Design, synthesis and biological evaluation of benzimidazole derivatives as novel human Pin1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.11.045

In this work, a series of novel benzimidazole derivatives were designed and synthesized as Pin1 inhibitors. Protease-coupled assay was used to investigate the Pin1 inhibitory potency of all synthesized compounds. Thirteen of them showed preferable Pin1 inhibitory effects with IC₅₀ values lower than 5 μM, and 12a, 15b, 15d and 16c exhibited the most promising Pin1 inhibitory activity at low micromolar level (0.33–1.00 μM) than the positive control compound Juglone. Flow cytometry results showed that treating PC-3 cells with 16c caused slight cycle arrest in a concentration-dependent manner. The structure-activity relationships of R¹, R², R³ and linker of the benzimidazole derivatives were analyzed in detail, which would help further exploration of new Pin1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2018.11.045

Accepted Manuscript
Design, synthesis and biological evaluation of benzimidazole derivatives as
novel human Pin1 inhibitors
Tianyi Ma, Min Huang, Aihua Li, Feng Zhao, Deyi Li, Dan Liu, Linxiang Zhao
PII:
S0960-894X(18)30922-3
https://doi.org/10.1016/j.bmcl.2018.11.045
BMCL 26156
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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28 September 2018
19 November 2018
22 November 2018
Please cite this article as: Ma, T., Huang, M., Li, A., Zhao, F., Li, D., Liu, D., Zhao, L., Design, synthesis and
biological evaluation of benzimidazole derivatives as novel human Pin1 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.045
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Design, synthesis and biological evaluation of benzimidazole derivatives as novel human Pin1
inhibitors
Tianyi Ma, Min Huang, Aihua Li, Feng Zhao, Deyi Li, Dan Liu* and Linxiang Zhao*
Key Laboratory of Structure-based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, P. R. China.
*Corresponding author:
Dan Liu: Tel: +86-024-43520218; E-mail: liudan@syphu.edu.cn
Linxiang Zhao: Tel: +86-024-43520221; E-mail: linxiang.zhao@vip.sina.com
ABSTRACT
In this work, a series of novel benzimidazole derivatives were designed and synthesized as Pin1
inhibitors. Protease-coupled assay was used to investigate the Pin1 inhibitory potency of all
synthesized compounds. Thirteen of them showed preferable Pin1 inhibitory effects with IC50 values
lower than 5 μM, and 12a, 15b, 15d and 16c exhibited the most promising Pin1 inhibitory activity at
low micromolar level (0.33~1.00 μM) than the positive control compound Juglone. Flow cytometry
results showed that treating PC-3 cells with 16c caused slight cycle arrest in a concentration-dependent
manner. The structure-activity relationships of R1, R2, R3 and linker of the benzimidazole derivatives
were analyzed in detail, which would help further exploration of new Pin1 inhibitors.
Keywords: Pin1 inhibitors, PPIase, 4-(1H-benzoimidazol-2-yl)-benzsulfamides, PC-3, Prostate cancer

Pin1 (Protein interaction with never in mitosis A1) is a kind of PPIase (peptidyl-prolyl cis-trans
isomerase) which specifically catalyzed the isomerization of pSer/Thr-Pro peptide bonds in its substrate
proteins to modulate their function and stability [1-3]. Abnormal expression of Pin1 often led to a
variety of diseases especially cancer due to that pSer/pThr-Pro sequence existed in many functional
proteins and regulated cell proliferation and differentiation [4-6]. Pin1 is overexpressed in various types
of cancer, including prostate cancer, hepatic cancer, esophageal cancer and so on [7]. The expression of
Pin1 correlates with malignant grade and poor prognosis in patients with cancer. Pin1 is reported to be
involved in regulating many signaling processes such as cell cycle controllers, which was known to be
regulated by Pin1 activity. In addition, Pin1 knockout mice developed normally to adulthood indicated
that Pin1 was expected to become an ideal antitumor drug target [8].
There has been persistent interest for developing Pin1 inhibitors, and numbers of promising
entities have been identified (Figure 1) [9-12]. Juglone was the first Pin1 inhibitor discovered by
high-throughput screening, which was used as positive control widely at present [13]. The α,
β-unsaturated ketone structure could interact with thiol of Cys113 in PPIase domain through Michael
addition and played irreversible inhibition activity against Pin1. Peptidomimetics Pin1 inhibitors
(PEP-3) exhibited strong enzyme inhibition activity at the molecular level [14-23], while it had the

poor stability and cell membrane permeability. Other small molecule inhibitors were designed based on
PPIase activity pocket of Pin1, just like PFI-1 [24-27]. Unfortunately, most of these compounds did not
show antitumor activity on cancer cells. A variety of strategies have been taken later to improve the
activity of the compounds in cell level without breakthrough [28]. Our objective was to identify some
small molecules as pin1 inhibitors with antiproliferative activity.
(Figure 1 should be listed here)
Figure 1. The structures of reported Pin1 inhibitors
Previously reported Pin1 inhibitors usually consisted of three fragments, forming necessary
interactions with prolyl pocket, hydrophobic superficial area and basic amino acids enrichment region
respectively. According to Jonathan D. Moore’s study, the 3-(1H-benzo[d]imidazol-2-yl) propanoic
acid moiety had been validated fitted well into the prolyl pocket and basic domain of Pin1. Furthermore,
the docking result confirmed that an extra hydrophobic fragment at N1-position of benzimidazole
occupied the shallow surface of Pin1 protein could enhance Pin1 inhibitory activity significantly
(Figure 2). Therefore, hydrophobicity aromatic fragments were introduced to make the target
compounds further combined with hydrophobic superficial PPIase domain. Besides, conformation
qualified of compounds can reduce entropy penalty in the process of the ligand molecules binding to a
receptor protein, and increase the stability of the complexes [29]. Therefore, we hoped to reduce
polarity while maintain Pin1 inhibitory activity through replacing fatty acids with aromatic
hydrophobic acids. As a result, the flexible propionic acid segments of benzimidazole C2-position were
replaced with the rigid benzoic acids. Since the carboxyl performed to be hydrophilic excessively
which was unfavorable to cell activity, we suspected that increasing hydrophobic moiety and replacing
with bioisosteres will give Pin1 inhibitors with cell activity.
(Figure 2 should be listed here)

Figure 2. The predicated binding modes of 3-(1H-benzo[d]imidazol-2-yl) propanoic acid moiety
The synthetic route adopted for preparation of target compounds 5a~5c was outlined in Scheme 1.
The cyclization reaction of succinic anhydride and succinic anhydride afforded 1 easily in high yields,
which was protected by a benzyl using benzyl chloride under basic conditions and then alkylated to
give the key intermediate 3. Subsequently, in the presence of EDCI, the coupling of 3 with differently
substituted aniline yield 4. An efficient deprotection was performed to afford compounds 5a~5c.
(Scheme 1 should be listed here)
Scheme 1. Synthesis of compounds 5a~5c. Reagents and conditions: a) succinic anhydride, dioxane,
80 oC; b) BnCl, K2CO3, DMF, r.t.; c) 3-bromopropionic acid, K2CO3, acetone/DMF/H2O, 70 oC; d)
substituted aniline, EDCI, HOBt, CH2Cl2, r.t.; e) LiOH, THF/H2O, 10 oC or H2, 5% Pd/C, MeOH, r.t..
The synthetic route of target compounds 10a~10i, 12a~12d, 15a~15d and 16a~16c was described
in Scheme 2~ Scheme 4. Start material 4-carboxybenzaldehyde was protected by benzyl, then
cyclization and alkylation were proceeded to provide 8, which was condensed with different arylamines
and debenzylation was performed to afford compounds 10a~10i. The coupling of 8 with different
phenol or naphthol and deprotection was performed to afford compounds 12a~12d. Nucleophilic
substitution and deprotection of key intermediate 7 underwent readily to give compounds 15a~15d,
which continued to react with differently substituted sulfonamide to afford compounds 16a~16c.
(Scheme 2 should be listed here)

Scheme 2. Synthesis of compounds 10a~10i. Reagents and conditions: a) BnBr, K2CO3, DMF, r.t.; b)
1,2-diaminobenzene, DMF/H2O, 80oC; c) 3-bromopropionic acid, K2CO3, acetone/DMF/H2O, 70 oC; d)
substituted arylamine, EDCI, HOBt, 4-methylmorpholine, CH2Cl2, r.t. or substituted arylamine, SOCl2,
CH2Cl2, r.t.; e) LiOH, THF/H2O, 10 oC or H2, 5% Pd/C, MeOH, r.t..
(Scheme 3 should be listed here)
Scheme 3. Synthesis of compounds 12a~12d. Reagents and conditions: a) SOCl2, CH2Cl2, r.t.,
substituted phenol or naphthol, 5% NaOH, 10 oC; b) H2, 5% Pd/C, MeOH, r.t..
(Scheme 4 should be listed here)
Scheme 4. Synthesis of compounds 15a~15d and 16a~16c. Reagents and conditions: a)
1,3-dibromopropane, K2CO3, MeCN, 82 oC; b) substituted phenol or naphthol, K2CO3, acetone, reflux;
c) LiOH, THF/H2O, r.t.; d) R2SO2NH2, EDCI, DMAP, DMF, 0 oC to r.t..
Protease-coupled assay was used to investigate the Pin1 inhibitory potency of all synthesized
compounds. As shown in Table 1, compounds 5a~5c which bearing hydrophobicity aromatic
fragments accompanied using ethyl as linker possessed moderate Pin1 inhibitory activity (7.56~19.72
μM). Phenyl amide derivative 5a displayed modest activity with IC50 value of 19.72 μM. The
inhibitory activities of compounds 5b and 5c with monosubstituted phenyl of side chain were increased
to 9.81 μM and 7.56 μM, respectively.
(Table 1 should be listed here)

Table 1. The structures and Pin1 inhibitory activities of all the target compounds.
Compound
5a
X
Y
R1
R2
R3
IC50 (μM) a
CO
N
H
19.72
5b
CO
CO
CO
CO
CO
CO
CO
CO
CO
N
N
N
N
N
N
N
N
N
H
H
H
H
H
H
H
H
H
9.81
7.56
7.27
1.10
18.68
11.28
13.62
2.89
3.57
5c
10a
10b
10c
10d
10e
10f
10g
10h
10i
CO
CO
N
N
1.53
5.40

12a
12b
12c
12d
15a
15b
15c
15d
CO
O
O
O
O
O
O
O
O
-
-
-
-
-
-
-
-
13.89
0.72
1.02
1.87
1.89
0.47
2.20
0.33
CO
CO
CO
CH2
CH2
CH2
CH2
16a
16b
CH2
CH2
O
O
-
-
7.83
2.26
16c
CH2
-
O
-
-
-
1.00
Juglone
-
-
10.81
aThe IC50 value was measured at two independent experiments.
The inhibition data of compounds 10a and 10b with aromatic acids substitution verified the
hypothesis that introduction of benzoic acid caused to significant improvement on pin1 inhibition (10a
vs. 5b, 10b vs. 5c). The compounds 10c~10g with meta- electron-withdrawing or electron-donating
substituents of phenyl were synthesised for SARs study. Among them, compounds 10f and 10g showed
improved potency with IC50 of 2.89 μM and 3.57 μM, but other modifications were not tolerable. In
addition, 10 h (1.53 μM) and 10i (5.40 μM) with bulkier and more lipophilic segments on nitrogen was
unable to further enhance the pin1 inhibition potency. The reason for this phenomenon might be the
stereo-hindrance effect from the interaction of two aromatic rings. Therefore, follow-up studies focused

on single hydrophobic aromatic ring. Meanwhile, other linkers such as ester bond or ether bond were
investigated. In addition, compounds 16a~16c were prepared to investigate effects of carboxylic acid
bioisosteres [30].
Compounds 12a~12d replacing amide with ester bond of linker at N1-position of benzimidazole
exhibited different pin1 inhibition potency. Among them, compounds 12b~12d displayed outstanding
activity with IC50 value ranged from 0.72 μM to 1.87 μM, whereas 12a are less active among them.
Compounds 15a~15d bearing ether bond linker were designed and synthesized. The results showed
that 15a~15d displayed excellent pin1 inhibition potency with IC50 value ranged from 0.33 μM to 2.20
μM, and 15d (0.33 μM) was most potent. Taken together, ether bond between benzimidazole and
substituention of N1-position were better. Compounds 16a (7.83 μM), 16b (2.26 μM), 16c (1.00 μM)
are sulfonamides bioisosteres synthesized from the corresponding carboxylic acid 15d, compound 16c
with benzene sulfonamide formyl was the most excellent which showed 1.00 μM values against Pin1.
Pin1 inhibitors were anticipated to interfere with multiple cellular processes including cell-cycle
regulation, we therefore tested whether 16c which has the potential to be more cell-permeable
interfered with cell cycle. 16c was evaluated for the antiproliferative activity against PC-3 cell lines
using MTT assay, and the results indicate that 16c showed excellent activity with GI50 value of 9.0 μM.
The impact to cell cycle of 16c at 2.5 μM, 5 μM, and 10 μM were investigated through flow cytometry
to verify the antiproliferative activity were correlated with inhibitory activity against Pin1 (Figure 3).
The results showed that there was slight cycle arrest in a concentration-dependent manner in G0/G1
phase of PC-3 cells after treatment with 16c for 24 h. Therefore, these data provided considerable
evidence that the antiproliferative effects on PC-3 cells of 16c were mediated, at least partly, via
inhibition of Pin1.
(Figure 3 should be listed here.)
Figure 3. The regulation of cell cycle in PC-3 cells treated with 16c for 24 h.
Molecular docking simulation studies were carried out to explore the binding modes of target
compound 16c with Pin1 protein. The results in Figure 4 indicated that 16c retained the key interaction

in the PPIase domain. The nitrogen at 3-position of imidazole ring formed hydrogen bond with Ser154,
and sulfonyl of benzene sulfonamide formyl participated in hydrogen bond formation with Arg69
mimic carboxyl. In addition, the naphthalene of benzimidazole at C2-position formed hydrophobic
interaction with the surrounding nonpolar amino acid. By analyzing the interactions mentioned above,
it was cogent that the benzene sulfonamide formyl and naphthalene moiety were greatly helpful to
enhance the Pin1 inhibition ability.
(Figure 4 should be listed here.)
Figure 4. The predicated binding modes of 16c with Pin1 PPIase domain
In summary, we synthesized a series novel 1,2-bis-substituted benzimidazoles derivatives as Pin1
inhibitors. The structure-activity relationship of these small-molecule inhibitors was discussed.
Compound 15d was the most potent compound with an IC50 value of 0.33 μM targeting Pin1. Flow
cytometry results indicated 16c induced slight cycle arrest in a concentration-dependent manner in
G0/G1 phase against PC-3 cells. The docking studies with pin1 protein showed that the benzimidazole
scaffold, benzene sulfonamide formyl moieties and naphthalene were necessary for target compounds
to possess potent antitumor activities. This study provides a different class of Pin1 inhibitors bearing
benzimidazole scaffold, which deserves further research.
Acknowledgment
This work was supported by the National Natural Science Foundation of China [Grant No.
81273360].

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Graphical abstract