Synthesis and biological evaluation of novel human Pin1 inhibitors with benzophenone skeleton

Article abstract of DOI:10.1016/j.bmc.2012.03.005

A series of novel benzophenone derivatives were prepared and their inhibitory activities were evaluated on hPin1. Of all the synthesized compounds, the most active compound displayed inhibitory activities with an IC ₅₀ value of 5.99 μmol/L. Pre

Full text of DOI:10.1016/j.bmc.2012.03.005

Bioorganic & Medicinal Chemistry 20 (2012) 2992–2999
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel human Pin1 inhibitors
with benzophenone skeleton
Chang Liu a,b, , Jing Jin a, , Liang Chen a,c, Jie Zhou a, Xiaoguang Chen a, Decai Fu b, Hongrui Song c,
Bailing Xu a,
⇑
a Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
b Hebei University of Science and Technology, Shijiazhuang 050018, China
c School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, 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 benzophenone derivatives were prepared and their inhibitory activities were evaluated
on hPin1. Of all the synthesized compounds, the most active compound displayed inhibitory activities
with an IC50 value of 5.99 lmol/L. Preliminary structure–activity relationships were analyzed in details
and the binding mode of the titled compounds was predicted using FlexX algorithm. The results of this
research will shed light on further design and optimization of novel small molecule Pin1 inhibitors.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 3 February 2012
Revised 1 March 2012
Accepted 1 March 2012
Available online 9 March 2012
Keywords:
Benzophenone
Pin1
Pin1 inhibitor
PPIase
Anti-cancer agents
1. Introduction
bound with Pin1, aryl indanyl ketones (B, Fig. 1) has been devel-
oped as reversible inhibitors with inhibition constants in sub-
Peptidyl-prolyl cis/trans isomerase (PPIase) consists of three
major families, namely, cyclophilins, FK-506 binding proteins
(FKBPs) and parvulins. Pin1 (Protein interaction with NIMA1) be-
longs to the parvulin family and has distinctive substrate specific-
ity. Pin1 specifically catalyzes the conformational switch between
cis- and trans- amide bond in pSer-Pro/pThr-Pro motif, which oc-
curred exclusively in many key protein kinases involved in cell cy-
cle.1–3 Pin1 is frequently up-regulated in various tumors and its
over-expression level is also associated with tumor grades and
clinical outcomes in prostate cancer.4,5 Depletion of Pin1 by over-
expression of Pin1antisense RNA induces mitotic arrest and apop-
tosis in human tumor cells.1,6 Therefore, it was expected that Pin1
may hold a promise as a novel anticancer target and inhibition of
Pin1 with small molecules may become a new anticancer treat-
ment strategy.
micromolar range.11 Structure-based strategy was successfully
employed to design the small molecule inhibitors of Pin1 by Ver-
nalis and Pfizer.12–15 The most potent inhibitor (C, Fig. 1) with
the aminophenylpropanol scaffold was found with nanomolar
inhibition activity. However, it is not active in cancer cells due to
its poor permeability.12 In the present, it is a utmost need for the
development of small molecules as Pin1 inhibitors to validate
Pin1 as an anticancer target.
In our early efforts to search for novel Pin1 inhibitors, quinazo-
line-based Pin1 inhibitors (D, Fig. 1) were discovered by screening
our in-house library.16 Another chemical entity (compound 7a)
containing benzophenone motif in the library was also identified
as Pin1 inhibitor with an IC50 value of 10.11 lM. In order to inves-
tigate the structure–activity relationships (SAR), a series of benzo-
phenone derivatives were synthesized by diversifying the
substituents at 4-position of A ring and 3- or 4-position of B ring.
The active site of Pin1 in its crystal structure featured two hydro-
phobic domains and one basic cluster formed by the side chains
of Lys63, Arg68 and Arg69 residues.12–15,17 The hydrophobic prolyl
pocket is formed by His59, His157, Met130 and Phe134 residues
and the slightly shallow hydrophobic shelf embraces His59,
Ala118 and Leu122 residues. We envisioned that the oxalic acid
group is anchored to the subpocket constructed by basic amino
acid residues, the hydrophobic aromatic fragments on A ring may
So far, a number of studies on small molecule inhibitors of Pin1
have been reported.7–16 Juglone (A, Fig. 1), identified by screening a
collection of pure secondary metabolites, irreversibly inhibited the
enzymatic activity of several parvulins including human Pin1.10
Mimicking the ‘twisted-amide’ transition state of the substrate
⇑
Corresponding author. Tel./fax: +86 10 63166764.
E-mail address: xubl@imm.ac.cn (B. Xu).
The first two authors contributed equally.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.005

C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
2993
O
OH O
OH
O2N
F
S
OH O
HN
N
O2N
Cl
Cl
N
O
HN
O
O
OH
O
N
H
N
H
F
P
NO2
O
HO OH
A
B Ki = 0.2 µM
C Ki = 0.006 µM
D IC50 = 2.9 µM
Figure 1. Several known Pin1 inhibitor structures.
occupy the two hydrophobic domains. Herein, the chemical syn-
thesis of these new benzophenone derivatives is described in de-
tails. The inhibitory activities on Pin1 of these new chemical
entities are presented along with their SAR analysis as follows.
reaction conditions, compounds 6a–6k were hydrolyzed into the
corresponding oxoacetic acid derivatives 7a–7k in 39–91% yields.
3. Biological results and discussion
2. Chemistry
The inhibitory activities on Pin 1 of all target compounds (7a–
7k) were evaluated by a protease-coupled enzyme assay with
Suc-Ala-Glu-Pro-Phe-pNA as the substrate.18,19 The corresponding
results are expressed as IC50 values and presented in Table 1.
As shown in Table 1, the SAR was investigated initially on the
substituents at 4-position of A ring. The target compounds with
aromatic hydrophobic fragments (compound 7a–7f) exhibited var-
ied inhibitory activities on Pin1 with an IC50 value ranging from
5.99 lM to 18.30 lM. Among which, 7-nitro benzothiophene
(compound 7d) and indole (compound 7e) motifs are the most
favorable variations and lead to inhibitory activities with an IC50
value of 5.99 lM and 6.31 lM, respectively. Substitutions at 4-po-
The synthesis of benzophenone derivatives (7a–7k) was de-
picted in Scheme 1. The benzophenone scaffolds (2a and 2b) were
constructed through Friedel–Crafts reaction between 2-chloro-5-
nitrobenzoyl chloride and substituted benzene. Amination of inter-
mediates 2a and 2b with aqueous ammonia under microwave
irradiation furnished compounds 3b and 3c in 54% and 47% yields,
respectively. The acylation of amines 3a–3c with ethyl oxalyl
monochloride in toluene underwent smoothly to provide the cor-
responding products in 64–83% yields. Compounds 4a–4c were re-
duced under catalytic hydrogenation in trifluoroethanol to the
corresponding amines 5a–5c in 76–91% yields. It was noted that
the reduction of compound 4b with SnCl2, Fe/CH3COOH, or cata-
lytic hydrogenation in methanol gave rise to compound 5b in rel-
atively low yield (30–50%). Nevertheless, compound 5b could be
prepared in excellent yield (91%) when the catalytic hydrogenation
was performed in trifluoroethaonl. It has been demonstrated that
the trifluoroethanol or hexafluoroisopropanol was a better solvent
for the catalytic hydrogenation reduction of nitro compounds.16
The conversion of amines 5a–5c to amides 6a–6k with various car-
boxylic chloride in the presence of hunig’s base proceeded
smoothly in moderate to good yields. Upon treatment with basic
sition with benzothiophene-2-carboxamido (compound 7a),
benzothiophene-3-carboxamido (compound 7b) and 3-chloro-
benzothiophene-2-carboxamido (compound 7c) functional groups
produced comparable inhibition effects on Pin1 at the 10 lM level.
In comparison with indole-2-carboxamido substitution (com-
pound 7e), the inhibitory potency of compound 7f with benzofu-
ran-2-carboxamido fragment decreased to about 1/3. When the
4-aromatic group was replaced by the acetyl substituent (com-
pound 7g), the inhibition effect was completely lost. It was indi-
cated that introduction of bulky aromatic group to the 4-position
of A ring is beneficial to the binding affinity.
O
O
O
Cl
O
NH
O
NH2
NO2
O
Cl
R1
R2
R1
R2
R1
R2
COCl
c
a
b
NO2
NO2
NO2
4a
3a R1 = R2 = H
R1 = R2 = H
2a R1 = OCH3, R2 = OCH3
1
3b R1 = R2 = OCH3
4b R1 = R2 = OCH3
2b
R1 = F, R2 = OCH3
3c
4c
R
R1 = F, R2 = OCH3
1 = F, R2 = OCH3
O
O
O
O
O
HO
O
O
O
NH
O
NH
O
NH
O
R1
R1
R1
e
d
f
R2
R2
R2
NH2
HN
HN
R3
R3
7a 7g
6a 6g
5a
-
R1 = R2 = H
6h - 6i R1 = R2 = OCH3
6j 6k
R
1 = R2 = H
5b R1 = R2 = OCH3
5c
-
R
1 = R2 = H
7h - 7i R1 = R2 = OCH3
7j 7k
R1 = F, R2 = OCH3
-
R1 = F, R2 = OCH3
R1 = F, R2 = OCH3
-
Scheme 1. Reagents and conditions: (a) AlCl3, 1,2-dimethoxybenzene or o-fluoroanisole; (b) NH3/H2O, microwave; (c) ethyl oxalyl monochloride, toluene, reflux; (d) H2/10%
Pd-C, trifluoroehtanol, rt; (e) RCOCl, DIEA, toluene, reflux; (f) NaOH, THF/H2O, rt.

2994
C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
Table 1
The chemical structures and inhibitory activities on hPin1 of compounds 7a–7k.
HO
O
O
NH
A
O
O
R1
R2
2
3
4
B
4
HN
R3
a
Compound
R1
H
R2
R3
IC50 (lM)
7a
H
H
10.11
11.49
S
7b
7c
H
H
Figure 2. FlexX-modeled binding mode of compound 7e (carbon atoms colored
green) in comparison with the crystal structure (3JYJ in PDB)13 of a carboxylate Pin1
inhibitor (carbon atoms colored violet). H-Bonding interactions are presented with
blue line. The chemical structure of reference molecule is presented. Molecular
image was generated with UCSF Chimera.21
S
Cl
H
8.93
S
7d
7e
H
H
H
H
5.99
6.31
S
O2N
gen bond was formed between 2-carbonyl oxygen of oxalic acid
and hydroxyl group of Ser114. We rationalized that 2-oxoacetic
acid may serve as an alternative for the phosphorus acid group
generally used in Pin1 inhibitor. The benzene ring A of benzophe-
none situated in a small hydrophobic area consisting of His59 and
Cys113 residues. The benzene ring B did not occupy the prolyl
pocket in which the benzene ring of reference molecule located.
The carbonyl oxygen between A and B ring formed a hydrogen
bond with the hydroxyl group of Ser154. It was suggested that
installment of a hydrogen bond acceptor on the appropriate posi-
tion of A ring may have a positive contribution to the binding affin-
ity. The bulky aromatic fragment on A ring extended to the
hydrophobic shelf including the side chains of Leu122 and
Met130. The orientation of this aromatic hydrophobic group was
somewhat different from the naphthalene ring of the reference
molecule. Nonetheless, both of them could bring important hydro-
phobic interaction with Pin1 due to a large shallow hydrophobic
region existed.
N
H
7f
H
H
H
H
18.30
>100
O
7g
CH3CO–
7h
OCH3
OCH3
OCH3
OCH3
OCH3
54.33
53.55
10.36
14.52
S
7i
OCH3
S
S
7j
F
F
7k
S
a
Compound dose (lM) required to inhibit the Pin1 activity by 50%.
4. Conclusion
The preliminary SAR of 3- and 4-substituents on B ring was also
In summary, a series of novel benzophenone-based chemical
entities was synthesized as potent Pin1 inhibitors with IC50 values
at micromolar level. The SAR and binding mode of the titled com-
pounds were explored preliminarily, and that will shed light for
the discovery of more potent novel small molecule Pin1 inhibitors.
explored. When 3,4-dimethoxyl benzene was used as B ring in-
stead of benzene, the inhibitory potency dramatically decreased
to 1/5 (compounds 7h and 7i vs 7a and 7b). While 3-fluoro-4-
methoxylbenzene served as B ring, compounds 7j and 7k displayed
somewhat similar inhibition effect to that of compounds 7a and
7b. It was rationalized that the bulky methoxyl group on 3-position
may result in unfavorable contact with Pin1. In comparison with 3-
position of B ring, the variation on 4-position was allowed to a
greater extent.
5. Experimental
5.1. Chemistry
In order to get some insights for further structure-based modi-
fication of benzophenones Pin1 inhibitor, the binding mode was
investigated using FlexX algorithm implemented in SYBYL 7.2.21
The coordinates of X-ray co-crystal structure of a carboxylate
inhibitor (reference molecule) with Pin1 (PDB code: 3JYJ) reported
by Pfizer in 201013 was employed for docking the synthesized
compounds. The binding modes of the synthesized benzophenones
are somewhat similar and exemplified by the representative com-
pound 7e as shown in Figure 2. The carboxylate of 2-oxoacetic acid
interacted with the positive charged amino side chain of Arg69 and
Lys63 via the key charge-charge interaction. In addition, the hydro-
5.1.1. General
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. 1H NMR (300 MHz) on a Varian
Mercury 300 spectrometer was recorded in DMSO-d6 or CDCl3.
Chemical shifts are reported in d (ppm) units relative to the inter-
nal standard tetramethylsilane (TMS). High resolution mass spec-
tra (HRMS) were obtained on an Agilent Technologies LC/MSD
TOF spectrometer. All chemicals and solvents used were of reagent
grade without purified or dried before use. All the reactions were
monitored by thin-layer chromatography (TLC) on pre-coated sil-

C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
2995
ica gel G plates at 254 nm under a UV lamp. Column chromatogra-
phy separations were performed with silica gel (200–300 mesh).
7.71 (t, J = 7.2 Hz, 1H, ArH), 7.58 (t, J = 7.5 Hz, 2H, ArH), 7.44–7.52
(m, 2H, ArH); HRMS (ESI): m/z, Calcd for C24H17N2O5S [M + H+]:
445.0853, Found 445.0863.
5.1.2. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-benzoylphe
nylamino)-2-oxoacetic acid (7a)
5.1.2.1. Ethyl 2-(2-benzoyl-4-nitrophenylamino)-2-oxoacetate
5.1.3. 2-(4-(benzo[b]thiophene-3-carboxamido)-2-
benzoylphenylamino)-2-oxoacetic acid (7b)
(4a).
To
a
stirred
solution
of
(2-amino-5-nitro-
5.1.3.1. Ethyl 2-(4-(benzo[b]thiophene-3-carboxamido)-2-ben-
phenyl)(phenyl)methanone (5.0 g, 0.02 mol) dissolved in toluene
(70 mL) at 110 °C was added chlorooxalic acid ethyl ester (2.82 g,
0.02 mol). The reaction mixture was stirred for 3 h and then di-
luted with EtOAc (200 mL) and saturated NaHCO3 aqueous solution
(200 mL). The organic layer was washed with water (100 mL Â 2)
and brine (100 mL Â 2), and dried over anhydrous Na2SO4. The
crude product was obtained after concentration and recrystalized
with EtOAc to afford compound 4a as white solid (4.53 g, 64%);
mp 157–159 °C; 1H NMR (acetone-d6) d (ppm): 12.13 (brs, 1H,
NH), 8.86 (d, J = 9.3 Hz, 1H, ArH), 8.56 (dd, J1 = 9.0 Hz, J2 = 2.7 Hz,
1H, ArH), 8.49 (d, J = 2.7 Hz, 1H, ArH), 7.85–7.88 (m, 2H, ArH),
7.75 (t, J = 7.2 Hz, 1H, ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), 4.39 (q,
J = 6.9 Hz, 2H, CH2), 1.38 (t, J = 6.9 Hz, 3H, CH3); HRMS (ESI): m/z,
Calcd for C17H15N2O6 [M+H+]: 343.0925, Found 343.0919.
zoylphenylamino)-2-oxoacetate (6b).
Following the prepa-
ration protocol of Section 5.1.2.3, the reaction mixture of
compound 5a (0.20 g, 0.64 mmol), benzo[b]thiophene-3-carboxa-
chloride (0.19 g, 0.96 mmol) and DIEA (0.12 g, 0.96 mmol) in tolu-
ene (4 mL) was heated at 110 °C for 1.0 h to give compound 6b as
yellow solid (0.22 g, 73%); mp 204–206 °C; 1H NMR (DMSO-d6) d
(ppm): 11.36 (s, 1H, NH), 10.55 (s, 1H, NH), 8.56 (s, 1H, ArH),
8.37–8.40 (m, 1H, ArH), 7.98–8.15 (m, 4H, ArH), 7.76 (d,
J = 7.2 Hz, 1H, ArH), 7.85 (d, J = 6.9 Hz, 1H, ArH), 7.69 (t, 1H,
J = 7.5 Hz, ArH), 7.56 (t, J = 7.2 Hz, 2H, ArH), 7.43–7.49 (m, 2H,
ArH), 4.26 (q, J = 7.5 Hz, 2H, CH2), 1.28 (t, J = 7.5 Hz, 3H, CH3);
HRMS (ESI): m/z, Calcd for C26H21N2O5S [M + H+]: 473.1166, Found
473.1174.
5.1.3.2. 2-(4-(Benzo[b]thiophene-3-carboxamido)-2-benzoylphe
5.1.2.2. Ethyl 2-(4-amino-2-benzoylphenylamino)-2-oxoacetate
nylamino)-2-oxoacetic acid (7b).
Following the preparation
(5a).
The mixture of compound 4a (2.0 g, 5.84 mmol) and 10%
protocol of Section 5.1.2.4, the reaction mixture of compound 6b
(0.10 g, 0.21 mmol) and NaOH (0.042 g, 1.05 mmol) in water
(2.0 mL) and THF (2.0 mL) was stirred at room temperature for
50 min to give compound 7b as yellow solid (0.081 g, 86%); mp
206–208 °C; 1H NMR (acetone-d6) d (ppm): 11.80 (s, 1H, NH),
9.86 (s, 1H, NH), 8.60 (d, J = 8.7 Hz, 1H, ArH), 8.51–8.54 (m, 1H,
ArH), 8.43 (s, 1H, ArH), 8.21 (d, J = 8.7 Hz, 2H, ArH), 7.98–8.01 (m,
1H, ArH), 7.84 (d, J = 7.2 Hz, 2H, ArH), 7.63–7.72 (m, 1H, ArH),
7.56 (t, J = 7.5 Hz, 2H, ArH), 7.42–7.49 (m, 2H, ArH); HRMS (ESI):
m/z, Calcd for C24H17N2O5S [M + H+]: 445.0853, Found 445.0861.
Pd-C (0.4 g) in trifluroethanol (50 mL) was hydrogenated at room
temperature and 1 atm for 3 h. Pd-C was filtered off and the filtrate
was concentrated to give the crude product, which was recrystal-
ized with EtOAc to afford compound 5a as yellow solid (1.49 g,
81.6%); mp 129–131 °C; 1H NMR (DMSO-d6) d (ppm): 10.96 (s,
1H, NH), 7.62–7.69 (m, 4H, ArH), 7.51 (t, J = 7.5 Hz, 2H, ArH), 6.79
(dd, J1 = 8.7 Hz, J2 = 2.4 Hz, 1H, ArH), 6.67(d, J = 2.4 Hz, 1H, ArH),
5.38 (s, 2H, NH2), 4.20 (q, J = 7.2 Hz, 2H, CH2), 1.24 (t, J = 7.2 Hz,
3H, CH3); HRMS (ESI): m/z, Calcd for C17H17N2O4 [M+H+]:
313.1183, Found 313.1197.
5.1.4. 2-(2-Benzoyl-4-(3-chlorobenzo[b]thiophene-2-carboxa
mido)phenylamino)-2-oxoacetic acid (7c)
5.1.2.3. Ethyl 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-ben-
zoylphenylamino)-2-oxoacetate (6a).
To a stirred solution of
5.1.4.1. Ethyl 2-(2-benzoyl-4-(3-chlorobenzo[b]thiophene-2-
compound 5a (0.20 g, 0.64 mmol) dissolved in toluene (4 mL) at
110 °C was added benzo[b]thiophene-2-carboxachloride (0.19 g,
0.96 mmol) and DIEA (0.12 g, 0.96 mmol). The reaction mixture
was heated for 2 h and then cooled to room temperature. The reac-
tion mixture was diluted with EtOAc (80 mL) and saturated NaH-
CO3 aq (80 mL). The organic layer was separated and washed
with water (80 mL Â 1) and brine (80 mL Â 1), and then dried over
anhydrous Na2SO4. The crude product was obtained after concen-
tration and recrystalized with EtOAc to afford compound 6a as yel-
low solid (0.21 g, 69%); mp 228–230 °C; 1H NMR (DMSO-d6) d
(ppm): 11.39 (s, 1H, NH), 10.72 (s, 1H, NH), 8.34 (s, 1H, ArH),
7.99–8.14 (m, 5H, ArH), 7.66 (d, J = 7.2 Hz, 2H, ArH), 7.70 (t,
J = 7.5 Hz, 1H, ArH), 7.56 (t, J = 7.5 Hz, 2H, ArH), 7.46–7.50 (m,
2H, ArH), 4.26 (q, J = 7.2 Hz, 2H, CH2), 1.28 (t, J = 7.2 Hz, 3H, CH3);
HRMS (ESI): m/z, Calcd for C26H21N2O5S [M+H+]: 473.1166, Found
473.1165.
carboxamido)phenylamino)-2-oxoacetate (6c).
Following
the preparation protocol of Section 5.1.2.3, the reaction mixture
of compound 5a (0.10 g, 0.32 mmol), benzo[b]thiophene-3-
chloro-2-carboxachloride (0.11 g, 0.48 mmol) and DIEA (0.06 g,
0.48 mmol) in toluene (4 mL) was heated at 110 °C for 0.5 h to give
compound 6c as yellow solid (0.18 g, 74%); mp 238–240 °C; 1H
NMR (DMSO-d6) d (ppm): 11.39 (s, 1H, NH), 10.78 (s, 1H, NH),
8.14–8.17 (m, 1H, ArH), 8.00–8.08 (m, 2H, ArH), 7.92–7.97 (m,
2H, ArH), 7.54–7.77 (m, 5H, ArH), 4.26 (q, J = 7.2 Hz, 2H, CH2),
1.28 (t, J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for
C
26H20ClN2O5S [M+H+]: 507.0776, Found 507.0781.
5.1.4.2. 2-(2-Benzoyl-4-(3-chlorobenzo[b]thiophene-2-carboxa-
mido)phenylamino)-2-oxoacetic acid (7c). Following the
preparation protocol of Section 5.1.2.4, the reaction mixture of
compound 6c (0.05 g, 0.10 mmol) and NaOH (0.02 g, 0.49 mmol)
in water (2.0 mL), THF (2 mL) and acetone (4.0 mL) was stirred at
room temperature for 30 min to give compound 7c as yellow solid
(0.038 g, 81%); mp 235–237 °C; 1H NMR (DMSO-d6) d (ppm): 11.41
(s, 1H, NH), 10.77 (s, 1H, NH), 8.09–8.17 (m, 2H, ArH), 7.92–8.03
(m, 3H, ArH), 7.76 (d, J = 6.9 Hz, 2H, ArH), 7.54–7.71 (m, 5H,
5.1.2.4. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-benzoylphe
nylamino)-2-oxoacetic acid (7a).
The reaction mixture of
compound 6a (0.10 g, 0.32 mmol) and NaOH (0.064 g, 1.6 mmol)
in water (2.0 mL), THF (2.0 mL) and acetone (4.0 mL) was stirred
at room temperature for 1 h. The solvent was removed by evapora-
tion and the residue was dissolved in water (10.0 mL), which was
cooled in ice-water bath and adjusted to pH = 2.0 with diluted
HCl solution. The resulting mixture was stirred for 0.5 h and the
precipitate was filtered to give the title compound 7a as yellow so-
lid (0.075 g, 53%); mp 223–225 °C; 1H NMR (acetone-d6) d (ppm):
11.40 (s, 1H, NH), 10.72 (s, 1H, NH), 8.34 (s, 1H, ArH), 8.13 (m,
2H, ArH), 7.99–8.07 (m, 3H, ArH), 7.78 (d, J = 7.2 Hz, 2H, ArH),
ArH); HRMS (ESI): m/z, Calcd for
C
24H16ClN2O5S [M+H+]:
479.0463, Found 479.0470.
5.1.5. 2-(2-Benzoyl-4-(6-nitrobenzo[b]thiophene-2-carboxa
mido)phenylamino)-2-oxoacetic acid (7d)
5.1.5.1. Ethyl 2-(2-benzoyl-4-(6-nitrobenzo[b]thiophene-2-car-
boxamido)phenylamino)-2-oxoacetate (6d).
preparation protocol of Section 5.1.2.3, the reaction mixture of
Following the

2996
C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
compound 5a (0.20 g, 0.64 mmol), benzo[b]thiophene-6-nitro-2-
carboxachloride (0.19 g, 0.76 mmol) and DIEA (0.10 g, 0.76 mmol)
in toluene (4 mL) was heated at 110 °C for 0.5 h to give compound
6d as yellow solid (0.29 g, 90%); mp > 300 °C; 1H NMR (DMSO-d6) d
(ppm): 11.39 (s, 1H, NH), 11.18 (s, 1H, NH), 8.92 (d, J = 2.1 Hz, 1H,
ArH), 8.61(s, 1H, ArH), 8.35 (d, 1H, J = 8.7 Hz, ArH), 8.27 (dd,
J1 = 8.7 Hz, J2 = 2.4 Hz, 1H, ArH), 8.15 (d, J = 8.7 Hz, 1H, ArH), 8.06
(d, 1H, J = 8.7 Hz, ArH), 8.03 (d, J = 2.4 Hz, 2H, ArH), 7.77 (d,
J = 7.5 Hz, 2H, ArH), 7.69 (t, J = 7.5 Hz, 1H, ArH), 7.58 (t, J = 7.5 Hz,
2H, ArH), 4.27 (q, J = 7.2 Hz, 2H, CH2), 1.28 (t, J = 7.2 Hz, 3H, CH3);
HRMS (ESI): m/z, Calcd for C26H20N3O7S [M+H+]: 518.1016, Found
518.1048.
and one drop DMF in CH2Cl2 (5 mL) was refluxed for 5 h. The crude
product of benzofuran-2-carboxachloride was obtained after evap-
oration and directly used in the next step without further
purification.
Following the preparation protocol of Section 5.1.2.3, the reac-
tion mixture of compound 5a (0.26 g, 0.82 mmol), benzofuran-2-
carboxachloride and DIEA (0.16 g, 1.23 mmol) in toluene (7 mL)
was heated at 110 °C for 1 h to give compound 6f as yellow solid
(0.34 g, 90%); mp 231–232 °C; 1H NMR (DMSO-d6) d (ppm):
11.40 (s, 1H, NH), 10.82 (s, 1H, NH), 8.17 (dd, J1 = 9.0 Hz,
J2 = 2.4 Hz,1H, ArH), 8.02–8.09 (m, 2H, ArH), 7.76–7.83 (m, 4H,
ArH), 7.69–7.71 (m, 2H, ArH), 7.49–7.60 (m, 3H, ArH), 7.37 (d,
J = 7.2 Hz, 1H, ArH), 4.26 (q, J = 6.9 Hz, 2H, CH2), 1.28 (t, J = 7.2 Hz,
3H, CH3); HRMS (ESI): m/z, Calcd for C26H21N2O6 [M+H+]:
457.1394, Found 457.1405.
5.1.5.2. 2-(2-Benzoyl-4-(6-nitrobenzo[b]thiophene-2-carboxa-
mido)phenylamino)-2-oxoacetic acid (7d).
Following the
preparation protocol of Section 5.1.2.4, the reaction mixture of
compound 6d (0.15 g, 0.29 mmol) and NaOH (0.058 g, 1.45 mmol)
in water (2.0 mL), THF (2 mL) and acetone (2.0 mL) was stirred at
room temperature for 60 min to give compound 7d as yellow solid
(0.12 g, 84%); mp 200–201 °C; 1H NMR (DMSO-d6) d (ppm): 11.39
(s, 1H, NH), 10.92 (s, 1H, NH), 8.93 (d, J = 1.8 Hz, 1H, ArH), 8.49 (s,
1H, ArH), 8.34 (d, J = 9.0 Hz, 1H, ArH), 8.26 (dd, J1 = 9.3 Hz,
J = 1.8 Hz, 1H, ArH), 8.12 (m, 2H, ArH), 7.98 (s, 1H, ArH), 7.77 (d,
J = 7.2 Hz, 2H, ArH), 7.69 (d, J = 7.2 Hz, 1H, ArH), 7.57 (t,
J = 7.2 Hz, 2H, ArH); HRMS (ESI): m/z, Calcd for C24H16N3O7S
[M+H+]: 490.0703, Found 490.0707.
5.1.7.2. 2-(2-Benzoyl-4-(1H-indole-2-carboxamido)phenylami-
no)-2-oxoacetic acid (7f).
Following the preparation protocol
of Section 5.1.2.4, the reaction mixture of compound 6f (0.15 g,
0.33 mmol) and NaOH (0.066 g, 1.64 mmol) in water (5.0 mL) and
THF (5.0 mL) was stirred at room temperature for 30 min to give
compound 7f as yellow solid (0.095 g, 67%); mp 210–212 °C; 1H
NMR (DMSO-d6) d (ppm): 11.41 (s, 1H, NH), 10.78 (s, 1H, NH),
8.08–8.13 (m, 3H, ArH), 7.69–7.83 (m, 6H, ArH), 7.58 (t,
J = 7.5 Hz, 2H, ArH), 7.51 (d, J = 7.2 Hz, 1H, ArH), 7.37 (d,
J = 7.8 Hz, 1H, ArH); HRMS (ESI): m/z, Calcd for
C24H17N2O6
[M+H+]: 429.1081, Found 429.1092.
5.1.6. 2-(2-Benzoyl-4-(1H-indole-2-carboxamido)phenylamino)
-2-oxoacetic acid (7e)
5.1.6.1. Ethyl 2-(2-benzoyl-4-(1H-indole-2-carboxamido)phe-
5.1.8. 2-(4-Acetamido-2-benzoylphenylamino)-2-oxoacetic acid
(7g)
nylamino)-2-oxoacetate (6e).
The mixture of 1H-indole-2-
5.1.8.1. Ethyl 2-(4-acetamido-2-benzoylphenylamino)-2-oxoac-
carboxalic acid (0.1 g, 0.62 mmol), SOCl2 (0.22 g, 1.86 mmol) and
one drop DMF in CH2Cl2 (2 mL) was refluxed for 2 h. The crude
product of 1H-indole-2-carboxachloride was obtained as yellow
solid after evaporation and directly used in the next step without
further purification.
etate (6g).
The reaction mixture of compound 5a (0.10 g,
0.32 mmol) and acetic anhydride (0.065 g, 0.64 mmol) in acetic
acid (5 mL) was stirred at 110 °C for 1 h. Then the reaction mixture
was poured into ice water, after filtration and recrystalization, the
title compound 6g was obtained as yellow solid (0.083 g, 73%); mp
181–182 °C; 1HNMR (DMSO-d6) d (ppm): 11.29 (s,1H, NH), 10.14
(s,1H, NH), 7.93 (d, J = 8.7 Hz, 1H, ArH), 7.84 (d, J = 8.7 Hz, 1H,
ArH), 7.66 (m, 4H, ArH), 7.53 (m, 2H, ArH), 4.24 (q, J = 6.9 Hz, 2H,
CH2), 2.00 (s,3H, CH3), 1.25 (t, J = 7.2 Hz, 3H, CH3); HRMS (ESI):
m/z, Calcd for C19H19N2O5 [M+H+]: 355.1288, found 355.1282.
Following the preparation protocol of Section 5.1.2.3, the reac-
tion mixture of compound 5a (0.13 g, 0.41 mmol), 1H-indole-2-
carboxachloride and DIEA (0.08 g, 0.62 mmol) in toluene (3 mL)
was heated at 110 °C for 2.0 h to give compound 6e as yellow solid
(0.135 g, 66%); mp 118–120 °C; 1H NMR (DMSO-d6) d (ppm): 11.76
(s, 1H, NH), 11.41 (s, 1H, NH), 10.44 (s, 1H, NH), 8.09–8.15 (m, 3H,
ArH), 7.78 (d, J = 7.2 Hz, 2H, ArH), 7.68 (t, J = 7.5 Hz, 2H, ArH), 7.58
(t, J = 7.8 Hz, 2H, ArH), 7.40–7.46 (m, 2H, ArH), 7.22 (t, J = 6.9 Hz,
1H, ArH), 7.06 (t, J = 7.5 Hz, 1H, ArH), 4.27 (q, J = 6.9 Hz, 2H, CH2),
1.28 (t, J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for
5.1.8.2.
acid (7g).
2-(4-Acetamido-2-benzoylphenylamino)-2-oxoacetic
Following the preparation protocol of Section
5.1.2.4, the reaction mixture of compound 6g (0.05 g, 0.14 mmol)
and NaOH (0.028 g, 0.70 mmol) in water (1.5 mL) and THF
(3.0 mL) was stirred at room temperature for 90 min to give com-
pound 7g as yellow solid (0.039 g, 85%); mp 178–179 °C; 1HNMR
(DMSO-d6) d (ppm): 11.32 (s,1H, NH), 10.14 (s,1H, NH), 8.01 (d,
J = 8.7 Hz, 1H, ArH), 7.84 (d, J = 8.7 Hz, 1H, ArH), 7.78 (s, 1H, ArH),
7.64–7.72 (m, 3H, ArH), 7.51–7.56 (m, 2H, ArH), 2.00 (s,3H, CH3);
HRMS (ESI): m/z, Calcd for C17H15N2O5 [M+H+] 327.0975, Found
327.0992.
C
26H22N3O5 [M+H+]: 456.1554, Found 456.1564.
5.1.6.2. 2-(2-Benzoyl-4-(1H-indole-2-carboxamido)phenylami-
no)-2-oxoacetic acid (7e). Following the preparation proto-
col of Section 5.1.2.4, the reaction mixture of compound 6e
(0.05 g, 0.11 mmol) and NaOH (0.022 g, 0.55 mmol) in water
(2.0 mL), THF (1 mL) and acetone (2.0 mL) was stirred at room tem-
perature for 60 min to give compound 7e as brown solid (0.043 g,
91%); mp 197–199 °C; 1H NMR (acedone-d6) d (ppm): 11.77 (s, 1H,
NH), 11.43 (s, 1H, NH), 10.45(s, 1H, NH), 8.10–8.14 (m, 3H, ArH),
7.79 (d, J = 6.9 Hz, 2H, ArH), 7.70 (t, J = 8.1 Hz, 2H, ArH), 7.58 (t,
J = 8.7 Hz, 2H, ArH), 7.43 (m, 2H, ArH), 7.22 (t, J = 7.2 Hz, 1H,
ArH), 7.06 (t, J = 7.2 Hz, 1H, ArH); HRMS (ESI): m/z, Calcd for
5.1.9. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-(3,4-dim
ethoxybenzoyl)phenylamino)-2-oxoacetic acid (7h)
5.1.9.1. (2-Chloro-5-nitrophenyl)(3,4-dimethoxyphenyl)metha-
none (2a).
The mixture of 2-chloro-5-nitrobenzoyl chloride
(3.0 g, 13.6 mmol), 1,2-dimethoxybenzene (10 mL, 78 mmol) and
AlCl3 (2.72 g, 20.5 mmol) was stirred for a while, which was sepa-
rated into three reaction tubes and heated by microwave (power
50 W, temperature 50 °C) for 30 min. The reaction mixture was
poured into 10% HCl aqueous solution cooled in ice bath, which
was extracted with EtOAc (100 mL Â 3). The combined organic
layer was washed with brine (50 mL Â 1) and dried over anhydrous
Na2SO4. The crude product was provided after evaporation and
C
24H18N3O5 [M+H+]: 428.1241, Found 428.1252.
5.1.7. 2-(4-(Benzofuran-2-carboxamido)-2-benzoylphenylami
no)-2-oxoacetic acid (7f)
5.1.7.1. Ethyl 2-(4-(benzofuran-2-carboxamido)-2-benzoylphe-
nylamino)-2-oxoacetate (6f).
The mixture of benzofuran-2-
carboxalic acid (0.20 g, 1.23 mmol), SOCl2 (0.44 g, 3.70 mmol)

C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
2997
recrystalized with EtOAc to give compound 2a as white solid
(0.71 g, 16%); mp 135–137 °C; 1H NMR (CDCl3) d (ppm): 8.29 (dd,
J1 = 8.4 Hz, J2 = 2.7 Hz, 1H, ArH), 8.24 (d, J = 2.4 Hz, 1H, ArH), 7.67
(d, J = 9.0 Hz, 1H, ArH), 7.59 (d, J = 2.1 Hz, 1H, ArH), 7.15 (dd,
J1 = 8.1 Hz, J2 = 1.5 Hz, 1H, ArH), 6.86 (d, J = 8.4 Hz, 1H, ArH), 3.97
(s, 3H, OCH3), 3.96 (s, 3H, OCH3); HRMS (ESI): m/z, Calcd for
lowing the preparation protocol of Section 5.1.2.4, the reaction
mixture of compound 6h (0.10 g, 0.19 mmol) and NaOH (0.025 g,
0.58 mmol) in water (5.0 mL) and acetone (5.0 mL) was stirred at
room temperature for 2 h to give compound 7h as yellow solid
(0.038 g, 39%); mp 225–227 °C; 1H NMR (DMSO-d6) d (ppm):
11.26 (s, 1H, NH), 10.74 (brs, 1H, NH), 8.36 (s, 1H, ArH), 7.99–
8.18 (m, 5H, ArH), 7.45–7.49 (m, 2H, ArH), 7.37–7.41 (m, 2H,
ArH), 7.13 (d, J = 8.4 Hz, 1H, ArH), 3.88 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3); HRMS (ESI): m/z, Calcd for C26H21N2O7S [M+H+]:
505.1064, Found 505.1066.
C
15H13ClNO5 [M+H+]: 322.0477, Found 322.0460.
5.1.9.2
(2-Amino-5-nitrophenyl)(3,4-dimethoxyphenyl)metha-
none (3b).
The reaction mixture of compound 2a (0.50 Â 2 g, 1.55 mmol)
and aqueous ammonia (2 Â 2 mL) was loaded into two reaction
tubes and irradiated by microwave (power 100 W, temperature
150 °C, pressure 180 psi) for 3 h respectively. The reaction mixture
was diluted with water (20 mL) and adjusted to pH = 2.0 with 10%
HCl aqueous solution. After stirring for 30 min, the precipitate was
filtered and recrystalized with EtOAc to afford compound 3b as
yellow solid (0.51 g, 54%); mp 224–226 °C; 1H NMR (acetone-d6)
5.1.10. 2-(4-(Benzo[b]thiophene-3-carboxamido)-2-(3,4-dim
ethoxybenzoyl)phenylamino)-2-oxoacetic acid (7i)
5.1.10.1. Ethyl 2-(4-(benzo[b]thiophene-3-carboxamido)-2-(3,4-
dimethoxybenzoyl)phenyl-amino)-2-oxoacetate (6i).
Fol-
lowing the preparation protocol of Section 5.1.2.3, the reaction
mixture of compound 5b (0.05 g, 0.13 mmol), benzo[b]thiophene-
3-carboxachloride (0.032 g, 0.16 mmol) and DIEA (0.021 g,
0.16 mmol) in toluene (3 mL) was heated at 110 °C for 2 h to give
compound 6i as yellow solid (0.022 g, 31%); mp 237–239 °C; 1H
NMR (DMSO-d6) d (ppm): 11.22 (s, 1H, NH), 10.54 (s, 1H, NH),
8.56 (s, 1H, ArH), 8.38 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H, ArH), 8.07
(m, 4H, ArH), 7.44–7.46 (m, 2H, ArH), 7.37 (d, J = 7.2 Hz, 2H,
ArH), 7.11 (d, J = 8.4 Hz, 1H, ArH), 4.26 (q, J = 6.9 Hz, 2H, CH2),
3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 1.27 (t, J = 6.9 Hz, 3H,
CH3); HRMS (ESI): m/z, Calcd for C28H25N2O7S [M+H+]: 533.1377,
Found 533.1380.
d
(ppm): 8.44 (d, J = 2.4 Hz, 1H, ArH), 8.13 (dd, J1 = 9.3 Hz,
J2 = 2.7 Hz, 1H, ArH), 7.61 (brs, 2H, NH), 7.30–7.36 (m, 2H, ArH),
7.10 (d, J = 7.5 Hz, 1H, ArH), 7.02 (d, J = 9.3 Hz, 1H, ArH), 3.93 (s,
3H, OCH3), 3.88 (s, 3H, OCH3); HRMS (ESI): m/z, Calcd for
C15H15N2O5 [M+H+]: 303.0975, Found 303.0945.
5.1.9.3. Ethyl 2-(2-(3,4-dimethoxybenzoyl)-4-nitrophenylami-
no)-2-oxoacetate (4b).
Following the preparation protocol
of Section 5.1.2.1, the reaction mixture of compound 3a (0.50 g,
1.66 mmol), chlorooxalic acid ethyl ester (0.23 g, 1.66 mol) in tolu-
ene (10 mL) was heated at 110 °C for 3 h to give compound 4b as
yellow solid (0.51 g, 76%); mp 134–136 °C; 1H NMR (acetone-d6)
d (ppm): 11.87 (brs, 1H, NH), 8.82 (d, J = 9.3 Hz, 1H, ArH), 8.52–
8.56 (m, 2H, ArH), 7.50 (d, J = 1.5 Hz, 1H, ArH), 7.44 (dd,
J1 = 8.7 Hz, J2 = 2.1 Hz, 1H, ArH), 7.12 (d, J = 8.1 Hz, 1H, ArH), 4.38
(q, J = 6.9 Hz, 2H, CH2), 3.95 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
1.37 (t, J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for
5.1.10.2. 2-(4-(Benzo[b]thiophene-3-carboxamido)-2-(3,4-dim-
ethoxybenzoyl)phenylamino)-2-oxoacetic acid (7i).
Fol-
lowing the preparation protocol of Section 5.1.2.4, the reaction
mixture of compound 6i (0.08 g, 0.16 mmol) and NaOH (0.031 g,
0.78 mmol) in water (3.0 mL) and THF (5.0 mL) was stirred at room
temperature for 2 h to give compound 7i as yellow solid (0.062 g,
78%); mp 232–234 °C; 1H NMR (DMSO-d6) d (ppm): 10.54 (brs,
1H, NH), 8.57 (s, 1H, ArH), 8.37 (m, 1H, ArH), 8.07–8.17 (m, 4H,
ArH), 7.37–7.46 (m, 4H, ArH), 7.12 (d, J = 8.7 Hz, 1H, ArH), 3.86 (s,
3H, OCH3), 3.83 (s, 3H, OCH3); HRMS (ESI): m/z, Calcd for
C
19H19N2O8 [M + H+]: 403.1136, Found 403.1139.
5.1.9.4. Ethyl 2-(4-amino-2-(3,4-dimethoxybenzoyl)phenylami-
no)-2-oxoacetate (5b). Following the preparation protocol of
C
26H21N2O7S [M+H+]: 505.1064, Found 505.1066.
Section 5.1.2.2, the reaction mixture of compound 4b (0.05 g,
0.12 mmol) and 10% Pd-C (0.01 g) in trifluoroethanol (5.0 mL)
was hydrogenated at room temperature and 1 atm for 10 h to pro-
vide compound 5b as yellow solid (0.042 g, 91%); mp 187–189 °C;
1H NMR (DMSO-d6) d (ppm): 7.65 (d, J = 8.7 Hz, 1H, ArH), 7.27–7.30
(m, 1H, ArH), 7.07 (d, J = 8.1 Hz, 1H, ArH), 6.77 (dd, J1 = 8.7 Hz,
J2 = 2.7 Hz, 1H, ArH), 6.72 (d, J = 2.4 Hz, 1H, ArH), 4.21 (q,
J = 7.2 Hz, 2H, CH2), 3.84 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 1.24 (t,
J = 6.9 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for C19H21N2O6
[M+H+]: 373.1394, Found 373.1355.
5.1.11. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-(3-fluoro-4-
methoxybenzoyl)phenyl-amino)-2-oxoacetic acid (7j)
5.1.11.1.
(2-Chloro-5-nitrophenyl)
(3-fluoro-4-methoxy-
phenyl)methanone (2b).
The mixture of 2-chloro-5-nitro-
benzoyl chloride (4.0 g, 18.18 mmol), 2-fluoroanisole (5.0 mL)
and AlCl3 (3.63 g, 27.27 mmol) was stirred at room temperature
for 24 h. The reaction mixture was poured into 10% HCl aqueous
solution cooled in ice bath, which was extracted with EtOAc
(100 mL Â 3). The combined organic layer was washed with brine
(100 mL Â 1) and dried over anhydrous Na2SO4. The crude product
was provided after evaporation and recrystalized with EtOAc to
give compound 2b as white solid (5.40 g, 96%); mp 113–115 °C;
1H NMR (DMSO-d6) d (ppm): 8.41 (dd, J1 = 8.7 Hz, J2 = 2.4 Hz, 1H,
ArH), 8.35 (d, J = 2.4 Hz, 1H, ArH), 7.88 (d, J = 8.7 Hz, 1H, ArH),
7.60–7.67 (m, 2H, ArH), 7.28 (t, J = 8.4 Hz, 1H, ArH), 4.01 (s, 3H,
OCH3); HRMS (ESI): m/z, Calcd for C14H10ClFNO4 [M+H+]:
310.0277, Found 310.0297.
5.1.9.5. Ethyl 2-(4-(benzo[b]thiophene-2-carboxamido)-2-(3,4-
dimethoxybenzoyl)phenyl-amino)-2-oxoacetate (6h).
Fol-
lowing the preparation protocol of Section 5.1.2.3, the reaction
mixture of compound 5b (0.05 g, 0.13 mmol), benzo[b]thiophene-
2-carboxachloride (0.032 g, 0.16 mmol) and DIEA (0.021 g,
0.16 mmol) in toluene (3 mL) was heated at 110 °C for 2 h to give
compound 6h as yellow solid (0.03 g, 42%); mp 262–263 °C; 1H
NMR (DMSO-d6) d (ppm): 11.23 (s, 1H, NH), 10.72 (s, 1H, NH),
8.34 (s, 1H, ArH), 7.98–8.05 (m, 5H, ArH), 7.43–7.49 (m, 2H, ArH),
7.37 (d, J = 7.8 Hz, 2H, ArH), 7.12 (d, J = 8.4 Hz, 1H, ArH), 4.25 (q,
J = 7.2 Hz, 2H, CH2), 3.86 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 1.27 (t,
J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for C28H25N2O7S
[M+H+]: 533.1377, Found 533.1388.
5.1.11.2. (2-Amino-5-nitrophenyl) (3-fluoro-4-methoxyphenyl)
methanone (3c).
The reaction mixture of compound 2b
(2.9 g, 9.38 mmol) and aqueous ammonia (12 mL) was separated
into six tubes and irradiated by microwave (power 100 W, temper-
ature 150 °C, pressure 180 psi) for 3 h respectively. The reaction
mixture was diluted with water (20 mL) and adjusted to pH = 7.0
with 10% HCl aqueous solution. After stirring for 30 min, the pre-
cipitate was filtered and recrystalized with EtOAc to afford com-
5.1.9.6. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-(3,4-dim-
ethoxybenzoyl)phenylamino)-2-oxoacetic acid (7h).
Fol-

2998
C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
pound 3c as yellow solid (1.28 g, 47%); mp 182–184 °C; 1H NMR
(acetone-d6) d (ppm): 8.39 (d, J = 2.4 Hz, 1H, ArH), 8.14 (dd,
J1 = 9.3 Hz, J2 = 2.7 Hz, 1H, ArH), 7.54–7.58 (m, 2H, ArH), 7.31 (t,
J = 8.1 Hz, 1H, ArH), 7.03 (d, J = 9.0 Hz, 1H, ArH), 4.02 (s, 3H,
OCH3); HRMS (ESI): m/z, Calcd for C14H12FN2O4 [M+H+]:
291.0776, Found 291.0762.
toluene (3 mL) was heated at 110 °C for 1 h to give compound 6l
as yellow solid (0.058 g, 80%); mp 210–211 °C; 1H NMR (DMSO-
d6) d (ppm): 11.19 (brs, 1H, NH), 10.56 (brs, 1H, NH), 8.58 (s, 1H,
ArH), 8.38 (m, 1H, ArH), 8.06–8.10 (m, 2H, ArH), 7.98 (d, 1H,
J = 1.8 Hz, ArH), 7.93 (d, 1H, J = 8.4 Hz, ArH), 7.58 (d, J = 10.2 Hz,
2H, ArH), 7.44 (m, 2H, ArH), 7.33 (t, J = 7.2 Hz, 1H, ArH), 4.24 (q,
J = 6.9 Hz, 2H, OCH2), 3.94 (s, 3H, OCH3), 1.26 (t, J = 6.9 Hz, 3H,
CH3); HRMS (ESI): m/z, Calcd for C27H22FN2O6S [M+H+]:
521.1177, Found 521.1196.
5.1.11.3. Ethyl 2-(2-(3-fluoro-4-methoxybenzoyl)-4-nitrophe-
nylamino)-2-oxoacetate (4c).
Following the preparation pro-
tocol of Section 5.1.2.1, the reaction mixture of compound 3b
(0.85 g, 2.93 mmol), chlorooxalic acid ethyl ester (0.40 g,
2.93 mol) in toluene (25 mL) was heated at 110 °C for 2 h to give
compound 4c as white solid (0.95 g, 83%); mp 152–154 °C; 1H
NMR (400 MHz, acetone-d6) d (ppm): 11.83 (brs, 1H, NH), 8.82
(d, J = 9.2 Hz, 1H, ArH), 8.56 (d, J = 9.2 Hz, 1H, ArH), 8.52 (d,
J = 2.4 Hz, 1H, ArH), 7.71 (m, 2H, ArH), 7.35 (t, J = 8.4 Hz, 1H,
ArH), 4.39 (q, J = 7.2 Hz, 2H, CH2), 4.05 (s, 3H, OCH3), 1.38 (t,
J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for C18H16FN2O7
[M+H+]: 391.0936, Found 391.0948.
5.1.12.2. 2-(4-(Benzo[b]thiophene-3-carboxamido)-2-(3-fluoro-
4-methoxybenzoyl)phenyl-amino)-2-oxoacetic
acid
(7k). Following the preparation protocol of Section 5.1.2.4,
the reaction mixture of compound 6k (0.04 g, 0.08 mmol) and
NaOH (0.015 g, 0.38 mmol) in water (4.0 mL) and THF (3.0 mL)
was stirred at room temperature for 2 h to give compound 7k as
yellow solid (0.027 g, 71%); mp 262–264 °C; 1H NMR (DMSO-d6)
d (ppm): 11.20 (brs, 1H, NH), 10.55 (brs, 1H, NH), 8.58 (s, 1H,
ArH), 8.38 (m, 1H, ArH), 8.00–8.11 (m, 4H, ArH), 7.61 (d,
J = 9.9 Hz, 2H, ArH), 7.44–7.49 (m, 2H, ArH), 7.33 (t, J = 8.4 Hz,
1H, ArH), 3.94 (s, 3H, OCH3); HRMS (ESI): m/z, Calcd for
5.1.11.4. Ethyl 2-(4-amino-2-(3-fluoro-4-methoxybenzoyl)phe-
nylamino)-2-oxoacetate (5c).
Following the preparation pro-
C
25H18FN2O6S [M+H+]: 493.0864, Found 493.0878.
tocol of Section 5.1.2.2, the reaction mixture of compound 4c
(0.05 g, 0.12 mmol) and 10% Pd-C (0.01 g) in trifluoroethanol
(5.0 mL) was hydrogenated at room temperature and 1 atm for
10 h to provide compound 5c as yellow solid (0.035 g, 76%); mp
194–195 °C; 1H NMR (DMSO-d6) d (ppm): 10.77 (s, 1H, NH),
7.48–7.57 (m, 3H, ArH), 7.29 (t, J = 8.4 Hz, 1H, ArH), 6.78 (dd,
J1 = 8.4 Hz, J2 = 2.1 Hz, 1H, ArH), 6.67(d, J = 2.4 Hz, 1H, ArH), 5.37
(brs, 1H, NH), 4.20 (q, J = 7.2 Hz, 2H, CH2), 3.92 (s, 3H, OCH3),
1.23 (t, J = 7.2 Hz, 3H, CH3); HRMS (ESI): m/z, Calcd for C18H18FN2O5
[M+H+]: 361.1194, Found 361.1197.
5.2. Biological evaluation
5.2.1. Protein expression and purification
The Pet28a-Pin1 plasmid was a gift from Professor Joseph P.
Noel (The Salk Institute for Biological Studies, La Jolla, California).
The N-terminally His6-tagged Pin1 was expressed at 22 °C in
E. coli strain BL21 following induction at an optical density of 0.6
(600 nm) with 0.5 mM IPTG for 20 h in terrific broth. Cells were
resuspended in 25 mM Tris–Cl, 500 mM NaCl, 10 mM imidazole,
100 lg/mL cocktail, 0.5 mg/mL lysozyme. Following sonication at
5.1.11.5. Ethyl 2-(4-(benzo[b]thiophene-2-carboxamido)-2-(3-
fluoro-4-methoxybenzoyl)-phenylamino)-2-oxoacetate
4 °C, the soluble supernatant was loaded onto an Ni-NTA (Qiagen)
column and washed with 10 bed volumes of washing buffer
(50 mM imidazole, 500 mM NaCl, 20 mM Tris–Cl). His6-Pin1 was
eluted with 400 mM imidazole, 500 mM NaCl, 20 mM Tris–Cl and
condensed by ultrafiltration (Millipore 5 kDa) with 20 mM
Tris–Cl, 100 mM NaCl, 5 mM DTT.
(6j).
Following the preparation protocol of Section 5.1.2.3, the
reaction mixture of compound 5c (0.20 g, 0.56 mmol) and
benzo[b]thiophene-2-carboxachloride (0.11 g, 0.56 mmol) in tolu-
ene (10 mL) was heated at 110 °C for 2 h to give compound 6j as
yellow solid (0.23 g, 79%); mp 242–244 °C; 1H NMR (DMSO-d6) d
(ppm): 11.21 (brs, 1H, NH), 10.71 (brs, 1H, NH), 8.34 (s, 1H, ArH),
7.96–8.09 (m, 5H, ArH), 7.59 (d, J = 9.6 Hz, 2H, ArH), 7.43–7.51
(m, 2H, ArH), 7.34 (t, J = 8.4 Hz, 1H, ArH), 4.25 (q, J = 6.9 Hz, 2H,
CH2), 3.95 (s, 3H, OCH3), 1.26 (t, J = 6.9 Hz, 3H, CH3); HRMS (ESI):
m/z, Calcd for C27H22FN2O6S [M+H+]: 521.1177, Found 521.1166.
5.2.2. Pin1 PPIase assay and IC50 measurements of Pin1
inhibitors
PPIase activities were measured at 10 °C JASCO V-650 spectro-
photometer using protease-coupled assay according to Wang
et al.18,19 Suc-Ala-Glu-Pro-Phe-4-nitroanilide in 0.47 M LiCl/trifluo-
roethanol was used as the substrate. In brief, the assay buffer
5.1.11.6. 2-(4-(Benzo[b]thiophene-2-carboxamido)-2-(3-fluoro-
(840
solution), and inhibitors (10
were preequilibrated in the cuvette at 10 °C for 30 min. Then,
100 L of ice-cooled chymotrypsin (60 mg/mL in 0.001 M HCl)
was added and mixed immediately. Additional 40 L of substrate
was added to start the assay and the reaction was monitored by
absorbance at 390 nM for 90 s. The data was analyzed by Graphpad
Prism 5.01.
l
L of 35 mM HEPES at PH 7.8), Pin1(10
lL of 850 lg/mL stock
4-methoxybenzoyl)phenyl-amino)-2-oxoacetic
acid
lL of varying concentrations in DMSO)
(7j). Following the preparation protocol of Section 5.1.2.4,
the reaction mixture of compound 6j (0.10 g, 0.20 mmol) and
NaOH (0.038 g, 0.96 mmol) in water (5.0 mL) and THF (10.0 mL)
was stirred at room temperature for 2 h to give compound 7j as
yellow solid (0.065 g, 68%); mp > 300 °C; 1H NMR(DMSO-d6) d
(ppm): 11.24 (brs, 1H, NH), 10.73 (brs, 1H, NH), 8.36 (s, 1H, ArH),
8.15 (m, 1H, ArH), 7.97–8.07 (m, 4H, ArH), 7.60–7.64 (m, 2H,
ArH), 7.43–7.49 (m, 2H, ArH), 7.34 (t, J = 8.7 Hz, 1H, ArH), 3.95 (s,
3H, OCH3); HRMS (ESI): m/z, Calcd for C25H18FN2O6S [M+H+]:
493.0864, Found 493.0883.
l
l
5.3. Computational studies
All calculations and manipulations were performed using FlexX
software package integrated in Sybyl 7.220, running on SGI Fuel
workstation. The X-ray crystal structure of Pin1 complexed with
a carboxylate inhibtor was retrieved from PDB (PDB code: 3JYJ).13
In FlexX docking, the Receptor Description File (RDF) described
the active site environment. It contains the information about
the protein, its amino acids, the active site, non-amino acid resi-
dues, and specific torsion angles. The active site was defined as
all residues within 6.5 Å radius of the cocrystallized reference
5.1.12. 2-(4-(Benzo[b]thiophene-3-carboxamido)-2-(3-fluoro-4-
methoxybenzoyl)phenyl-amino)-2-oxoacetic acid (7k)
5.1.12.1. Ethyl 2-(4-(benzo[b]thiophene-3-carboxamido)-2-(3-
fluoro-4-methoxybenzoyl)-phenylamino)-2-oxoacetate
(6k).
Following the preparation protocol of Section 5.1.2.3,
the reaction mixture of compound 5c (0.05 g, 0.14 mmol) and
benzo[b]thiophene-3-carboxachloride (0.027 g, 0.14 mmol) in

C. Liu et al. / Bioorg. Med. Chem. 20 (2012) 2992–2999
2999
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9. Mori, T.; Uchida, T. Curr. Enz. Inhib. 2010, 6, 46.
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molecule. The default Sybyl/FlexX parameters were used. The syn-
thesized title compounds was built using the Sybyl Sketcher model
and fully minimized with the Powell method (Tripos force field and
Gasteiger–Huckel charges) to an energy gradient of 0.05 kcal/
(mol Å). The 30 final docked conformations were ranked according
to their binding free energy. The docking mode was chosen on the
basis of binding affinity rank.
13. Dong, L.; Marakovits, J.; Hou, X.; Guo, C.; Greasley, S.; Dagostino, E.; Ferre, R.;
Johnson, M. C.; Kraynov, E.; Thomson, J.; Pathak, V.; Murry, B. W. Bioorg. Med.
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Acknowledgments
14. Potter, A. J.; Ray, S.; Gueritz, L.; Nunns, C. L.; Bryant, C. J.; Scrace, S. F.;
Matassova, N.; Baker, L.; Dokurno, P.; Robinson, D. A.; Surgenor, A. E.; Davis, B.;
Murry, J. B.; Richardson, C. M.; Moore, J. D. Bioorg. Med. Chem. Lett. 2010, 20,
586.
This work is supported by the National Natural Science Founda-
tion of China (No. 30500634), NSF of Beijing (No. 7102112) and
‘‘863’’ Program of China (No. 2012AA020302).
15. Potter, A. J.; Oldfield, V.; Nunns, C.; Fromont, C.; Ray, S.; Northfield, C. J.; Bryant,
C. J.; Scrace, S. F.; Robinson, D.; Matossova, N.; Baker, L.; Dokurno, P.; Surgenor,
A. E.; Davis, B.; Richardson, C. M.; Murray, J. B.; Moore, J. D. Bioorg. Med. Chem.
Lett. 2010, 20, 6483.
16. Zhu, L.; Jin, J.; Liu, C.; Zhang, C.; Sun, Y.; Guo, Y.; Fu, D.; Chen, X.; Xu, B. Bioorg.
Med. Chem. 2011, 19, 2797.
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