Discovery of 4,6-bis(benzyloxy)-3-phenylbenzofuran as a novel Pin1 inhibitor to suppress hepatocellular carcinoma via upregulating microRNA biogenesis

Article abstract of DOI:10.1016/j.bmc.2019.04.028

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)participates in diverse cancer-associated signaling pathways, playing an oncogenic role in multiple human cancers, including hepatocellular carcinoma (HCC). Our recent works clarify that Pin1 modulates miRNAs biogenesis by interacting with ERK-phosphorylated exportin-5 (XPO5)and changing XPO5 conformation, giving a potential target for HCC treatment. Herein, we discover 4,6-bis(benzyloxy)-3-phenylbenzofuran (TAB29)as a novel Pin1 inhibitor that targets Pin1 PPIase domain. TAB29 potently inhibits Pin1 activity with the IC₅₀ value of 874 nM and displays an excellent selectivity toward Pin1 in vitro. Cell-based biological evaluation reveals that TAB29 significantly suppresses cell proliferation of HCC cells through restoring the nucleus-to-cytoplasm export of XPO5 and upregulating mature miRNAs expression. Collectively, this work provides a promising small molecule lead compound for Pin1 inhibition, highlighting the therapeutic potential of miRNA-based treatment for human cancers.

Full text of DOI:10.1016/j.bmc.2019.04.028

Accepted Manuscript
Discovery of 4,6-bis(benzyloxy)-3-phenylbenzofuran as a novel Pin1 inhibitor
to suppress hepatocellular carcinoma via upregulating microRNA biogenesis
Xin Fan, Huaiyu He, Jiao Li, Guoyong Luo, Yuanyuan Zheng, Jian-Kang Zhou,
Juan He, Wenchen Pu, Yun Zhao
PII:
S0968-0896(19)30124-5
https://doi.org/10.1016/j.bmc.2019.04.028
BMC 14882
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2019
9 April 2019
18 April 2019
Please cite this article as: Fan, X., He, H., Li, J., Luo, G., Zheng, Y., Zhou, J-K., He, J., Pu, W., Zhao, Y., Discovery
of 4,6-bis(benzyloxy)-3-phenylbenzofuran as a novel Pin1 inhibitor to suppress hepatocellular carcinoma via
upregulating microRNA biogenesis, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.04.028
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Discovery of 4,6-bis(benzyloxy)-3-
phenylbenzofuran as a novel Pin1 inhibitor
to suppress hepatocellular carcinoma via
upregulating microRNA biogenesis
Xin Fan, Huaiyu He, Jiao Li, Guoyong Luo, Yuanyuan Zheng, Jian-Kang Zhou, Juan He, Wenchen Pu and Yun
Zhao

Bioorganic & Medicinal Chemistry
Discovery of 4,6-bis(benzyloxy)-3-phenylbenzofuran as a novel Pin1 inhibitor to
suppress hepatocellular carcinoma via upregulating microRNA biogenesis
Xin Fana,c, Huaiyu Hea,c, Jiao Lia, Guoyong Luob, Yuanyuan Zhenga, Jian-Kang Zhoua, Juan Hea, Wenchen
a,
a,


Pu and Yun Zhao
a Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Cancer Center, West China Hospital, Sichuan
University, Chengdu 610041, China.
b Guiyang College of Traditional Chinese Medicine, Guiyang 550025, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) participates in diverse cancer-
associated signaling pathways, playing an oncogenic role in multiple human cancers, including
hepatocellular carcinoma (HCC). Our recent works clarify that Pin1 modulates miRNAs
biogenesis by interacting with ERK-phosphorylated exportin-5 (XPO5) and changing XPO5
conformation, giving a potential target for HCC treatment. Herein, we discover 4,6-
bis(benzyloxy)-3-phenylbenzofuran (TAB29) as a novel Pin1 inhibitor that targets Pin1 PPIase
domain. TAB29 potently inhibits Pin1 activity with the IC50 value of 874 nM and displays an
excellent selectivity toward Pin1 in vitro. Cell-based biological evaluation reveals that TAB29
significantly suppresses cell proliferation of HCC cells through restoring the nucleus-to-cytoplasm
export of XPO5 and upregulating mature miRNAs expression. Collectively, this work provides a
promising small molecule lead compound for Pin1 inhibition, highlighting the therapeutic
potential of miRNA-based treatment for human cancers.
Keywords:
Pin1
hepatocellular carcinoma
microRNA
benzofuran
XPO5
2009 Elsevier Ltd. All rights reserved.
1. Introduction
biogenesis is the reason for the downregulation of mature
miRNAs.9 During the process of miRNA biogenesis, exportin-5-
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1),
belongs to the peptidyl-prolyl isomerase (PPIase) family, is the
only known enzyme that catalyzes the cis-trans isomerization of
phosphorylated Serine/Threonine-Proline (pS/T-P) motif of
substrate.1,2 Pin1 is widely overexpressed in human cancers and
participates in diverse cancer-associated signaling pathways. For
instance, Pin1 binds to CDK4-phosphoralated p53-R249S mutant
and activates c-myc, facilitating the gain of function of mutant p53
in hepatocellular carcinoma (HCC).3 Cullin3 (Cul3) substrate
adaptor KLHL20 coordinates with the actions of CDK1/2 and Pin1
to mediate hypoxia-induced PML proteasomal degradation,
potentiating prostate cancer progression.4 Thus, Pin1 plays an
oncogenic role in multiple human cancers.
Hepatocellular carcinoma (HCC) is the most critical type of
primary liver cancer in adults, and is one of the leading causes of
cancer death worldwide.5 In HCC, microRNAs (miRNAs), a class
of small noncoding RNAs that regulate gene expression by
repressing protein translation or destabilizing target mRNAs,6 are
aberrantly downregulated.7,8 Importantly, compromised miRNAs
mediated (XPO5-mediated) nucleus-to-cytoplasm export of
precursor miRNA (pre-miRNA) is the rate-limiting step.10-12
Recently, we have demonstrated that Pin1 changes the
conformation of XPO5 phosphorylated by ERK at
T345/S416/S497, prevents microRNAs biosynthesis, thereby
contributing to HCC development.13,14 Therefore, Pin1 is an
attractive drug target for HCC treatment.
Increasing endeavor have been devoted to the investigation of
Pin1 inhibition. Early stage small molecule Pin1 inhibitors, such
as juglone, EGCG and PiB, have been discovered by low
throughput screening and exhibited moderate Pin1-inhibitory
activity.15 The juglone analogue KPT-6566 covalently interacts
with Pin1 and promotes its structural change and degradation,
inhibiting cancer cell proliferation.16 All-trans retinoic acid
(ATRA) selectively inhibits and degrades active Pin1 in acute
promyelocytic leukemia and breast cancer cells by directly binding
to the Pin1 active site.17 Recently, 6-O-benzylguanine derivative
API-1 has been found to locate into Pin1 PPIase domain and
———
* Corresponding author. E-mail addresses: zhaoyun@scu.edu.cn (Y. Zhao), pu_vincent@scu.edu.cn (W. Pu)
c These authors contributed equally to this work.

2. Results and Discussion
Based on the computer-aided virtual screening model
established in our previous work,18 we noticed that 6-methoxy-3-
phenylbenzofuran was a lead structure targeting Pin1 PPIase
domain (Fig. 1). Thus, we performed PPIase activity assay to
characterize the Pin1 inhibitory function of 6-methoxy-3-
phenylbenzofuran in vitro. In PPIase activity assay, a protease-
coupled assay, the p-nitroaniline label added at the C terminus of
peptide (Suc-AEPF-pNA) is subjected to cleavage by α-
chymotrypsin after Pin1-induced cis/trans isomerization of Suc-
AEPF-pNA, releasing free p-nitroaniline as a Pin1 activity
reporter.18 The data of in vitro PPIase activity assay demonstrated
the Pin1 inhibitory activity of 6-methoxy-3-phenylbenzofuran
with an IC50 value of 2.23 μM (Fig. 1). Thus, we planned to
investigate the potential of 3-phenylbenzofuran derivatives as Pin1
inhibitors.
Firstly, a series of substituted 3-phenylbenzofurans, with
diverse functional groups, were designed and submitted to
chemical synthesis. Considering the commercial availability of
starting materials and substrate scope of organic reactions,
multiple synthetic strategies were utilized to prepare 3-
phenylbenzofurans. Starting from benzophenones and
dimethylacetamide, 3-phenylbenzofuran TAB1-9 were afforded
via copper-mediated cascade formation of furan O1-C2 and C2-
C3 bonds (Scheme 1).19 To prepare 5-methoxyl 3-
phenylbenzofurans, 4-methoxyphenol and substituted 2-bromo-1-
phenylethan-1-ones were used to conduct condensation and
cyclization to obtain TAB10-14 with 60-81% yields (Scheme 2).
Furthermore, 5-hydroxyl 3-phenylbenzofuran TAB15-19 were
Fig. 1. 6-Methoxyl-3-phenylbenzofuran docked into PPIase domain of Pin1
and inhibited Pin1 activity with an IC50 value of 2.23 μM.
prevent its function both in vitro and in vivo.18 However, to date,
no Pin1 inhibitors have been applied to clinical treatment against
solid tumor, including HCC, summoning an urgent need for novel
Pin1 inhibitors.
Here, we identified 4,6-bis(benzyloxy)-3-phenylbenzofuran
(TAB29) as a novel Pin1 inhibitor via computer-aided virtual
screening and in vitro PPIase activity assay. TAB29 was prepared
with high yield via only two steps from commercially available
materials. TAB29 significantly suppressed HCC through restoring
XPO5 function and upregulating miRNAs biogenesis, providing a
valuable lead compound for Pin1 inhibition.
Scheme 1. Synthesis of TAB1-9 from benzophenones and dimethylacetamide via copper-promoted cascade formation of furan O1-C2 and C2-C3 bonds.
Scheme 2. Synthesis of 5-methoxyl or 5-hydroxyl 3-phenylbenzofurans (TAB10-19) from 4-methoxyphenol and substituted 2-bromo-1-phenylethan-1-ones.

Scheme 3. Synthesis of TAB20-28 from cinnamic acids through C-H olefination of phenols.
Scheme 4. Synthesis of TAB29-42 from (a) 4-phenylcoumarins via copper-catalyzed decarboxylative intramolecular C-O coupling, (b) Suzuki coupling of
heteroaryl bromides, (c) copper-catalyzed aerobic oxidative cyclization of phenols and alkynes.
synthesized by demethylation of corresponding 5-methoxyl 3-
phenylbenzofurans with high yields (Scheme 2).20 Selective
palladium-catalyzed C-H olefination of phenol is favorable for the
synthesis of 3-phenylbenzofurans with 5-nitro, 5-cyano or 6-alkyl
groups.21 Thus, TAB20-28 were prepared under this protocol from
cinnamic acids and phenols with acceptable yields (Scheme 3).
compounds (TAB1-42) were successfully synthesized through a
variety of synthetic methodologies.
With these compounds in hand, we further evaluated their
potential binding affinity toward Pin1 PPIase domain via
molecular docking and Pin1 inhibitory activity via in vitro PPIase
activity assay. As in Table 1, TAB29 exhibited the most potent
Pin1 inhibitory function in vitro with 75.38% of Pin1 inhibition
and the high predictive affinity toward PPIase domain with 57.02
GOLDScore.Fitness. TAB29 exhibited stronger activity than
juglone in vitro with the IC50 value of 0.874 μM (Fig. 2a).
Furthermore, to identify the selectivity of TAB29 against Pin1
protein, we conducted PPIase activity assay using other PPIases
including Pin4, FKBP12 and cyclophilin A, which were expressed
and purified from E. coli. system. The results indicated that
TAB29 was insensitive to Pin4, FKBP12 and cyclophilin A,
showing excellent selectivity to Pin1 over other PPIases (Fig. 2b).
In order to give the binding mode of Pin1 and TAB29, we
performed flexible docking based on the crystal structure of Pin1
(PDB ID: 3IKD). The result indicated that the oxygen of
Our
self-developed
copper-catalyzed
decarboxylative
intramolecular C-O coupling is preferable for C-4- and/or C-6-
substituted 3-phenlbenzofurans synthesis.22 TAB29-32 were
smoothly afforded from corresponding 4-phenlycoumarins with
65-91% yields (Scheme 4a). To evaluate the influence of C-3
heteroaryl and C-2 phenyl substitutions of benzofurans, 3-
heteroaryl benzofuran TAB34-36 as well as TAB33 were obtained
via palladium-catalyzed Suzuki coupling of aryl bromides and
benzofuran-3-ylboronic acid (Scheme 4b).23 And then, to prepare
2,3-diphenylbenzofurans, phenols and alkynes were aerobic
oxidative cyclized in the presence of copper species to afford
TAB37-42 (Scheme 4c).24 Together, forty-two 3-arylbenzofuran

Table 1. Pin1 inhibition of 3-arylbenzofurans at 10 μM in PPIase activity assay and their potential binding affinity toward PPIase
domain in GOLD molecular docking.
Pin1
inhibition (%)
Pin1
inhibition (%)
Pin1
inhibition (%)
Compound
GOLD Score Compound
-
GOLDScore
Compound
GOLDScore
Juglone
TAB1
TAB2
TAB3
TAB4
TAB5
TAB6
TAB7
TAB8
TAB9
TAB10
TAB11
TAB12
TAB13
TAB14
52.58
12.38
19.16
19.48
10.97
16.23
59.53
22.25
28.87
18.29
26.98
18.03
30.74
29.83
23.67
38.29
40.21
36.48
22.67
30.24
56.84
47.61
54.83
47.29
40.88
48.74
39.58
42.11
52.84
TAB15
TAB16
TAB17
TAB18
TAB19
TAB20
TAB21
TAB22
TAB23
TAB24
TAB25
TAB26
TAB27
TAB28
28.02
13.82
23.76
33.07
27.82
13.97
32.62
23.36
43.73
38.21
46.87
63.72
64.64
30.32
55.86
39.38
52.94
48.97
34.32
37.18
39.27
46.20
62.01
52.69
54.71
53.82
43.58
35.76
TAB29
TAB30
TAB31
TAB32
TAB33
TAB34
TAB35
TAB36
TAB37
TAB38
TAB39
TAB40
TAB41
TAB42
75.38
65.08
45.91
69.58
54.75
34.83
43.51
22.18
15.75
13.86
25.72
22.91
13.61
19.63
57.02
52.83
54.98
59.17
59.02
55.82
38.77
50.32
47.30
35.33
50.37
43.45
54.82
45.90
Fig. 2. TAB29 is a potent and selective Pin1 inhibitor. (a) PPIase activity assay of TAB29 at varied concentrations using Pin1 as enzyme and juglone as positive
control. Graphic data were run in duplicate and were shown as the means ± SD. (b) PPIase activity assay of TAB29 at the concentration of 1, 10, 50 μM using
Pin1, Pin4, Cyclophilin A or FKBP12 as enzyme. Graphic data were run in duplicate and were shown as the means ± SD.
benzyloxy group at C-6 position interacted with Ser114 amino acid
residue through hydrogen bond (Fig. 3). Benzofuran motif
associated with both Arg68 and Arg69 via cation-π interaction,
while this interaction was also existed between the benzene ring of
benzyloxy group at C-6 position and Arg69 residue (Fig. 3), giving
potential structural basis for the interaction of Pin1 and TAB29.
To further evaluate the activity of the small molecular TAB29
in HCC cells, the cell viability of HCC cells was detected by MTT
assay. The results indicated TAB29 inhibited cell proliferation of
HCC cells with an IC50 value of 1.140 μM in SK-Hep-1 cells and
2.732 μM in SNU-423 cells (Fig. 4a and Table 2). Moreover, to
check whether this anti-proliferative effect was under time- and
dose- dependent manner, the cell viability of SK-Hep-1 cells was
measured after 0, 24, 48, 72 and 96 hours of TAB29 treatment at
the concentrations of 5, 10 and 25 μM, respectively. The results
showed the cell viability was decreased with the prolongation of
inhibitor treatment time (Fig. 4b). The data of colony formation
assay also confirmed the anti-proliferation activity of TAB29 in
HCC cells (Fig. 5). These data indicated the Pin1 inhibitor TAB29
Fig. 3. Flexible docking of TAB29 and Pin1 (PDB ID: 3IKD) in Discovery
Studio software.
could effectively inhibit cell proliferation of HCC cells in a dose-
and time-dependent manner.

Fig. 4. TAB29 suppresses HCC cell proliferation. (a) MTT assay of TAB29 at varied concentrations in SK-Hep-1 and SNU-423 cells. Graphic data were run in
duplicate and were shown as the means ± SD. (b) MTT assay of TAB29 in SK-Hep-1 at the concentrations of 5 (blue), 10 (green) and 25 μM (red) measured in
the indicated times. Graphic data were run in duplicate and were shown as the means ± SD.
Fig. 5. TAB29 suppresses HCC colony formation. (A) The images of colony formation assay in SK-Hep-1 cells with or without TAB29 (2.5 μM) treatment. (B)
The quantification of colony formation assay in SK-Hep-1 cells with or without TAB29 (2.5 μM) treatment. The data were normalized to the colony area of control.
** P < 0.01.
been reported that delivery of miR-122, a liver-specific and
dominant microRNA (about 70% of total miRNAs in normal liver
Table 2. The IC50 values of TAB29 in MTT assay against SK-
cells), to a myc-driven mouse model of HCC strongly inhibits
tumorigenesis.25 Moreover, systemic administration of miR-29b
significantly suppresses angiogenesis and tumorigenesis in vivo.26
Owing to the tumor suppressive role of miR-122 and miR-29b,
these results gave a potential mechanism for the anti-cancer
activity of TAB29.
Broad pharmacological activity of benzofuran and widespread
existence in nature have aroused people's attention. Studies have
shown various biological activities of benzofuran, such as
antitumor and antioxidant activities.27,28 However, the mechanism
of antitumor benzofurans remain obscure. In our study, we
identified that 3-phenylbenzofuran TAB29 suppressed HCC cell
proliferation by targeting Pin1 isomerase (Fig. 2, 3, 4 and 5), which
is significantly overexpressed in HCC and is the only enzyme
catalyzing cis-trans isomerization of pS/T-P motif of substrate,
suggesting a potential mechanism for the anticancer effect of
benzofuran compounds.
Hep-1 and SNU-423 cells.
Cell line
IC50 (µM)
SK-Hep-1
SNU-423
1.140
2.732
Our previous research suggested the nucleus-to-cytoplasm
transport of XPO5 and pre-miRNAs was inhibited by Pin1-
induced inactivation of XPO5: after ERK induced phosphorylation
of XPO5, the conformation of phosphorylated XPO5 was changed
by Pin1, leading to the reduction of miRNAs biogenesis;13,14 Pin1
inhibition restored XPO5 exportation and upregulated miRNAs
expression.14,18 Thus, to evaluate the function of TAB29 toward
miRNAs biogenesis, confocal microscopy experiment and real-
time quantitative PCR (qPCR) were performed in SK-Hep-1 cells.
As in Fig. 6a, compared with DMSO-treated SK-Hep-1 cells, the
cytoplasm distribution of XPO5 was significantly increased upon
TAB29 incubation, indicating TAB29 could restore the nucleus-
to-cytoplasm export of XPO5 in HCC cells. Furthermore, the
results of qPCR showed TAB29 could remarkably promote the
biosynthesis of miRNAs (miR-122 and miR-29b) (Fig. 6b). It has
We previously discovered a novel small-molecular Pin1
inhibitor API-1, exhibiting promising anti-HCC activity in vitro
and in vivo.18 However, unfavorable yield of API-1 (23% overall
yield) via a four-steps method makes it difficult to conduct
structural optimization and SAR study.18 3-Phenylbenzofuran-
type Pin1 inhibitor TAB29 has a concise chemical structure, which

Fig. 6. TAB29 regulates miRNAs biogenesis via restoring XPO5 function. (a) Confocal microscopy of XPO5 nucleus/cytoplasm distribution in SK-Hep-1 cells
with or without TAB29 (2.5 µM) treatment. (b) Mature miRNAs expression detected by quantitative PCR in SK-Hep-1 cells with or without TAB29 (2.5 µM)
treatment. Graphic data were run in duplicate and were shown as the means ± SD. Statistical significance was determined by the Student t-test. * P < 0.01.
could be synthesized with 77% overall yield via a two-steps
strategy including Pechmann reaction and subsequent
decarboxylative intramolecular C-O coupling from commercial
available materials (Scheme 4a), facilitating the SAR study and
structural modification. Importantly, TAB29, with the IC50 value
of 874 nM (Fig. 2a), performs a comparable Pin1 inhibitory
function with other Pin1 inhibitors, providing a valuable lead
structure for Pin1 inhibition.
HCC is one of the leading causes of cancer death worldwide.
Although curative and pharmacological treatments have been
developed in recent years, unfortunately, most individuals
diagnosed with HCC are at advanced stage,29 in which these
treatments are less-effective,30 miRNAs are globally
downregulated in many tumors and tightly associated with tumor
development, including HCC,31,32 suggesting a potential miRNA-
based therapy for HCC.33,34 Our previous study showed that the
conformation of phosphorylated XPO5 was changed by Pin1,
resulting in the reduction of miRNA biogenesis and promoting
HCC development.13,14,18 In this study, we identified TAB29 as a
novel Pin1 inhibitor to enhance the biogenesis of tumor suppressor
miRNAs (miR-122 and miR-29b) and suppresses HCC cell
proliferation (Fig. 4, 5 and 6), providing experimental data to Pin1-
targeted therapy for HCC and highlighting the therapeutic
perspective of miRNA-based approach for human cancers.
dimethylacetamide (2.5 mL). The mixture was stirred for 24 h at
140℃under oxygen atmosphere. The mixture was cooled to room
temperature and water (5.0 mL) was added. The resulting aqueous
suspension was extracted with ethyl acetate (3 x 5 mL). The
organic layer was then washed with water (15 mL) and brine (15
mL), dried over anhydrous sodium sulfate, and concentrated under
reduced pressure. The crude product was purified by flash
chromatography using ethyl acetate/hexane as eluent to give 3-
phenylbenzofurans.
3-Phenylbenzofuran (TAB1): yellow oil with 66% HPLC
1
yields. H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 7.7 Hz, 1H),
7.81 (s, 1H), 7.67 (d, J = 6.6 Hz, 2H), 7.54 (d, J = 7.1 Hz, 1H),
7.48 (t, J = 7.7 Hz, 2H), 7.38 (t, J = 6.9 Hz, 1H), 7.36-7.30 (m,
2H). 13C NMR (100 MHz, CDCl3) δ 155.9, 141.3, 132.0, 128.9,
127.5, 127.5, 124.5, 122.8, 122.3, 120.2, 111.7. HR-ESIMS:
217.0628 [M+Na]+ (calc. for C14H10NaO, 217.0624).
5-Chloro-3-phenylbenzofuran (TAB2): yellow oil with 52%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.79 (s,
1H), 7.61 (d, J = 7.4 Hz, 2H), 7.51-7.45 (m, 3H), 7.38 (t, J = 7.4
Hz, 1H), 7.31 (dd, J = 8.7, 2.0 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 154.3, 142.5, 131.5, 129.1, 128.6, 127.9, 127.8, 127.4,
124.7, 122.2, 120.2, 112.7. HR-ESIMS: 251.0231 [M+Na]+ (calc.
for C14H935ClNaO, 251.0235).
3. Conclusion
5-Bromo-3-phenylbenzofuran (TAB3): yellow oil with 59%
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.79 (s,
1H), 7.60 (d, J = 7.6 Hz, 2H), 7.49 (t, J = 7.6 Hz, 2H), 7.45-7.38
(m, 3H). 13C NMR (100 MHz, CDCl3) δ 154.7, 142.5, 131.3,
129.2, 128.7, 128.5, 127.7, 127.5, 123.2, 122.0, 116.2, 113.2. HR-
ESIMS: 294.9727 [M+Na]+ (calc. for C14H979BrNaO, 294.9729).
This study screened out TAB29 as a novel small-molecular Pin1
inhibitor with an IC50 value of 874 nM. TAB29 had a potent and
specific inhibitory activity toward Pin1 isomerase. MTT and
colony formation assays confirmed that TAB29 suppressed HCC
cell proliferation in a dose- and time-dependent manner.
Mechanistically, TAB29 modulated the subcellular distribution of
XPO5 and promoted mature miRNA biogenesis. Therefore, this
work provided a promising lead compound for HCC treatment,
highlighting the miRNA-targeted cancer therapy in human
cancers.
1
5-tert-Butyl-3-phenylbenzofuran (TAB4): yellow oil with 88%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.76 (s,
1H), 7.65 (d, J = 8.3 Hz, 2H), 7.54-7.37 (m, 5H), 1.42 (s, 9H), 13C
NMR (100 MHz, CDCl3) δ 154.2, 146.2, 141.5, 132.3, 129.1,
127.6, 127.2, 126.0, 122.4, 116.3, 111.0, 34.8, 31.9. HR-ESIMS:
273.1254 [M+Na]+ (calc. for C18H18NaO, 273.1250).
4. Experimental
4.1. Chemistry
5,7-Dichloro-3-phenylbenzofuran (TAB5): yellow oil with 87%
HPLC. 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.70 (d, J = 1.9
Hz, 2H), 7.62-7.52 (m, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.42 (d, J =
7.6 Hz, 1H), 7.38 (d, J = 1.9 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ 151.5, 143.1, 132.9, 130.7, 129.1, 129.2, 128.6, 128.1, 127.6,
4.1.1. Synthesis of TAB1-9
TAB1-9 were synthesized via the reported protocols.19 In brief,
a round-bottom flask was added 2-hydroxybenzophenones (0.252
mmol), copper acetate (0.123 mmol), 8-hydroxyquinoline (15 mg,
0.103 mmol), potassium carbonate (0.252 mmol) and anhydrous

124.7, 119.6, 118.9. HR-ESIMS: 284.9842 [M+Na]+ (calc. for
C14H835Cl2NaO, 284.9845).
5-Methoxy-3-phenylbenzofuran (TAB10): yellow oil with 83%
HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.64-7.58
(m, 2H), 7.53-7.41 (m, 3H), 7.40-7.32 (m, 1H), 7.26 (d, J = 2.6 Hz,
1H), 6.96 (dd, J = 8.9, 2.6 Hz, 1H), 3.85 (s, 3H); 13C NMR (100
MHz, CDCl3) δ 156.4, 150.8, 142.2, 132.2, 129.1, 127.5, 127.3,
127.0, 122.4, 113.3, 112.2, 102.8, 56.0. HR-ESIMS: 247.0728
[M+Na]+ (calc. for C15H12NaO2, 247.0730).
4-Methoxy-3-phenylbenzofuran (TAB6): yellow oil with 22%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.72-7.63 (m, 4H),
7.51-7.35 (m, 3H), 7.07 (d, J = 2.3 Hz, 1H), 6.95 (dd, J = 8.7, 2.3
Hz, 1H), 3.86 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 158.4, 156.7,
140.2, 132.2, 128.8, 127.4, 127.3, 122.1, 120.4, 112.1, 96.2, 55.7.
HR-ESIMS: 247.0733 [M+Na]+ (calc. for C15H12NaO2, 247.0730).
3-(4-Chlorophenyl)-5-methoxybenzofuran (TAB11): yellow oil
with 74% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H),
7.59-7.55 (m, 2H), 7.50-7.45 (m, 3H), 7.24 (d, J = 2.6 Hz, 1H),
7.01 (dd, J = 8.9, 2.6 Hz, 1H), 3.89 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 156.4, 150.7, 142.3, 133.2, 130.6, 129.2, 128.6, 126.7,
121.4, 113.3, 112.3, 102.7, 56.0. HR-ESIMS: 281.0343 [M+Na]+
(calc. for C15H1135ClNaO2, 281.0340).
7-Methoxy-3-phenylbenzofuran (TAB7): yellow oil with 47%
HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.66 (d,
J = 8.1 Hz, 2H), 7.51-7.33 (m, 5H), 6.87 (d, J = 7.5 Hz, 1H), 4.04
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 145.9, 145.3, 141.4, 132.0,
128.9, 128.1, 127.5, 127.5, 123.7, 122.6, 112.6, 106.6, 56.1. HR-
ESIMS: 247.0731 [M+Na]+ (calc. for C15H12NaO2, 247.0730).
3-(4-Fluorophenyl)-5-methoxybenzofuran (TAB12): yellow oil
with 76% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.31 (s, 1H),
7.26-7.17 (m, 5H), 6.91 (t, J = 8.7 Hz, 2H), 6.85 (dd, J = 8.7, 2.6
Hz, 1H). HR-ESIMS: 265.0638 [M+Na]+ (calc. for C15H11FNaO2,
265.0636).
3-(4-Bromophenyl)-5-tert-butylbenzofuran (TAB8): yellow oil
with 72% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H),
7.74 (d, J = 8.5 Hz, 1H), 7.64-7.58 (m, 2H), 7.51-7.40 (m, 4H),
1.39 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 154.1, 146.4, 141.5,
132.1, 129.1, 124.3, 122.8, 121.5, 121.2, 121.1, 116.1, 111.2, 34.9,
31.9. HR-ESIMS: 351.0351 [M+Na]+ (calc. for C18H1779BrNaO,
351.0355).
5-Methoxy-3-(p-tolyl)benzofuran (TAB13): yellow oil with
60% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.65
(d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.9 Hz, 1H), 7.42-7.40 (m, 3H),
7.10 (dd, J = 8.9, 2.6 Hz, 1H), 3.97 (s, 3H), 2.54 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 156.4, 150.9, 142.2, 137.3, 129.8, 129.3,
127.4, 127.3, 122.4, 113.2, 112.2, 103.1, 56.0, 21.3. HR-ESIMS:
261.0888 [M+Na]+ (calc. for C16H14NaO2, 261.0886).
3-(4-Chlorophenyl)benzofuran (TAB9): yellow oil with 67%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.82-7.77 (m, 2H),
7.60-7.54 (m, 3H), 7.46-7.43 (m, 2H), 7.41-7.32 (m, 2H), 13C
NMR (100 MHz, CDCl3) δ 151.5, 143.0, 132.9, 130.4, 129.0,
128.7, 128.2, 127.6, 124.8, 119.3, 118.9. HR-ESIMS: 251.0231
[M+Na]+ (calc. for C14H935ClNaO, 251.0235).
3-(3,4-Dimethylphenyl)-5-methoxybenzofuran
(TAB14):
1
yellow oil with 81% HPLC yield. H NMR (400 MHz, CDCl3) δ
7.45 (s, 1H), 7.16-7.02 (m, 5H), 6.76 (dd, J = 8.6, 2.4 Hz, 1H),
2.25 (s, 3H), 2.22 (s, 3H). HR-ESIMS: 275.1045 [M+Na]+ (calc.
for C17H16NaO2, 275.1043).
4.1.2. Synthesis of TAB10-19
TAB10-19 were synthesized via the reported protocols.20 In
brief, potassium carbonate (5 mmol) and potassium iodide (5
mmol) were added to a mixture of 2-bromo-1-arylethanone (50
mmol), 4-methoxyphenol (5 mmol) and anhydrous acetone (60
mL). The mixture was stirred at room temperature in dark for 16 h
under nitrogen atmosphere, and then was diluted with
dichloromethane (120 mL). The solids were filtered off and the
organic layer was washed with water, dried over anhydrous
sodium sulfate and concentrated under reduced pressure. The
crude products were directly used for the next reactions without
further purification.
3-Phenylbenzofuran-5-ol (TAB15): yellow oil with 85% HPLC
1
yield. H NMR (400 MHz, CDCl3) δ 7.76 (s, 1H), 7.61-7.58 (m,
2H), 7.47-7.43 (m, 2H), 7.39 (d, J = 9.0 Hz 1H), 7.37-7.34 (m,
1H), 7.24 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 9.0, 2.4 Hz, 1H), 4.88
(s, 1H). 13C NMR (100 MHz, CDCl3) δ 151.3, 150.6, 142.1, 131.7,
128.7, 127.1, 126.9, 121.9, 113.2, 112.0, 105.5. HR-ESIMS:
209.0594 [M-H]- (calc. for C14H9O2, 209.0598).
3-(4-Chlorophenyl)benzofuran-5-ol (TAB16): yellow oil with
84% HPLC yield. 1H NMR (400 MHz, D6-acetone) δ 8.05 (s, 1H),
7.69 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 9.0
Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 9.0, 2.4 Hz, 1H).
13C NMR (100 MHz, D6-acetone) δ 154.1, 150.3, 143.2, 132.6,
131.3, 129.2, 128.8, 126.8, 120.9, 113.8, 112.2, 104.9. HR-
ESIMS: 243.0210 [M-H]- (calc. for C14H835ClO2, 243.0208).
A mixture of 2-(4-methoxyphenoxy)-1-arylethanones (about 10
mmol), polyphosphoric acid (20 g), and toluene (60 mL) was
refluxed for 6 h. The mixture was then cooled to room temperature,
poured into water, and extracted with diethyl ether (3 x 50 mL).
The organic extracts were dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The solid residue obtained was
purified by silica gel column chromatography to give TAB10-14.
3-(4-Fluorophenyl)benzofuran-5-ol (TAB17): yellow oil with
90% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 7.28
(dd, J = 8.7, 5.5 Hz, 2H), 7.25 (d, J = 8.7 Hz, 1H), 7.11 (d, J = 2.7
Hz, 1H), 6.91 (t, J = 8.7 Hz, 2H), 6.84 (dd, J = 8.7, 2.7 Hz, 1H),
6.66 (s,1H). 13C NMR (100 MHz, CDCl3) δ 161.8 (d), 151.1,
150.5, 142.0, 128.4 (d), 127.5, 126.9, 121.0, 115.5 (d), 113.3,
112.1, 105.4. HR-ESIMS: 227.0501 [M-H]- (calc. for C14H8FO2,
227.0503).
3-Aryl-5-methoxybenzofuran (2 mmol) was added to mixed
solution (12 mL) of 48% hydrobromic acid and acetic anhydride
(1:1, v/v). The mixture was refluxed at 140℃for 4 h and cooled to
room temperature, neutralized with saturated sodium bicarbonate
solution and extracted with diethyl ether (3 x 20 mL). The organic
layer was washed with brine, dried over anhydrous sodium sulfate
and evaporated under reduced pressure. The solid residue was
purified by chromatography on silica gel column chromatography
to give TAB15-19.
3-p-Tolylphenylbenzofuran-5-ol (TAB18): yellow oil with 80%
HPLC yield. 1H NMR (400 MHz, D6-acetone) δ 7.96 (s, 1H), 7.55
(d, J = 7.8 Hz, 2H), 7.38 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 2.4 Hz,
1H), 7.26 (d, J = 7.8 Hz, 2H), 6.94 (dd, J = 9.0, 2.4 Hz, 1H), 2.37

(s, 3H). 13C NMR (100 MHz, D6-acetone) δ 153.9, 150.4, 142.4,
137.0, 129.8, 129.5, 127.3, 127.2, 122.0, 113.6, 112.0, 105.2, 20.8.
HR-ESIMS: 223.0752 [M-H]- (calc. for C15H11O2, 223.0754).
7.55-7.48 (m, 2H), 7.07-7.02 (m, 2H), 3.87 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 159.8, 157.5, 142.7, 128.9, 128.3, 127.8, 125.9,
122.9, 122.2, 119.6, 114.9, 113.1, 107.1, 55.6. HR-ESIMS:
272.0685 [M+Na]+ (calc. for C16H11NNaO2, 272.0682).
3-(3,4-Dimethylphenyl)benzofuran-5-ol (TAB19): yellow oil
with 91% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H),
7.29-7.06 (m, 5H), 6.81 (dd, J = 8.7, 2.4 Hz, 1H), 2.23 (s, 3H),
2.20 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 151.2, 150.6, 141.8,
136.9, 135.7, 129.9, 129.1, 128.3, 127.3, 124.5, 121.8, 113.0,
111.9, 105.6, 19.8, 19.5. HR-ESIMS: 237.0914 [M-H]- (calc. for
C16H13O2, 237.0911).
3-(Benzo[d][1,3]dioxol-5-yl)benzofuran-5-carbonitrile
(TAB25): white solid with 66% HPLC yield. 1H NMR (400 MHz,
CDCl3) δ 8.12 (t, J = 1.2 Hz, 1H), 7.82 (s, 1H), 7.65-7.60 (m, 2H),
7.09-7.02 (m, 2H), 6.95 (d, J = 7.9 Hz, 1H), 6.04 (s, 2H). 13C NMR
(100 MHz, CDCl3) δ 157.5, 148.7, 147.9, 142.9, 128.4, 127.6,
125.8, 124.3, 121.4, 119.5, 113.2, 109.3, 108.12, 107.2, 101.8,
101.6. HR-ESIMS: 286.0477 [M+Na]+ (calc. for C16H9NNaO3,
286.0475).
4.1.3. Synthesis of TAB20-28
TAB20-28 were synthesized via the reported protocols.21 In
brief, to a screw cap reaction tube charged with a magnetic stir-
bar, palladium (II) acetate (0.025 mmol), 1,10-phenonthroline
(0.05 mmol), copper (II) acetate (0.25 mmol), phenols (0.75
mmol) and cinnamic acids (0.25 mmol) were added under air
atmosphere. In the reaction tube, dichloroethane (4 mL) was added
and oxygen was purged in the mixture for 15 min. Then the
mixture was vigorously stirred in a preheated oil bath at 130℃for
24h. After completion, the mixture was filtered through a celite
with ethyl acetate as the washing solvent. The organic layer was
washed with brine, dried over anhydrous sodium sulfate and
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography to afford TAB20-28.
5-(6-tert-Butylbenzofuran-3-yl)benzo[d][1,3]dioxole (TAB26):
white oil with 23% HPLC yield. H NMR (400 MHz, CDCl3) δ
1
7.73 (d, J = 8.3 Hz, 1H), 7.69 (s, 1H), 7.57 (d, J = 1.7 Hz, 1H),
7.38 (dd, J = 8.3, 1.7 Hz, 1H), 7.16-7.08 (m, 2H), 6.94-6.90 (m,
1H), 6.02 (s, 2H), 1.40 (s, 9H). 13C NMR (100 MHz, CDCl3) δ
156.3, 148.8, 148.3, 147.2, 140.8, 126.3, 124.1, 122.0, 121.0,
119.7, 109.0, 108.6, 108.1, 101.3, 35.1, 31.9. HR-ESIMS:
317.1146 [M+Na]+ (calc. for C19H18NaO3, 317.1149).
6,7-Dimethyl-3-p-tolylbenzofuran (TAB27): white solid with
19% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.59
(dd, J = 8.0, 4.1 Hz, 3H), 7.31 (d, J = 7.8 Hz, 2H), 7.16 (d, J = 8.0
Hz, 1H), 2.51 (s, 3H), 2.45 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 155.5, 140.5, 137.2, 133.0, 129.7, 129.7, 127.4, 125.2, 124.1,
122.4, 120.4, 117.1, 21.4, 19.3, 11.8. HR-ESIMS: 259.1096
[M+Na]+ (calc. for C17H16NaO, 259.1094).
3-(Naphthalen-1-yl)-5-nitrobenzofuran (TAB20): yellow solid
1
with 80% HPLC yield. H NMR (400 MHz, CDCl3) δ 8.28-8.35
(m, 2H), 8.02-7.94 (m, 3H), 7.85 (m, 1H), 7.70 (d, J = 8.9 Hz, 1H),
7.64-7.54 (m, 3H), 7.48 (ddd, J = 8.3, 6.8, 1.4 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 158.2, 145.8, 144.6, 134.1, 132.1, 129.4,
129.1, 128.9, 128.2, 127.4, 126.9, 126.6, 125.7, 125.3, 122.0,
120.8, 117.80, 112.40. HR-ESIMS: 312.0635 [M+Na]+ (calc. for
C18H11NNaO3, 312.0632).
7-Chloro-5-nitro-3-p-tolylbenzofuran (TAB28): yellow solid
with 39% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 8.64 (d, J =
2.1 Hz, 1H), 8.32 (d, J = 2.1 Hz, 1H), 7.95 (s, 1H), 7.51-7.47 (m,
2H), 7.35-7.32 (m, 2H), 2.44 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 154.6, 144.7, 144.4, 138.9, 130.3, 128.4, 127.7, 126.8, 124.4,
120.6, 118.3, 115.9, 21.5. HR-ESIMS: 310.0247 [M+Na]+ (calc.
for C15H1035ClNNaO3, 310.0242).
5-Nitro-3-(p-tolyl)benzofuran (TAB21): yellow solid with 76%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 2.4 Hz,
1H), 8.28 (dd, J = 9.1, 2.4 Hz, 1H), 7.90 (s, 1H), 7.63 (d, J = 9.0
Hz, 1H), 7.54-7.50 (m, 2H), 7.35-7.32 (m, 2H), 2.44 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 158.7, 144.6, 143.9, 138.5, 130.2,
127.6, 127.4, 125.4, 123.5, 120.6, 117.5, 112.3, 21.5. HR-ESIMS:
276.0635 [M+Na]+ (calc. for C15H11NNaO3, 276.0632).
4.1.4. Synthesis of TAB29-32
TAB29-32
were
prepared
via
copper-catalyzed
decarboxylative intramolecular C-O formation.22 In brief, a 25 mL
flask was charged with coumarins (1 mmol), cupric chloride (0.15
mmol), phenathroline (0.15 mmol), DMSO (10 mL) and 4 Å
molecular sieve (300 mg). The reaction mixture was stirred and
primarily heated to 110℃for 1h. The temperature was then raised
to 150℃and maintained for 24 h. Keep the mixture exposing to air
during all reaction time. After cooling to room temperature,
hydrochloric acid (2 mol/L, 10 mL) and water (20 mL) were
poured to terminate the reaction, which, simultaneously, brought
about the generation of brown solid and bubble. The suspension
was then extracted with chloroform (3 x 20 mL). The combined
organic layer was washed in turn with water (20 mL) and brine (20
mL), dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure. The solid residue obtained
was purified by silica gel column chromatography.
5-(5-Nitrobenzofuran-3-yl)benzo[d][1,3]dioxole
(TAB22):
1
yellow solid with 79% HPLC yield. H NMR (400 MHz, CDCl3)
δ 8.71 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 9.0, 2.4 Hz, 1H), 7.85 (s,
1H), 7.61 (d, J = 9.0 Hz, 1H), 7.14-7.03 (m, 2H), 6.96 (d, J = 7.9
Hz, 1H), 6.05(s, 2H). 13C NMR (100 MHz, CDCl3) δ 158.6, 148.7,
148.0, 144.6, 143.7, 134.8, 127.4, 124.1, 121.4, 120.7, 117.3,
112.4, 109.3, 108.2, 101.6. HR-ESIMS: 306.0376 [M+Na]+ (calc.
for C15H9NNaO5, 306.0373).
3-(4-Methoxyphenyl)-5-nitrobenzofuran (TAB23): yellow solid
with 55% HPLC yield. 1H NMR (400 MHz, CDCl3) δ 8.73 (d, J =
2.3 Hz, 1H), 8.28 (dd, J = 9.1, 2.3 Hz, 1H), 7.87 (s, 1H), 7.62 (d, J
= 9.0 Hz, 1H), 7.59-7.53 (m, 2H), 7.08-7.04 (m, 2H), 3.89 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 159.9, 158.7, 144.6, 143.5, 129.0,
127.5, 123.2, 122.7, 120.6, 117.4, 115.0, 112.3, 55.6. HR-ESIMS:
292.0580 [M+Na]+ (calc. for C15H11NNaO4, 292.0581).
4,6-Bis(benzyloxy)-3-phenylbenzofuran (TAB29): white solid
1
with 88% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.57-7.62
(m, 2H), 7.51-7.45 (m, 3H), 7.41 (t, J = 7.4 Hz, 2H), 7.38-7.32 (m,
1H), 7.31-7.24 (m, 7H), 7.20-7.14 (m, 2H), 6.78 (d, J = 1.9 Hz,
1H), 6.54 (d, J = 1.9 Hz, 1H), 5.11 (s, 2H), 5.06 (s, 2H). 13C NMR
(100 MHz, CDCl3) δ 158.1, 157.7, 153.7, 139.9, 136.7, 136.3,
132.1, 129.5, 128.6, 128.2, 128.1, 127.8, 127.6, 127.6, 127.1,
3-(4-Methoxyphenyl)benzofuran-5-carbonitrile
yellow solid with 45% HPLC yield. H NMR (400 MHz, CDCl3)
(TAB24):
1
δ 8.14 (t, J = 1.2 Hz, 1H), 7.83 (s, 1H), 7.62 (d, J = 1.2 Hz, 2H),

126.9, 122.8, 110.5, 96.4, 89.9, 70.6, 70.2. HR-ESIMS: 429.1461
[M+Na]+ (calc. for C28H22NaO3, 429.1462).
3-Furan-2-yl-benzofuran (TAB36): colorless oil with 84%
HPLC yield. 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.93-7.89
(m, 1H), 7.56-7.50 (m, 2H), 7.41-7.32 (m, 2H), 6.68-6.67 (m, 1H),
6.55-6.53 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 155.5, 147.1,
141.4, 140.9, 124.7, 124.6, 123.1, 120.7, 113.5, 111.7, 111.2,
106.0. HR-ESIMS: 207.0419 [M+Na]+ (calc. for C12H8NaO2,
207.0417).
6-Methoxy-3-methylbenzofuran (TAB30): white solid with 69%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 8.5 Hz,
1H), 7.32 (d, J = 1.2 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.88 (dd, J
= 8.5, 2.2 Hz, 1H), 3.85 (s, 3H), 2.21 (d, J = 1.3 Hz, 3H). HR-
ESIMS: 185.0578 [M+Na]+ (calc. for C10H10NaO2, 185.0573).
4.1.6. Synthesis of TAB37-42
4,6-Dimethoxy-3-methylbenzofuran (TAB31): white solid with
65% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 1.3
Hz, 1H), 6.57 (d, J = 1.9 Hz, 1H), 6.27 (d, J = 1.8 Hz, 1H), 3.86
(s, 3H), 3.83 (s, 3H), 2.30 (d, J = 1.2 Hz, 3H). HR-ESIMS:
215.0677 [M+Na]+ (calc. for C11H12NaO3, 215.0679).
1
TAB37-42 were synthesized via the reported protocols.24 In
brief, in a 20 mL Schlenk tube was charged with phenol (1.5
mmol), 1,2-diphenylethyne (1.0 mmol), zinc chloride (1.5 mmol),
copper (II) triflate (0.1 mmol) and nitrobenzene (2 mL) under
oxygen atmosphere. The mixture was heated at 120℃, and the
reaction was allowed to stir for 24 h. After reaction completion,
the mixture was washed with brine and extracted with ethyl
acetate. The organic layer was dried with anhydrous magnesium
sulfate, concentrated under reduced pressure and purified by silica
gel column chromatography to give TAB37-42.
4,6-Dimethoxy-3-phenylbenzofuran (TAB32): white solid with
1
91% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.65-7.60 (m,
2H), 7.49 (s, 1H), 7.43-7.37 (m, 2H), 7.36-7.31 (m, 1H), 6.69 (d,
J = 2.0 Hz, 1H), 6.37 (d, J = 2.0 Hz, 1H), 3.87 (s, 3H), 3.81 (s,
3H). 13C NMR (100 MHz, CDCl3) δ 159.1, 157.8, 154.6, 139.8,
132.3, 129.2, 127.9, 127.0, 122.7, 109.7, 94.5, 88.2, 55.7, 55.3.
HR-ESIMS: 277.0837 [M+Na]+ (calc. for C16H13NaO3, 277.0836).
2,3-Diphenylbenzofuran (TAB37): yellow oil with 88% HPLC
yield. 1H NMR (400 MHz, CDCl3) δ 7.65-7.67 (m, 2H), 7.55 (d, J
= 8.0Hz, 1H), 7.44-7.51 (m, 5H), 7.38-7.41 (m, 1H), 7.28-7.34 (m,
4H), 7.23 (t, J = 8.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 154.0,
150.6, 132.9, 130.7, 130.3, 129.8, 129.0, 128.4, 128.4, 127.7,
127.1, 124.7, 122.9, 120.1, 117.5, 111.1. HR-ESIMS: 293.0939
[M+Na]+ (calc. for C20H14NaO, 293.0937).
4.1.5. Synthesis of TAB33-36
TAB33-36 were synthesized via the reported protocols.23 In
brief, in an argon-filled glove box, to a 25 mL Schlenk tube was
charged with palladium (II) acetate (0.010 mmol),
tributylphosphonium tetrafluoroborate (0.012 mmol), heteroaryl
bromide (0.50 mmol), organoboronic acid (0.60 mmol), n-
dodecane (40 μL) and n-butanol (2.80 mL). The mixture was
prestirred at room temperature for 15 min, and then a solution of
sodium hydroxide (0.85 mmol) in 0.68 mL of degassed water was
added to initiate the Suzuki reaction. The Schlenk tube was capped
tightly and the reaction mixture was stirred vigorously at room
temperature until the heteroaryl bromide was fully consumed. At
the end of the reaction, the organic phase was separated and the
aqueous phase was further extracted with ether (3 x 3 mL). The
combined organic extracts were concentrated under reduced
pressure. The resulting residue was purified by silica gel column
chromatography to obtain TAB33-36.
5-Methyl-2,3-diphenylbenzofuran (TAB38): yellow oil with
1
78% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.64-7.64 (m,
2H), 7.36-7.47 (m, 6H), 7.25-7.26 (m, 4H), 7.09-7.11 (m, 1H),
2.38 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 152.6, 150.8, 133.2,
132.5, 130.9, 130.5, 129.9, 129.2, 128.5, 128.3, 127.7, 127.2,
126.1, 119.9, 117.6, 110.7, 21.5. HR-ESIMS: 307.1092 [M+Na]+
(calc. for C21H16NaO, 307.1094).
5-Methoxy-2,3-diphenylbenzofuran (TAB39): yellow solid with
1
92% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.62 (d, J =
8.0Hz, 2H), 7.39-7.49 (m, 6H), 7.27-7.29 (m, 3H), 6.91-6.93 (m,
2H), 3.78 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 156.3, 151.4,
149.1, 133.0, 130.8, 130.7, 129.8, 129.1, 128.4, 128.3, 127.6,
127.0, 117.8, 113.6, 111.7, 102.3, 56.0. HR-ESIMS: 323.1046
[M+Na]+ (calc. for C21H16NaO2, 323.1043).
3-(4-Methoxyphenyl)benzofuran (TAB33): yellow oil with 83%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.84-7.80 (m, 1H),
7.75 (s, 1H), 7.59-7.54 (m, 3H), 7.36-7.30 (m, 2H), 7.04-7.01 (m,
2H), 3.87 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 160.6, 156.5,
142.4, 129.7, 126.3, 124.6, 123.5, 122.3, 121.3, 114.7, 111.5, 55.8.
HR-ESIMS: 247.0732 [M+Na]+ (calc. for C15H12NaO2, 247.0730).
5-Chloro-2,3-diphenylbenzofuran (TAB40): orange solid with
1
72% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.64-7.61 (m,
2H), 7.46-7.40 (m, 7H), 7.29-7.24 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 152.4, 152.0, 132.2, 131.7, 130.2, 129.7, 129.1, 128.5,
128.0, 127.1, 124.9, 119.6, 117.2, 112.1. HR-ESIMS: 327.0542
[M+Na]+ (calc. for C20H1335ClNaO, 327.0548).
2-(Benzofuran-3-yl)-6-methoxypyridine (TAB34): yellow oil
1
with 93% HPLC yield. H NMR (400 MHz, CDCl3) δ 8.34-8.31
(m, 1H), 8.10 (s, 1H), 7.63 (dd, J = 8.3, 7.3 Hz, 1H), 7.56-7.54 (m,
1H), 7.37-7.34 (m, 2H), 7.29 (dd, J = 7.3, 0.7 Hz, 1H), 6.69 (d, J
= 8.3 Hz, 1H), 4.09 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.0,
156.2, 149.8, 144.1, 139.9, 125.8, 124.7, 123.3, 122.3, 121.9,
113.4, 111.7, 109.0, 53.6. HR-ESIMS: 248.0685 [M+Na]+ (calc.
for C14H11NNaO2, 248.0682).
7-Chloro-2,3-diphenylbenzofuran (TAB41): orange solid with
75% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.76-7.72 (m,
1
2H), 7.54-7.48 (m, 5H), 7.43-7.41 (m, 1H), 7.38-7.36 (m, 4H),
7.21 (t, J = 8.0 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 151.5,
149.8, 132.4, 131.9, 130.1, 129.7, 129.1, 128.8, 128.5, 127.9,
127.2, 124.8, 123.8, 118.5, 118.0, 116.6. HR-ESIMS: 327.0549
[M+Na]+ (calc. for C20H1335ClNaO, 327.0548).
3-(Thiophen-3-yl)benzofuran (TAB35): colorless oil with 90%
1
HPLC yield. H NMR (400 MHz, CDCl3) δ 7.93-7.90 (m, 1H),
7.86 (s, 1H), 7.57-7.54 (m, 1H), 7.40-7.33 (m, 4H), 7.18-7.15 (m,
1H). 13C NMR (100 MHz, CDCl3) δ 155.7, 141.2, 133.5, 127.8,
126.1, 125.0, 124.4, 124.5, 123.3, 120.6, 116.4, 111.8. HR-
ESIMS: 223.0185 [M+Na]+ (calc. for C12H8NaOS, 223.0189).
7-Methyl-2,3-diphenylbenzofuran (TAB42): yellow solid with
83% HPLC yield. H NMR (400 MHz, CDCl3) δ 7.68-7.66 (m,
2H), 7.50-7.48 (m, 2H), 7.43 (t, J = 8.0 Hz, 2H), 7.38-7.37 (m,
1H), 7.32-7.25 (m, 4H), 7.13-7.11 (m, 2H), 2.61 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 153.2, 150.3, 133.2, 131.0, 129.9, 129.8,
1

129.0, 128.5, 128.2, 127.6, 127.1, 125.7, 123.1, 121.4, 117.8,
117.6, 15.1. HR-ESIMS: 307.1097 [M+Na]+ (calc. for C21H16NaO,
307.1094).
was removed. Then, DMSO (100 μL) was added into each well,
followed by thorough shaking. The absorbance of the formazan
product was measured at 570 nm on Thermo Scientific Varioskan
Flash Multimode Reader. The IC50 value was obtained by fitting
dose-response data to a three parametric nonlinear regression
model using GraphPad Prism 6.0 software.
4.2. Computer-aided virtual screening and flexible docking
The computer-aided virtual screening and flexible docking
calculation were performed by using the crystal structures of Pin1
(PDB ID: 3IKD) via GOLD docking and flexible docking
protocols in Discovery Studio v3.1 software by using default
parameters. The images of molecular docking were processed by
PyMOL v1.8 software.
4.7. Colony formation assay
SK-Hep-1 cells were seeded in a 6-well culture plate and
allowed to grow for 24 hours in DMEM medium supplemented
with 10% FBS and 1% penicillin/streptomycin sulfate. The cells
were then treated with TAB29 (2.5 μM) or vehicle and incubated
in a 5% CO2 incubator. After 7 days, the cells were dyed with
crystal violet solution (0.5%) for 20 minutes. After removing
crystal violet solution, the cells were washed by water and colony
formation was recorded by high-resolution scanner.
4.3. Preparation of recombinant proteins
Full-length Pin1 sequences with N-terminal 6 His tag followed
by a thrombin cleavage site were subcloned into pET28 vector. In
E. coli cells, protein expressions were induced by IPTG overnight
at 16℃. The cells were then pelleted by centrifugation at 4000 rpm
for 15 minutes and resuspended in 20 mL buffer (20 mM Tris-HCl,
150 mM NaCl, 25 mM imidazole pH 8.0). His-tag proteins were
purified by nickel-affinity chromatography followed by thrombin
cleavage. The proteins were further purified by size-exclusion
chromatography using a Superdex 75 column (GE Healthcare).
4.8. Confocal microscopy
SK-Hep-1 cells were plated in a 6-well plate and treated with
TAB29 (2.5 μM) or vehicle control at 37℃ for 36 hours. For
confocal microscopy, cells after treatments were fixed in 4%
paraformaldehyde, permeabilized with 0.5% Triton X-100,
blocked with 5% bovine serum albumin, and then incubated with
anti-XPO5 at 4℃ overnight, followed by incubation with the
appropriate secondary antibody. Nuclei were stained with DAPI
before mounting. Confocal fluorescence images were captured
using Leica TCS SP5 II confocal spectral.
4.4. PPIase activity assay
The typical PPIase activity assay was established by Jiang et.
al.35 and described in our previous work.18 In brief, a typical PPIase
activity assay (total volume of 150 μL) involves PPIases (80
nmol/L), ɑ-chymotrypsin (6 mg/mL, Sigma), Suc-AEPF-pNA (50
μmol/L, Abcam), and test samples for reaction. Sample and PPIase
protein were preincubated for 10 minutes, and the mixture was
added to the reaction buffer containing ɑ-chymotrypsin. The
reaction was initiated by the addition of the Suc-AEPF-pNA, and
the reaction progress was monitored at 390 nm on Thermo
Scientific Varioskan Flash Multimode Reader at 4℃ for 180
seconds. The results of the Pin1 inhibition (%) in Table 1 were
calculated by the following formula based on the OD values of
tests at the time of 180s:
4.9. Real-time quantitative PCR
The primers for miR-122 (catalog number: MQPS0000467-1-
100) were ordered from RiboBio (Guangzhou, China). The
sequences of primers for miR-29b were showed as Table S1 in
Supplementary Information. SK-Hep-1 cells were plated in a 6-
well plate and treated with TAB29 (2.5 μM) or vehicle control at
37℃ for 36 hours. Total RNA was isolated by TRIzol Reagent
(Thermo fisher) according to manufacturer’s instruction. The
concentrations of total RNAs were determined by Thermo
NanoDrop 2000 UV-Vis spectrophotometer. cDNAs were
generated from total RNAs by cDNA Reverse Transcription kit
(Thermo fisher), and the microRNA (miRNA) level was
determined by Applied Biosystems StepOne Plus Real-Time PCR
Systems with SYBR Green reagent (Thermo fisher). Small
endogenous nucleolar RNU6 was used as control for miRNA
normalization.
Pin1 inhibition (%) = (ODPin1-ODinhibitor)/(ODPin1-ODabs)*100
ODPin1 is the value of assay with Pin1 pretreated with vehicle.
ODinhibitor is the value of assay with Pin1 pretreated with inhibitor.
ODabs is the value of assay in the absence of Pin1, inhibitor and
vehicle.
Acknowledgments
4.5. Cell culture
This work was supported by National Natural Science
Foundation of China (No.81702980 to W.P.), China Postdoctoral
Science Foundation (No.2018M640925 to W.P.) and Post-Doctor
Research Project, West China Hospital, Sichuan University
(No.2018HXBH014 to W.P.).
SK-Hep-1 cells were cultured in DMEM medium
supplemented with 10% fetal bovine serum (FBS). SNU-423 cells
were cultured in RPMI 1640 medium with 10% FBS at 37℃ in a
5% CO2 incubator.
4.6. Cell viability assay
Appendix A. Supplementary data
Supplementary data to this article can be found online at
HCC cells were seeded in a 96-well culture plate and allowed
to grow for 24 hours in DMEM or RPMI 1640 medium
supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin sulfate. The cells were then treated with
samples or vehicle and incubated in a 5% CO2 incubator for
indicated time. At the end of incubation, 10 μL of MTT stock
solution (5 mg/mL) was added into each well. The plate was
continued to be incubated at 37℃ for 4 hours before the medium
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