Design, synthesis, and biological evaluation of novel 2-ethyl-5- phenylthiazole-4-carboxamide derivatives as protein tyrosine phosphatase 1B inhibitors with improved cellular efficacy

Article abstract of DOI:10.1016/j.ejmech.2013.09.017

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a key negative regulator of the insulin and leptin signal-transduction pathways. PTP1B inhibitors have emerged as attractive and potent pharmaceutical agents for the treatment of type 2 diabetes and obesity. We identified a series of 2-ethyl-5-phenylthiazole-4-carboxamide (PTA) derivatives, inspired from the ACT scaffold of Scleritodermin A, as a novel class of PTP1B inhibitors. Structure-activity relationship (SAR) analysis and docking studies revealed the molecular basis of PTP1B inhibition by these compounds. PTA derivative 18g was capable of inhibiting intracellular PTP1B and subsequently activating the insulin signaling pathway. Treatment of cells with 18g markedly increased the phosphorylation levels of IRβ and Akt as well as the rate of glucose uptake.

Full text of DOI:10.1016/j.ejmech.2013.09.017

European Journal of Medicinal Chemistry 69 (2013) 399e412
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, and biological evaluation of novel 2-ethyl-5-
phenylthiazole-4-carboxamide derivatives as protein tyrosine
phosphatase 1B inhibitors with improved cellular efficacy
Yue-Ting Chen 1, Chun-Lan Tang 1, Wei-Ping Ma, Li-Xin Gao, Yi Wei, Wei Zhang,
**
**
*
Jing-Ya Li , Jia Li , Fa-Jun Nan
Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein tyrosine phosphatase 1B (PTP1B) is implicated as a key negative regulator of the insulin and
leptin signal-transduction pathways. PTP1B inhibitors have emerged as attractive and potent pharma-
ceutical agents for the treatment of type 2 diabetes and obesity. We identified a series of 2-ethyl-5-
phenylthiazole-4-carboxamide (PTA) derivatives, inspired from the ACT scaffold of Scleritodermin A, as
a novel class of PTP1B inhibitors. Structureeactivity relationship (SAR) analysis and docking studies
revealed the molecular basis of PTP1B inhibition by these compounds. PTA derivative 18g was capable of
inhibiting intracellular PTP1B and subsequently activating the insulin signaling pathway. Treatment of
Received 5 April 2013
Received in revised form
6 August 2013
Accepted 10 September 2013
Available online 19 September 2013
Keywords:
Protein tyrosine phosphatase 1B
Type 2 diabetes
2-Ethyl-5-phenylthiazole-4-carboxamide
cells with 18g markedly increased the phosphorylation levels of IRb and Akt as well as the rate of glucose
uptake.
Ó 2013 Elsevier Masson SAS. All rights reserved.
(PTA)
SAR
1. Introduction
antisense oligonucleotides exhibited normalized blood glucose
level, improved insulin sensitivity and modulated fat storage and
lipogenesis in adipose tissue [21,22]. Therefore, PTP1B inhibitors
have emerged as attractive and potent pharmaceutical agents and
may provide a novel strategy for the treatment of type 2 diabetes
and obesity [23e25].
Various PTP1B inhibitors have been developed over the past
decade. Some compounds display high binding affinity, but many
do not show inhibitory potency against intracellular PTP1B.
Because of the highly cationic character of the active site of PTP1B
enzyme, most pTyr mimetics are negatively charged at physiolog-
ical pH, and this highly polar nature provides these compounds
with poor physiochemical properties and therefore poor cell
permeability and low oral bioavailability. Although developing
potent and specific PTP1B inhibitors with high cell permeability
and orally bioavailability remains challenging, there are a number
of examples of PTP1B inhibitors with cellular and/or in vivo activity,
which may lead to opportunities to develop new treatment stra-
tegies for type 2 diabetes and obesity [26e30].
Type 2 diabetes and obesity are characterized by resistance to
the hormones insulin and leptin [1]. Impaired insulin and leptin
signaling play a key role in the pathogenesis of type 2 diabetes.
Protein tyrosine phosphatase 1B (PTP1B) is an intracellular non-
receptor phosphatase (PTPase) that is implicated as a key negative
regulator in signal transduction for both insulin [2e4] and leptin
[5e9] pathways. It acts by dephosphorylating specific phospho-
tyrosine (pTyr) residues of the insulin receptor (IR) [10e12], IR
substrates (IRS) [13] and Janus kinase 2 (JAK2) [14,15]. PTP1B
expression and activity are increased in pathologically insulin-
resistant and obese humans [16e18]. In two landmark PTP1B
knockout studies, PTP1B-deficient mice displayed increased insulin
sensitivity and resistance to diet-induced obesity [19,20]. Consis-
tent with this finding, diabetic mice treated with specific PTP1B
* Corresponding author. Fax: þ86 21 50800954.
** Corresponding authors. Fax: þ86 21 50801552.
E-mail addresses: jyli@mail.shcnc.ac.cn (J.-Y. Li), jli@mail.shcnc.ac.cn (J. Li),
fjnan@mail.shcnc.ac.cn (F.-J. Nan).
We previously reported our discovery of novel PTP1B inhibitors
with an ACT (2-(1-amino-2-p-hydroxyphenylethane)-4-(4-
carboxy-2,4-dimethyl-2Z,4E-propadiene)-thiazole) skeleton [31]
inspired by the key structure of the natural product
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.017

400
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
Scleritodermin A [32]. Structural modification at sites A, B, and C
(R1, R2 and R3), and the conjugated diene moiety (Fig. 1) led to a
series of ACT derivatives with improved inhibitory activity (low
micromolar IC50 value) compared with the original hit and mod-
erate selectivity over TCPTP. In this study, considering the reactive
properties of the conjugated diene ester moiety as a Michael
acceptor and the difficulties of synthesis, we carried out further
structural optimization to replace the conjugated diene moiety.
And also several substituent groups were introduced to the C5
position of the thiazole moiety (site D) to investigate the role of site
D in PTP1B inhibitory activity. We focused on site D and the con-
jugated diene moiety (Fig.1), and subsequently identified a series of
2-ethyl-5-phenylthiazole-4-carboxamide (PTA) derivatives as
PTP1B inhibitors with improved in vitro efficacy.
17b and 18b were then obtained via hydrolysis of ester 17a or 18a
with LiOH.
Furthermore, in order to determine the effect of the sub-
stitutions on the phenyl ring at site C, compounds 18cen were
synthesized as shown in Scheme 4. Deprotection of the Boc-
protected amine 3c with TFA afforded amine 19. Amine 19 was
reacted with 3-indolepropionic acid and then hydrolyzed to afford
acid 21. Acid 21 reacted with several substituted methyl amino-
benzoates followed by hydrolysis to afford various PTA derivatives
18cen.
3. Results and discussion
3.1. Structureeactivity relationship
2. Chemistry
Structureeactivity relationship studies, focused on site D and
the conjugated diene moiety, were carried out based on our pre-
vious work [31]. First, to investigate the role of site D in the ACT
skeleton, several substituent groups were introduced to the C5
position of the thiazole moiety to afford compounds 9aed. The C5
position of the thiazole moiety was tolerant to several substituents
(Table 1). Introduction of bulky substituents retained PTP1B
The synthesis of the ACT-diene derivatives was performed as
shown in Scheme 1. Boc-L-Tyr(Bn)-OH was reacted with R4C(O)
CH(NH2)COOEt to give amides 2aec. A thiazole ring was then
constructed using Lawesson’s reagent to obtain intermediates 3ae
c. The conjugated double bonds were constructed via Hornere
WadswortheEmmons reaction and StilleGennari phosphonate
reaction after converting the ester groups of 3aec and 6aec to
aldehyde groups (5aec and 7aec) with LiAlH4 and IBX, respec-
tively. The resulting ACT derivatives 8aec were deprotected using
TFA/CH2Cl2 and then reacted with cyclohexanecarbonyl chloride to
furnish the desired analogs 9aec.
The synthesis of 2-ethylthiazole-4-carboxamide derivatives
commenced with removal of the Boc group of intermediate 10,
followed by acylation with cyclohexanecarbonyl chloride to afford
amide 12. Compound 12 was hydrolyzed to afford the corre-
sponding acid 13. Condensation of acid 13 and methyl amino-
benzoates afforded amides 14aec, which were then hydrolyzed to
afford acids 14def (Scheme 2).
inhibitory activity. The isobutyl derivative 9b (IC50 9.54 ꢀ 1.99
and the phenyl derivative 9c (IC50 8.44 ꢀ 1.68 M) exhibited similar
inhibitory activity to the unsubstituted analog 1a (IC50
mM)
m
9.32 ꢀ 0.42
m
M) [31], while the methyl derivative 9a (IC50
13.46 ꢀ 1.95
m
M) displayed slightly decreased inhibitory activity.
Compounds 1a and 9c were then hydrolyzed to give acids 1b and
9d, respectively. Both acids displayed similar potency (IC50
14.69 ꢀ 1.16
compared to their corresponding esters (IC50 9.32 ꢀ 0.42
8.44 ꢀ 1.68 M for 1a and 9c, respectively).
m
M and 10.89 ꢀ 1.08
m
M for 1b and 9d, respectively)
mM and
m
In our previous research, we found that the thiazole-derived
conjugated diene moiety was essential for inhibitory activity
against PTP1B and the cisetrans isomers retained the activities. This
result allowed us to replace the conjugated diene moiety with an
aryl group by considering its instability as a Michael acceptor and
the long synthetic route of constructing the conjugated double
bonds. Therefore, a series of 2-ethylthiazole-4-carboxamide de-
rivatives (Fig. 1) were synthesized as the analogs of ACT derivatives
1a, b. As shown in Table 2, all the 2-ethylthiazole-4-carboxamide
The PTA derivatives 17a, b and 18a, b were synthesized as shown
in Scheme 3. Intermediate 3c was hydrolyzed and then reacted
with methyl 3-aminobenzoate hydrochloride to afford amide 16.
The Boc group of amide 16 was deprotected by 4 M HCl/EtOAc, and
the resulting amine was reacted with cyclohexanecarbonyl chloride
or 3-indolepropionic acid to afford compound 17a and 18a. Acid
Fig. 1. Structural optimization of PTP1B inhibitors inspired from the ACT scaffold.

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
401
Scheme 1. Reagents and conditions: (a) R4C(O)CH(NH2)COOEt, EDCI, DMAP, Et3N, CH2Cl2; (b) Lawesson’s reagent, THF, reflux; (c) LiAlH4, THF, 0 ꢁC; (d) IBX, DMSO, toluene; (e)
(EtO)2P(O)CH(CH3)COOEt, NaH, THF, 0 ꢁC / rt; (f) (CF3CH2O)2P(O)CH(CH3)COOEt, KHMDS, 18-crown-6, THF, ꢂ78 ꢁC; (g) TFA, CH2Cl2; (h) cyclohexanecarbonyl chloride, DIPEA,
CH2Cl2, rt; (i) LiOH$H2O, 1,4-dioxane/H2O, reflux.
derivatives (14aef) displayed moderate PTP1B inhibitory activities,
which demonstrated that replacing the conjugated diene moiety
with an aryl group could lead to retention of the inhibitory activity.
The ortho- and para-carboxyl-substituted analogs 14d and f (IC50
reported to act as a negative regulator of the insulin signaling
pathway through the dephosphorylation of the IR. Hence, inhibi-
tion of PTP1B activity should enhance insulin action and signaling.
The above compounds were tested in Chinese hamster ovary cells
overexpressing the human IR (CHO-hIR), and IR phosphorylation in
whole-cell lysates was examined by western blot analysis. Both the
ACT and 2-ethylthiazole-4-carboxamide derivatives were investi-
gated, but only acid 9d, which has a phenyl group substituted at the
C5 position of the thiazole moiety, increased IR phosphorylation
weakly in CHO-hIR cells (as shown in Fig. 2a). All the C5-
unsubstituted analogs, including the ACT derivatives and 2-
ethylthiazole-4-carboxamide derivatives, lacked cellular efficacy.
Acid 9d demonstrated a slight increase in IR phosphorylation in the
8.12 ꢀ 0.98
inhibitory activity to their corresponding esters 14a and c (IC50
8.79 ꢀ 1.60 M and 4.42 ꢀ 0.85 M, respectively). However, the
meta-carboxyl-substituted analogs 14e (IC50 6.00 ꢀ 0.43 M) dis-
played slightly improvement compared with its corresponding
ester 14b (IC50 10.55 ꢀ 1.48 M). Comparing the two skeletons, the
2-ethylthiazole-4-carboxamide derivatives 14c, e, and (IC50
4.42 ꢀ 0.85 M, 6.00 ꢀ 0.43 M and 4.88 ꢀ 0.60 M, respectively)
exhibited lower IC50 values than the ACT derivatives 1a and b (IC50
M, respectively).
mM and 4.88 ꢀ 0.60
mM, respectively) showed similar
m
m
m
m
f
m
m
m
9.32 ꢀ 0.42
m
M and 14.69 ꢀ 1.16
m
concentration range of 1.25e10 mM. Notably, the inhibitory con-
Given the observed inhibitory activity of both ACT and 2-
ethylthiazole-4-carboxamide derivatives, we proceeded to assess
their capacity to inhibit intracellular PTP1B. PTP1B has been
centration values against intracellular PTP1B of compound 9d and
its IC50 value observed in the enzyme assay were of an equal order
of magnitude. By contrast, quite a few PTP1B inhibitors lack cellular

402
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2, 0 ꢁC / rt; (b) cyclohexanecarbonyl chloride, DIPEA, CH2Cl2; (c) LiOH$H2O, 1,4-dioxane/H2O; (d) H2NC6H4COOMe$HCl, EDCI,
DMAP, Et3N, CH2Cl2, rt.
efficacy because of their poor membrane permeability [27]. This
result indicated that compound 9d was membrane permeable and
capable of blocking PTP1B activity in cells. However, its analogs,
ACT derivatives 1a, b and 2-ethylthiazole-4-carboxamide de-
rivatives 14aef were devoid of cellular efficacy. This result implied
that the introduction of a phenyl group at the C5 position of the
thiazole moiety improved the membrane permeability of the ACT
derivatives and improved their cellular efficacy.
Although compounds 14aef lacked cellular efficacy compared
with the ACT-diene skeleton, these compounds could be synthe-
sized easily via a simple amidation of substituted thiazole-4-
carboxylic acid with amines. Hence, we decided to focus our ef-
forts on further structural modification based on the 2-
ethylthiazole-4-carboxamide skeleton. To improve the cellular ac-
tivities, a phenyl group was introduced onto the C5 position of the
thiazole ring, which afforded the 2-ethyl-5-phenylthiazole-4-
carboxamide (PTA) skeleton, to give compound 17b as the analog
of acid 14e. As shown in Table 3, ester 17a (IC50 3.79 ꢀ 0.67 M) was
twice as potent as the ACT and 2-ethylthiazole-4-carboxamide
analogs (esters 9c and 14b, IC50 8.44 1.68 and
M, respectively). The corresponding acid 17b (IC50
M) showed a 5.1- to 9.3-fold increase in potency
M and
M, respectively). This improvement in inhibitory ac-
tivity encouraged us to try to further optimize the structure of the
PTA scaffold. In our previous report, the 2-(1H-indol-3-yl)-ethyl
derivatives showed preferable inhibitory activity compared with its
analogs [31]. Hence, we induced the 2-(1H-indol-3-yl) ethyl group
to the PTA scaffold to give ester 18a and acid 18b, both of which
displayed low micromolar IC50 values against PTP1B (IC50
m
ꢀ
mM
10.55 ꢀ 1.48
1.17 ꢀ 0.09
m
m
compared with its analogs 9d and 14e (IC50 10.89 ꢀ 1.08
m
6.00 ꢀ 0.43
m
2.68 ꢀ 0.18
m
M for 18a and 1.01 ꢀ 0.07
mM for 18b). These results
demonstrated the feasibility of introducing a phenyl group to the
Scheme 3. Reagents and conditions: (a) LiOH$H2O, THF/H2O, 50 ꢁC; (b) methyl 3-aminobenzoate hydrochloride, HATU, NMM, CH2Cl2; (c) 4 M HCl/EtOAc; (d) cyclohexanecarbonyl
chloride, Et3N, CH2Cl2; or 3-indolepropionic acid, EDCI, DMAP, CH2Cl2, rt; (e) LiOH$H2O, THF/MeOH/H2O.

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
403
Scheme 4. Reagents and conditions: (a) TFA, CH2Cl2, 0 ꢁC / rt; (b) 3-indolepropionic acid, EDCI, DMAP, CH2Cl2, rt; (c) K2CO3, MeOH/H2O, rt; (d) ArNH2, HATU, NMM, CH2Cl2; (e)
LiOH$H2O, THF/MeOH/H2O.
C5 position of the thiazole ring of the 2-ethylthiazole-4-
carboxamide scaffold because all the resulting PTA derivatives
retained inhibitory activity against PTP1B.
The 2-(1H-indol-3-yl)-ethyl-substituted acid 18b emerged as
the most potent PTP1B inhibitor among all of the compounds
mentioned above. To determine whether the inhibitory effect of
PTA derivatives observed in the PTP1B enzyme assay could be
recapitulated in a cellular assay, we tested these compounds in
CHO-hIR and examined IR phosphorylation by western blot anal-
ysis. As shown in Fig. 2b, acid 18b in the low micromolar range (5e
(18g and h) displayed submicromolar IC50 values against PTP1B.
The introduction of strong electron-withdrawing F and Cl atoms in
the para position of the carboxyl group lowers the pKa values of
compounds 18g and h, which provides more favorable interactions
with the highly cationic phosphatase active site of PTP1B. The 4-
carboxy-2-fluoro-substituted analog 18m, with a submicromolar
IC50 value (IC50 0.77 ꢀ 0.12
inhibitory activity compared with the corresponding nonhaloge-
nated acid 18d (IC50 2.28 ꢀ 0.67 M).
mM), also exhibited increased enzyme
m
These results reveal some interesting structureeactivity re-
lationships for PTP1B inhibition by PTA derivatives. Introduction of
a phenyl group at the C5 position of the thiazole moiety benefits the
inhibitory activity, membrane permeability and cellular efficacy.
Halogen substitutions on the right-hand phenyl ring also affect the
potency of compounds; 2-carboxy-5-fluoro-substitution appeared
to be the best.
20
mM) elicited a concentration-dependent increase in IR phos-
phorylation level. However, its corresponding ester 18a was inac-
tive in cells. Compared with acid 9d as a closely related control,
compound 18b was more effective in stimulating IR phosphoryla-
tion. These results suggested that PTA derivative 18b possessed
improved cell permeability and could stimulate the insulin
signaling pathway in cells. These results confirmed our hypothesis
and the feasibility of introducing a phenyl group to the C5 position
of the thiazole ring to improve the membrane permeability and
cellular efficacy of the ACT-derived compounds.
After confirming the cellular efficacy of the PTA skeleton, we
continued further structural modification by changing the substi-
tution position of the carboxyl group or by introducing a halogen to
the phenyl ring at site C to afford compounds 18cen. As shown in
Table 4, either ortho- or para-carboxyl-substituted analogs (18c and
3.2. Selectivity against other phosphatases
The PTPase domains of all PTPases are highly conserved. T-cell
protein tyrosine phosphatase (TCPTP), the most homologous
phosphatase to PTP1B, has 74% sequence identity in the catalytic
domain. TCPTP knockout mice are born healthy but die at 3e5
weeks of age because of impaired B cell and T cell function [33].
While heterozygous deficiency of TCPTP doesn’t result in any overt
immune phenotype [33] and may be beneficial in the context of
type 2 diabetes, as TCPTP is also an important regulator of insulin
18d, IC50 5.56 ꢀ 0.68
less potency than the meta-carboxyl-substituted analog 18b (IC50
M). The introduction of halogen atoms caused different
mM and 2.28 ꢀ 0.67
mM, respectively) showed
1.01 ꢀ0.07
m
effects on these three acids. Halogen substitution at the 5-position
of the 2-carboxyl analog led to a 5- to 10-fold increase in inhibitory
Table 2
activity (18gei, IC50 0.54 ꢀ 0.18
m
M, 0.71 ꢀ 0.11
mM, and
PTP1B inhibition data for compounds 14aef.
1.18 ꢀ 0.21
m
M, respectively) compared with the parent compound
18c (IC50 5.56 ꢀ 0.68
mM). Both fluorinated and chlorinated analogs
Table 1
PTP1B inhibition data for compounds 1a, b and 9aed.
R4
R3
IC50
(m
M)
Compd.
Ar
IC50
(
m
M)
a
a
Compd.
1a
1b
9a
9b
9c
9d
H
H
Et
H
Et
Et
Et
H
9.32 ꢀ 0.42
14.69 ꢀ 1.16
13.46 ꢀ 1.95
9.54 ꢀ 1.99
8.44 ꢀ 1.68
10.89 ꢀ 1.08
14a
14b
14c
14d
14e
14f
2-COOMe-Ph
3-COOMe-Ph
4-COOMe-Ph
2-COOH-Ph
3-COOH-Ph
4-COOH-Ph
8.79 ꢀ 1.60
10.55 ꢀ 1.48
4.42 ꢀ 0.85
8.12 ꢀ 0.98
6.00 ꢀ 0.43
4.88 ꢀ 0.60
CH3
CH2CH(CH3)2
Ph
Ph
a
a
Values are the mean ꢀ SD (n ¼ 3). Oleanolic acid was employed as a positive
M).
Values are the mean ꢀ SD (n ¼ 3). Oleanolic acid was employed as a positive
M).
control (IC50 ¼ 2.01 ꢀ 0.26
m
control (IC50 ¼ 2.01 ꢀ 0.26
m

404
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
Table 4
PTP1B inhibition data for compounds 18cen.
Compd. Ar
18c 2-COOH-Ph
a
IC50
(mM)
5.56 ꢀ 0.68
2.28 ꢀ 0.67
4.18 ꢀ 0.50
2.82 ꢀ 0.72
0.54 ꢀ 0.18
0.71 ꢀ 0.11
1.18 ꢀ 0.21
1.62 ꢀ 0.51
1.42 ꢀ 0.41
1.12 ꢀ 0.20
0.77 ꢀ 0.12
2.56 ꢀ 0.61
18d
18e
18f
18g
18h
18i
18j
18k
18l
4-COOH-Ph
2-COOH-3F-Ph
2-COOH-4F-Ph
2-COOH-5F-Ph
2-COOH-5Cl-Ph
2-COOH-5Br-Ph
3-COOH-4F-Ph
3-COOH-4Cl-Ph
5-COOH-2F-Ph
4-COOH-2F-Ph
4-COOH-3F-Ph
18m
18n
a
Values are the mean ꢀ SD (n ¼ 3). Oleanolic acid was employed as a positive
M).
control (IC50 ¼ 2.01 ꢀ 0.26
m
inside cells. As shown in Fig. 3a, incubation of CHO-hIR cells with
18g in 5e20 M concentration range increased insulin-mediated
IR phosphorylation in a concentration-dependent manner. Meta-
Fig. 2. Immunoblot analysis of PTP1B inhibitors in CHO-hIR cells. (a) Effect of 9d on IR
phosphorylation in CHO-hIR cells. (b) Effect of 18a and 18b on IRb phosphorylation in
CHO-hIR cells. CHO-hIR cells were incubated with 1 mM sodium orthovanadate (V),
0.2% DMSO, or compound 9d, 18a, or 18b for 2 h, and then treated with 10 nM insulin
b
m
b
bolic insulin signal transduction occurs through insulin binding to
its cell surface receptor (IR), upon which IR undergoes autophos-
phorylation on several tyrosine residues located in its activation
loop [12]. Autophosphorylation increases IR kinase activity and
leads to the recruitment of IRS proteins, which causes activation of
phosphatidylinositol 3-kinase (PI3K) and downstream protein ki-
nase B (Akt) [36]. The PI3KeAkt pathway is responsible for most of
the metabolic action of insulin including the translocation of
glucose transporter 4 (GLUT4) to the plasma membrane, which
allows the uptake of extracellular glucose into skeletal muscle [37].
PTP1B inhibitors are thought to enhance the insulin-induced PI3Ke
Akt pathway [2]. We used western blot analysis to examine
whether 18g could augment the insulin-mediated PI3KeAkt
pathway. Akt phosphorylation level was compared between un-
treated and 18g-treated differentiated L6 myotubes. As shown in
Figure 3b, 18g treatment increased insulin-induced Akt phosphor-
ylation. To investigate the role of compound 18g in activating Akt,
we treated L6 myotubes with wortmannin, a potent and selective
PI3K inhibitor [38]. As shown in Fig. 3c, 18g-stimulated insulin-
dependent Akt phosphorylation in L6 myotubes was suppressed
by 250 nM wortmannin, indicating that compound 18g effectively
inhibited intracellular PTP1B and subsequently activated the
insulin-induced PI3KeAkt signaling pathways.
for 10 min. Cell lysates were probed with anti-pTyr antibody. b-Actin was used as a
loading control. V: 1 mM sodium orthovanadate used as a positive control.
signaling and glucose homeostasis [34]. Although it’s proposed that
the combined inhibition of PTP1B and TCPTP may prove useful in
ameliorating glucose homeostasis and preventing obesity and
diabetes [35], achieving pharmacological inhibition clinically at a
corresponding level would be challenging and the clinical outcome
of pharmacological inhibition of TCPTP continues to be debated.
Hence, although challenging, selectivity over TCPTP remains a
desirable profile for PTP1B inhibitors. To examine the selectivity
profile of PTA derivatives, several representative compounds, 17b,
18b, g, k and l, were tested as inhibitors against a variety of
different phosphatases, including TCPTP, CDC25B, SHP-1, SHP-2 and
LAR. As shown in Table 5, the representative compounds showed
modest selectivity (2.6- to 3.5-fold) over TCPTP, which has the
highest homology to PTP1B, with 74% sequence identity in the
catalytic domain. Compound 18g showed 2.6-, 3.0- and 18.3-fold
selectivity for PTP1B over TCPTP, CDC25B and SHP-2, respectively,
and greater than 20-fold selectivity over SHP-1 and LAR.
3.3. Cellular activity
In insulin signaling pathway, a key action of insulin is the pro-
motion of glucose uptake into cells by inducing the translocation of
GLUT4 from intracellular storage to the plasma membrane. Previ-
ous studies have demonstrated that Akt mediates insulin-
stimulated GLUT4 translocation and Akt activation should lead to
increased glucose uptake [37]. To further evaluate the biological
Given the observed inhibitory activity of compound 18g, which
emerged as the most potent inhibitor of PTP1B among all the an-
alogs tested, we proceeded to evaluate its ability to inhibit PTP1B
consequences of the increased IRb and Akt phosphorylation levels
Table 3
stimulated by 18g, we tested this compound in differentiated L6
myotubes to assess its ability to stimulate glucose uptake. As shown
in Fig. 3d, L6 myotubes treated with 18g displayed a concentration-
PTP1B inhibition data for compounds 17a, b and 18a, b.
Table 5
Inhibition of PTPases by selected PTA derivatives.
IC50
(
m
M)
a
Compd.
R1
R4
IC50
(
m
M)
PTP1B
TCPTP
CDC25B
SHP-1
SHP-2
LAR
17a
17b
18a
18b
Cyclohexyl
Cyclohexyl
2-(1H-Indol-3-yl)-ethyl
2-(1H-Indol-3-yl)-ethyl
Me
H
Me
H
3.79 ꢀ 0.67
1.17 ꢀ 0.09
2.68 ꢀ 0.18
1.01 ꢀ 0.07
17b 1.17 ꢀ 0.09 3.97 ꢀ 0.50 2.51 ꢀ 0.02 6.93 ꢀ 0.33 11.66 ꢀ 0.91 >20
18b 1.01 ꢀ 0.07 3.38 ꢀ 0.42 7.37 ꢀ 0.71 7.53 ꢀ 0.74 5.30 ꢀ 0.46 >20
18g 0.54 ꢀ 0.18 1.42 ꢀ 0.11 1.62 ꢀ 0.11 12.64 ꢀ 0.41 9.89 ꢀ 2.18 >20
18k 1.42 ꢀ 0.41 3.36 ꢀ 0.46 4.94 ꢀ 0.22 8.79 ꢀ 0.32 6.79 ꢀ 0.50 >20
18l 1.12 ꢀ 0.20 3.98 ꢀ 0.50 9.74 ꢀ 0.81 10.61 ꢀ 0.97 10.15 ꢀ 0.97 >20
a
Values are the mean ꢀ SD (n ¼ 3). Oleanolic acid was employed as a positive
M).
control (IC50 ¼ 2.01 ꢀ 0.26
m

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
405
Fig. 3. Compound 18g stimulates the insulin signaling pathway in cells. (a) Effect of 18g on insulin-mediated IRb phosphorylation in CHO-hIR cells. CHO-hIR cells were incubated
with 0.2% DMSO or compound 18g for 2 h and then incubated with or without 10 nM insulin for 10 min. Cell lysates were probed with anti-pTyr antibody. (b) Effect of 18g on Akt
phosphorylation in L6 myotubes. Differentiated L6 myotubes were incubated with 0.2% DMSO or compound 18g for 2 h and then incubated with or without 10 nM insulin for
10 min. Cell lysates were immunoblotted with anti-phospho-Akt (Ser473) antibody. (c) The 18g-stimulated phosphorylation of Akt is PI3K-dependent. L6 myotubes were pretreated
with 250 nM wortmannin and then with 20
phospho-Akt (Ser473) antibody.
mM 18g for 2 h, after which the cells were stimulated with or without 10 nM insulin for 30 min. Cell lysates were probed with anti-
b
-Actin was used as a loading control. (d) Effect of 18g on glucose uptake in L6 myotubes. L6 myotubes were serum starved for 2 h and then
incubated with 18g for 2 h followed by stimulation with 10 nM insulin for 30 min. The rate of glucose uptake was measured following the addition of 2-deoxy-[3H]-deoxyglucose at
37 ꢁC for 15 min. The graph shows the average of at least three independent replicates.
dependent increase in the rate of glucose uptake. The rate of
glucose uptake was increased by 40% and 86% in the presence of 10
and 20 mM 18g, respectively.
3.4. Molecular docking
The full-length PTP1B consists of 435 amino acids, of which
residues 30e278 comprise the catalytic domain. The main struc-
tural features of PTP1B catalytic domain are the phosphate-
binding loop, the WPD loop and the second aryl phosphate-
binding site. The phosphate-binding loop (P-loop, His214e
Arg221), which contains the catalytic residue Cys215, mediates the
substrate recognition and binding of the phosphotyrosine deep in
the catalytic site [39]. Upon substrate binding, the WPD loop
(Thr177ePro185) closes down onto the substrate and thereby
positions the thiolate of Cys215 for the nucleophilic attack upon
the phosphotyrosine [40,41]. The second aryl phosphate-binding
site adjacent to the catalytic site is lined by Arg24, Asp48, Val49,
Ile219, Arg254, Met258, Gly259 and Gln262 [42]. It’s proposed that
dual-binding inhibitors that occupy both the active site and sec-
ond aryl phosphate-binding site would provide not only increased
binding affinity but also the opportunity for improved selectivity
over highly homologous phosphatase TCPTP [24,27,42,43]. To
obtain structural information about the binding mode of inhibitors
of PTP1B for further structural optimization, we used the LibDock
protocol, available in Discovery Studio 2.1, to examine the mo-
lecular docking of compounds 18g and 9d into the active site of
PTP1B in the open conformation. The X-ray crystal structure of
PTP1B (PDB-ID: 1NNY) was retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org). As shown in Fig. 4a, compound 18g
bound both the catalytic site and the second aryl phosphate-
binding site. Three hydrogen bonds were formed between the
carboxylate group and the side chains of Gln266 and Arg221, and
one hydrogen bond was formed between the fluorine atom and
the side chain of Lys116. The indole side chain of compound 18g
fully occupied the second aryl phosphate-binding site and formed
Fig. 4. Proposed binding modes of (a) 18g and (b) 9d. Ligands are shown in stick
representation with carbon atoms in yellow. Hydrogen bonds are displayed as a green
dashed line. The crystal structure of PTP1B, available from the RCSB Protein Data Bank
(PDB: 1NNY), was used to examine the docking. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
a cationep interaction with the guanidine side chain of Arg24. By

406
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
contrast, compound 9d did not fully occupy the second aryl
phosphate-binding site (Fig. 4b). Four hydrogen bonds were
formed between the carboxylate group of 9d and the side chains
of Gln266 and Arg221. These proposed binding modes show that
compound 18g positioned itself effectively in the PTP1B active site
pocket and fully occupied both the catalytic site and the second
aryl phosphate-binding site. This correlated well with the signifi-
cantly improved inhibitory activity of 18g compared with its ACT-
derived analogs.
5.1.2. (2Z,4E)-5-(2-((S)-2-(4-(Benzyloxy)phenyl)-1-
(cyclohexanecarboxamido)ethyl) thiazol-4-yl)-2,4-dimethylpenta-
2,4-dienoic acid (1b)
To a solution of 1a (202 mg, 1.0 eq, 0.353 mmol) in dioxane/H2O
(2 mL/2 mL) was added LiOH/H2O (148 mg, 10 eq, 3.53 mmol) and
the solution was refluxed overnight. It was cooled and diluted with
ethyl acetate and the water layer was acidified with 1 M HCl. The
organic layer was then washed with water and brine, dried over
Na2SO4 and concentrated in vacuo. The residue was purified by
chromatography (CH2Cl2/MeOH ¼ 7/1) to afford acid 1b (192 mg,
4. Conclusion
100%). 1H NMR (300 MHz, CDCl3):
d 1.30e1.44 (m, 4H), 1.66e1.80
(m, 6H), 2.03 (s, 3H), 2.07 (m, 1H), 2.15 (s, 3H), 3.19 (br, 2H), 5.00 (s,
2H), 5.53 (d, 1H, J ¼ 7.8 Hz), 6.36 (s, 1H), 6.58 (s, 1H), 6.84 (d, 2H,
J ¼ 8.4 Hz), 6.98 (d, 2H, J ¼ 8.4 Hz), 7.01 (s, 1H), 7.30e7.42 (m, 5H).
In this work, we identified and characterized a novel structural
class of PTP1B inhibitors inspired from the key ACT scaffold of
Scleritodermin A: the 2-ethyl-5-phenylthiazole-4-carboxamide
(PTA) derivatives (as exemplified by compounds 18b and g).
Replacement of the conjugated diene moiety with the amide-
linked aryl group provided a concise route for the synthesis of
these PTP1B inhibitors. In the PTA skeleton, the phenyl group at
the C5 position of the thiazole moiety contributed to the
improved inhibitory activity of PTA derivatives compared with
ACT or 2-ethylthiazole-4-carboxamide derivatives and improved
their membrane permeability and cellular efficacy. In addition,
the introduction of halogen atoms in the phenyl ring at site C
further improved the inhibitory activity of PTA derivatives. The
fluorinated analog 18g displayed a submicromolar IC50 value
against PTP1B and remarkable potency in cell-based assays.
Compound 18g dose dependently increased the insulin-induced
13C NMR (75 MHz, CDCl3):
d 18.0, 21.8, 25.7, 29.4, 29.5, 40.6, 45.3,
51.9, 67.1, 70.0, 114.9, 117.0, 124.1, 127.6, 128.0, 128.6, 128.9, 129.3,
130.5, 136.9, 137.0, 138.5, 152.7, 157.7, 170.3, 173.6, 176.1. HRMS (ESI)
m/z calc for C32H36N2O4S [M þ H]þ is 545.2469, found 545.2466.
5.1.3. Ethyl2-((S)-3-(4-(benzyloxy)phenyl)-2-(tert-
butoxycarbonylamino)propanamido)-3-oxo-3-phenylpropanoate
(2c)
To a solution of Boc-L-Tyr(Bn)-OH (7.6 g, 1.0 eq, 20.5 mmol) in
CH2Cl2 (100 mL) was added EDC hydrochloride (5.9 g, 1.5 eq,
30.8 mmol), DMAP (0.5 g, 0.2 eq, 4.1 mmol), Et3N (5.9 mL, 2.0 eq,
41.0 mmol) and PhC(O)CH(NH2)COOEt (5.0 g, 1.0 eq, 20.5 mmol).
The reaction mixture was stirred at room temperature for 2 h. After
the solvent was concentrated in vacuo, the residue was purified by
chromatography (petroleum ether/ethyl acetate ¼ 4/1) to afford 2c
phosphorylation of IRb and Akt, the key molecules in the insu-
lin signaling pathway, in the low micromolar range and effec-
tively stimulated glucose uptake in L6 myotubes. Further
improvements of compound 18g and its structurally related an-
alogs may lead to the development of a novel class of therapeutic
agents for antidiabetes treatment.
as colorless gum (7.8 g, 68%). 1H NMR (300 MHz, CDCl3):
d 1.35 (t,
3H, J ¼ 6.9 Hz), 1.41 (s, 9H), 2.98e3.09 (m, 2H), 4.14 (q, 2H,
J ¼ 6.9 Hz), 4.46 (m, 1H), 5.00 (s, 1H), 5.02 (s, 2H), 6.12 (d, 1H,
J ¼ 10.8 Hz), 6.84e6.89 (m, 4H), 7.09 (t, 2H, J ¼ 8.4 Hz), 7.29e7.40
(m, 3H), 7.50 (t, 2H, J ¼ 7.8 Hz), 7.64 (t, 1H, J ¼ 7.8 Hz), 8.01 (t, 2H,
J ¼ 7.8 Hz).
5. Experimental section
5.1.4. (S)-Ethyl 2-(2-(4-(benzyloxy)phenyl)-1-(tert-
5.1. Chemistry
butoxycarbonylamino)ethyl)-5-phenylthiazole-4-carboxylate (3c)
A solution of compound 2c (509 mg, 1.0 eq, 0.91 mmol) and
Lawesson’s reagent (918 mg, 2.5 eq, 2.3 mmol) in dry THF (30 mL)
was heated at 65 ꢁC for 2 h. Then the reaction mixture was allowed
to reach room temperature and concentrated in vacuo. The residue
was purified by chromatography (petroleum ether/ethyl
acetate ¼ 6:1) to afford 3c as white solid (418 mg, 82%). 1H NMR
5.1.1. General methods
Starting materials, reagents and solvents were purchased from
commercial suppliers and used without further purification, un-
less otherwise stated. Anhydrous THF and CH2Cl2 were obtained
from a distillation over sodium wire or CaH2. All non-aqueous
reactions were run under an inert atmosphere (nitrogen or
argon) with rigid exclusion of moisture from reagents and all re-
action vessels were oven-dried. The progress of reactions was
monitored by silica gel thin layer chromatography (TLC) plates,
visualized under UV or charred using phosphomolybdic acid so-
lution followed by heating. Products were purified by flash column
chromatography (FCC) on 200e300 mesh silica gel. Petroleum
ether refers to the fraction with boiling range 60e90 ꢁC or 30e
60 ꢁC. Proton nuclear magnetic resonance spectra (1H NMR) were
recorded on a spectrometer operating at 300 MHz. Data is re-
(300 MHz, CDCl3):
d
1.19 (t, 3H, J ¼ 7.2 Hz), 1.40 (s, 9H), 3.29 (m, 2H),
4.28 (q, 2H, J ¼ 7.2 Hz), 5.04 (s, 2H), 5.24 (br, 1H), 6.90 (d, 2H,
J ¼ 8.4 Hz), 7.08 (d, 2H, J ¼ 8.4 Hz), 7.32e7.43 (m, 10H).
5.1.5. (S)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(4-formyl-5-
phenylthiazol-2-yl) ethylcarbamate (5c)
LiAlH4 (30 mg, 1.0 eq, 0.75 mmol) was added in several portions
to a solution of ester 3c (419 mg, 1.0 eq, 0.75 mmol) in dry THF
(15 mL) at 0 ꢁC. The mixture reaction was stirred at room temper-
ature for 2 h and then quenched with Na2SO4$10H2O. The mixture
was extracted with ethyl acetate and the organic layers were dried
over Na2SO4 and concentrated in vacuo to obtain alcohol 4c
(340 mg, 88%), which was used in the next step without further
purification. A solution of alcohol 4c (340 mg, 1.0 eq, 0.66 mmol)
and IBX (222 mg, 1.2 eq, 7.9 mmol) in toluene/DMSO (5 mL/2 mL)
was heated at 50 ꢁC for 2 h. The white solid was removed by
filtration and the filtrate was diluted with ethyl acetate and washed
successively with water, saturated sodium bicarbonate and brine.
The organic layers were dried over Na2SO4 and concentrated in
vacuo to give aldehyde 5c (289 mg, 85%). 1H NMR (300 MHz,
ported as follows: chemical shift, multiplicity (s
¼
singlet,
d ¼ doublet, dd ¼ double doublet, t ¼ triplet, q ¼ quartet,
br ¼ broad, m ¼ multiplet) and integration. Carbon nuclear
magnetic resonance spectra (13C NMR) were recorded on a spec-
trometer operating at 75 MHz. High-resolution mass data were
obtained on a Micromass Q-Tof UltimaTM spectrometer. Purity
was evaluated by analytical HPLC chromatograms using Agilent
1200 series LC system equipped with Zorbax SB C18 column,
4.6 ꢃ 150 mm, 5
mm particle size, at room temperature. Mobile
phase: MeOH: 0.1% TFA in H2O (80:20). Flow rate: 1.0 mL/min. UV
detection: 285 nm.
CDCl3): d 1.41 (s, 9H), 3.29 (m, 2H), 5.04 (s, 2H), 5.22 (br, 2H), 6.90 (d,

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
407
2H, J ¼ 8.4 Hz), 7.07 (d, 2H, J ¼ 8.4 Hz), 7.31e7.44 (m, 5H), 7.48 (s,
17.2, 22.1, 25.8, 29.7, 29.8, 40.6, 45.5, 52.2, 60.9, 70.2, 100.1, 115.0,
123.2, 127.7, 128.1, 128.3,128.7, 128.9,129.8, 130.8, 131.9, 137.3, 137.6,
157.9, 167.6, 170.6, 175.5. HRMS (ESI) m/z calc for C40H44N2O4S
[M þ H]þ is 649.3095, found 649.3092.
5H), 9.91 (s, 1H).
5.1.6. (S,E)-Ethyl 3-(2-(2-(4-(benzyloxy)phenyl)-1-(tert-
butoxycarbonylamino)ethyl)-5-phenylthiazol-4-yl)-2-
methylacrylate (6c)
5.1.10. (2Z,4E)-5-(2-((S)-2-(4-(Benzyloxy)phenyl)-1-
(cyclohexanecarboxamido) ethyl)-5-phenylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoic acid (9d)
(EtO)2P(O)CH(CH3)CO2Et (614 mg, 6.0 eq, 2.58 mmol) in dry THF
(2 mL) was added to a suspension of NaH (189 mg, 11.0 eq,
4.73 mmol, 60% in mineral oil) in dry THF (8 mL) at 0 ꢁC. The re-
action mixture was stirred for 30 min at room temperature. Then
aldehyde 5c (221 mg, 1.0 eq, 0.43 mmol) in THF (5 mL) was added.
The mixture was stirred at room temperature for 4 h. The reaction
was quenched with saturated NH4Cl and extracted with ethyl ac-
etate. The organic layer was washed with brine, dried over Na2SO4
and concentrated in vacuo. The crude product was purified by
chromatography (petroleum ether/ethyl acetate ¼ 12:1) to give
Compound 9d was prepared using similar procedures as for 1b.
Yield 90% from 9c. M.p. 103e106 ꢁC. 1H NMR (300 MHz, CDCl3):
d
1.10e1.42 (m, 7H), 1.68e1.88 (m, 6H), 2.05 (s, 3H), 2.09 (m, 1H),
2.17 (s, 3H), 3.24 (d, 2H, J ¼ 7.8 Hz), 5.00 (s, 2H), 5.54 (q, 1H,
J ¼ 8.4 Hz), 6.30 (s,1H), 6.36 (br,1H), 6.86 (d, 2H, J ¼ 7.8 Hz), 6.90 (m,
1H), 7.06 (d, 2H, J ¼ 8.4 Hz), 7.30e7.39 (m, 9H). 13C NMR (75 MHz,
CDCl3):
d 17.6, 23.0, 26.1, 26.2, 40.4, 45.5, 52.4, 52.6, 70.5, 115.3,
121.7, 128.1, 128.5, 128.6, 129.1, 129.2, 129.8, 130.2, 131.0, 131.1, 132.3,
ester 6c (224 mg, 87%). 1H NMR (300 MHz, CDCl3):
d
1.29 (t, 3H,
135.5, 137.6, 140.2, 148.1, 158.2, 169.4, 176.7. HRMS (ESI) m/z calc for
J ¼ 7.8 Hz), 1.42 (s, 9H), 2.36 (m, 3H), 3.28 (d, 2H, J ¼ 6.9 Hz), 4.22 (q,
2H, J ¼ 7.8 Hz), 5.03 (s, 2H), 5.20 (br, 2H), 6.88 (d, 2H, J ¼ 9.0 Hz),
7.07 (d, 2H, J ¼ 9.0 Hz), 7.33e7.43 (m, 9H), 7.55 (d, 1H, J ¼ 2.7 Hz).
C
38H40N2O4S [M þ H]þ is 621.2782, found 621.2780.
5.1.11. Compounds 2ae9a, 2be9b were prepared using similar
procedures as for 2ce9c
5.1.7. (S,E)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(4-(2-methyl-3-
oxoprop-1-enyl)-5-phenylthiazol-2-yl) ethylcarbamate (7c)
Compound 7c was prepared using similar procedures as for 5c.
5.1.11.1. Ethyl
nylamino) propanamido)-3-oxobutanoate (2a). Yield 47% from Boc-
-Tyr(Bn)-OH. 1H NMR (300 MHz, CDCl3):
2-((S)-3-(4-(benzyloxy)phenyl)-2-(tert-butoxycarbo
L
d
1.29 (td, 3H, J ¼ 2.1,
Yield 80% for 2 steps from 6c. 1H NMR (300 MHz, CDCl3):
d 1.42 (s,
7.8 Hz), 1.40 (s, 9H), 2.35 (s, 3H), 3.04 (m, 2H), 4.25 (dq, 2H, J ¼ 2.1,
7.8 Hz), 4.42 (br, 1H), 5.02 (s, 2H), 5.19 (dd, 1H, J ¼ 2.1, 7.8 Hz), 6.90
(d, 2H, J ¼ 8.1 Hz), 7.12 (d, 2H, J ¼ 8.1 Hz), 7.31e7.43 (m, 5H).
9H), 2.30 (s, 3H), 3.30 (m, 2H), 5.02 (s, 2H), 5.23 (br, 2H), 6.89 (d, 2H,
J ¼ 8.4 Hz), 7.09 (d, 2H, J ¼ 8.4 Hz), 7.11 (s, 1H), 7.32e7.46 (m, 10H),
9.49 (s, 1H).
5.1.11.2. Ethyl
nylamino) propanamido)-5-methyl-3-oxohexanoate (2b). Yield 60%
from Boc- 0.90 (dd, 6H,
-Tyr(Bn)-OH. 1H NMR (300 MHz, CDCl3):
2-((S)-3-(4-(benzyloxy)phenyl)-2-(tert-butoxycarbo
5.1.8. (2Z,4E)-Ethyl 5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-(tert-
butoxycarbonylamino) ethyl)-5-phenylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (8c)
L
d
J ¼ 6.3,13.2 Hz),1.26 (t, 3H, J ¼ 7.8 Hz),1.39 (s, 9H), 2.17 (m,1H), 2.55
(t, 2H, J ¼ 7.8 Hz), 3.04 (m, 2H), 4.22 (m, 2H), 4.42 (m, 1H), 5.00 (s,
2H), 5.16 (br, 2H), 6.88 (d, 2H, J ¼ 8.4 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz),
7.28e7.41 (m, 5H).
To a solution of (CF3CH2O)2P(O)CH(CH)3CO2Et (197 mg, 3.0 eq,
0.53 mmol) and 18-crown-6 (713 mg, 15.0 eq, 2.70 mmol) in THF
at ꢂ78 ꢁC was added KHMDS (1.06 mL, 3.0 eq, 0.5 mmol/mL,
0.53 mmol) and the reaction mixture was stirred for 1 h at ꢂ78 ꢁC.
Then aldehyde 7c (99 mg, 1.0 eq, 0.18 mmol) was added and the
mixture was stirred at ꢂ78 ꢁC for 2 h. The reaction was quenched
with saturated NH4Cl and extracted with ethyl acetate. The organic
layer was washed with brine, dried over Na2SO4 and concentrated
in vacuo. The residue was purified by chromatography (petroleum
ether/acetone ¼ 15:1) to give ester 8c (115 mg, 39%). 1H NMR
5.1.11.3. (S)-Ethyl 2-(2-(4-(benzyloxy)phenyl)-1-(tert-butoxycarbon
ylamino)ethyl)-5-methylthiazole-4-carboxylate (3a). Yield 76% from
2a. 1H NMR (300 MHz, CDCl3):
d 1.25e1.43 (m, 12H), 2.69 (s, 3H),
3.22 (m, 2H), 4.21 (q, 2H, J ¼ 7.8 Hz), 5.02 (s, 2H), 5.19 (m, 2H), 6.87
(d, 2H, J ¼ 8.4 Hz), 7.03 (d, 2H, J ¼ 8.4 Hz), 7.30e7.42 (m, 5H).
(300 MHz, CDCl3):
d
1.26 (t, 3H, J ¼ 7.8 Hz), 1.60 (s, 9H), 2.00 (s, 3H),
5.1.11.4. (S)-Ethyl 2-(2-(4-(benzyloxy)phenyl)-1-(tert-butoxycarbon
2.18 (s, 3H), 3.27 (m, 2H), 4.17 (q, 2H, J ¼ 7.8 Hz), 5.03 (s, 2H), 5.21
(br, 2H), 6.20 (s, 1H), 6.33 (s, 1H), 6.88 (d, 2H, J ¼ 8.4 Hz), 7.05 (t, 2H,
J ¼ 8.4 Hz), 7.31e7.43 (m, 10H).
ylamino)ethyl)-5-isobutylthiazole-4-carboxylate (3b). Yield 81%
from 2b. 1H NMR (300 MHz, CDCl3):
d
0.90 (dd, 6H, J ¼ 6.6, 13.8 Hz),
1.26 (t, 3H, J ¼ 7.8 Hz), 1.39 (s, 9H), 2.17 (m, 1H), 2.55 (t, 2H,
J ¼ 7.8 Hz), 3.04 (m, 2H), 4.22 (q, 2H, J ¼ 7.8 Hz), 4.44 (m,1H), 5.00 (s,
2H), 5.16 (br, 2H), 6.88 (d, 2H, J ¼ 8.4 Hz), 7.11 (d, 2H, J ¼ 8.4 Hz),
7.28e7.41 (m, 5H).
5.1.9. (2Z,4E)-Ethyl 5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-
(cyclohexanecarboxamido) ethyl)-5-phenylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (9c)
To a solution of 8c (30 mg, 1.0 eq, 0.047 mmol) in CH2Cl2 (2 mL),
trifluoroacetic acid (0.4 mL) was added and the mixture was stirred
for 2 h at room temperature. The reaction mixture evaporated in
vacuo to afford the crude amine, which was used without further
purification. A solution of the amine and DIPEA (0.1 mL, 10 eq,
0.47 mmol) in dry CH2Cl2 (5 mL) was cooled in an ice bath and then
5.1.11.5. (S)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(4-formyl-5-
methylthiazol-2-yl) ethylcarbamate (5a). Yield 81% for 2 steps
from 3a. 1H NMR (300 MHz, CDCl3):
d 1.41 (s, 9H), 2.73 (s, 3H), 3.22
(d, 2H, J ¼ 6.6 Hz), 5.03 (s, 2H), 6.88 (d, 2H, J ¼ 7.8 Hz), 7.03 (d, 2H,
J ¼ 7.8 Hz), 7.35e7.41 (m, 5H), 10.11 (s, 1H).
cyclohexanecarbonyl chloride (11
mL, 1.5 eq, 0.070 mmol) was
added. The reaction mixture was stirred at room temperature
overnight. After the solvent was concentrated in vacuo, the residue
was purified by chromatography (petroleum ether/acetone ¼ 9/1)
to afford 9c (21 mg, 69%). M.p. 130e132 ꢁC. 1H NMR (300 MHz,
5.1.11.6. (S)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(4-formyl-5-
isobutylthiazol-2-yl) ethylcarbamate (5b). Yield 91% for 2 steps
from 3b. 1H NMR (300 MHz, CDCl3):
9H),1.88 (m, 1H), 3.06 (d, 2H, 7.8 Hz), 3.21 (d, 2H, J ¼ 6.9 Hz), 5.02 (s,
2H), 5.20 (m, 2H), 6.87 (d, 2H, J ¼ 9.0 Hz), 7.10 (d, 2H, J ¼ 9.0 Hz),
7.31e7.43 (m, 5H), 10.09 (s, 1H).
d
0.94 (d, 6H, J ¼ 6.9 Hz),1.41 (s,
CDCl3):
d
1.25 (t, 3H, J ¼ 7.8 Hz), 1.28e1.45 (m, 4H), 1.68e1.88 (m,
6H), 2.00 (s, 3H), 2.09 (m, 1H), 2.17 (s, 3H), 3.27 (m, 2H), 4.17 (q, 2H,
J ¼ 7.8 Hz), 5.03 (s, 2H), 5.55 (q, 1H, J ¼ 8.1 Hz), 6.20 (s, 1H), 6.30 (d,
1H, J ¼ 8.1 Hz), 6.33 (s, 1H), 6.87 (d, 2H, J ¼ 8.4 Hz), 7.05 (d, 2H,
5.1.11.7. (S,E)-Ethyl
3-(2-(2-(4-(benzyloxy)phenyl)-1-(tert-butox-
J ¼ 8.4 Hz), 7.31e7.43 (m, 10H). 13C NMR (75 MHz, CDCl3):
d 14.2,
ycarbonylamino) ethyl)-5-methylthiazol-4-yl)-2-methylacrylate (6a).

408
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
Yield 73% from 5a. 1H NMR (300 MHz, CDCl3):
d
1.35 (t, 3H,
7.8 Hz), 4.20 (q, 2H, J ¼ 7.8 Hz), 5.01 (s, 2H), 5.47 (q, 1H, J ¼ 7.8 Hz),
6.25 (br, 2H), 6.36 (d, 1H, J ¼ 8.4 Hz), 6.82 (d, 2H, J ¼ 8.4 Hz), 6.96 (d,
J ¼ 7.8 Hz), 1.55 (s, 9H), 2.34 (d, 3H, J ¼ 2.7 Hz), 2.45 (s, 3H), 3.22 (d,
2H, J ¼ 6.9 Hz), 4.27 (q, 2H, J ¼ 7.8 Hz), 5.03 (s, 2H), 5.13 (br 2H), 6.86
(d, 2H, J ¼ 9.0 Hz), 7.02 (d, 2H, J ¼ 9.0 Hz), 7.31e7.43 (m, 4H), 9.50 (d,
1H, J ¼ 2.7 Hz).
2H, J ¼ 8.4 Hz), 7.31e7.42 (m, 5H). 13C NMR (75 MHz, CDCl3):
d 14.3,
17.1, 22.0, 22.3, 25.6, 25.8, 25.9, 25.9, 29.1, 29.6, 29.7, 31.2, 35.5, 40.8,
45.5, 52.1, 60.8, 70.1, 114.8, 122.2, 127.6, 128.1, 128.7, 129.0, 130.7,
136.2, 136.3, 137.2, 137.7, 148.6, 157.8, 160.8, 165.9, 169.8, 170.6,
175.5. HRMS (ESI) m/z calc for C38H48N2O4S [M þ H]þ is 629.3408,
found 629.3420.
5.1.11.8. (S,E)-Ethyl
ycarbonylamino) ethyl)-5-isobutylthiazol-4-yl)-2-methylacrylate
(6b). Yield 75% from 5b. 1H NMR (300 MHz, CDCl3):
0.92 (d, 6H,
3-(2-(2-(4-(benzyloxy)phenyl)-1-(tert-butox-
d
J ¼ 7.8 Hz), 1.34 (t, 3H, J ¼ 7.8 Hz), 1.42 (s, 9H), 1.81 (m, 1H), 2.35 (d,
3H, J ¼ 2.1 Hz), 2.69 (d, 2H, J ¼ 7.8 Hz), 3.21 (br 2H), 4.27 (q, 2H,
J ¼ 7.8 Hz), 5.02 (s, 2H), 5.16 (br, 2H), 6.85 (d, 2H, J ¼ 8.4 Hz), 7.00 (d,
2H, J ¼ 8.4 Hz), 7.31e7.43 (m, 4H), 7.51 (d, 1H, J ¼ 2.1 Hz).
5.1.12. Compound 10
Compound 10 was synthesized as previously reported [44].
5.1.13. (S)-Ethyl 2-(2-(4-(benzyloxy)phenyl)-1-
(cyclohexanecarboxamido)ethyl) thiazole-4-carboxylate (12)
Compound 12 was prepared using similar procedures from 10 as
for 9c. Yield 86% for 2 steps from 10. 1H NMR (300 MHz, CDCl3):
5.1.11.9. (S,E)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(5-methyl-4-
(2-methyl-3-oxoprop-1-enyl)thiazol-2-yl)ethylcarbamate
(7a).
1.42 (s,
Yield 80% for 2 steps from 6a. 1H NMR (300 MHz, CDCl3):
d
d
1.41 (t, 3H, J ¼ 7.2 Hz), 1.44e1.77 (m, 5H), 2.05e2.12 (m, 1H), 3.19e
9H), 2.27 (s, 3H), 2.53 (s, 3H), 3.23 (d, 2H, J ¼ 6.6 Hz), 5.03 (s, 2H),
5.14 (m, 2H), 6.87 (d, 2H, J ¼ 8.4 Hz), 7.03 (d, 2H, J ¼ 8.4 Hz), 7.06
(s, 1H), 7.30e7.43 (m, 5H), 9.60 (s, 1H).
3.33 (m, 2H), 4.43 (q, 2H, J ¼ 6.9 Hz), 5.02 (s, 2H), 5.55 (q, 1H,
J ¼ 8.1 Hz), 6.24 (d, 1H, J ¼ 7.8 Hz), 6.86 (d, 2H, J ¼ 8.4 Hz), 6.99 (d,
2H, J ¼ 8.4 Hz), 7.27e7.43 (m, 5H), 8.02 (s, 1H).
5.1.11.10. (S,E)-tert-Butyl 2-(4-(benzyloxy)phenyl)-1-(5-isobutyl-
5.1.14. (S)-2-(2-(4-(Benzyloxy)phenyl)-1-
4-(2-methyl-3-oxoprop-1-enyl)thiazol-2-yl)ethylcarbamate (7b).
(cyclohexanecarboxamido)ethyl)thiazole-4-carboxylic acid (13)
Yield 60% for 2 steps from 6b. 1H NMR (300 MHz, CDCl3):
d
0.95
Yield 99% from 12. 1H NMR (300 MHz, CDCl3):
d 1.25e1.35 (m,
(d, 6H, J ¼ 7.8 Hz), 1.42 (s, 9H), 1.86 (m, 1H), 2.28 (s, 3H), 2.76 (d,
2H, J ¼ 7.8 Hz), 3.23 (d, 2H, J ¼ 6.6 Hz), 5.02 (s, 2H), 5.16 (br, 2H),
6.86 (d, 2H, J ¼ 8.4 Hz), 7.00 (s, 1H), 7.04 (d, 2H, J ¼ 8.4 Hz), 7.31e
7.43 (m, 5H), 9.60 (s, 1H).
10H), 1.75e1.78 (m, 1H), 3.26 (d, 1H), 5.03 (s, 2H), 5.56 (q, 1H,
J ¼ 8.1 Hz), 6.11 (d, 1H, J ¼ 8.4 Hz), 6.88 (d, 2H, J ¼ 9.0 Hz), 6.99 (d,
1H, J ¼ 8.7 Hz), 7.27e7.40 (m, 5H), 8.15 (s, 1H).
5.1.15. General procedure for the synthesis of 14aec
5.1.11.11. (2Z,4E)-Ethyl
5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-(tert-
To a solution 13 (30 mg, 1.0 eq, 0.065 mmol) in CH2Cl2 (100 mL)
was added EDC hydrochloride (15 mg, 1.2 eq, 0.078 mmol), DMAP
(2 mg, 0.2 eq, 0.013 mmol), Et3N (20 mL, 2.0 eq, 0.156 mmol) and
butoxycarbonylamino)ethyl)-5-methylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (8a). Yield 67% from 7a. 1H NMR
(300 MHz, CDCl3):
d
1.30 (t, 3H, J ¼ 7.8 Hz), 1.41 (s, 9H), 2.03 (s, 3H),
HCl$H2NC6H4COOMe (15 mg, 1.2 eq, 0.078 mmol). The reaction
mixture was stirred at room temperature overnight. After the sol-
vent was concentrated in vacuo, the residue was purified by chro-
matography (petroleum ether/ethyl acetate ¼ 4/1) to afford 14aec.
2.14 (s, 3H), 2.32 (s, 3H), 3.20 (d, 2H, J ¼ 6.6 Hz), 4.20 (q, 2H,
J ¼ 7.8 Hz), 5.02 (s, 2H), 5.16 (br, 2H), 6.27 (br, 2H), 6.85 (d, 2H,
J ¼ 9.0 Hz), 7.02 (d, 2H, J ¼ 9.0 Hz), 7.31e7.43 (m, 5H).
5.1.11.12. (2Z,4E)-Ethyl
5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-(tert-
5.1.15.1. (S)-Methyl
hexanecarboxamido)ethyl) thiazole-4-carboxamido)benzoate (14a).
Yield 47%. M.p. 214e217 ꢁC. 1H NMR (300 MHz, CDCl3):
1.48e1.93
2-(2-(2-(4-(benzyloxy)phenyl)-1-(cyclo-
butoxycarbonylamino)ethyl)-5-isobutylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (8b). Yield 36% from 7b. 1H NMR
d
(300 MHz, CDCl3):
d
0.90 (d, 6H, J ¼ 6.6 Hz), 1.28 (t, 3H, J ¼ 7.8 Hz),
(m, 10H), 2.04e2.08 (m, 1H), 3.09 (t, 1H, J ¼ 6.3 Hz), 3.20e3.46 (m,
1H), 3.97 (s, 3H), 5.02 (d, 2H, J ¼ 7.8 Hz), 5.61 (q, 1H, J ¼ 6.3 Hz), 5.88
(d, 1H), 6.68 (d, 1H, J ¼ 7.2 Hz), 6.85e7.06 (m, 5H), 7.15 (t, 1H,
J ¼ 6.3 Hz), 7.35e7.39 (m, 5H), 7.61 (t, 1H, J ¼ 8.4 Hz), 8.10 (d, 1H,
J ¼ 7.5 Hz), 8.91 (d, 1H, J ¼ 8.4 Hz). HRMS (ESI) m/z calc for
1.42 (s, 9H), 1.87 (m, 1H), 2.03 (s, 3H), 2.17 (s, 3H), 2.58 (d, 2H,
J ¼ 7.8 Hz), 3.21 (d, 2H, J ¼ 6.6 Hz), 4.20 (q, 2H, J ¼ 7.8 Hz), 5.01 (s,
2H), 5.18 (br, 2H), 6.26 (br, 2H), 6.84 (d, 2H, J ¼ 9.0 Hz), 6.95 (d, 2H,
J ¼ 8.7 Hz), 7.31e7.43 (m, 5H).
C
34H35N3O5S [M þ H]þ is 598.2370, found 598.2368.
5.1.11.13. (2Z,4E)-Ethyl 5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-(cyclo-
hexanecarboxamido)ethyl)-5-methylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (9a). Yield 36% for 2 steps from 8a. 1H
5.1.15.2. (S)-Methyl 3-(2-(2-(4-(benzyloxy)phenyl)-1-(cyclohexane
carboxamido)ethyl)
thiazole-4-carboxamido)benzoate
(14b).
1.32e1.46
NMR (300 MHz, CDCl3):
d
1.27 (t, 3H, J ¼ 7.9 Hz), 1.29e1.43 (m, 4H),
Yield 73%. M.p. 130e131 ꢁC. 1H NMR (300 MHz, CDCl3):
d
1.66e1.86 (m, 6H), 2.03 (s, 3H), 2.07 (m, 1H), 2.11 (s, 1H), 2.32 (s,
3H), 3.20 (d, 2H, J ¼ 6.6 Hz), 4.20 (q, 2H, J ¼ 7.2 Hz), 5.02 (s, 2H), 5.45
(q, 1H, J ¼ 8.4 Hz), 6.26 (br, 2H), 6.31 (d, 1H, J ¼ 8.4 Hz), 6.84 (d, 2H,
J ¼ 9.0 Hz), 6.99 (d, 2H, J ¼ 9.0 Hz), 7.31e7.43 (m, 5H). 13C NMR
(m, 6H), 1.68e1.86 (m, 4H), 2.09e2.19 (m, 1H), 3.28 (d, 2H,
J ¼ 6.6 Hz), 3.89 (s, 3H), 5.02 (s, 2H), 5.60 (q, 2H, J ¼ 8.1 Hz), 6.07 (d,
1H, J ¼ 8.1 Hz), 6.89 (d, 2H, J ¼ 8.4 Hz), 7.01 (d, 2H, J ¼ 8.4 Hz), 7.31e
7.49 (m, 7H), 7.83 (d, 1H, J ¼ 7.8 Hz), 8.09 (s, 1H), 8.14 (d, 1H,
J ¼ 9.0 Hz), 8.19 (s, 1H), 9.22 (s, 1H). HRMS (ESI) m/z calc for
(75 MHz, CDCl3):
d 11.8, 14.2, 17.1, 21.9, 25.8, 29.6, 29.7, 40.6, 45.5,
52.0, 60.9, 70.1, 114.9, 122.0, 127.6, 128.1, 128.7, 129.0, 129.1, 130.7,
131.1, 136.4, 137.2, 137.6, 148.6, 157.8, 165.8, 175.5. HRMS (ESI) m/z
calc for C35H42N2O4S [M þ H]þ is 587.2938, found 587.2934.
C
34H35N3O5S [M þ H]þ is 598.2370, found 598.2370.
5.1.15.3. (S)-Methyl
ecarboxamido)ethyl)
4-(2-(2-(4-(benzyloxy)phenyl)-1-(cyclohexan
thiazole-4-carboxamido)benzoate
(14c).
5.1.11.14. (2Z,4E)-Ethyl 5-(2-((S)-2-(4-(benzyloxy)phenyl)-1-(cyclo-
hexanecarboxamido)ethyl)-5-isobutylthiazol-4-yl)-2,4-
dimethylpenta-2,4-dienoate (9b). Yield 36% for 2 steps from 8b. 1H
Yield 61%. M.p. 148e151 ꢁC. 1H NMR (300 MHz, CDCl3):
d
1.38e1.81
(m, 10H), 2.13e2.20 (m, 1H), 3.28 (d, 2H), 3.91 (s, 3H), 5.02 (s, 2H),
5.60 (m, 1H), 6.04 (d, 1H), 6.89 (d, 2H, J ¼ 7.8 Hz), 7.00 (d, 2H,
J ¼ 7.8 Hz), 7.32e7.53 (m, 5H), 7.80 (d, 2H, J ¼ 8.4 Hz), 8.06 (d, 2H,
J ¼ 8.7 Hz), 8.11 (s, 1H), 9.27 (s, 1H). HRMS (ESI) m/z calc for
NMR (300 MHz, CDCl3):
d
0.89 (dd, 6H, J ¼ 2.1, 7.8 Hz), 1.26 (t, 3H,
J ¼ 7.8 Hz), 1.29e1.44 (m, 4H), 1.66e1.85 (m, 6H), 2.03 (s, 3H), 2.08
(m, 1H), 2.13 (s, 3H), 2.56 (d, 2H, J ¼ 7.8 Hz), 3.20 (dd, 2H, J ¼ 2.7,
C
34H35N3O5S [M þ Na]þ is 620.2190, found 620.2190.

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
409
5.1.16. General procedure for the synthesis of 14def
5.1.19. (S)-Methyl 3-(2-(2-(4-(benzyloxy)phenyl)-1-
To a solution of 14aec (20 mg, 1.0 eq, 0.033 mmol) in dioxane/
H2O (0.5 mL/0.5 mL) was added LiOH/H2O (12 mg, 10 eq,
0.33 mmol) and the solution was stirred overnight at room tem-
perature. It was diluted with ethyl acetate and the water layer was
acidified with 1 M HCl. The organic layer was then washed with
water and brine, dried over Na2SO4 and concentrated in vacuo. The
residue was purified by chromatography (CH2Cl2/MeOH ¼ 7/1) to
afford acid 14def.
(cyclohexanecarboxamido)ethyl)-5-phenylthiazole-4-carboxamido)
benzoate (17a)
Compound 16 (131 mg, 0.20 mmol) was dissolved in 10 mL 4 M
HCl/EtOAc. The solution was stirred overnight at room temperature.
Then it was evaporated in vacuo to obtain the crude amine which
was used in the next step without further purification. A solution of
the crude amine (67 mg, 1.0 eq, 0.12 mmol) and Et3N (38
0.26 mmol) in dry CH2Cl2 (5 mL) was cooled in an ice bath and then
cyclohexanecarbonyl chloride (17.5 L, 1.1 eq, 0.13 mmol) was
mL, 2.2 eq,
m
5.1.16.1. (S)-2-(2-(2-(4-(Benzyloxy)phenyl)-1-(cyclo-
hexanecarboxamido)ethyl) thiazole-4-carboxamido)benzoic acid
(14d). Yield 98%. M.p. >240 ꢁC. 1H NMR (300 MHz, DMSO-d6):
added. The reaction mixture was stirred at room temperature
overnight. After the solvent was concentrated in vacuo, the residue
was purified by chromatography (petroleum ether/acetone ¼ 3/1)
to afford 17a as light brown solid (51 mg, 60%). M.p. 178e180 ꢁC. 1H
d
1.47e1.66 (m, 10H), 2.08e2.20 (m, 1H), 2.99e3.59 (m, 2H), 5.02 (s,
2H), 5.28 (m, 1H), 6.92 (d, 2H, J ¼ 8.1 Hz), 7.10 (t, 2H), 7.24 (d, 2H,
J ¼ 8.1 Hz), 7.29e7.48 (m, 5H), 8.06 (d, 1H, J ¼ 8.4 Hz), 8.31 (s, 1H),
8.59 (d, 1H, J ¼ 8.1 Hz), 8.75 (d, 1H, J ¼ 8.1 Hz). HRMS (ESI) m/z calc
for C33H33N3O5S [M þ Na]þ is 606.2033, found 606.2031.
NMR (300 MHz, CDCl3): d 1.36e1.47 (m, 2H), 1.68e1.96 (m, 8H),
2.09e2.18 (m, 1H), 3.23e3.38 (m, 2H), 3.90 (m, 3H), 5.03 (m, 2H),
5.59 (q, 1H, J ¼ 7.8 Hz), 6.13 (d, 1H, J ¼ 7.8 Hz), 6.93 (d, 2H,
J ¼ 8.7 Hz), 7.09 (d, 2H, J ¼ 8.4 Hz), 7.30e7.42 (m, 8H), 7.55e7.59 (m,
2H), 7.78 (d, 1H, J ¼ 7.5 Hz), 8.07 (d, 1H, J ¼ 8.1 Hz), 8.13 (s, 1H), 9.42
(s, 1H). HRMS (ESI) m/z calc for C40H39N3O5S [M þ H]þ is 674.2683,
found 674.2685.
5.1.16. 2. (S)-3-(2-(2-(4-(Benzyloxy)phenyl)-1-(cyclo-
hexanecarboxamido)ethyl) thiazole-4-carboxamido)benzoic acid
(14e). Yield 98%. M.p. 201e203 ꢁC. 1H NMR (300 MHz, DMSO-d6):
d
1.48e1.71 (m, 10H), 2.14 (m, 1H), 3.05 (t, 2H), 5.07 (s, 2H), 5.31 (m,
5.1.20. (S)-Methyl 3-(2-(1-(3-(1H-indol-3-yl)propanamido)-2-(4-
(benzyloxy)phenyl) ethyl)-5-phenylthiazole-4-carboxamido)
benzoate (18a)
1H), 6.94 (d, 2H, J ¼ 6.6 Hz), 7.23 (d, 2H, J ¼ 6.6 Hz), 7.32e7.52 (m,
7H), 7.70 (d, 1H, J ¼ 7.2 Hz), 8.08 (d, 1H, J ¼ 8.1 Hz), 8.36 (s, 1H), 8.47
(s, 1H), 8.61 (d, 1H, J ¼ 7.5 Hz), 10.33 (s, 1H). HRMS (ESI) m/z calc for
Compound 18a was prepared using similar procedures as for 20
C
33H33N3O5S [M þ Na]þ is 606.2033, found 606.2033.
in 88% yield for 2 steps from 16. M.p. 77e79 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
2.63 (t, 2H, J ¼ 7.2 Hz), 3.09e3.13 (m, 2H), 3.89 (s, 3H), 4.99
5.1.16. 3. (S)-4-(2-(2-(4-(Benzyloxy)phenyl)-1-(cyclo-
hexanecarboxamido)ethyl) thiazole-4-carboxamido)benzoic acid
(14f). Yield 92%. M.p. 173e176 ꢁC. 1H NMR (300 MHz, CD3OD):
(s, 2H), 5.52 (q, 1H, J ¼ 8.1 Hz), 6.12 (d, 1H, J ¼ 8.1 Hz), 6.78e6.87 (m,
5H), 7.07e7.59 (m, 14H), 7.78 (d, 1H, J ¼ 7.8 Hz), 8.02 (d, 1H,
J ¼ 8.4 Hz), 8.13 (s, 1H), 9.31 (s, 1H). HRMS (ESI) m/z calc for
d
1.32e1.68 (m, 10H), 2.06e2.14 (m, 1H), 2.97e3.05 (m, 2H), 5.07 (s,
C
44H38N4O5S [M þ H]þ is 735.2636, found 735.2631.
2H), 5.29 (m, 1H), 6.94 (d, 2H, J ¼ 8.7 Hz), 7.22 (d, 2H, J ¼ 8.4 Hz),
7.31e7.45 (m, 5H), 7.93e8.00 (m, 4H), 8.39 (s, 1H), 8.61 (d, 1H,
J ¼ 8.4 Hz), 10.37 (s, 1H). HRMS (ESI) m/z calc for C33H33N3O5S
[M þ Na]þ is 606.2033, found 606.2029.
5.1.21. (S)-3-(2-(2-(4-(Benzyloxy)phenyl)-1-
(cyclohexanecarboxamido)ethyl)-5-phenylthiazole-4-carboxamido)
benzoic acid (17b)
To a solution of 17a (41 mg, 1.0 eq, 0.061 mmol) in THF/MeOH/
H2O (1:1:1, 10 mL) was added LiOH/H2O (26 mg, 10.0 eq,
0.61 mmol) and the solution was stirred at 50 ꢁC for 2 h. It was
diluted with CH2Cl2 and the water layer was acidified with 1 M HCl.
The organic layer was then washed with water and brine, dried over
Na2SO4 and concentrated in vacuo to afford acid 17b as white solid
5.1.17. (S)-2-(2-(4-(Benzyloxy)phenyl)-1-(tert-
butoxycarbonylamino)ethyl)-5-phenylthiazole-4-carboxylic acid
(15)
To a solution of 3c (508 mg, 1.0 eq, 0.91 mmol) in THF/H2O
(10 mL/10 mL) was added LiOH/H2O (230 mg, 6 eq, 6.0 mmol) and
the solution was stirred overnight at 50 ꢁC. It was cooled and
diluted with ethyl acetate and the water layer was acidified with
1 M HCl. The organic layer was then washed with water and brine,
dried over Na2SO4 and concentrated in vacuo to afford acid 15
in 99% yield. M.p. 217e219 ꢁC. 1H NMR (300 MHz, CDCl3):
d 1.28e
2.04 (m, 10H), 2.11e2.18 (m, 1H), 3.29e3.31 (m, 2H), 5.04 (s, 2H),
5.55 (q, 1H, J ¼ 7.2 Hz), 6.04 (d, 1H, J ¼ 8.1 Hz), 6.93 (d, 2H,
J ¼ 7.5 Hz), 7.09 (d, 3H), 7.28e7.56 (m, 8H), 7.82 (d, 1H, J ¼ 8.1 Hz),
8.11 (s, 1H), 8.17 (d, 1H, J ¼ 8.1 Hz), 9.41 (s, 1H). 13C NMR (75 MHz,
(483 mg, 100%). 1H NMR (300 MHz, CDCl3):
d 1.40 (s, 9H), 3.21 (m,
2H), 5.00 (s, 2H), 5.14 (m, 1H), 6.87 (d, 2H, J ¼ 8.4 Hz), 7.04 (d, 2H,
CDCl3): d 25.5, 25.6, 25.8, 29.4, 40.2, 45.2, 52.0, 70.1, 115.1, 121.1,
J ¼ 8.7 Hz), 7.28e7.46 (m, 10H).
124.5, 125.7, 127.5, 128.0, 128.2, 128.6, 129.2, 129.3, 129.9, 130.2,
130.4, 131.3, 133.6, 136.9,138.0,141.0, 144.1, 158.0,159.7, 168.6, 169.1,
176.7, 180.5. HRMS (ESI) m/z calc for C39H37N3O5S [M þ Na]þ is
682.2346, found 682.2342.
5.1.18. (S)-Methyl 3-(2-(2-(4-(benzyloxy)phenyl)-1-(tert-
butoxycarbonylamino)ethyl)-5-phenylthiazole-4-carboxamido)
benzoate (16)
To a solution 15 (490 mg, 1.0 eq, 0.92 mmol) in CH2Cl2 (50 mL)
was added HATU (525 mg, 1.5 eq, 1.38 mmol), 4-methylmorpholine
(0.30 mL, 3.0 eq, 2.8 mmol) and methyl 3-aminobenzoate hydro-
chloride (208 mg, 1.2 eq, 1.1 mmol). The reaction mixture was
stirred at room temperature overnight. After the solvent was
concentrated in vacuo, the residue was purified by chromatography
(petroleum ether/acetone ¼ 3/1) to afford compound 16 as white
5.1.22. (S)-3-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-
(benzyloxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)benzoic
acid (18b)
Compound 18b was prepared using similar procedures as for
17b in 91% yield from 18a. M.p. 168e170 ꢁC. 1H NMR (300 MHz,
CDCl3):
d
2.63 (t, 2H, J ¼ 6.9 Hz), 3.02e3.15 (m, 4H), 4.98 (s, 2H), 5.53
(q, 1H, J ¼ 7.5 Hz), 6.18 (d, 1H, J ¼ 8.1 Hz), 6.73e6.85 (m, 5H), 7.05e
solid (360 mg, 59%). 1H NMR (300 MHz, CDCl3):
d 1.44 (s, 9H), 3.28
7.15 (m, 2H), 7.21 (d, 1H, J ¼ 7.5 Hz), 7.30e7.44 (m, 7H), 7.50e7.57
(m, 2H), 3.91 (s, 3H), 5.04 (s, 2H), 5.15 (m, 1H), 6.93 (d, 2H,
J ¼ 8.7 Hz), 7.10 (d, 2H, J ¼ 8.7 Hz), 7.31e7.46 (m, 8H), 7.54e7.57 (m,
2H), 7.75 (d, 1H, J ¼ 8.4 Hz), 8.06 (d, 1H, J ¼ 8.4 Hz), 8.14 (s, 1H), 9.43
(s, 1H).
(m, 3H), 7.80 (d, 1H, J ¼ 7.5 Hz), 8.07e8.09 (m, 2H), 9.28 (s, 1H). 13
C
NMR (75 MHz, CDCl3):
d 21.4, 37.4, 40.3, 52.1, 70.2, 108.4, 111.5,
114.5,115.2,118.8,119.6,121.4,122.2,122.3,123.2,125.2,125.9,127.2,
127.7, 128.2, 128.4, 128.8, 129.5, 130.0, 130.4, 130.6, 136.5, 137.0,

410
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
138.3, 141.0, 143.4, 145.1, 158.0, 159.6, 163.7, 168.7, 170.5, 173.0.
HRMS (ESI) m/z calc for C43H36N4O5S [M þ Na]þ is 743.2299, found
743.2292.
5.1.25.3. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-6-fluorobenzoic
acid (18e). Yield 66% for 2 steps from 21. M.p. 149e151 ꢁC. 1H NMR
(300 MHz, CDCl3/CD3OD):
d 2.74 (t, 2H), 2.95e3.08 (m, 4H), 4.88 (s,
5.1.23. (S)-Ethyl 2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-
2H), 5.58 (m, 1H), 6.66e6.72 (m, 4H), 6.90e7.14 (m, 6H), 7.22e7.49
(benzyloxy)phenyl)ethyl)-5-phenylthiazole-4-carboxylate (20)
To a solution of 3c (1.904 g, 1.0 eq, 3.41 mmol) in CH2Cl2
(15 mL), trifluoroacetic acid (3 mL) was added and the mixture
was stirred for 2 h at room temperature. The reaction mixture
evaporated in vacuo to afford the crude amine 19, which was used
without further purification. To a solution of the amine 19 in
CH2Cl2 (50 mL) was added 3-indolepropionic acid (0.645 g, 1.0 eq,
3.41 mmol), EDC hydrochloride (0.784 g, 1.2 eq, 4.1 mmol), DMAP
(83 mg, 0.2 eq, 0.68 mmol) and Et3N (1 mL, 2.0 eq, 6.82 mmol).
The reaction mixture was stirred overnight at room temperature.
The reaction mixture was extracted with ethyl acetate and the
organic layer was washed successively with 1 M HCl, saturated
NaHCO3 and brine, dried over Na2SO4 and concentrated in vacuo.
The crude product was purified by chromatography (petroleum
ether/ethyl acetate ¼ 1:1) to give compound 20 as white solid
(m, 11H), 8.38 (d, 1H, J ¼ 8.4 Hz); 13C NMR (75 MHz, CDCl3/CD3OD):
d
21.1, 29.8, 36.6, 38.9, 51.7, 70.0, 110.9, 111.2, 111.4, 113.8, 114.9,
116.4, 118.4, 119.0, 121.8, 122.1, 122.2, 126.9, 127.7, 128.1, 128.2, 128.5,
128.7, 129.3, 130.1, 130.3, 130.4, 132.3, 136.2, 136.3, 136.9, 139.8,
141.3, 144.9, 157.7, 160.1, 160.2, 161.6, 163.6, 167.0, 167.4, 170.1, 173.9.
HRMS (ESI) m/z calc for C43H35FN4O5S [M þ Na]þ is 761.2204,
found 761.2205.
5.1.25.4. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-5-fluorobenzoic
acid (18f). Yield 79% for 2 steps from 21. M.p. 180e182 ꢁC. 1H NMR
(300 MHz, CDCl3/CD3OD):
d
2.68 (t, 2H, J ¼ 6.6 Hz), 3.07e3.26 (m,
4H), 4.97 (s, 2H), 5.48 (t, 1H, J ¼ 6.0 Hz), 6.78 (d, 2H, J ¼ 8.1 Hz), 6.89
(s,1H), 6.95 (d, 2H, J ¼ 8.4 Hz), 7.01e7.12 (m, 2H), 7.20e7.42 (m,10H),
7.52e7.58 (m, 3H), 7.78 (dd, 1H, J ¼ 2.7, 9.6 Hz), 8.79 (dd, 1H, J ¼ 5.1,
(1.935 g, 90% for 2 steps). 1H NMR (300 MHz, CDCl3):
d
1.26 (t, 3H,
9.3 Hz); 13C NMR (75 MHz, CDCl3/CD3OD):
d 21.4, 37.1, 40.1, 52.2,
J ¼ 7.2 Hz), 2.59 (t, 2H, J ¼ 7.5 Hz), 3.07e3.22 (m, 4H), 4.28 (q, 2H,
J ¼ 7.2 Hz), 5.01 (s, 2H), 5.50 (q, 1H, J ¼ 8.4 Hz), 6.17 (d, 1H,
J ¼ 8.1 Hz), 6.79 (d, 2H, J ¼ 8.7 Hz), 6.88 (d, 2H, J ¼ 8.7 Hz), 6.92 (s,
1H), 7.08e7.21 (m, 2H), 7.29e7.43 (m, 12H), 7.58 (d, 1H, J ¼ 7.2 Hz),
8.00 (s, 1H).
76.8, 111.4, 114.3, 115.0, 117.7, 118.0, 118.6, 119.2, 121.2, 121.5, 121.9,
122.1, 127.1, 127.6, 128.1, 128.2, 128.7, 128.7, 129.3, 129.6, 130.0, 130.3,
130.6, 133.2, 134.9, 135.0, 136.4, 137.0, 137.6, 141.2, 157.8, 160.1, 165.0,
167.0,168.8,173.4. HRMS (ESI) m/z calc for C43H35FN4O5S [M þ H]þ is
739.2385, found 739.2383.
5.1.24. (S)-2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzyloxy)
phenyl)ethyl)-5-phenylthiazole-4-carboxylic acid (21)
5.1.25.5. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-4-
fluorobenzoic acid (18g). Yield 78% for 2 steps from 21. M.p. 240e
To a solution of 20 (1.53 g, 1.0 eq, 2.43 mmol) in MeOH/H2O (1:1,
50 mL) was added K2CO3 (1.0 g, 3.0 eq, 7.29 mmol) and the solution
was stirred overnight at room temperature. It was diluted with
CH2Cl2 and the water layer was acidified with 1 M HCl. The organic
layer was then washed with water and brine, dried over Na2SO4 and
concentrated in vacuo to afford acid 21 as white solid (1.46 g, 100%).
242 ꢁC. 1H NMR (300 MHz, CDCl3/CD3OD):
d 2.53 (t, 2H), 2.98e3.00
(t, 4H), 4.88 (S, 2H), 5.52 (m, 1H), 6.58e6.71 (m, 4H), 6.89e7.16 (m,
6H), 7.26e7.48 (m,10H), 8.06 (s,1H), 8.54 (d,1H); 13C NMR (75 MHz,
CDCl3/CD3OD):
d 21.1, 36.7, 38.9, 51.7, 70.0, 91.2, 109.3, 109.6, 111.2,
113.7, 114.8, 118.2, 118.7, 121.5, 121.9, 126.9, 127.5, 128.0, 128.1, 128.5,
128.7, 129.1, 129.9, 130.1, 130.3, 133.8, 134.9, 136.3, 136.8, 141.5,
145.0, 157.6, 160.5, 164.2, 167.5, 174.0. HRMS (ESI) m/z calc for
1H NMR (300 MHz, CDCl3):
d 2.58 (t, 2H), 3.00e3.15 (m, 2H), 5.01 (s,
2H), 5.47 (q, 1H, J ¼ 8.1 Hz), 6.69e6.85 (m, 4H), 7.07e7.19 (m, 2H),
7.26e7.46 (m, 8H), 7.56 (d, 1H, J ¼ 7.8 Hz), 7.98 (s, 1H).
C
43H35FN4O5S [M þ H]þ is 739.2385, found 739.2380. HPLC purity:
98.5%.
5.1.25. Compounds 18cen were prepared using similar procedures
as for 18b from compound 21
5.1.25.1. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
5.1.25.6. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-4-chlorobenzoic
acid (18h). Yield 63% for 2 steps from 21. M.p. 239e241 ꢁC. 1H NMR
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)benzoic
(18c). Yield 74% for 2 steps from 21. M.p. 190e192 ꢁC. 1H NMR
(300 MHz, CDCl3/CD3OD): 2.51 (t, 2H), 2.96e3.01 (m, 4H), 3.73 (s,
acid
(300 MHz, CDCl3/CD3OD):
d
2.60 (t, 2H), 3.02 (t, 2H, J ¼ 6.9 Hz), 3.13
d
(d, 2H, J ¼ 6.9 Hz), 4.94 (s, 2H), 5.43 (t, 1H, J ¼ 6.3 Hz), 6.75 (d, 2H,
J ¼ 8.1 Hz), 6.87 (s, 1H), 6.93e7.11 (m, 6H), 7.20e7.51 (m, 13H), 8.00
(d, 1H, J ¼ 8.4 Hz), 8.82 (s, 1H). 13C NMR (75 MHz, CDCl3/CD3OD):
3H), 4.92 (s, 2H), 5.59 (t, 1H), 6.70e6.77 (m, 3H), 6.98e7.49 (m,
17H), 8.14 (d, 1H, J ¼ 7.2 Hz), 8.67 (d, 1H, J ¼ 8.1 Hz); 13C NMR
(75 MHz, CDCl3/CD3OD):
d
21.1, 36.7, 38.8, 51.6, 70.0, 111.3, 113.6,
d 21.3, 29.7, 37.0, 39.8, 52.1, 70.1, 111.3, 114.1, 114.9, 118.5, 119.0, 120.0,
114.9, 118.3, 118.8, 120.0, 121.6, 122.1, 122.7, 126.9, 127.6, 128.0, 128.1,
128.6, 129.1, 130.2, 130.2, 130.3, 131.7, 132.6, 135.5, 135.5, 136.2,
136.8, 140.2, 142.1, 144.4, 157.6, 160.4, 167.4, 174.2. HRMS (ESI) m/z
calc for C43H36N4O5S [M þ H]þ is 721.2479, found 721.2480.
121.7, 122.0,122.9,127.0,127.6,128.1, 128.2, 128.6, 128.8,129.4, 129.9,
130.2, 130.5, 131.4, 132.8, 136.4, 136.9, 141.2, 141.9, 145.4, 157.7, 160.4,
167.2, 173.6. HRMS (ESI) m/z calc for C43H35ClN4O5S [M þ Na]þ is
777.1909, found 777.1907.
5.1.25.2. (S)-4-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
5.1.25.7. (S)-2-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-4-bromobenzoic
acid (18i). Yield 63% for 2 steps from 21. M.p. 110e113 ꢁC. 1H NMR
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)benzoic
(18d). Yield 77% for 2 steps from 21. M.p. 147e149 ꢁC. 1H NMR
(300 MHz, CDCl3/CD3OD): 2.63 (t, 2H), 3.09e3.14 (m, 4H), 4.99 (s,
acid
d
(300 MHz, CDCl3):
d
2.53 (t, 2H), 2.98 (t, 2H, J ¼ 7.2 Hz), 3.08 (d, 2H,
2H), 5.53 (q, 1H, J ¼ 7.5 Hz), 6.11 (d, 1H, J ¼ 8.4 Hz), 6.77e6.87 (m,
5H), 7.07e7.17 (m, 2H), 7.25 (d, 1H, J ¼ 7.8 Hz), 7.31e7.59 (m, 10H),
7.69 (d, 2H, J ¼ 8.7 Hz), 8.03 (d, 2H, J ¼ 8.7 Hz), 9.34 (s, 1H); 13C NMR
J ¼ 7.2 Hz), 4.90 (s, 2H), 5.48 (t,1H, J ¼ 6.9 Hz), 6.72 (d, 2H, J ¼ 8.1 Hz),
6.79 (s, 1H), 6.91e7.11 (m, 4H), 7.19 (d, 1H, J ¼ 7.8 Hz), 7.23e7.47 (m,
12H), 8.19 (s, 1H), 8.60 (d, 1H, J ¼ 9.0 Hz). 13C NMR (75 MHz, CDCl3):
(75 MHz, CDCl3/CD3OD):
d
21.3, 37.2, 39.9, 52.2, 70.0, 107.8, 111.4,
d 21.3, 36.9, 38.6, 51.8, 70.0, 111.3, 113.9, 114.9, 115.2, 118.4, 119.0,
113.8, 114.9, 118.4, 118.9, 119.0, 121.7, 122.1, 125.8, 127.0, 127.5, 128.0,
128.2, 128.5, 129.3, 129.8, 130.1, 130.3, 131.0, 136.4, 136.8, 140.7,
142.0, 145.1, 157.8, 159.7, 168.5, 169.0, 173.6. HRMS (ESI) m/z calc for
121.8, 122.0,127.0,127.6, 128.1, 128.2,128.5,128.6,129.3,130.0,130.3,
130.5, 134.4, 136.0, 136.3, 136.9, 139.8, 141.6, 145.0, 157.7, 160.4, 167.3,
173.9. HRMS (ESI) m/z calc for C43H35BrN4O5S [M þ Na]þ is 821.1404,
found 821.1394.
C
43H36N4O5S [M þ H]þ is 721.2479, found 721.2473.

Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
411
5.1.25.8. (S)-5-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-2-fluorobenzoic
acid (18j). Yield 65% for 2 steps from 21. M.p. 123e125 ꢁC. 1H NMR
133.0,136.4,136.8,140.4,143.4,143.6,145.5,157.7, 159.8,161.2,164.6,
166.2, 169.1, 173.7, 173.8. HRMS (ESI) m/z calc for C43H35FN4O5S
[M þ Na]þ is 761.2204, found 761.2204.
(300 MHz, CDCl3/CD3OD):
d
2.58 (t, 2H, J ¼ 6.9 Hz), 3.04e3.12 (m,
4H), 4.95 (s, 2H), 5.44 (t, 1H, J ¼ 6.3 Hz), 6.76 (d, 2H, J ¼ 8.7 Hz), 6.84
(d, 2H), 6.99e7.10 (m, 2H), 7.21 (d, 1H, J ¼ 7.8 Hz), 7.25e7.54 (m,
12H), 7.93e7.96 (m, 2H), 9.38 (s, 1H); 13C NMR (75 MHz, CDCl3/
5.2. Biological assays
5.2.1. Enzyme-based assay of PTP1B
CD3OD):
d
21.4, 29.8, 31.0, 37.1, 40.2, 52.1, 70.2,111.5,114.1,115.1,117.7,
A colorimetric high throughput assay to measure inhibition
against PTP1B was performed in 96-well plates. Briefly, the tested
compounds were solubilized in DMSO and serially diluted into
concentrations for the inhibitory test. The assays were carried out
118.6, 119.2, 122.0, 123.5, 126.4, 127.6, 128.7, 129.4, 130.2, 131.6, 132.3,
133.9, 137.0, 137.8, 140.8, 145.0, 157.0, 158.0, 160.0, 166.1, 168.8, 173.3.
HRMS (ESI) m/z calc for C43H35FN4O5S [M þ H]þ is 739.2385, found
739.2384.
in a final volume of 100 mL containing 50 mmol/L MOPS, pH 6.5,
2 mmol/L pNPP, 30 nmol/L GST-PTP1B, and 2% DMSO, and the
catalysis of pNPP was continuously monitored on a SpectraMax 340
microplate reader at 405 nm for 2 min at 30 ꢁC. The IC50 value was
calculated from the nonlinear curve fitting of the percent inhibition
[inhibition (%)] vs the inhibitor concentration [I] using the
following equation: % inhibition ¼ 100/{1 þ (IC50/[I]) k}, where k is
the Hill coefficient.
5.1.25.9. (S)-5-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-2-chlorobenzoic
acid (18k). Yield 54% for 2 steps from 21. M.p. 217e219 ꢁC. 1H
NMR (300 MHz, CDCl3/CD3OD):
d
2.58 (t, 2H, J ¼ 6.9 Hz), 3.02e
3.11 (m, 4H), 4.94 (s, 2H), 6.77 (d, 2H, J ¼ 8.7 Hz), 6.84e6.87 (m,
3H), 6.94e7.07 (m, 2H), 7.20 (d, 1H, J ¼ 7.5 Hz), 7.29e7.53 (m,
12H), 7.78 (d, 1H, J ¼ 8.7 Hz), 7.98 (s, 1H), 9.43 (s, 1H); 13C NMR
(75 MHz, CDCl3/CD3OD):
d
21.4, 29.7, 37.0, 40.1, 52.2, 70.1, 111.5,
5.2.2. Materials
114.0, 115.0, 118.5, 119.1, 121.9, 122.1, 122.3, 122.7, 122.8, 123.9,
127.1, 127.6, 128.1, 128.2, 128.4, 128.5, 128.6, 129.4, 129.8, 130.2,
130.4, 130.6, 131.6, 136.5, 136.6, 136.9, 140.7, 157.8, 159.7, 167.5,
173.5. HRMS (ESI) m/z calc for C43H35ClN4O5S [M þ Na]þ is
777.1909, found 777.1905.
Insulin and wortmannin were obtained from Sigma Aldrich (St.
Louis, MO). Radiochemical 2-deoxy-[3H]-deoxy-glucose, Western
blotting detection kits (enhanced chemiluminescence) and
Hyperfilm were purchased from Amersham Biosciences (Uppsala,
Sweden).
5.1.25.10. (S)-3-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-4-fluorobenzoic
acid (18l). Yield 58% for 2 steps from 21. M.p. 138e140 ꢁC. 1H NMR
5.2.3. Cell culture
CHO-hIR cells were cultured in Ham’s F-12 medium (Invitrogen,
Carlsbad, CA) supplemented with 10% (v/v) FBS, 100 units/mL
(300 MHz, CDCl3):
d
2.62 (t, 2H, J ¼ 7.2 Hz), 3.08e3.19 (m, 4H), 5.00
penicillin and 100 m
g/mL streptomycin at 37 ꢁC in 5% CO2. L6
(s, 2H), 5.53 (q, 1H, J ¼ 8.4 Hz), 5.96 (d, 1H, J ¼ 8.4 Hz), 6.79 (d, 2H,
J ¼ 9.0 Hz), 6.85 (d, 2H, J ¼ 9.0 Hz), 6.89 (s, 1H), 7.09e7.21 (m, 2H),
7.31e7.42 (m, 9H), 7.53e7.59 (m, 2H), 7.83 (m, 1H), 7.94 (s, 1H), 9.10
myoblasts were cultured in Dulbecco’s modified Eagle medium
(DMEM) (Invitrogen, Carlsbad, CA) containing 10% (v/v) FBS, 4.5 g/L
glucose, 100 units/mL of penicillin and 100 mg/mL streptomycin at
(d,1H), 9.52 (s,1H); 13C NMR (75 MHz, CDCl3):
d
21.3, 22.5, 23.6, 29.7,
37 ꢁC in 5% CO2. For L6 myoblast differentiation, the concentration
of FBS was decreased to 2%. Myotubes were used for experiments
5e7 days after differentiation.
33.9, 37.0, 39.5, 48.2, 48.5, 48.8, 49.0, 49.3, 49.6, 52.0, 70.1, 89.1, 99.2,
111.4, 113.8, 115.0, 118.4, 119.0, 121.7, 122.1, 123.7, 127.0, 127.5, 128.0,
128.2, 128.6, 129.4, 130.2, 130.4, 130.7, 137.0, 147.1, 148.5, 157.8, 160.3,
161.4, 167.9, 173.8, 185.7. HRMS (ESI) m/z calc for C43H35FN4O5S
[M þ H]þ is 739.2385, found 739.2391.
5.2.4. Measurement of 2-deoxy-[3H]-deoxyglucose uptake in L6
myotubes
2-Deoxyglucose uptake was measured as described previously
[45]. Briefly, after treatment with 18g and subsequent stimulation
with 10 nM insulin, cells were washed three times with HEPES-
buffered saline solution (20 mM HEPES, 136 mM NaCl, 4.7 mM
KCl, 1.25 mM mgSO4, 1.2 mM CaCl2, pH 7.4) followed by incubation
with 2-deoxy-[3H]-deoxy-glucose for 15 min. Then cells were
washed three times with ice-cold PBS and lysed using 0.1% Triton-X
100 and radioactivity counted.
5.1.25.11. (S)-4-(2-(1-(3-(1H-indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-3-fluorobenzoic
acid (18m). Yield 67% for 2 steps from 21. M.p. 212e214 ꢁC. 1H NMR
(300 MHz, THF-d8):
d
2.53 (t, 2H, J ¼ 7.2 Hz), 3.04 (t, 2H, J ¼ 7.5 Hz),
3.15 (dd, 1H, J ¼ 8.4, 14.1 Hz), 3.39 (dd, 1H, J ¼ 6.3, 14.1 Hz), 5.00 (s,
2H), 5.55 (t, 1H, J ¼ 6.3 Hz), 6.87 (d, 2H, J ¼ 8.4 Hz), 6.93e7.05 (m,
3H), 7.14 (d, 2H, J ¼ 8.7 Hz), 7.22e7.40 (m, 8H), 7.54 (d, 1H,
J ¼ 7.8 Hz), 7.61 (m, 1H), 7.75e7.84 (m, 2H), 8.59 (t, 1H, J ¼ 8.4 Hz),
9.88 (d, 1H); 13C NMR (75 MHz, THF-d8):
d
22.2, 37.7, 39.9, 53.2,
5.2.5. Immunoblotting
53.3, 70.6, 112.0, 115.6, 116.6, 116.9, 119.3, 119.3, 120.7, 120.8, 122.0,
122.9, 123.1, 127.4, 128.3, 128.5, 128.8, 129.2, 129.9, 130.6, 130.6,
130.7, 131.2, 138.8, 141.5, 146.3, 159.0, 160.0, 166.5, 171.8, 172.7, 172.8.
HRMS (ESI) m/z calc for C43H35FN4O5S [M þ Na]þ is 761.2204,
found 761.2199.
CHO-hIR cells or differentiated L6 myotubes were starved in
serum-free medium for 2 h. Then the cells were treated with PTP1B
inhibitors for 2 h, followed by stimulation with or without 10 nM
insulin for 10 min. Then cells were washed three times with ice-
cold PBS and lysed with lysis buffer (50 mM TriseHCl, pH 8.0,
150 mM NaCl, 1% NP-40, 1 mM Na3VO4, 1 mM PMSF, 1 mM DTT,
1 mM EDTA, 1 mM EGTA) containing complete protease inhibitors
(Roche). Cell lysates were subjected to electrophoresis by 8% SDS-
PAGE, electrotransferred to nitrocellulose membranes and blotted
with anti-pTyr antibody from anta Cruz Biotechnology or anti-
phospho-Akt (Ser473) antibody purchased from Cell Signaling
5.1.25.12. (S)-4-(2-(1-(3-(1H-Indol-3-yl)propanamido)-2-(4-(benzy-
loxy)phenyl)ethyl)-5-phenylthiazole-4-carboxamido)-2-fluorobenzoic
acid (18n). Yield 79% for 2 steps from 21. M.p. 118e120 ꢁC. 1H NMR
(300 MHz, CDCl3/CD3OD):
d
2.63 (t, 2H, J ¼ 6.9 Hz), 3.02e3.13 (m,
4H), 5.00 (s, 2H), 5.48 (t,1H, J ¼ 6.9 Hz), 6.76e6.90 (m, 5H), 7.06e7.17
(m, 2H), 7.26e7.59 (m, 12H), 7.73 (d, 1H, J ¼ 13.2 Hz), 7.92 (t, 1H,
Technology (Beverly, MA) and anti-b-actin antibody from Upstate
J ¼ 8.4 Hz), 9.55 (s, 1H). 13C NMR (75 MHz, CDCl3/CD3OD):
d
21.3,
(Billerica, MA). The immunoblots were visualized by chem-
iluminescence using the enhanced chemiluminescence Western
Blotting System.
36.9, 40.0, 52.3, 70.0,107.4,107.7,111.3,113.7,113.9,114.6,114.9,118.3,
118.8, 121.6, 122.1, 122.2, 127.0, 128.0, 128.5, 129.4, 129.7, 130.0, 130.3,

412
Y.-T. Chen et al. / European Journal of Medicinal Chemistry 69 (2013) 399e412
[20] L.D. Klaman, Increased energy expenditure, decreased adiposity, and tissue-
Acknowledgments
specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient
mice, Mol. Cell. Biol. 20 (2000) 5479e5489.
This work was supported by The National Natural Science
Foundation of China (Grants 81125023, 30725049, 30901850), the
National Basic Research Program of China (Grant 2012CB524906),
and the National Science and Technology Major Projects for Major
New Drugs Innovation and Development (Grant 2013ZX09507002).
[21] B.A. Zinker, Reduction of protein tyrosine phosphatase 1B normalizes glucose
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