Novel thiophene derivatives as PTP1B inhibitors with selectivity and cellular activity

Article abstract of DOI:10.1016/j.bmc.2010.01.055

A series of novel thiophene derivatives was designed, synthesized and their activities as competitive inhibitors of protein tyrosine phosphatase (PTPs) 1B (PTP1B) inhibitors were evaluated. All the compounds showed inhibitory potencies, and 10 of these exhibited moderate inhibitory activities with IC₅₀ values less than 10 μM. The activity of the most potent compound P28 (IC₅₀ = 2.1 μM) was 15 times higher than that of the hit compound P01. Further, four representative compounds (P19, P22, P28, and P31) demonstrated remarkably high selectivities against other PTPs (e.g., PTPα, LAR, CD45, and TCPTP); P19 exhibited greater than sixfold selectivity over highly homologous TCPTP. More importantly, these compounds are permeable to cell membranes. The treatment of CHO-K1 cells with P28 (10 μM) resulted in increased phosphorylation of AKT, which suggested extensive cellular activity of this compound. The novel chemical entities reported in this study could be used for overcoming the poor selectivity and low cellular activity of PTP1B inhibitors and might represent a starting point for development of therapeutic PTP inhibitors.

Full text of DOI:10.1016/j.bmc.2010.01.055

Bioorganic & Medicinal Chemistry 18 (2010) 1773–1782
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel thiophene derivatives as PTP1B inhibitors with selectivity and
cellular activity
Deju Ye a, , Yu Zhang a, , Fei Wang a, , Mingfang Zheng a, Xu Zhang a,b, Xiaomin Luo a, Xu Shen a,
,
*
Hualiang Jiang a,c, Hong Liu a,b,
*
a Drug Discovery and Design Centre, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, PR China
b Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong jia Xiang, Nanjing 210009, PR China
c School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel thiophene derivatives was designed, synthesized and their activities as competitive
inhibitors of protein tyrosine phosphatase (PTPs) 1B (PTP1B) inhibitors were evaluated. All the com-
pounds showed inhibitory potencies, and 10 of these exhibited moderate inhibitory activities with IC50
Received 8 December 2009
Accepted 22 January 2010
Available online 28 January 2010
values less than 10
higher than that of the hit compound P01. Further, four representative compounds ( P19, P22, P28,
and P31) demonstrated remarkably high selectivities against other PTPs (e.g., PTP , LAR, CD45, and
TCPTP); P19 exhibited greater than sixfold selectivity over highly homologous TCPTP. More importantly,
these compounds are permeable to cell membranes. The treatment of CHO-K1 cells with P28 (10 M)
lM. The activity of the most potent compound P28 (IC50 = 2.1 lM) was 15 times
Keywords:
a
Protein tyrosine phosphatase 1B (PTP1B)
Inhibitors
Thiophene
l
resulted in increased phosphorylation of AKT, which suggested extensive cellular activity of this com-
pound. The novel chemical entities reported in this study could be used for overcoming the poor selec-
tivity and low cellular activity of PTP1B inhibitors and might represent a starting point for development
of therapeutic PTP inhibitors.
Structure–activity relationships (SAR)
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
et al.5a used a focused library approach to identify a non-hydrolyz-
able bisdifluorophosphonate (compound 1, Fig. 1) which is the
Protein tyrosine phosphatase 1B (PTP1B), an intracellular pro-
tein tyrosine phosphatase (PTPs), has been implicated in the nega-
tive regulation of insulin and leptin signal transduction pathways
among other biological functions.1 Mice lacking functional PTP1B
exhibit increased insulin sensitivity and improved glycemic con-
trol, and are resistant to diet-induced obesity.2 Treatment of dia-
betic mice with PTP1B antisense oligonucleotides resulted in
reduced PTP1B expression, and subsequent decreases in the adi-
pose tissue mass, plasma insulin, and blood glucose levels.3 It has
been suggested that PTP1B inhibitors may be used for designing
drugs for the treatment of type II diabetes and obesity. Moreover,
since PTP1B has been reported to function as an oncogene in breast
cancer, PTP1B-specific inhibitors can be used for effective treat-
ment of breast cancer.4
most potent compound to date; Liu et al.5b employed a linked-frag-
ment approach to identify compound 2, with a less polar phospho-
tyrosine (pTyr) mimetic and a more rigid linker, which displayed a
30-fold selectivity compared with TCPTP; Wiesmann et al.5c were
the first to discover a series of allosteric inhibitors (compound 3
as the representative); these inhibitors might be used for
developing selective inhibitors with acceptable pharmacological
properties. Most of the reported compounds containing nega-
tively-charged non-hydrolyzable pTyr mimetics, have exhibited
excellent potency (at nanomolar concentrations) in vitro studies;
however, the low cell permeability and low bioavailability of these
compounds have limited their application for the development of
effective drugs.6 Thus, new potential drug candidates with im-
proved physicochemical properties and bioavailability need to be
developed.7 Very recently, a selective noncompetitive PTP1B inhib-
itor, namely, trodusquemine,8 developed by Genaera company, has
proceeded to the phase I clinical trials. The results of the preclinical
and early clinical trials were promising, and trodusquemine has
been shown to possess both appetite suppressing effects and a
hypoglycemic and hypocholesterolemic properties.
Given the compelling evidence that PTP1B is associated with
numerous human diseases, a large number of PTP1B inhibitors
have been developed over the last decade with the aim of develop-
ing potent and selective compounds as drug candidates.5 Shen
* Corresponding authors. Tel.: +86 21 50807042; fax: +86 21 50807088.
E-mail address: hliu@mail.shcnc.ac.cn (H. Liu).
These authors equally contributed to this work.
Compounds with a thiophene core have been widely studied
due to their ability to bind to many receptors with high affinity.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.055

1774
D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
O
F
F
OH
OH
HO
P
N
O
O
O
H
N
NH2
F
O
O
O
P
N
H
O
HO
HO
O
O
O
OH
F
F
HO
2 Ki = 6.9 µM (PTP1B)
Ki = 164 µM (TCPTP)
1 Ki = 2.4 nM (PTP1B)
Br
Ki = 24 nM (TCPTP)
OH
Br
O
O
H
N
O
2
3
4
H
N
Et
O
HOOC
S
S
O
S
5
O
O
N
P01 (IC50 = 30 µM)
3 IC50 = 8.0 µM (PTP1B)
allosteric inhibitor
Figure 1. Structures of reported PTP1B inhibitors and the screening hit P01.
Recently, Wyeth pharmaceuticals have performed structure-based
optimization and identified a series of monocyclic, bicyclic, and tri-
cyclic thiophenes that can be used as highly potent PTP1B inhibi-
tors.9,6b In the present study, we performed the in vitro screening
of abundant substituted thiophene compounds from our in-house
collection, and identified P01 as a competitive PTP1B inhibitor,
5 were prepared from simple ketones by microwave-assisted Ge-
wald’s reaction in a solvent-free medium, which was optimized
previously.10 Direct reaction of 5 with benzoyl chloride afforded
compound P07. We performed overnight refluxing of 5 with a vari-
ety of acid anhydrides in dry CH2Cl2 under Ar and obtained com-
pound 6 (P01–P06, P09–P22, and P32) in moderate to high
yields. Compound P21 was esterified with MeOH to yield P08. Fi-
nally, P23–P28 with substituted biphenyl-4-yl moieties and com-
pounds P29–P31 were obtained in excellent yields using the
microwave-assisted Suzuki coupling reaction of halogenated ben-
zene derivatives 7 with boronic acid derivatives in H2O.11
with an IC50 value of 30 lM (Fig. S1, see in ESI). Molecular model-
ing suggested that P01 simultaneously binds to the active site and
the second pTyr binding site of PTP1B (Fig. S2). Further optimiza-
tion of P01 by extension of the molecule from the enzyme active
site into the second pTyr binding site was explored to identifica-
tion of novel moderately potent, selective, and cellular active
PTP1B inhibitors.
3. Results and discussion
2. Chemistry
3.1. PTP1B inhibitory activities and structure–activity
relationship
2.1. Design of substituted thiophene derivatives
For the primary assay, we measured the percentage inhibitory
rates of all the synthetic compounds at concentrations of 100
Based on the structure of thiophene derivative P01 and mole-
cule docking model of P01 with PTP1B, 31 new thiophene-based
analogues (P02–P32) (Table 1) were designed and synthesized
for the first round. Replacing the cyclic acid moiety in P01 with
other cyclic acids or acyclic acids, we obtained analogues P02–
P06. Removal or esterification of the cyclic acid led to compounds
P07 and P08. Keeping the 2-naphthalenyl ring, we changed the i-
propyl ester for different alkyl ester, and obtained compounds
P09–P12. Replacement of the 2-naphthalenyl ring with the phenyl
ring and substituted phenyl ring led to five compounds (P13–P17).
By cycling the R3 and R4 group in thiophene ring with –(CH2)4–
group, we got P18. Compounds P19–P21 were obtained by substi-
tuting the 2-naphthalenyl with biphenyl-4-yl group. On the basis
of the structure features of compounds P19 and P21, we designed
analogues P22–P28 via introduction of various electronic, hydro-
phobic groups to the ortho-, meta-, or para-position of the terminal
phenyl ring of biphenyl-4-yl group. Displacing the biphenyl-4-yl
group of P21 to biphenyl-3-yl or biphenyl-2-yl group, we afforded
compounds P29 and P30. The introduction of a methyl group to 5-
position in thiophene ring of P21 led to compound P31. At last,
moving the phenyl group from R3 to R4 in compound P13, we ob-
tained analogue P32.
and 10
lM. Compounds with good inhibition rates (>50% at
10 M) were selected for further determination of IC50 values
l
(Table 1). The binding mode of P01 to PTP1B revealed the orienta-
tion of the cyclic acid moiety, which is located deep inside the
active site (Fig. S2). A slightly improving potency was observed
when the bicycle[2.2.1]heptane-2-carboxylic acid moiety in P01
was replaced with other cyclic acid moieties, such as bicy-
clo[2.2.1]hept-5-ene-2-carboxylic acid (P02), 1,2-cyclohexanedi-
carboxylic acid (P03), or 1,2-benzenedicarboxylic acid (P04). In
contrast, the potency was remarkably decreased if acyclic acid
moieties were used (P05 vs P01–P04 and P06 vs P19–P21). As ex-
pected, the removal or esterification of the carboxylic acid in com-
pound P21 resulted in a loss of activity (e.g., P07 and P08). This
indicated that cyclic acid moiety favors the maintenance of the
inhibitory activity. A comparison of the potencies of compounds
P03, P09, and P10 suggested that a smaller alkyl ester group
(Me, Et, and i-Pr) in the 3-thiophenencarboxylic acid moiety did
not change the potency against the PTP1B enzyme to remarkable
extent, whereas, the incorporation of a bulky benzyl ester in the
3-thiophenencarboxylic acid moiety, as in the case of compound
P11, decreased the activity of the compound.
Furthermore, the 2-naphthalenyl ring of P01 can pass through
the ‘gateway’ between the active site and the second pTyr binding
site of PTP1B, and extend to the second binding site as shown in
Figure S2. The comparison of the compounds P13–P21 suggested
that the inhibitory activity was affected greatly by the substituents
2.2. Synthesis of substituted thiophene derivatives
Scheme 1 describes the straightforward synthesis of the ana-
logues P01–P32. The substituted 2-aminothiophene intermediates

D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
1775
Table 1
Structures and inhibition of PTP1B of thiophene derivatives P01–P32
R2
O
O
H
R1
N
(C)
(G)
(A)
(E)
(B) HOOC
(F)
(D)
(H)
O
COOH
HOOC
HOOC
R1 =
R3
S
R4
HOOC
HOOC
P01-P32
COOMe
a
Compd
R1
R2
R3
R4
% Inhibition at 100
lM
% Inhibition at 10
lM
IC50 (lM)
P01
P02
P03
P04
P05
P06
P07
P08
P09
P10
P11
P12
P13
P14
P15
P16
P17
P18
P19
P20
P21
P22
P23
P24
P25
P26
P27
P28
P29
P30
P31
P32
3c
A
B
C
D
E
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Et
Me
Bn
Et
Et
i-Pr
Et
2-Naphthenyl
2-Naphthenyl
2-Naphthenyl
2-Naphthenyl
2-Naphthenyl
Biphenyl-4-yl
Biphenyl-4-yl
Biphenyl-4-yl
2-Naphthenyl
2-Naphthenyl
2-Naphthenyl
2-Naphthenyl
Phenyl
4-NO2-phenyl
4-F-phenyl
Phenyl
4-Br-phenyl
–(CH2)4–
Biphenyl-4-yl
Biphenyl-4-yl
Biphenyl-4-yl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
67.0
98.7
100
3.5
6.8
16.0
23.3
—
16.7
—
—
20.4
15.4
—
2.4
—
—
—
—
3.4
—
63.2
23.4
91.8
62.0
61.3
95.6
92.3
61.6
24.9
95.3
84.7
2.0
30.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
b
100
21.5
98.4
18.7
19.2
96.4
92.6
72.1
95.9
10.7
45.0
30.0
26.6
97.3
27.5
90.6
84.4
99.5
90.5
100.0
99.4
99.2
100.0
86.3
99.3
98.6
96.6
100.0
15.5
99.4
F
G
H
C
C
C
B
B
C
B
D
D
B
B
C
D
B
D
D
D
D
D
D
D
D
D
B
i-Pr
Et
—
—
6.7
—
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Et
Et
Et
Et
Et
Et
i-Pr
Et
Et
i-Pr
H
H
H
H
H
H
H
H
H
H
H
H
Me
Ph
5.4
6.8
8.5
4.8
4.6
5.0
—
2.1
8.7
—
40-Cl-biphenyl-4-yl
20-MeO-biphenyl-4-yl
30-MeO-biphenyl-4-yl
40-MeO-biphenyl-4-yl
40-F-biphenyl-4-yl
40-CF3-biphenyl-4-yl
30-Cl-40-F-biphenyl-4-yl
Biphenyl-3-yl
Biphenyl-2-yl
Biphenyl-4-yl
H
95.3
—
79.5
3.2
—
3.0
a
b
c
Values are means of three determinations and deviation from the mean is <10% of the mean value.
‘—’: The IC50 value or inhibitory rate at 10 lM was not determined.
3: Positive control.
on 4-position of the thiophene template. The introduction of rings
smaller than the 2-naphthalenyl ring, such as simple phenyl ring
(e.g., P13 and P16) or substituted phenyl ring (e.g., P14, P15, and
P17), resulted in decrease or loss of potency. Conversely, when a
biphenyl-4-yl group was introduced, a remarkable enhancement
in activity was achieved (P21 vs P04). The increase in potency
maybe ascribed to the length of the biphenyl-4-yl group that ex-
tends toward Met258 and forms a stronger van der Waals interac-
tion with its side chain.
In order to optimize van der Waals interaction between the
compound and the Met258 side chain of PTP1B, various electronic
and hydrophobic groups were introduced at the ortho-, meta- or
para-positions of the unsubstituted phenyl ring of biphenyl-4-yl
group to afford compounds P22–P28. It was observed that com-
pounds with meta- or para-methoxyl substituents were more po-
tent than those with ortho-methoxyl substituents (P23–P25). The
introduction of a chloro- or fluoro-group at the para-position
(P22 and P26) did not translate into better inhibitory activity,
whereas simultaneous introduction of the chloro- and fluoro-
groups at the meta- and para-positions, respectively, afforded the
is ꢀ15-fold more potent than the initial hit compound P01. Con-
versely, compound P27 containing the electron-withdrawing
group of para-CF3 exhibited a remarkably reduced in enzyme po-
tency. While the compound P29 with a biphenyl-3-yl group was
approximately equipotent to the biphenyl-4-yl group substituted
compound, compound P30 with a biphenyl-2-yl substituent was
less active. Finally, we also found that a small substituent like
Me on the 5-position of the thiophene scaffold is beneficial for
improving potency (P31). The structure–activity relationships
(SAR) are summarized in Figure 2.
3.2. Selectivity against other phosphatases
In addition to potency improvements, we investigated the
selectivity of four representative compounds, namely, P19, P22,
P28, and P31 against other PTPs (TCPTP, CD45, PTP
As shown in Figure 3, all the four compounds demonstrated excel-
lent selectivity against PTP and LAR (>10-fold) and moderate
a, and LAR).
a
selectivity against CD45 (ꢀ3–5-fold). The differences in the selec-
tivities of PTP1B and CD45 may be partially attributed to the differ-
ences between the active site and the second phosphotyrosine
most potent compound P28 with an IC50 value of 2.1 lM, which

1776
D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
R2
O
O
H
N
O
R3
b
S
S
R2
O
O
R4
O
H2N
P07
O
COOMe
a
R4
R2
O
R2
O
O
R3
S
R3
H
N
H
R1
N
d
R4
5
c
O
O
4
R3
R3
S
R4
R4
6 (P01-06, P09-22, P32)
P08
COOH
COOH
R2
O
O
R2
O
O
H
N
H
N
e
O
O
S
S
R3
P23-31
Br
R4
R4
7
Scheme 1. Synthesis of compounds P01–P32. Reagents and conditions: (a) CNCH2COOR2, S, CH3COONH4, morpholine, basic Al2O3, MW, 320 W, 10 min; (b) PhCOCl, CH2Cl2,
TEA, rt; (c) acid anhydride, CH2Cl2, Ar, reflux; (d) MeOH, H2SO4, reflux; (e) ArB(OH)2, Na2CO3, TBAB, Pd(PPh3)4, H2O, MW, 150 °C, 10 min.
Me, Et, i-Pr
acid moieties required
> BnO
R2
O
O
H
N
R1
HOOC
HOOC
>
or
or
>
R'
O
>
R3
S
COOH
3' or 4'-position substitution is
HOOC
R4
better than 2'-position substitution;
>
HOOC
or
>
HOOC
H, Me > Ph
or
R''
Figure 2. Structure–activity relationships.
binding site at the gateway of these two enzymes.13 PTP1B has a
glycine (Gly259) lining the bottom of the gateway, while CD45
has a larger amino acid, leucine. Among these four compounds,
P19 showed greater than sixfold selectivity against TCPTP, the
most homologous PTPs, which is comparable with that of allosteric
inhibitor 3. Further, the selectivity of P28 and P31 (obtained by
replacing the bicycle[2.2.1]hept-5-ene-2-carboxylic acid moiety
in P19 with 1,2-benzenedicarboxylic acid) against TCPTP was low-
er (ꢀ2-fold) than that of P19; the introduction of a 40-Cl (P22) at
the biphenyl-4-yl group of P19 to function at the second pTyr bind-
ing site did not translate into better selectivity against TCPTP.
Selectivity against PTPs (IC50
)
>100
>100
>100
>100
>100
>100
>100
100
80
60
40
20
0
PTP1B
TCPTP
CD45
PTPa
LAR
66.7
49.7
48.6
43.0
33.0
32.6
23.1
6.7
21.8
9.7
7.0
3.2
6.6
6.8
6.7
3.3. Cellular activity of selected compound
5.4
2.1
3.0
P19
P22
P28
P31
3
Phosphatidylinositol 3-kinase/AKT (PI3 K/AKT) signaling is
known to be the major pathway affecting insulin-stimulated
Figure 3. The IC50 values of compounds P19, P22, P28, and P31 against PTPs.

D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
1777
glucose metabolism. PTP1B inhibitors have affected the activation
of AKT in CHO-K1 cells.7b,14 Hence, we next explored the possible
regulatory role of our most potent compound P28 in AKT activa-
tion. As shown in Figure 4, the treatment of the CHO-K1 cells with
compound P28 (10 lM) resulted in an expected increase in the
phosphorylation of AKT, which is consistent with the result of
the known PTP1B inhibitor 3. These results suggest that P28 pos-
sesses good membrane permeability, and could possibly induce
insulin signaling at the cellular level.
3.4. Binding models
To obtain structural information for further structural optimiza-
tion studies, we constructed the 3D binding models of compound
P28 to PTP1B in open conformation based on the docking simula-
tion, as shown in Figure 5. Similar to the binding model of P01, the
model of P28 revealed a dense network of hydrogen bonds be-
tween the carboxylic acid group of P28 and Ala217, Cys215,
Ser216, and Arg221 at the active site of PTP1B was observed as well
as hydrophobic interactions between the phenyl ring of the ligand
and the surrounding amino acid side chains (e.g., Asn111,
Lys120).15 Tyr46 was anticipated to establish aromatic stacking
interactions with the thiophene scaffold which are commonly ob-
served in many known PTP1B/inhibitor complexes.16,5b,6b,7a An
additional interaction through hydrogen bond between Tyr46
and the amide oxygen of compound P28 was possible. Further-
more, P28 fitted deeply into the second pTyr binding site and
established favorable hydrophobic interactions with the lipophilic
amino acid side chains (Met258, Gly259, and Phe52) of PTP1B.
Because of these multiple interactions, P28 positioned itself
Figure 5. Binding mode of P28 (B) to PTP1B derived from docking simulations. The
inhibitor P28 and key residues of PTP1B are shown in a capped stick representation.
Carbon is in green for ligands and gray for PTP1B, oxygen is in red, nitrogen is in
blue, sulfur is in yellow. All structure figures were prepared using PyMol (http://
pymol.sourceforge.net/).
5. Experimental section
The reagents (chemicals) were purchased from Lancaster, Acros
and Shanghai Chemical Reagent Company, and used without fur-
ther purification. Analytical thin-layer chromatography (TLC) was
performed on HSGF 254 (150–200 lm thickness, Yantai Huiyou
Company, China). Yields were not optimized. Melting points were
measured in capillary tube on a SGW X-4 melting point apparatus
without correction. Microwave experiments under solvent-free
conditions were carried out in a domestic microwave oven (Galanz
WG800DSL20II-K6). Microwave assisted Suzuki coupling reactions
were performed in an InitiatorTM EXP microwave system (Biotage,
Inc.) at the specified temperature using the standard mode of oper-
ation (the heating process: temperature: 0–250 °C; temperature
increase: 2–5 °C/s; pressure range: 0–20 bar; power range: 0–
400 W at 2.45 GHz; reaction vials (mL): 0.2–0.5 (EXP), 0.5–2, 2–5,
10–20 (EXP); reaction volumes: 0.2–20 mL). Column chromatogra-
phy was performed with CombiFlashÒ Companion system (Tele-
dyne Isco, Inc.). Nuclear magnetic resonance (NMR) spectra were
performed on a Brucker AMX-400 and AMX-300 NMR (IS as
TMS). Chemical shifts were reported in parts per million (ppm, d)
downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), triplet (t), quartet (q), multi-
plet (m), and broad (br). Low- and high-resolution mass spectra
(LRMS and HRMS) were given with electric, electrospray and ma-
trix-assisted laser desorption ionization (EI, ESI, and MALDI) pro-
duced by Finnigan MAT-95, LCQ-DECA spectrometer and IonSpec
4.7 Tesla. An Agilent 1100 Series HPLC with an Agilent Zorbax
effectively at the enzyme active site to serve as
inhibitor.
a potent
4. Conclusion
In conclusion, a series of novel thiophene derivatives were de-
signed and synthesized and their potency as competitive PTP1B
inhibitors was evaluated. After performing systematic SAR studies
of R1–R4 substituents of the screen hit P01, we identified 10 com-
pounds with IC50 values less than 10
sentative compounds ( P19, P22, P28, and P31) demonstrated
excellent selectivity against PTP and LAR (>10-fold), and moder-
lM. Among these, four repre-
a
ate selectivity against CD45 (ꢀ3–5-fold). More importantly, P19
exhibited greater than sixfold selectivity over TCPTP. Further stud-
ies on cellular activities revealed that P28 was membrane-perme-
able and exerted extensive cellular effects on the activation of
PI3K/AKT pathway in CHO-K1 cells. The novel chemical entities re-
ported in this study could provide a possible opportunity for devel-
oping novel PTP1B inhibitors with promising selectivity and
improved pharmacological properties.
Eclipse XDB-C18 (4.6 ꢁ 50 mm, 5
lm) reversed phase column was
used for analytical HPLC analyses. Elemental analyses were per-
formed on an Elementar vario EL I analyzer.
5.1. General procedure for the synthesis of compound 6 (P01–
P06, P09–P22, and P32)
To a solution of substituted 2-amino-thiophene-3-carboxylic
derivatives 5 (0.1 mmol) in 10 mL dry CH2Cl2 was added an equi-
molar amount of the appropriate acid anhydrides. The mixture
was kept at refluxing overnight under an atmosphere of Argon.
The reaction mixture was concentrated under reduced pressure
and purified by flash column chromatography. The pure products
(P01–P06, P09–P22, and P32) were recrystallized from CH2Cl2/
petroleum ether (PE).
Figure 4. P28 significantly activated phosphorylation of AKT in CHO-K1 cells. CHO-
K1 cells were serum-starved for 2 h and then incubated with 10 lM P28 or 3 for 2 h.
Cells were lysed and immuno-blotted with indicated antibody. GAPDH was used as
loading control.

1778
D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
5.1.1. 2-[(3-Carboxy-bicyclo[2.2.1]heptane-2-carbonyl)-amino]-
4-naphthalen-2-yl-thiophene-3-carboxylic acid isopropyl ester
(P01)
251 (100%); HRMS (EI) m/z calcd for C22H21NO5S (M+) 411.1140,
found 411.1145. Anal. (C22H21NO5S) C, H, N.
The product was purified by flash column chromatography with
ethyl acetate (EA)/PE (1/3, v/v) and recrystallized from CH2Cl2/PE
(1/3.5, v/v) to give a white solid. 63% yield; mp 164–165 °C; 1H
NMR (300 MHz, CDCl3): d 0.84 (d, J = 6.3 Hz, 6H), 1.54 (m, 4H),
1.77 (m, 1H), 2.11 (m, 1H), 2.64–2.70 (m, 2H), 3.04 (d, J = 11.4 Hz,
1H), 3.28 (d, J = 11.7 Hz, 1H), 4.96 (m, 1H), 6.64 (s, 1H), 7.39 (d,
J = 8.4 Hz, 1H), 7.48 (m, 2H), 7.73–7.86 (m, 4H), 11.5 (s, 1H); LRMS
(EI) m/z 477 (M+), 251 (100%); HRMS (EI) m/z calcd for C27H27NO5S
(M+) 477.1610, found 477.1609.
5.1.6. 4-Biphenyl-4-yl-2-((Z)-3-carboxy-acryloylamino)-thio-
phene-3-carboxylic acid isopropyl ester (P06)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/3, v/v) to give
a yellow solid. 83% yield; mp 154–155 °C; 1H NMR (300 MHz,
CDCl3): d 0.99 (d, J = 6.0 Hz, 6H), 5.03 (m, 1H), 6.54 (m, 2H), 6.88
(s, 1H), 7.34 (m, 3H), 7.48 (t, J = 7.2 Hz, 2H), 7.64 (m, 4H), 12.3 (s,
1H); LRMS (EI) m/z 465 (M+), 251 (100%); HRMS (EI) m/z calcd
for C24H21NO5S (M+) 435.1140, found 435.1148. Anal.
(C24H21NO5S) C, H, N.
5.1.2. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-amino]-
4-naphthalen-2-yl-thiophene-3-carboxylic acid isopropyl ester
(P02)
5.1.7. 2-[(2-Carboxy-cyclohexanecarbonyl)-amino]-4-naph-
thalen-2-yl-thiophene-3-carboxylic acid ethyl ester (P09)
The product was purified by flash column chromatography with
EA/PE (1/2.5, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to
give a white solid. 78% yield; mp 195–197 °C; 1H NMR (300 MHz,
CDCl3): d 0.81 (t, J = 7.2 Hz, 3H), 1.53–2.23 (m, 8H), 2.93–3.11 (m,
2H), 4.04 (q, J = 7.2 Hz, 2H), 6.66 (s, 1H), 7.39–7.49 (m, 3H), 7.76–
7.83 (m, 4H), 11.6 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 13.4,
22.9, 23.4, 25.5, 26.4, 41.7, 42.7, 60.3, 111.0, 115.8, 125.8, 126.0,
126.4, 126.9, 127.4, 127.8, 127.9, 131.9, 132.5, 134.8, 138.9,
149.2, 165.0, 171.6, 174.6; LRMS (EI) m/z 451 (M+), 251 (100%);
HRMS (EI) m/z calcd for C25H25NO5S (M+) 451.1453, found
451.1445. Anal. (C25H25NO5S) C, H, N.
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/3.5, v/v) to
give
a
yellow solid. 75% yield; mp 148–150 °C; 1H NMR
(300 MHz, CDCl3): d 0.85 (d, J = 6.3 Hz, 6H), 1.43 (d, J = 8.1 Hz,
1H), 1.57 (d, J = 8.4 Hz, 1H), 2.28 (m, 2H), 3.43 (d, J = 10.2 Hz, 1H),
3.52 (d, J = 10.2 Hz, 1H), 4.97 (m, 1H), 6.34 (m, 1H), 6.47 (m, 1H),
6.63 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.48 (m, 2H), 7.73–7.86 (m,
4H), 11.5 (s, 1H); LRMS (EI) m/z 475 (M+), 251 (100%); HRMS (EI)
m/z calcd for C27H25NO5S (M+) 475.1453, found 475.1457. Anal.
(C27H25NO5Sꢂ0.2H2O) C, H, N.
5.1.3. 2-[(2-Carboxy-cyclohexanecarbonyl)-amino]-4-naphtha-
len-2-yl-thiophene-3-carboxylic acid isopropyl ester (P03)
The product was purified by flash column chromatography with
EA/PE (1/2.5, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to
give a white solid. 57% yield; mp 204–205 °C; 1H NMR (300 MHz,
CDCl3): d 0.85 (d, J = 6.3 Hz, 6H), 1.14–2.24 (m, 8H), 2.93–3.14
(m, 2H), 4.98 (m, 1H), 6.66 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.48
(m, 2H), 7.74–7.87 (m, 4H), 11.7 (s, 1H); 13C NMR (75 MHz,
DMSO-d6): d 21.1, 22.9, 23.4, 25.4, 26.5, 41.7, 42.8, 67.9, 111.4,
115.7, 125.8, 126.1, 126.4, 127.0, 127.7, 128.0, 131.9, 132.5,
135.0, 138.9, 149.1, 164.6, 171.6, 174.6; LRMS (EI) m/z 465 (M+),
251 (100%); HRMS (EI) m/z calcd for C26H27NO5S (M+) 465.1610,
found 465.1598. Anal. (C26H27NO5S) C, H, N.
5.1.8. 2-[(2-Carboxy-cyclohexanecarbonyl)-amino]-4-naph-
thalen-2-yl-thiophene-3-carboxylic acid methyl ester (P10)
The product was purified by flash column chromatography with
EA/PE (1/2.5, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to
give a white solid. 70% yield; mp 99–101 °C; 1H NMR (300 MHz,
CDCl3): d 1.53–2.29 (m, 8H), 2.92–3.14 (m, 2H), 3.56 (s, 3H), 6.67
(s, 1H), 7.39–7.54 (m, 3H), 7.77–7.88 (m, 4H), 11.6 (s, 1H); LRMS
(EI) m/z 437 (M+), 251 (100%); HRMS (EI) m/z calcd for C24H23NO5S
(M+) 437.1297, found 437.1303.
5.1.9. 2-[(2-Carboxy-cyclohexanecarbonyl)-amino]-4-naph-
thalen-2-yl-thiophene-3-carboxylic acid benzyl ester (P11)
The product was purified by flash column chromatography with
EA/PE (1/5, v/v) and recrystallized from CH2Cl2/PE (1/3, v/v) to give
a white solid. 72% yield; mp 144–145 °C; 1H NMR (300 MHz,
CDCl3): 1.53–2.29 (m, 8H), 2.91–2.95 (m, 1H), 3.10 (br, 1H), 5.05
(s, 2H), 6.62–6.68 (m, 3H), 6.84 (t, J = 7.8 Hz, 2H), 7.04 (t,
J = 7.8 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.47–7.50 (m, 2H), 7.62 (d,
J = 7.8 Hz, 1H), 7.70–7.85 (m, 3H), 11.6 (s, 1H); 13C NMR (75 MHz,
DMSO-d6): d 22.9, 23.4, 25.4, 26.4, 41.7, 42.8, 51.4, 66.0, 110.9,
115.9, 125.8, 125.9, 126.1, 126.8, 127.3, 127.6, 127.7, 127.8,
132.1, 132.7, 134.8, 134.9, 135.0, 138.9, 149.2, 149.4, 164.7,
171.7, 174.6; LRMS (EI) m/z 513 (M+), 91 (100%); HRMS (EI) m/z
calcd for C30H27NO5S (M+) 513.1610, found 513.1620. Anal.
(C30H27NO5Sꢂ0.8H2O) C, H, N.
5.1.4. 2-(2-Carboxy-benzoylamino)-4-naphthalen-2-yl-thiophene-
3-carboxylic acid isopropyl ester (P04)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a white solid. 62% yield; mp 158–160 °C; 1H NMR (300 MHz,
CDCl3): d 0.85 (d, J = 6.0 Hz, 6H), 4.97 (m, 1H), 6.77 (s, 1H), 7.43–
7.89 (m, 10H), 8.13 (d, J = 7.5 Hz, 1H), 11.8 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 21.6, 68.2, 112.8, 116.2, 125.9, 126.1,
126.5, 127.0, 127.4, 127.6, 127.8, 127.9, 129.8, 130.6, 130.7,
132.0, 132.1, 132.5, 134.7, 135.8, 139.1, 148.4, 164.4, 165.7,
167.3; LRMS (ESI) m/z 458 [M+H]+; HRMS (ESI) m/z calcd for
C26H21NO5NaS [M+Na]+ 482.1038, found 482.1045. Anal.
(C26H21NO5S) C, H, N.
5.1.10. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-4-naphthalen-2-yl-thiophene-3-carboxylic acid ethyl
ester (P12)
5.1.5. 2-(3-Carboxy-propionylamino)-4-naphthalen-2-yl-thio-
phene-3-carboxylic acid isopropyl ester (P05)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/3.5, v/v) to
give a light yellow solid. 65% yield; mp 123–124 °C; 1H NMR
(300 MHz, CDCl3): d 0.81 (t, J = 7.2 Hz, 3H), 1.43 (d, J = 8.7 Hz,
1H), 1.57 (d, J = 8.7 Hz, 1H), 2.27 (m, 2H), 3.39–3.53 (m, 2H), 4.04
(q, J = 7.2 Hz, 2H), 6.34 (m, 1H), 6.46 (m, 1H), 6.63 (s, 1H), 7.39
(d, J = 8.4 Hz, 1H), 7.48 (m, 2H), 7.74–7.76 (m, 4H), 11.4 (s, 1H);
13C NMR (75 MHz, DMSO-d6): d 13.4, 45.7, 46.3, 48.4, 49.1, 49.2,
54.9, 60.1, 110.9, 115.7, 125.8, 126.0, 126.4, 126.8, 127.4, 127.8,
The product was purified by flash column chromatography with
EA/PE (1/2, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to give
a white solid. 48% yield; mp 154–155 °C; 1H NMR (300 MHz,
CDCl3): d 0.85 (d, J = 6.0 Hz, 6H), 2.87 (br, 4H), 4.97 (m, 1H), 6.69
(s, 1H), 7.39–7.62 (m, 3H), 7.75–7.87 (m, 4H), 11.8 (s, 1H); 13C
NMR (75 MHz, DMSO-d6): d 21.1, 28.6, 30.7, 67.9, 112.0, 115.8,
125.8, 126.1, 126.5, 126.9, 127.4, 127.7, 127.9, 131.9, 132.5,
134.8, 138.8, 148.2, 164.1, 169.7, 173.5; LRMS (EI) m/z 411 (M+),

D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
1779
131.9, 132.5, 134.1, 134.9, 135.8, 138.7, 149.0, 164.7, 169.8, 173.0;
LRMS (ESI) m/z 460 [MꢃH]ꢃ; HRMS (ESI) m/z calcd for C26H23NO5S-
Na [M+Na]+ 484.1195, found 484.1187. Anal. (C26H23NO5SꢂH2O) C,
H, N.
d6): d 13.3, 60.5, 112.3, 116.3, 120.3, 127.6. 129.8, 130.4, 130.7,
130.8, 131.0, 132.0, 135.7, 136.2, 137.8, 148.3, 164.5, 165.8,
167.3; LRMS (EI) m/z 475 (M+), 281 (100%); HRMS (EI) m/z calcd
for C21H16BrNO5S (M+) 472.9933, found 472.9946. Anal.
(C21H16BrNO5S) C, H, N.
5.1.11. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-4-phenyl-thiophene-3-carboxylic acid ethyl ester (P13)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/3, v/v) to give
a yellow solid. 71% yield; mp 162–163 °C; 1H NMR (300 MHz,
CDCl3): d 0.92 (t, J = 7.2 Hz, 3H), 1.42 (d, J = 7.8 Hz, 1H), 1.57 (d,
J = 8.1 Hz, 1H), 2.26 (m, 2H), 3.41 (d, J = 10.2 Hz, 1H), 3.50 (d,
J = 9.9 Hz, 1H), 4.06 (q, J = 7.2 Hz, 2H), 6.33 (m, 1H), 6.46 (m, 1H),
6.55 (s, 1H), 7.25–7.33 (m, 5H), 11.4 (s, 1H); 13C NMR (75 MHz,
DMSO-d6): d 13.3, 45.7, 46.2, 48.4, 49.0, 49.1, 60.1, 110.9, 115.2,
126.8, 127.4, 128.8, 134.1, 135.7, 137.3, 138.8, 148.7, 164.7,
169.7, 173.0; LRMS (ESI) m/z 411 (M+), 201 (100%); HRMS (EI) m/
z calcd for C22H21NO5S (M+) 411.1140, found 411.1143. Anal.
(C22H21NO5S) C, H, N.
5.1.16. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic
acid isopropyl ester (P18)
The product was purified by flash column chromatography with
EA/PE (1/2, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to give
a white solid. 81% yield; mp 181–182 °C; 1H NMR (300 MHz,
CDCl3): d 1.37 (d, J = 6.3 Hz, 6H), 1.39 (m, 1H), 1.52 (m, 1H), 1.77
(m, 2H), 2.26 (m, 2H), 2.60 (m, 2H), 2.74 (m, 2H), 3.23 (m, 2H),
3.33–3.47 (m, 2H), 5.20 (m, 1H), 6.27 (m, 1H), 6.41 (m, 1H), 11.5
(s, 1H); LRMS (EI) m/z 403 (M+), 179 (100%); HRMS (EI) m/z calcd
for C21H25NO5S (M+) 403.1453, found 403.1437.
5.1.17. 4-Biphenyl-4-yl-2-[(3-carboxy-bicyclo[2.2.1]hept-5-ene-
2-carbonyl)-amino]-thiophene-3-carboxylic acid isopropyl
ester (P19)
5.1.12. 2-[(2-Carboxy-cyclohexanecarbonyl)-amino]-4-(4-
nitro-phenyl)-thiophene-3-carboxylic acid isopropyl ester (P14)
The product was purified by flash column chromatography with
EA/PE (1/2, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a yellow solid. 73% yield; mp 195–196 °C; 1H NMR (300 MHz,
CDCl3): d 0.97 (d, J = 6.3 Hz, 6H), 1.48–2.23 (m, 8H), 2.93 (m, 1H),
3.12 (m, 1H), 5.03 (m, 1H), 6.64 (s, 1H), 7.45 (d, J = 6.9 Hz, 1H),
8.21 (d, J = 6.9 Hz, 1H), 11.6 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d 21.1, 22.8, 23.4, 25.3, 26.4, 41.7, 42.7, 68.4, 110.8, 116.9,
122.6, 130.4, 136.8, 144.4, 146.4, 149.6, 164.1, 171.7, 174.6; LRMS
(EI) m/z 460 (M+), 246 (100%); HRMS (EI) m/z calcd for C22H24N2O7S
(M+) 460.1304, found 460.1293. Anal. (C22H24N2O7S) C, H, N.
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a yellow solid. 53% yield; mp 172–173 °C; 1H NMR (300 MHz,
CDCl3): d 0.98 (d, J = 6.0 Hz, 6H), 1.43 (d, J = 8.1 Hz, 1H), 1.57 (d,
J = 8.1 Hz, 1H), 2.27 (m, 2H), 3.42 (d, J = 10.2 Hz, 1H), 3.51 (d,
J = 10.2 Hz, 1H), 5.00 (m, 1H), 6.34 (m, 1H), 6.46 (m, 1H), 6.59 (s,
1H), 7.35 (m, 3H), 7.46 (t, J = 7.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 2H),
7.63 (d, J = 7.2 Hz, 2H), 11.5 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d 21.1, 45.7, 46.3, 48.4, 49.1, 49.2, 67.9, 111.2, 115.1, 125.6,
126.5, 127.4, 128.9, 129.5, 134.1, 135.7, 136.6, 138.5, 138.7,
139.9, 148.8, 164.3, 169.7, 173.0; LRMS (ESI) m/z 500 [MꢃH]ꢃ;
HRMS (ESI) m/z calcd for C29H27NO5NaS [M+Na]+ 524.1508, found
524.1504. Anal. (C29H27NO5S) C, H, N.
5.1.13. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-4-(4-fluoro-phenyl)-thiophene-3-carboxylic acid ethyl
ester (P15)
5.1.18. 4-Biphenyl-4-yl-2-[(2-carboxy-cyclohexanecarbonyl)-
amino]-thiophene-3-carboxylic acid isopropyl ester (P20)
The product was purified by flash column chromatography with
EA/PE (1/2, v/v) and recrystallized from CH2Cl2/PE (1/3, v/v) to give
a yellow solid. 61% yield; mp 242–243 °C; 1H NMR (300 MHz,
DMSO-d6): d 0.98 (d, J = 6.3 Hz, 6H), 1.30–2.10 (m, 8H), 2.93 (m,
1H), 3.38 (m, 1H), 4.96 (m, 1H), 6.92 (s, 1H), 7.31–7.80 (m, 9H),
11.3 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 21.1, 22.9, 23.4,
25.4, 26.4, 41.7, 42.7, 54.9, 68.1, 111.4, 115.2, 125.6, 127.4, 128.9,
129.5, 136.5, 138.6, 138.7, 139.9, 149.0, 164.5, 171.5, 174.6; LRMS
(EI) m/z 491 (M+), 277 (100%); HRMS (EI) m/z calcd for C28H29NO5S
(M+) 491.1766, found 491.1772. Anal. (C28H29NO5Sꢂ0.7H2O) C, H, N.
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a yellow solid. 73% yield; mp 164–165 °C; 1H NMR (300 MHz,
CDCl3): d 0.96 (t, J = 7.2 Hz, 3H), 1.43 (m, 1H), 1.56 (m, 1H), 2.26
(m, 2H), 3.38–3.52 (m, 2H), 4.08 (q, J = = 7.2 Hz, 2H), 6.32 (m,
1H), 6.44 (m, 1H), 6.52 (s, 1H), 7.01 (m, 2H), 7.23 (m, 2H), 11.4
(s, 1H); LRMS (EI) m/z 429 (M+), 219 (100%); HRMS (EI) m/z calcd
for C22H20FNO5S (M+) 429.1046, found 429.1041.
5.1.14. 2-(2-Carboxy-benzoylamino)-4-phenyl-thiophene-3-
carboxylic acid isopropyl ester (P16)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a white solid. 68% yield; mp 190–191 °C; 1H NMR (300 MHz,
CDCl3): d 0.93 (d, J = 6.0 Hz, 6H), 4.95 (m, 1H), 6.66 (s, 1H), 7.25–
7.35 (m, 5H), 7.58–7.69 (m, 3H), 8.09 (d, J = 7.8 Hz, 1H), 11.8 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 21.1, 68.1, 112.8, 115.7,
126.9, 127.4, 127.5, 128.9, 129.8, 130.6, 130.8, 132.1, 135.8,
137.1, 139.2, 148.1, 164.4, 165.7, 167.3; LRMS (EI) m/z 409 (M+),
201 (100%); HRMS (EI) m/z calcd for C22H19NO5S (M+) 409.0984,
found 409.0984. Anal. (C22H19NO5S) C, H, N.
5.1.19. 4-Biphenyl-4-yl-2-(2-carboxy-benzoylamino)-thiophene-
3-carboxylic acid isopropyl ester (P21)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a white solid. 52% yield; mp 161–162 °C; 1H NMR (300 MHz,
CDCl3): d 0.96 (d, J = 6.0 Hz, 6H), 4.97 (m, 1H), 6.71 (s, 1H), 7.35
(m, 3H), 7.46 (t, J = 7.2 Hz, 2H), 7.56–7.68 (m, 7H), 8.09 (d,
J = 7.8 Hz, 1H), 11.8 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 21.1,
68.3, 112.8, 115.8, 125.7, 126.5, 127.4, 127.6, 129.0, 129.5, 129.9,
130.6, 130.8, 132.1, 135.8, 136.2, 138.8, 138.9, 139.9; LRMS (EI)
m/z 485 (M+), 277 (100%); HRMS (ESI) m/z calcd for C28H23NO5S
(M+) 485.1297, found 485.1299. Anal. (C28H23NO5S) C, H, N.
5.1.15. 4-(4-Bromo-phenyl)-2-(2-carboxy-benzoylamino)-
thiophene-3-carboxylic acid ethyl ester (P17)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/2, v/v) to give
a light green solid. 65% yield; mp 161–163 °C; 1H NMR (300 MHz,
CDCl3): d 0.96 (t, J = 7.2 Hz, 3H), 4.04 (q, J = 7.2 Hz, 2H), 6.64 (s, 1H),
7.20 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.1 Hz, 3H), 7.61–7.69 (m, 4H),
8.09 (d, J = 7.8 Hz, 1H), 11.7 (s, 1H); 13C NMR (75 MHz, DMSO-
5.1.20. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-4-(40-chloro-biphenyl-4-yl)-thiophene-3-carboxylic
acid isopropyl ester (P22)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/3, v/v) to give

1780
D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
a yellow solid. 75% yield; mp 192–194 °C; 1H NMR (300 MHz,
DMSO-d6): 0.98 (d, J = 6.3 Hz, 6H), 1.42–1.59 (dd, J = 8.4,
of argon. The vial was sealed and the mixture was then irradiated
for 10 min at 150 °C. After the reaction was cooled to ambient tem-
perature, the crude reaction mixture was extracted three times
with EA (15 mL ꢁ 3). The combined organic phase was washed
with brine, dried with Na2SO4, filtered, and concentrated. The res-
idue was purified by column chromatography on Combiflash, and
recrystallized to provide the desired products.
d
12.3 Hz, 2H), 2.28 (m, 2H), 3.41–3.53 (m, 2H), 5.01 (m, 1H), 6.32
(m, 1H), 6.44 (m, 1H), 6.58 (s, 1H), 7.33 (d, J = 7.8 Hz, 2H), 7.40–
7.57 (m, 6H), 11.49 (s, 1H); 13C NMR (75 MHz, CDCl3): d 21.4,
46.1, 47.3, 49.0, 49.1, 50.3, 68.4, 111.7, 115.1, 125.7, 128.2, 128.9,
129.8, 133.3, 133.8, 136.3, 137.5, 138.4, 139.1, 139.3, 149.9,
165.6, 169.3, 176.2; LRMS (EI) m/z 535 (M+), 311 (100%); HRMS
(EI) m/z calcd for C29H26ClNO5S (M+) 535.1220, found 535.1229.
5.4.1. 2-(2-Carboxy-benzoylamino)-4-(20-methoxy-biphenyl-4-
yl)-thiophene-3-carboxylic acid ethyl ester (P23)
5.1.21. 2-[(3-Carboxy-bicyclo[2.2.1]hept-5-ene-2-carbonyl)-
amino]-5-phenyl-thiophene-3-carboxylic acid isopropyl ester
(P32)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 85% yield; mp 192–194 °C; 1H NMR (300 MHz,
CDCl3): d 0.96 (t, J = 7.2 Hz, 3H), 3.83 (s, 3H), 4.09 (q, J = 6.6 Hz,
2H), 6.75 (s, 1H), 7.00–7.07 (m, 2H), 7.35 (m, 4H), 7.51 (d,
J = 8.1 Hz, 2H), 7.62–7.70 (m, 3H), 8.12 (d, J = 7.5 Hz, 1H), 11.8 (s,
1H); 13C NMR (75 MHz, CDCl3): d 13.2, 55.5, 60.6, 111.2, 112.6,
115.7, 120.8, 127.7, 128.5, 128.6, 128.8, 129.3, 130.5, 130.7,
131.0, 131.6, 132.8, 135.8, 136.1, 137.3, 139.9, 149.6, 156.5.
166.1, 166.2, 168.5; LRMS (EI) m/z 501 (M+), 307 (100%); HRMS
(ESI) m/z calcd for C28H23NO6S (M+) 501.1246, found 501.1252.
Anal. (C28H23NO6Sꢂ0.4H2O) C, H, N.
The product was purified by flash column chromatography with
EA/PE (1/2, v/v) and recrystallized from CH2Cl2/PE (1/4, v/v) to give
a yellow solid. 93% yield; mp 192–194 °C; 1H NMR (300 MHz,
CDCl3): d 1.36 (d, J = 6.3 Hz, 6H), 1.42 (m, 1H), 1.57 (m, 1H), 2.26
(m, 2H), 3.38–3.52 (m, 2H), 5.23 (m, 1H), 6.32 (m, 1H), 6.45 (m,
1H), 7.25–7.37 (m, 2H), 7.57 (d, J = 8.4 Hz, 2H), 11.2 (s, 1H); LRMS
(ESI) m/z 424 [MꢃH]ꢃ; HRMS (ESI) m/z calcd for C23H23NO5NaS
[M+Na]+ 448.1195, found 448.1175.
5.2. 2-Benzoylamino-4-biphenyl-4-yl-thiophene-3-carboxylic
acid isopropyl ester (P07)
5.4.2. 2-(2-Carboxy-benzoylamino)-4-(30-methoxy-biphenyl-4-
yl)-thiophene-3-carboxylic acid ethyl ester (P24)
To a solution of 2-amino-4-biphenyl-4-yl-thiophene-3-carbox-
ylic acid isopropyl ester (0.1 mmol) in dry CH2Cl2 was added with
0.105 mmol of benzoyl chloride and 0.15 mmol of triethylamine
(TEA). The mixture was stirred at room temperature for 2–3 h until
the starting material disappeared at TLC analysis (EA/PE = 1/8). The
reaction mixture was concentrated under reduced pressure and
the residue was purified by flash column chromatography to give
a white solid. Mp 131–133 °C; 1H NMR (300 MHz, CDCl3): d 1.02
(d, J = 6.3 Hz, 6H), 5.08 (m, 1H), 6.71 (s, 1H), 7.35 (m, 3H), 7.46 (t,
J = 7.2 Hz, 2H), 7.56–7.68 (m, 7H), 8.09 (m, 2H), 12.4 (s, 1H); LRMS
(ESI) m/z 442 [M+H]+; HRMS (ESI) m/z calcd for C27H23NO5SNa
[M+Na]+ 464.1296, found 464.1232.
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 86% yield; mp 210–211 °C; 1H NMR (300 MHz,
DMSO-d6):
d 0.94 (t, J = 7.5 Hz, 3H), 3.83 (s, 3H), 4.09 (q,
J = 7.5 Hz, 2H), 6.94–6.98 (dd, J = 1.2, 7.2 Hz, 1H), 7.07 (s, 1H),
7.21–7.29 (m, 2H), 7.38–7.43 (m, 3H), 7.62–7.75 (m, 5H), 7.95 (d,
J = 7.5 Hz, 1H), 11.4 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 13.4,
55.1, 60.5, 112.0, 112.7, 113.0, 116.0, 118.9, 125.9, 127.6, 129.4,
129.8, 130.0, 130.7, 130.8, 132.1, 135.9, 136.2, 138.7, 141.4,
148.0, 159.8, 164.7, 165.8, 167.3; LRMS (EI) m/z 501 (M+), 307
(100%); HRMS (ESI) m/z calcd for C28H23NO6S (M+) 501.1246, found
501.1241.
5.3. 2-(2-Methoxycarbonyl-benzoylamino)-4-biphenyl-4-yl-
thiophene-3-carboxylic acid isopropyl ester (P08)
5.4.3. 2-(2-Carboxy-benzoylamino)-4-(40-methoxy-biphenyl-4-
yl)-thiophene-3-carboxylic acid ethyl ester (P25)
The product was purified by flash column chromatography with
EA/PE (1/4, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 82% yield; mp 180–181 °C; 1H NMR (300 MHz,
CDCl3): d 0.94 (t, J = 6.9 Hz, 3H), 3.87 (s, 3H), 4.09 (q, J = 6.6 Hz,
2H), 6.75 (s, 1H), 7.02 (m, 2H), 7.39 (m, 2H), 7.53–7.70 (m, 7H),
8.12 (d, J = 7.5 Hz, 1H), 11.8 (s, 1H); 13C NMR (75 MHz, DMSO-
d6): d 13.4, 55.2, 60.5, 112.8, 114.4, 115.9, 125.3, 127.6, 129.3,
129.8, 130.7, 132.0, 132.2, 135.3, 135.8, 138.5, 138.8, 147.9,
158.9, 164.8, 165.8, 167.3; LRMS (EI) m/z 501 (M+), 307 (100%);
HRMS (ESI) m/z calcd for C28H23NO6S (M+) 501.1246, found
501.1239. Anal. (C28H23NO6Sꢂ0.3H2O) C, H, N.
To a solution of P21 (0.1 mmol) in methanol (10 mL) was added
300 lL concentrated sulfuric acid, and the mixture was kept stir-
ring under reflux for 2–3 h. The resulting solution was concen-
trated under vacuum, and the residue was dissolved in EA
(30 mL), washed with saturated NaHCO3 solution, brine, dried with
Na2SO4 and concentrated. The product was purified by flash col-
umn chromatography with EA/PE (1/10, v/v) and recrystallized
from CH2Cl2/PE (1/2, v/v) to give a light pink solid. 90% yield; mp
178–180 °C; 1H NMR (300 MHz, CDCl3): d 0.98 (d, J = 6.0 Hz, 6H),
3.90 (s, 3H), 4.97 (m, 1H), 6.72 (s, 1H), 7.35 (m, 2H), 7.48 (t,
J = 7.5 Hz, 2H), 7.56–7.68 (m, 7H), 7.98 (d, J = 7.8 Hz, 1H), 11.7 (s,
1H); 13C NMR (75 MHz, DMSO-d6): d 21.1, 52.6, 68.3, 113.3,
116.0, 125.8, 126.5, 127.4, 127.7, 128.9, 129.5, 129.7, 129.8,
131.1, 132.4, 135.4, 136.1, 138.8, 139.9, 147.8, 164.3, 165.1,
166.6; LRMS (ESI) m/z 500 [M+H]+; HRMS (ESI) m/z calcd for
C29H25NO5SNa [M+Na]+ 522.1351, found 522.1360.
5.4.4. 2-(2-Carboxy-benzoylamino)-4-(40-fluoro-biphenyl-4-yl)-
thiophene-3-carboxylic acid ethyl ester (P26)
The product was purified by flash column chromatography with
EA/PE (1/3.5, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to
give a white solid. 82% yield; mp 179–180 °C; 1H NMR (300 MHz,
CDCl3): d 0.94 (t, J = 7.2 Hz, 3H), 4.07 (q, J = 7.2 Hz, 2H), 6.73 (s,
1H), 7.14 (t, J = 8.4 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.51–7.70 (m,
8H), 8.12 (d, J = 7.5 Hz, 1H), 11.8 (s, 1H); 13C NMR (75 MHz,
DMSO-d6): d 13.4, 60.5, 112.7, 115.7, 115.9, 116.0, 125.7, 127.6,
128.5, 128.6, 129.4, 129.8, 130.6, 130.8, 132.1, 135.7, 136.0,
136.3, 137.8, 138.7, 148.0, 160.1, 163.1, 164.7, 165.7, 167.3; LRMS
(EI) m/z 489 (M+), 295 (100%); HRMS (ESI) m/z calcd for
C27H20FNO5S (M+) 489.1046, found 489.1064. Anal. (C27H20FNO5S)
C, H, N.
5.4. General procedure for the synthesis of compounds P23–
P31
A mixture of 4-(4-bromo-phenyl)-2-(2-carboxy-benzoylami-
no)-thiophene-3-carboxylic acid ethyl ester (P17) (0.1 mmol),
boronic acid derivatives (0.12 mmol), Pd(PPh3)4 (0.01 mmol), tet-
raethylammonium bromide (TBAB: 0.1 mmol), and Na2CO3
(0.2 mmol) was stirred in water (3–5 mL) under an atmosphere

D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
1781
5.4.5. 2-(2-Carboxy-benzoylamino)-4-(40-trifluoromethyl-
5.5. Biological assay
biphenyl-4-yl)-thiophene-3-carboxylic acid ethyl ester (P27)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 83% yield; mp 200–202 °C; 1H NMR (300 MHz,
CDCl3): d 0.94 (t, J = 7.2 Hz, 3H), 4.07 (q, J = 7.2 Hz, 2H), 6.74 (s,
1H), 7.42 (d, J = 8.1 Hz, 2H), 7.68–7.72 (m, 10H), 8.12 (d,
J = 6.9 Hz, 1H), 11.8 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d 13.4,
60.7, 112.3, 115.9, 125.7, 126.2, 127.2, 127.7, 128.9, 129.9, 130.9,
137.6, 132.9, 136.0, 137.7, 138.4, 139.2, 144.3, 149.9, 166.0,
166.1, 169.3; LRMS (EI) m/z 539 (M+), 345 (100%); HRMS (ESI) m/
z calcd for C28H20F3NO5S (M+) 539.1014, found 539.1041. Anal.
(C28H20F3NO5S) C, H, N.
5.5.1. Construction, expression, and purification of proteins
The vector pGEX4T-1-TC-PTP1–288 and pGEX4T-1-PTP1B1–321
were constructed with BamHI–HindIII and BamHI–EcoRI sites,
respectively, using a PTP1B or TC45 vector as templates which
were kindly provided by Professor Nicholas K. Tonks (Cold Spring
Harbor Laboratory, NY, USA). Vectors pGEX2T-LAR1282–1888 and
pGEX2T-CD45560–1256 were kindly donated by Professor Rafael
Pulido (Centro de Investigación Príncipe Felipe, Valencia 46013,
Spain). The plasmid pGEX2T-PTPa182–803 was kindly provided by
Professor Frank R. Jirik (Department of Biochemistry and Molecular
Biology, University of Calgary, Canada). Plasmids were transformed
into E. coli BL21 (DE3) separately and purification of the fusion pro-
teins was carried out following our previously reported method.12
Briefly, overnight cultures were diluted 1:100 into 1 L LB medium
and grew at 37 °C until the absorbance at 600 nm reached 0.6. The
5.4.6. 2-(2-Carboxy-benzoylamino)-4-(30-chloro-40-fluoro-
biphenyl-4-yl)-thiophene-3-carboxylic acid ethyl ester (P28)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 84% yield; mp 159–160 °C; 1H NMR (300 MHz,
CDCl3): d 0.94 (t, J = 6.9 Hz, 3H), 4.07 (q, J = 6.6 Hz, 2H), 6.71 (s,
1H), 7.19–7.69 (m, 10H), 8.10 (d, J = 7.5 Hz, 1H), 11.7 (s, 1H); 13C
NMR (75 MHz, CDCl3): d 13.4, 60.5, 112.6, 116.1, 117.2, 117.4,
120.0, 120.2, 125.9, 127.1, 127.2, 127.5, 128.4, 129.5, 129.8,
130.6, 130.8, 132.1, 135.8, 136.4, 136.6, 137.7, 138.4, 148.0,
155.6, 158.0, 164.7, 165.8, 167.3; LRMS (EI) m/z 523 (M+), 505
(100%); HRMS (ESI) m/z calcd for C27H19ClFNO5S (M+) 523.0656,
found 523.0678. Anal. (C27H19ClFNO5S) C, H, N.
recombinant proteins were induced by isopropyl b-D-thiogalacto-
side (IPTG) (0.2 mM) for another 6 h and cells were harvested by
centrifuging for 10 min at 4 °C. Then the cells were frozen at
ꢃ80 °C for storage. GST-fused proteins were thus purified accord-
ing to manufacturer’s instructions (Amersham Pharmacia).
5.5.2. Enzyme activity assay
The enzymatic system of PTP1B contained 5 mM p-nitrophenyl
phosphate (pNPP), 0.03 lM GST-PTP1B1–321, and reaction buffer
(20 mM HEPES, 150 mM NaCl, and 1 mM EDTA, pH 7.0). After incu-
bation of compounds for 10 min, reactions were initiated by add-
ing pNPP. The amount of produced pNP was measured by
detecting absorbance at 405 nm using microplate spectrophotom-
eter (Bio-Rad). IC50 value was calculated by fitting data with Origin
software. To determine inhibition type, DMSO, or compounds were
incubated with different concentration of pNPP (1 mM–5 mM),
respectively, and initial velocities were measured. The inhibition
type was determined using the double-reciprocal plots. In experi-
ments to determine the selectivity over PTPases, the enzymatic
systems for other PTPases were the same as it for PTP1B.
5.4.7. 4-Biphenyl-3-yl-2-(2-carboxy-benzoylamino)-thiophene-
3-carboxylic acid isopropyl ester (P29)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 84% yield; mp 131–133 °C; 1H NMR (300 MHz,
CDCl3): d 0.87 (d, J = 6.3 Hz, 6H), 4.93 (m, 1H), 6.72 (s, 1H), 7.26–
7.69 (m, 12H), 8.10 (d, J = 7.2 Hz, 1H), 11.8 (s, 1H); 13C NMR
(75 MHz, DMSO-d6): d 21.0, 68.1, 112.9, 115.9, 125.3, 126.7,
127.3, 127.5, 127.6, 128.0, 128.2, 129.0, 129.8, 130.6, 130.8,
132.1, 135.8, 136.4, 137.7, 139.1, 139.4, 140.0, 148.2, 164.4,
165.8, 167.3; LRMS (EI) m/z 485 (M+), 277 (100%); HRMS (ESI) m/
z calcd for C28H23NO5S (M+) 485.1297, found 485.1249. Anal.
(C28H23NO5Sꢂ0.2H2O) C, H, N.
5.5.3. AKT activation assay
CHO-K1 cells were cultured in F-12 Ham medium supple-
mented with 10% FBS, penicillin–streptomycin (50 U/mL) at 37 °C
in a humidified atmosphere with 5% CO2. Cells were starved and
incubated subsequently with P28 (10 lM) or 3 (10 lM) for 2 h.
5.4.8. 4-Biphenyl-2-yl-2-(2-carboxy-benzoylamino)-thiophene-
3-carboxylic acid ethyl ester (P30)
AKT phosphorylation and total AKT protein levels were immuno-
blotted with anti-phospho-AKT (Ser473) and anti-AKT primary
antibodies. The loading volumes were normalized by anti-GAPDH.
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 80% yield; mp 99–101 °C; 1H NMR (300 MHz, DMSO-
d6): d 0.78 (t, J = 6.9 Hz, 3H), 3.64 (q, J = 6.9 Hz, 2H), 6.89 (s, 1H),
7.12–7.45 (m, 9H), 7.61–7.72 (m, 3H), 7.82 (d, J = 7.2 Hz, 1H),
11.2 (s, 1H); LRMS (EI) m/z 471 (M+); HRMS (ESI) m/z calcd for
C27H21NO5S (M+) 471.1140, found 471.1160.
Acknowledgments
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (Grants 20721003 and
20872153), International Collaboration Projects (Grant 20720
102040) and the 863 Hi-Tech Program of China (Grants 2006AA
020402 and 2006AA020602), National S&T Major Projects
(2009ZX09501-001 and 2009ZX09501-010).
5.4.9. 4-Biphenyl-4-yl-2-(2-carboxy-benzoylamino)-5-methyl-
thiophene-3-carboxylic acid ethyl ester (P31)
The product was purified by flash column chromatography with
EA/PE (1/3, v/v) and recrystallized from CH2Cl2/PE (1/1, v/v) to give
a white solid. 82% yield; mp 200–201 °C; 1H NMR (300 MHz,
CDCl3): d 0.80 (d, J = 6.9 Hz, 3H), 2.28 (s, 3H), 3.96 (q, J = 6.9 Hz,
2H), 6.72 (s, 1H), 7.26–7.69 (m, 12H), 8.12 (d, J = 6.0 Hz, 1H), 11.6
(s, 1H); 13C NMR (75 MHz, CDCl3): d 13.0, 13.2, 60.4, 113.4,
126.2, 126.9, 127.2, 127.7, 128.8, 129.2, 130.2, 130.9, 131.6,
132.9, 134.7, 135.9, 136.3, 139.6, 139.4, 141.0, 146.4, 164.4,
165.8, 166.1, 169.7; LRMS (EI) m/z 485 (M+), 277 (100%);
HRMS (ESI) m/z calcd for C28H23NO5S (M+) 485.1297, found
485.1292.
Supplementary data
Supplementary data (Figs. S1 and S2, Table S1 of element anal-
ysis, and Table S2 of HPLC purity) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2010.01.055.
References and notes
1. (a) Cheng, A.; Dubé, N.; Gu, F.; Tremblay, M. L. Eur. J. Biochem. 2002, 269, 1050;
(b) Zhang, Z.-Y.; Lee, S. Y. Expert Opin. Invest. Drugs 2003, 12, 223.

1782
D. Ye et al. / Bioorg. Med. Chem. 18 (2010) 1773–1782
2. (a) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.; Loy, A. L.;
Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan, C.-C.; Ramachandran, C.;
Gresser, M. J.; Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544; (b)
Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J. K.; Martino, J. L.; Zabolotny, J. M.;
Moghal, N.; Lubkin, M.; Kim, Y. B.; Sharpe, A. H.; Stricker-Krongrad, A.;
Shulman, G. I.; Neel, B. G.; Kahn, B. B. Mol. Cell. Biol. 2000, 20, 5479.
3. Zinker, B. A.; Rondinone, C. M.; Trevillyan, J. M.; Gum, R. J.; Clampit, J. E.;
Waring, J. F.; Xie, N.; Wilcox, D.; Jacobson, P.; Frost, L.; Kroeger, P. E.; Reilly, R.
M.; Koterski, S.; Opgenorth, T. J.; Ulrich, R. G.; Crosby, S.; Butler, M.; Murray, S.
F.; McKay, R. A.; Bhanot, S.; Monia, B. P.; Jirousek, M. R. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 11357.
Yeleswaram, S.; Li, Y.; Wynn, R.; Burn, T. C.; Hollis, G.; Liu, P. C.; Metcalf, B. J.
Med. Chem. 2006, 49, 3774; (b) Sun, T.; Wang, Q.; Yu, Z.; Zhang, Y.; Guo, Y.;
Chen, K.; Shen, X.; Jiang, H. L. ChemBioChem 2007, 8, 187; (c) Stuible, M.; Zhao,
L.; Aubry, I.; Schmidt-Arras, D.; Böhmer, F. D.; Li, C. J.; Tremblay, M. L.
ChemBioChem 2007, 8, 179; (d) Liu, S.; Zeng, L.-F.; Wu, L.; Yu, X.; Xue, T.;
Gunawan, A. M.; Long, Y.-Q.; Zhang, Z.-Y. J. Am. Chem. Soc. 2008, 130, 17075; (e)
Park, H.; Bhattarai, B. R.; Ham, S. W.; Cho, H. Eur. J. Med. Chem. 2009, 44, 2380.
8. Mclane, M.; Ruiz-White, I.; Wolfe, H. WO 2009032321, 2009.
9. (a) Moretto, A. F.; Kirincich, S. J.; Xu, W. X.; Smith, M. J.; Wan, Z.-K.; Wilson, D.
P.; Follows, B. C.; Binnun, E.; Joseph-McCarthy, D.; Foreman, K.; Erbe, D. V.;
Zhang, Y. L.; Tam, S. K.; Tam, S. Y.; Lee, J. Bioorg. Med. Chem. 2006, 14, 2162; (b)
Wan, Z.-K.; Lee, J.; Xu, W. X.; Erbe, D. V.; Joseph-McCarthy, D.; Follows, B. C.;
Zhang, Y. L. Bioorg. Med. Chem. Lett. 2006, 16, 4941; (c) Wan, Z.-K.; Follows, B.;
Kirincich, S.; Wilson, D.; Binnun, E.; Xu, W. X.; Joseph-McCarthy, D.; Wu, J.;
Smith, M.; Zhang, Y. L.; Tam, M.; Erbe, D.; Tam, S.; Saiah, E.; Lee, J. Bioorg. Med.
Chem. Lett. 2007, 17, 2913.
10. Huang, W.; Li, J.; Tang, J.; Liu, H.; Shen, J. H.; Jiang, H. L. Synth. Commun. 2005,
35, 1351.
11. Tromp, R. A.; van Ameijde, S.; Pütz, C.; Sundermann, C.; Sundermann, B.; von
Frijtag Drabbe Künzel, J. K.; Ijzerman, A. P. J. Med. Chem. 2004, 47, 5441.
12. Zhang, Y.; Li, Y.; Guo, Y. W.; Jiang, H. L.; Shen, X. Acta Pharmacol. Sin. 2009, 30,
333.
13. Iversen, L. F.; Andersen, H. S.; Moller, K. B.; Olsen, O. H.; Peters, G. H.; Branner,
S.; Mortensen, S. B.; Hansen, T. K.; Lau, J.; Ge, Y.; Holsworth, D. D.; Newman, M.
J.; Hundahl Moller, N. P. Biochemistry 2001, 40, 14812.
14. Hill, M. M.; Clark, S. F.; Tucker, D. F.; Birnbaum, M. J.; James, D. E.; Macaulay, S.
L. Mol. Cell. Biol. 1999, 19, 7771.
4. (a) Julien, S. G.; Dubé, N.; Read, M.; Penney, J.; Paquet, M.; Han, Y.; Kennedy, B.
P.; Muller, W. J.; Tremblay, M. L. Nat. Genet. 2007, 39, 338; (b) Bentires-Alj, M.;
Neel, B. G. Cancer Res. 2007, 67, 2420.
5. (a) Shen, K.; Keng, Y.-F.; Wu, L.; Guo, X.-L.; Lawrence, D. S.; Zhang, Z.-Y. J. Biol.
Chem. 2001, 276, 47311; (b) Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-
Zapatero, C.; Hutchins, C. W.; Zhao, H.; Lubben, T. H.; Ballaron, S. J.; Haasch, D.
L.; Kaszubska, W.; Rondinone, C. M.; Trevillyan, J. M.; Jirousek, M. R. J. Med.
Chem. 2003, 46, 4232; (c) Wiesmann, C.; Barr, K. J.; Kung, J.; Zhu, J.; Erlanson,
D. A.; Shen, W.; Fahr, B. J.; Zhong, M.; Taylor, L.; Randal, M.; McDowell, R. S.;
Hansen, S. K. Nat. Struct. Mol. Biol. 2004, 11, 730; (d) Johnson, T. O.; Ermolieff,
J.; Jirousek, M. R. Nat. Rev. Drug Disc. 2002, 1, 696; (e) Lee, S.; Wan, Q. Med.
Res. Rev. 2007, 27, 553; (f) Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007,
12, 373.
6. (a) Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Frederickson, M.; Tickle, I. J.;
Jhoti, H. J. Med. Chem. 2005, 48, 403; (b) Wilson, D. P.; Wan, Z. K.; Xu, W. X.;
Kirincich, S. J.; Follows, B. C.; Joseph-McCarthy, D.; Foreman, K.; Moretto, A.;
Wu, J.; Zhu, M.; Binnun, E.; Zhang, Y. L.; Tam, M.; Erbe, D. V.; Tobin, J.; Xu, X.;
Leung, L.; Shilling, A.; Tam, S. Y.; Mansour, T. S.; Lee, J. J. Med. Chem. 2007, 50,
4681; (c) Hussain, M.; Ahmed, V.; Hill, B.; Ahmed, Z.; Taylor, S. D. Bioorg. Med.
Chem. 2008, 16, 6764.
15. Asante-Appiah, E.; Patel, S.; Desponts, C.; Taylor, J. M.; Lau, C.; Dufresne, C.;
Therien, M.; Friesen, R.; Becker, J. W.; Leblanc, Y.; Kennedy, B. P.; Scapin, G. J.
Biol. Chem. 2006, 281, 8010.
16. Liu, G.; Xin, Z.; Liang, H.; Abad-Zapatero, C.; Hajduk, P. J.; Janowick, D. A.;
Szczepankiewicz, B. G.; Pei, Z.; Hutchins, C. W.; Ballaron, S. J.; Stashko, M. A.;
Lubben, T. H.; Berg, C. E.; Rondinone, C. M.; Trevillyan, J. M.; Jirousek, M. R. J.
Med. Chem. 2003, 46, 3437.
7. (a) Combs, A. P.; Zhu, W.; Crawley, M. L.; Glass, B.; Polam, P.; Sparks, R. B.; Modi,
D.; Takvorian, A.; McLaughlin, E.; Yue, E. W.; Wasserman, Z.; Bower, M.; Wei,
M.; Rupar, M.; Ala, P. J.; Reid, B. M.; Ellis, D.; Gonneville, L.; Emm, T.; Taylor, N.;