg. Med. Chem. Lett. 2010, 20, 2210.
35. Potter, A.; Oldfield, V.; Nunns, C.; Fromont, C.; Ray, S.; Northfield, C. J.; Bryant, C. J.; Scrace, S.
F.; Robinson, D.; Matossova, N.; Baker, L.; Dokurno, P.; Surgenor, A. E.; Davis, B.; Richardson, C.
M.; Murray, J. B.; Moore, J. D. Bioorg. Med. Chem. Lett. 2010, 20, 6483.
36. Potter, A. J.; Ray, S.; Gueritz, L.; Nunns, C. L.; Bryant, C. J.; Scrace, S. F.; Matassova, N.; Baker,
L.; Dokurno, P.; Robinson, D. A.; Surgenor, A. E.; Davis, B.; Murray, J. B.; Richardson, C. M.; Moore,
J. D. Bioorg. Med. Chem. Lett. 2010, 20, 586.
37. Zhu, L.; Jin, J.; Liu, C.; Zhang, C.; Sun, Y.; Guo, Y.; Fu, D.; Chen, X.; Xu, B. Bioorgan. Med.
Chem. 2011, 19, 2797.
38. Liu, C.; Jin, J.; Chen, L.; Zhou, J.; Chen, X.; Fu, D.; Song, H.; Xu, B. Bioorgan. Med. Chem.
2012, 20, 2992.
39. Xi, Y. Y.; Jin, J.; Sun, Y.; Chen, X. G.; Song, H. R.; Xu, B. L. Acta pharmaceutica Sinica 2013,
48, 1266.
40. Wei, S.; Kozono, S.; Kats, L.; Nechama, M.; Li, W.; Guarnerio, J.; Luo, M.; You, M.; Yao, Y.;
Kondo, A.; Hu, H.; Bozkurt, G.; Moerke, N. J.; Cao, S.; Reschke, M.; Chen, C.; Rego, E. M.; Lo-Coco,
F.; Cantley, L. C.; Lee, T. H.; Wu, H.; Zhang, Y.; Pandolfi, P. P.; Zhou, X. Z.; Lu, K. P. Nat. Med.
2015, 21, 457.
41. D'Amico, J. J.; Campbell, R. H. J. Org. Chem. 1967, 32, 2567.
42. Kofron, J. L.; Kuzmic, P.; Kishore, V.; Colon-Bonilla, E.; Rich, D. H. Biochemistry 1991, 30,
6127.
43. Wang, X. J.; Xu, B.; Mullins, A. B.; Neiler, F. K.; Etzkorn, F. A. J. Am. Chem. Soc. 2004, 126,
15533.
44. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.;
Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.
45. Wu, G.; Robertson, D. H.; Brooks, C. L.; Vieth, M. J. Comput. Chem. 2003, 24, 1549.

Graphical Abstract:
Synthesis and Pin1 inhibitory activity of thiazole derivatives
Hailong Zhao, Guonan Cui , Jing Jin, Xiaoguang Chen , Bailing Xu
O
OH
R1 = small hydrophobic groups
HN X
4
Ar = bulky aromatic substituents
X = CH2 or CO
IC50 = 3.22 -23.4 uM
H
N
N
Ar
2
R1
5
O
S
O