Design and synthesis of novel 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives as potent Mnks inhibitors by fragment-based drug design

Article abstract of DOI:10.1016/j.bmc.2018.05.004

A novel series of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives featured with various C-2 substituents were designed and synthesized as Mnks inhibitors through fragment-based drug design. Among them, 5b, 5i, 5o and 8k showed the best Mnk2 inhibitory activity with IC₅₀ values of 1.45, 1.16, 3.55 and 0.27 μM, respectively. And these compounds inhibited the activity of Mnk1 at the same time. Furthermore, compounds 5o and 8k exhibited anti-proliferative effects to human leukemia cancer THP-1 and MOLM-13 cell lines and colon cancer HCT-116 cell line. Moreover, Western blot assay suggested that 8k could decrease the levels of p-eIF4E in a dose-dependent manner in HCT-116 cells. Docking studies demonstrated strong interactions between 8k and Mnk2. Therefore, this unique benzofuran scaffold demonstrated great potential to be further explored as potent Mnks inhibitors with improved potency.

Full text of DOI:10.1016/j.bmc.2018.05.004

Accepted Manuscript
Design and synthesis of novel 6-hydroxy-4-methoxy-3-methylbenzofuran-7-
carboxamide derivatives as potent Mnks inhibitors by fragment-based drug
design
Shuxiang Wang, Bo Li, Bo Liu, Min Huang, Deyi Li, Lihong Guan, Jie Zang,
Dan Liu, Linxiang Zhao
PII:
S0968-0896(18)30219-0
https://doi.org/10.1016/j.bmc.2018.05.004
BMC 14346
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 February 2018
1 May 2018
3 May 2018
Please cite this article as: Wang, S., Li, B., Liu, B., Huang, M., Li, D., Guan, L., Zang, J., Liu, D., Zhao, L., Design
and synthesis of novel 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives as potent Mnks
inhibitors by fragment-based drug design, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.bmc.2018.05.004
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Design and synthesis of novel 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide
derivatives as potent Mnks inhibitors by fragment-based drug design
Shuxiang Wanga, Bo Lia, Bo Liua, Min Huanga, Deyi Lia, Lihong Guana, Jie Zanga, Dan Liua* and
Linxiang Zhaoa*
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China
*Corresponding authors.
Tel./Fax: +86 024 4352 0221.
E-mail address: sammyld@163.com (D. Liu);
linxiang.zhao@vip.sina.com (L. Zhao)

Abstract
A novel series of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives featured
with various C-2 substituents were designed and synthesized as Mnks inhibitors through fragment-
based drug design. Among them, 5b, 5i, 5o and 8k showed the best Mnk2 inhibitory activity with IC50
values of 1.45, 1.16, 3.55 and 0.27 μM, respectively. And these compounds inhibited the activity of
Mnk1 at the same time. Furthermore, compounds 5o and 8k exhibited anti-proliferative effects to
human leukemia cancer THP-1 and MOLM-13 cell lines and colon cancer HCT-116 cell line.
Moreover, western blot assay suggested that 8k could decrease the levels of p-eIF4E in a dose-
dependent manner in HCT-116 cells. Docking studies demonstrated strong interactions between 8k and
Mnk2. Therefore, this unique benzofuran scaffold demonstrated great potential to be further explored
as potent Mnks inhibitors with improved potency.
Keywords: 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide derivatives, fragment-based
drug design, anti-proliferative effects, Mnks inhibitors.

1. Introduction
Dys-regulation of protein synthesis is implicated in the progression of various pathologies, most
notably cancer.1 As a key rate-limiting step in protein synthesis, initiation of cap-dependent translation
is contingent on the availability and activity of eukaryotic initiation factor 4E (eIF4E).2 eIF4E, the most
prominent substrate of mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks), is
phosphorylated exclusively by Mnks at the conserved serine 209.3 Mnks comprise a subfamily of
Ser/Thr kinases and belong to the group of Ca2+/calmodulin-dependent kinases. Mnks are present in
two isoforms: Mnk1 and Mnk2, the catalytic domains of Mnk1/2 are highly homologous with ~80%
sequence identity between them. Despite this, Mnk2 possesses high constitutive basal activity
comparing to Mnk1, which has been shown in in vitro studies.4 Though the activity of Mnks is
necessary for eIF4E-mediated tumorigenesis, it is dispensable for normal tissue development, which
has been proved by the experiments of silencing Mnk1 with small hairpin RNA (shRNA) or knocking
out Mnk1/2.5 Thus, inhibiting Mnks offers an exciting opportunity for effectively treating cancer with
low toxicity.
Despite the fact that Mnks are potential targets for cancer therapy with better outcomes and fewer
side effects, the role of Mnk1/2 in cancer still remains elusive due to the absence of potent and
selective probes. In many instances, hypotheses have been built upon unspecific Mnk1/2 inhibitors
such as CGP-57380 or cercosporamide. Lately, the first two clinical programs targeting Mnks in
oncology have been revealed (eFT508 and BAY1143269, the structure of BAY1143269 has not been
disclosed).6
Figure 1. Chemical structures of known Mnks inhibitors.
ATP-competitive kinase inhibitors typically form 1~3 hydrogen bonds with the hinge region of
the kinase, aryl carbamoyl became one of new fragments binding to the hinge region of kinases
appeared in the structure of many candidate compounds. For instance, the VEGFR inhibitor lenvatinib

approved for advanced thyroid carcinoma at 2015 showed the potential advantages of the fragment of
aryl carbamoyl (Figure 2).7-14
Figure 2. Kinase inhibitors in development with carbamoyl moiety as hinge binder.
The carbamoyl group could form two hydrogen bonds with the hinge region of Mnks via carbonyl
and NH2. Ideally, introducing a hydroxyl group to ortho position of carbamoyl could form another
hydrogen bond with the hinge. In our efforts to discover novel Mnks inhibitors, bioactive benzofuran
scaffold was chosen as the mimics of adenine core, and introduces 6-hydroxy and 7-carbamoyl, a hit
compound of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide (FY0413) was prepared as a
potential Mnks inhibitor. FY0413 showed an IC50 of 0.749 μM for Mnk2 and 49% inhibition ratio for
Mnk1 at 1 μM, the ligand efficiency of FY0413 is 0.53. Besides, FY0413 showed moderate anti-
proliferative activity to leukemia and colon cancer cells (IC50 THP-1: 32.35 μM, MOLM-13: 24.81 μM,
HCT-116: 26.05 μM) in vitro. The docking mode (Figure 3) of FY0413 with Mnk2 (PDB: 2HW7)
indicated that only two hydrogen bonds were formed between FY0413 and Mnk2. Besides, there is still
a lot of space in the ATP-binding pocket, introducing a substituent at C-2 position of FY0413 may
occupy it with additional interactions and result better potency. To illuminate the Mnks inhibitory

activity by this unique and proprietary scaffold in a systemic way, a series of novel 6-hydroxy-4-
methoxy-3-methylbenzofuran-7-carboxamide derivatives featured with various C-2 substituents were
described. Their inhibitory activities toward Mnks as well as the in vitro anti-proliferative activity
toward two leukemia cell lines (THP-1, MOLM-13) and one colon cancer cell line (HCT-116) were
studied. Finally, the inhibition toward the phosphorylation of eIF4E was also investigated with
representative compound in HCT-116 cells. The proposed binding mode in the ATP pocket of Mnk2
was illustrated.
Figure 3. The structure of FY0413 and the docking mode of FY0413 in the ATP binding pocket of the
Mnk2 kinase domain (2HW7 in PDB). H-Bonding interactions were presented with green line.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5a-5r and FY0413 was described in Scheme 1. Commercial starting
material 1-(2,4,6-trihydroxyphenyl)ethan-1-one was reacted with two equivalents saturated
dimethylsulfate to afford dimethylated intermediate 1, the key intermediate 2 was obtained by
condensation and cyclization with ethyl bromoacetate. Introducing carbamoyl at the 7-position of 2
with CSI to afford the intermediate ethyl 7-carbamoyl-4,6-dimethoxy-3-methylbenzofuran-2-
carboxylate 3. Followed by ester hydrolysis and reaction of the resulted acid with amino to yield the
corresponding amide, treatment of the resulted amide with BBr3 afforded 5b-5r. Treatment of 3 with
either 30% hydrobromic acid or BBr3 afforded the hit compound FY0413 and 5a.

Scheme 1. Synthesis of 5a-5r and FY0413. Reagents and Conditions: (a) (CH3)2SO4, K2CO3, acetone;
(b) BrCH2COOC2H5, K2CO3, DMF, 70-90°C; (c) CSI, CH3CN, 0°C to r.t., then 1N HCl; (d) NaOH,
CH3OH/H2O, 40°C; (e) RNH2, HATU, DIEA, DMSO, r.t.; (f) BBr3, CH2Cl2, -40°C to r.t.; (g)
HBr(30%), CH3COOH, reflux.
The synthesis of compounds 8a-8p and 17a was described in Scheme 2. The intermediate 4
reacted with 4-amino-1-Boc-piperidine or 4-aminomethyl-1-Boc-piperidine, then deprotection of Boc
group afforded 7, 7 reacted with substituted benzyl chloride or substituted benzoyl chloride, then
deprotection of methyl group afforded 8a-8p. 7a reacted with 3-trifluoromethyl benzyl chloride to
afford 17a.
Scheme 2. Synthesis of 8a-8p and 17a. Reagents and Conditions: (a) 4-amino-1-Boc-piperidine or 4-
aminomethyl-1-Boc-piperidine, HATU, DIEA, DMSO, r.t.; (b) HCl in ethyl acetate, r.t.; (c) substituted
benzyl chloride or benzoyl chloride, K2CO3, DMF, 80°C; (d) BBr3, CH2Cl2, -40°C to r.t..
Treatment of 8k with BBr3 afforded 17b, which was described in Scheme 3.

Scheme 3. Synthesis of 17b. Reagents and Conditions: (a) BBr3, CH2Cl2, -40°C to r.t..
The synthesis of 17c was described in Scheme 4. The intermediate 9 was synthesized by ester
hydrolysis of 2. Treatment of 9 with 1-Boc-piperidine-4-amino, deprotection of Boc group and reacted
with 3-trifluromethylbenzyl bromide to afford 17c.
Scheme 4. Synthesis of 17c. Reagents and Conditions: (a) NaOH, H2O/CH3OH, reflux; (b) 4-amino-1-
Boc-piperidine, HATU, DIEA, DMSO, r.t.; (c) HCl in ethyl acetate, r.t.; (d) 3-trifluromethylbenzyl
bromide, Cs2CO3, DMF, 80°C.
The synthesis of compounds 17d and 17e were described in Scheme 5. Selecting 1-(2,4-
dihydroxyphenyl)ethanone (12) as starting material, the 4-hydroxy of 12 was protected selectively by
methyl. The synthesis of 15 was conducted through cyclization and ester hydrolysis of 13. Scaffold 15
was further reacted with 1-Boc-piperidine-4-amino to yield 16, deprotection the Boc group and then
reacted with 3-trifluromethylbenzyl bromide to afford 17d. Treatment of 17d with BBr3 afforded 17e.

Scheme 5. Synthesis of 17d and 17e. Reagents and Conditions: (a) (CH3)2SO4, K2CO3, acetone; (b)
BrCH2COOC2H5, K2CO3, DMF, 70-90°C; (c) NaOH, CH3OH/H2O, 40°C; (d) RNH2, HATU, DIEA,
DMSO, r.t.; (e) HCl in ethyl acetate, r.t.; (f) 3-trifluromethylbenzyl bromide, Cs2CO3, DMF. 80°C; (g)
BBr3, CH2Cl2, -40°C to r.t..
2.2. Biological activity
At the initial stage of the study, only the inhibition ratio at 10 μM and 1 μM against Mnk2 were
determined for each compound with an advanced HTRF method at Cisbio. CGP-57380 exhibited
inhibitory effect with IC50 value of 1.53 μM in the tested system (the reported value is 1.60 μM), and
the results for all target compounds were shown in Table 1 ~ Table3. The results indicated that portion
of the target compounds exhibited potent activities towards Mnk2. Among which, compounds 5b, 5i, 5l,
5o and 8k displayed a inhibition ratio > 50% at 10 μM; compounds 5a, 5c, 5n, 8a, 8c, 8e, 8g, 8h, 8i, 8l,
and 8p displayed a inhibition ratio > 30% at 10 μM. 5a with ethyl ester substituted at C-2 position
exhibited little potency (41% at 10 μM, inhibition ratio at 10 μM was chosen to analysis the SAR
below); N-methylformamide substituent (5b) led to elevated inhibitory activity, indicating that amide
bond is favorable at C-2 position than ester bond. So the followed compounds employed amide bond as
linker, and a more systematic study of the substituents on nitrogen atoms were carried out.
The potency of 5c with N, N-dimethyl substituted was inferior to 5b, indicated that the presence of
hydrogen atom at amide of C-2 is essential for the activity. Prolonged the substituent (5d) resulting the
decrease of potency, indicating that the larger flexible chain system was not suitable for the kinase
pocket. 5e and 5f with cyclopentyl and cyclohexyl substituted showed little potency. Contracted the
exo nitrogen atom of amide into an endo one resulted 5g-5i. 5g and 5h exhibited little potency to Mnk2,

but 5i with N-ethylpiperazinyl substituted was found to be more potent than 5g and 5h, which indicated
that piperazinyl is helpful to the inhibitory activity. The substituted phenyl was introduced to the 4-
position of piperazinyl, subsequently. The results indicated that the substituents on phenyl influence the
potency by the trends: 3-CF3 (5l: 61%) > H (5j: 15%) > 4-F (5k: 10%). 5m-5r showed the effect of
aromatic amine substituents on activities. 5m without substituent on phenyl lost potency completely.
However, when the substituents were introduced into the 3- and 4-position of phenyl, its potencies
were greatly improved (5n and 5o). Among them, 5o with 4-methyl-3-trifluoromethyl substituents on
phenyl exhibited better activity. Replacing the phenyl by benzyl (5p) resulted improved activity, and
when the hydrogen atom on amide was substituted by methyl (5q), the activity lost completely, which
was similar to 5b.
Table 1. The structure and inhibitory activities of compounds FY0413 and 5a-5r on Mnk2 kinase.
Inhibition ratio/%a
Inhibitionratio/%a
Compd.
FY0413
R
Compd.
5j
R
10 μM
1 μM
10 μM
1 μM
-
77
43
15
8
41
21
10
5
5a
5k
73
45
66
24
61
0
28
0
5b
5c
5l
5m
24
22
19
6
35
57
18
75
5d
5e
5n
5o
28
24
20
12
28
0
24
0
5f
5p
5q
5g

23
79
19
36
23
11
5h
5i
5r
a All results represent the mean of duplicate experiments.
5i with N-ethylpiperazinyl substituted exhibited best inhibitory activity in the above optimization.
Considering the importance of hydrogen atom on the amide, piperazinyl was replaced with 4-
aminepiperidinyl and ethyl was replaced with lager substituents such as pyridyl, substituted pyrimidyl,
substituted benzoyl or substituted benzyl, which afforded 8a-8p. The inhibitory activity indicated that
aromatic heterocyclic substituents on piperidinyl had little effect on enzyme binding (8a, 8b), so the
following compounds were designed to substituted benzyl. The inhibitory activity indicated that the
substituents on phenyl influenced the activity by the trends: 3-CF3 >> 4-Cl ≈ 4-CF3 ≈ 4-CH3 > 2-F > 4-
F (8k >> 8h ≈ 8i ≈ 8g > 8c > 8d). 8k with 3-trifluoromethyl substituted exhibited the best inhibitory
activity on Mnk2, which is similar to 5l and 5o. Replacing the substituted benzyl with substituted
benzoyl decreased the kinase inhibitory activity (8f < 8e, 8j < 8i). The inhibitory activity of di-
substituted compounds was weaker than mono-substituted compounds, for example, 8m ~ 8p < 8h.
The above results showed that the type of substituents is more important than the number of
substituents, and 3-trifluoromethyl substituted (8k) is the best.
Table 2. The structure and inhibitory activities of compounds 8a-8p on Mnk2.
Inhibition ratio/%a
Inhibitionratio/%a
Compd.
8a
R
n
Compd.
8i
R
n
10 μM
1 μM
10 μM
1 μM
0
32
15
4-CF3-benzyl
0
43
24
0
11
0
4-CF3- benzoyl
0
29
26
8b
8j
2-F-benzyl
4-F-benzyl
0
0
1
1
0
31
15
41
23
40
29
8
3-CF3-benzyl
3-CF3-benzyl
0
1
0
0
0
90
49
27
0
85
23
1
8c
8d
8e
8f
8k
8l
4-F-benzyl
32
16
27
2,6-diCl-benzyl
2,4-diCl-benzyl
2,3-diCl-benzyl
8m
8n
8o
4-F-benzoyl
4-CH3-benzyl
0
21
18
8g

4-Cl-benzyl
0
45
29
3,4-diCl-benzyl
1
38
21
8h
8p
a All results represent the mean of duplicate experiments.
To prove the idea of the ortho hydroxyl carboxamide substituents is beneficial to the binding, the
substituents on C-2 was fixed and compounds 17a-17e were designed to discuss the optimal
substituents on position C-4, C-6 and C-7(Table 3). The inhibitory activities indicated that compound
with 6-hydroxy-7-carboxamide substituents (8k) exhibited best Mnk2 inhibitory activity, removing the
carbamoyl on position C-7 (17c-17e) or replacing hydroxyl to methoxy on position C-6 (17a) sharply
decreased the kinase inhibitory activity, which indicated the significance of 6-hydroxy-7-carbamoyl
substituents for inhibitory activity. Meanwhile, replacement the methoxy on position C-4 to hydroxyl
led to depressed activity, most strikingly elucidated by 17b, which indicated that 4-methoxy substituent
is essential to Mnk2 inhibitory activity. All these data confirmed that the substituents on hit compound
(FY0413) are necessary for Mnk2 inhibitory activity, and this rule is appropriate for compounds with
substituents on C-2.
Table 3. The structure and inhibitory activities of compounds 8k and 17a-17e on Mnk2.
Inhibition ratio/%a
Compd.
8k
R1
carbamoyl
carbamoyl
carbamoyl
H
R2
R3
10 μM
1 μM
hydroxy
methoxy
hydroxy
methoxy
methoxy
H
methoxy
methoxy
hydroxy
methoxy
H
90
85
29
34
35
27
28
2
1
6
0
0
17a
17b
17c
H
17d
17e
H
H
a All results represent the mean of duplicate experiments.
Compounds 5b, 5i, 5o and 8k showed potent inhibitory activity against Mnk2, the IC50 values
against Mnk2 and the inhibition ratio at 10 μM, 1 μM and 0.1 μM against Mnk1 were determined.
Meanwhile, the anti-proliferative activity of these compounds against THP-1, MOLM-13 and HCT-116
cell lines was further evaluated using MTT assay. The results fitted well with the Mnk2 inhibition data.
5b, 5i, 5o and 8k exhibited potent Mnk2 inhibitory activity with the IC50 values of 1.45, 1.16, 3.55 and

0.27 μM, respectively, which demonstrated Mnk1 inhibition at the same time (inhibition ratio at 10 μM:
39%, 30%, 23% and 59%, respectively). 8k showed a three-fold potency improvement compared with
FY0413, similar to pan-kinases inhibitor sorafinib, and it was more potent than CGP-57380, a widely
used Mnks inhibitor. The anti-proliferative activity of 8k is superior to CGP-57380.
Table 4. Inhibition effects of selected compounds towards Mnks and cancer cells.
Mnk1 inhibition ratio/%a
IC50/μM
THP-1b MOLM-13b
Compd.
HCT-116b
10 μM 1 μM
0.1 μM
Mnk2a
39
39
30
23
59
49
26
7
34
0.749
32.35
24.81
26.05
FY0413
11
3
1.45
1.16
>50
n.d.
n.d.
15.72
n.d.
5b
18.76
>50
13.54
n.d.
5i
5o
23
41
n.d.c
12
20
3.55
0.273
0.262
1.53
6.30
n.d.
3.15
n.d.
2.77
n.d.
8k
Sorafinib
CGP-57380c
IC50=0.85 μMd
11.68
8.24
10.21
a All results represent the mean of duplicate experiments.
b Data shown are means of three independent experiments.
c Not determined;
d CGP-57380 was demonstrated as IC50 values towards Mnk1.
To gain further insights into the mechanisms of action of these compounds, compound 8k, which
possessed significant Mnks and cellular inhibitory effects, was selected to examine the inhibitory effect
on the phosphorylation level of eIF4E at Ser209 with western bolt analysis in HCT-116 cells. eIF4E,
the most prominent substrate of mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks),
is phosphorylated exclusively by Mnks at the conserved serine 209, thus the phosphorylation level of
eIF4E could reflect the residual activity of Mnks indirectly. HCT-116 cells were treated with 8k at
different concentrations or DMSO as negative control for 24 h (Figure 4). The results indicated that
treatment with 8k decreasing the levels of phosphorylation of eIF4E at Ser209, but barely affected the
levels of eIF4E, confirming their cellular Mnks inhibitory activity.

Figure 4. 8k decreased the expression of p-eIF4E in HCT-116 cells.
In order to understand the binding mode of 8k with Mnk2 kinase, the docking experiments were
conducted with Discovery studio software based on the X-ray co-crystal structure of Mnk2 with
staurosporine (PDB: 2HW7). Supporting the docking results, staurosporine was first docked and fitted
well with that in crystal structure. Then 8k was docked with the same parameters. The binding mode
(Figure 5) demonstrated strong interactions between 8k and Mnk2. Three hydrogen bonds were
established between hydroxyl and carbamoyl and Glu160 and Met162 as expected. Besides, the
carbonyl of amide formed a unique hydrogen bond with Ser166. Apart from hydrogen bonding, the
benzofuran scaffold and 3-trifluoromethylbenzyl showed hydrophobic and van der Waals interactions
with residues Val98, Ala111, Leu168 and Leu212 of the receptor protein, which likely provided a
significant contribution to Mnk2 binding and inhibition.
Figure 5. The proposed binding mode of 8k in the ATP binding pocket of the Mnk2 kinase domain
(2HW7 in PDB). H-Bonding interactions were presented with green line.
3. Conclusion
We report herein the discovery and synthesis of novel benzofuran derivatives as potent Mnks
inhibitors. Among the 39 compounds, 5b, 5i, 5o and 8k were the most potent Mnk2 inhibitor with IC50

values of 1.45, 1.16, 3.55 and 0.27 μM, respectively. And these compounds inhibited the activity of
Mnk1 at the same time. 5o and 8k exhibited low micromolar anti-proliferative activity toward leukemia
cancer THP-1, MOLM-13 and colon cancer HCT-116 cell lines. In addition, 8k could block the
phosphorylation of eIF4E in a dose-dependent manner in HCT-116 cells. Molecular modeling
illustrated a strong binding effect of 8k within Mnk2 ATP-binding site. Together, these results provide
compelling evidence that the anti-proliferative activity of these compounds in THP-1, MOLM-13 and
HCT-116 cell lines was mediated, at least in part, by the inhibition of Mnks. Therefore, this unique
benzofuran scaffold demonstrated great potential to be further explored as potent Mnks inhibitors with
improved potency. Efforts are ongoing to further optimize and provide a systematic structure-activity
analysis at the proper position and the results will be reported in due course.
4. Experimental
4.1. Chemistry
Melting point was measured with X-4 digital micro melting point apparatus with temperature
1
unrevised. Infra-red (IR) spectra were recorded on Bruker IR-27G spectrometer with KBr pellets. H-
NMR spectra were measured with a Bruker ARX (400 MHz) spectrometer and 13C-NMR with a Bruker
AV (100 MHz) instrument. Chemical shifts are recorded in δ units using tetramethylsilane as the
standard (NMR peak description: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad
peak). Low resolution mass spectrometry (MS) were recorded with an Agilent 1100 ion trap liquid
chromatography-mass spectrometer. Organic solutions were dried over anhydrous MgSO4 during
workup. Column chromatography was carried out on TELEDYNE ISCO Combiflash Rf+ preparative
liquid chromatograph. Silica gel 60 (200-300 mesh) and TLC were purchased from Qingdao Haiyang
chemical Co. Ltd. All commercial reagents and solvents were used without further purification unless
otherwise noted. The purity of all compounds screened in biological assays was >95% pure as judged
by HPLC. HPLC analysis was obtained on an Hitachi L-2400 system, using a AichromBond-AQ C18
column (150 mm × 4.6 mm, 5 μm) with a 1.5 mL/min flow rate using acetonitrile and water solution
(v/v=70:30) as the eluent over 30 min.
4.1.1. General procedure for the synthesis of 5a-5r
Preparation of 1-(2-hydroxy-4,6-dimethoxyphenyl)ethanone (1)

To a solution of 1-(2,4,6-trihydroxyphenyl)ethanone (11.16 g, 0.06 mol) in dry acetone (150 mL),
potassium carbonate (24.84g, 0.18 mol) was added and stirred for 30 min at room temperature. Then
dimethyl sulphate (12 mL, 0.12 mol) was added drop-wise, the reaction solution was stirred for 4-5 h at
room temperature and monitored by thin-layer chromatograph (TLC). Poured the reaction solution to
ice-water(1000 mL) and the precipitate was filtered, washed with water, dried to afford 1 (11.0 g) as
1
white solid in 93.5% yield. H-NMR (400 MHz, DMSO-d6) δ: 13.79 (s, 1H, OH), 6.11 (d, 1H, J=2.3
Hz), 6.08 (d, 1H, J=2.3 Hz), 3.86 (s, 1H), 3.81 (s, 1H), 2.55 (s, 1H). 13C-NMR (100 MHz, DMSO-d6) δ:
203.2, 166.7, 166.4, 163.2, 105.9, 94.1, 91.2, 56.5, 56.1, 33.1.
Preparation of ethyl 4,6-dimethoxy-3-methylbenzofuran-2-carboxylate (2)
To a solution of 1 (3.92 g, 0.02 mol) in dry DMF (50 mL), potassium carbonate (8.28 g, 0.06 mol)
was added, the reaction solution was warmed up to 70℃ and stirred for 30 min, then ethyl 2-
bromoacetate (3.3 mL, 0.03 mol) was added drop-wise and the reaction solution was stirred for another
2 h at 90°C. Poured the reaction solution to ice-water (500 mL) and the precipitate was filtered, washed
with water, dried to afford 2 as brown solid, the resulting residue was then purified by column
chromatography on silica to give 2 (1.8 g) as white solid in 34.0% yield. LC-MS m/z: 265.1 [M+H]+，
287.1 [M+Na]+. 1H-NMR (400 MHz, DMSO-d6) δ: 6.82 (d, 1H, J= 1.84 Hz), 6.42 (d, 1H, J= 1.84 Hz),
4.30 (q, 2H, J= 7.1 Hz), 3.87 (s, 3H), 3.82 (s, 3H), 2.61 (s, 3H), 1.32 (t, 3H, J=7.1 Hz). 13C-NMR (100
MHz, DMSO-d6) δ: 162.2, 159.9, 156.5, 138.7, 126.6, 112.2, 95.4, 88.6, 60.8, 56.2, 14.6, 11.4.
Preparation of ethyl 7-carbamoyl-4,6-dimethoxy-3-methylbenzofuran-2-carboxylate (3)
To a solution of 2 (2.64 g, 0.01 mol) in dry acetonitrile, CSI (2.21 g, 0.015 mol) was added drop-
wise under nitrogen atmosphere at 0°C, the solution was warmed up to room temperature and stirred
for 3 h. Then 20 mL 2N HCl was added to the reaction solution and stirred for additional 3 h. The
precipitate was filtered, washed with water, dried to afford 3 as white solid in 94.0% yield. LC-MS m/z:
1
308.2 [M+H]+, 330.1 [M+Na]+. H-NMR (400 MHz, DMSO-d6) δ: 7.63 (s, 1H), 7.47 (s, 1H), 6.61 (s,
1H), 4.31 (q, 2H, J= 7.2 Hz), 3.97 (s, 3H), 3.91(s, 3H), 2.62(s, 3H), 1.32(t, 3H, J= 7.2 Hz). 13C-NMR
(100 MHz, DMSO-d6) δ: 164.7, 159.8, 158.5, 156.9, 153.3, 139.2, 126.3, 112.2, 104.8, 92.0, 60.9, 57.2,
56.6, 14.7, 11.5
Preparation of 6-hydroxy-4-methoxy-3-methylbenzofuran-7-carboxamide (FY0413)
To a solution of 3 (0.31 g, 1 mmol) in acetic acid, 30%HBr was added, the solution was stirred for
5-7 h at 110°C and monitored by thin-layer chromatograph (TLC). Sodium bicarbonate solution was

added to the solution to adjust pH to 7, extracted with ethyl acetate for 3 times, the organic phase was
washed with water and brine, dried over Na2SO4, concentrated and purified by column chromatography
on silica (petroleum ether: ethyl acetate = 3:1) to give FY0413 as white solid in 39.0% yield. M.p.:
208.2-210.1°C. LC-MS m/z: 222.1 [M+H]+, 244.1 [M+Na]+. IR (KBr, cm-1 ν): 3455, 3323, 3195, 2924,
2852, 1633, 1519, 1443, 1328, 1292, 903. 1H-NMR (400 MHz, DMSO-d6) δ: 13.76 (s, 1H, 6-OH), 8.17
(s, 1H, 7-CONH2), 7.60 (d, 1H, J= 7.6 Hz, 2-H), 7.53 (s, 1H, 7-CONH2), 6.34 (s, 1H, 5-H), 3.89 (s, 3H,
4-OCH3), 2.24 (d, 3H, 3-CH3, J= 2.2 Hz). 13C-NMR (100 MHz, DMSO-d6) δ: 170.1, 163.2, 158.4,
153.5, 139.4, 115.5, 109.9, 94.7, 93.5, 56.0, 9.4.
Preparation of 7-carbamoyl-4,6-dimethoxy-3-methylbenzofuran-2-carboxylic acid (4)
Intermediate 3 (4.61 g, 0.015 mol) was placed on 50 mL mixed solution of methanol and NaOH
(1.8 g/ 5 mL H2O), the solution was stirred for 2 h at 60°C, then poured the solution into 500 mL cold
water and adjusted the pH to 2 with 2N HCl, filtrated and washed the cake with water, dried to afford 4
as white solid in 90.0% yield. LC-MS m/z: 277.9 [M-H]-.
Preparation of ethyl 7-carbamoyl-6-hydroxy-4-methoxy-3-methylbenzofuran-2-carboxylate (5a)
To a solution of 3 (1.0 g, 3.26 mmol) in dry DCM, BBr3 (0.6 mL, 6.5 mmol) was added drop-wise
under nitrogen atmosphere at -40°C and stirred for 4 h, then warmed up to room temperature and
stirred for additional 2 h. The reaction was quenched with water and the DCM was removed by
evaporators, then sodium bicarbonate solution was added to the solution to adjust pH to 7. The
precipitate was filtered and recrystallized with ethyl alcohol to afford 5a (0.45 g) as white solid in 47.0%
yield. M.p.: 198.0-199.6°C. IR (KBr, cm-1 ν): 3457, 3330, 3210, 2923, 2851, 1721, 1633, 1520, 1435,
1327,1280, 926. LC-MS: 294.2 [M+H]+, 316.1 [M+Na]+, 332.0 [M+K]+. 1H-NMR (400 MHz, DMSO-
d6) δ: 13.98 (s, 1H, 6-OH), 8.42 (s, 1H, 7-CONH2), 7.16 (s, 1H, 7-CONH2), 6.39 (m, 1H, 5-H), 4.33 (q,
2H, J= 4.3 Hz, -COOCH2CH3), 3.92 (d, 3H, J= 3.9 Hz), 2.56 (s, 3H, 3-CH3), 1.33 (t, 3H, J= 1.3 Hz).
13C-NMR (100 MHz, DMSO-d6) δ: 169.7, 166.0, 159.6, 158.9, 152.8, 138.3, 126.3, 110.4, 95.8, 93.0,
60.6, 56.3, 14.1, 10.7.
General procedure for the synthesis of 5b-5r
To a solution of 7-carbamoyl-6-hydroxy-4-methoxy-3-methylbenzofuran-2-carboxylic acid (4)
(0.279 g, 1 mmol) in DMSO, HATU (0.52 g, 1.3 mmol) and DIEA (0.3mL, 1.9 mmol) were added at
25°C, then corresponding amino side chain (1 mmol) was added. After thin-layer chromatograph (TLC)
indicating the reaction completed, poured the solution to 150 mL ice-water and stirred for 30 min, the

precipitate was filtered and dried. Then these residues were dissolved in dry DCM under nitrogen
atmosphere, BBr3 (2 eq.) was added drop-wise under nitrogen atmosphere at -40°C and stirred for 4 h,
then warmed up to room temperature and stirred for additional 2 h. The reaction was quenched with
water and the DCM was removed by evaporators, then sodium bicarbonate solution was added to the
solution to adjust pH to 7. The precipitate was filtered and recrystallized with ethyl alcohol to afford
5b-5r.
Preparation of 6-hydroxy-4-methoxy-N2,3-dimethylbenzofuran-2,7-dicarboxamide (5b)
Yield 58.0%. M.p.: 219.0-220.7°C. IR (KBr, cm-1 ν): 3431, 2923, 2852, 1622, 1536, 1451, 1329,
1290, 872. LC-MS m/z: 279.2 [M+H]+, 301.1 [M+Na]+, 276.9 [M-H]-. 1H-NMR (400 MHz, DMSO-d6)
δ: 14.50 (s, 1H, 6-OH), 8.71 (t, 1H, J= 8.7 Hz, 2-CONH-), 8.31 (s, 1H, 7-CONH2), 7.91 (s, 1H, 7-
CONH2), 6.39 (s, 1H, 5-H), 3.92 (s, 1H, 4-OCH3), 2.80 (d, 3H, J= 2.79 Hz, NHCH3), 2.61 (s, 3H, 3-
CH3). 13C-NMR (100 MHz, DMSO-d6) δ: 169.8, 165.7, 159.3, 151.7, 141.5, 122.0, 110.8, 95.7, 93.1,
56.1, 25.3, 10.6.
Preparation of 6-hydroxy-4-methoxy-N2,N2,3-trimethylbenzofuran-2,7-dicarboxamide (5c)
Yield 53.0%. M.p.: 227.3-229.9°C. IR (KBr, cm-1 ν): 3476, 3183, 2971, 2923, 1625, 1526, 1450,
1
1324, 1209, 821. LC-MS m/z: 293.2 [M+H]+, 315.1 [M+Na]+, 291.0 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 13.93 (s, 1H, 6-OH), 8.26 (s, 1H, 7-CONH2), 7.40 (s, 1H, 7-CONH2), 6.41 (s, 1H, 5-H),
3.92 (s, 3H, 4-OCH3), 3.05 (brs, 6H, -NMe2), 2.39 (s, 3H, 3-Me). 13C-NMR (100 MHz, DMSO-d6) δ:
169.9, 164.6, 160.4, 159.0, 151.9, 142.1, 119.6, 110.1, 95.4, 93.3, 56.2, 10.38.
Preparation of N2-(2-(dimethylamino)ethyl)-6-hydroxy-4-methoxy-3-methylbenzofuran-2,7-
dicarboxamide (5d)
Yield 64.0%. M.p.: 256.1-258.6°C. IR (KBr, cm-1 ν): 3434, 3340, 2922, 2852, 1621, 1511, 1455,
1
1329, 1208, 820. LC-MS m/z: 336.2 [M+H]+, 358.2 [M+Na]+, 334.0 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.50 (s, 1H, 6-OH), 8.75 (t, 1H, J= 8.7 Hz, 2-CONH-), 8.32 (s, 1H, 7-CONH2), 7.91 (s,
1H, 7-CONH2), 6.40 (s, 1H, 5-H), 3.92 (s, 3H, 4-OCH3), 3.36 (m, 4H), 2.61 (s, 3H, 3-CH3), 2.44 (t, 2H,
J= 2.4 Hz), 2.22 (s, 6H). 13C-NMR (100 MHz, DMSO-d6) δ: 170.3, 166.3, 159.8, 159.3, 152.2, 141.8,
122.8, 111.4, 96.2, 93.6, 58.8, 56.7, 45.7, 36.9, 11.1.
Preparation of N2-cyclopentyl-6-hydroxy-4-methoxy-3-methylbenzofuran-2,7-dicarboxamide (5e)
Yield 42.0%. M.p.: 229.5-230.8°C. IR (KBr, cm-1 ν): 3475, 2921, 2852, 1623, 1526, 1450, 1328,
1
1208, 821. LC-MS m/z: 333.2 [M+H]+, 355.2 [M+Na]+, 371.2 [M+K]+, 331.0 [M-H]-. H-NMR (400

MHz, DMSO-d6) δ: 14.41 (s, 1H, 6-OH), 8.53 (d, 1H, J=8.5 Hz, 2-CONH-), 8.28 (s, 1H, 7-CONH2),
8.00 (s, 1H, 7-CONH2), 6.39 (s, 1H, 5-H), 4.22 (m, 1H), 3.91 (s, 3H, 4-OCH3), 2.60 (s, 1H, 3-CH3),
1.93 (m, 2H), 1.71 (m, 2H), 1.55 (m, 4H). 13C-NMR (100 MHz, DMSO-d6) δ: 169.8, 165.6, 159.3,
158.7, 151.7, 141.6, 122.2, 111.0, 95.7, 93.3, 56.2, 50.2, 31.9, 23.7, 10.7.
Preparation of N2-cyclohexyl-6-hydroxy-4-methoxy-3-methylbenzofuran-2,7-dicarboxamide (5f)
Yield 51.0%. M.p.: 202.2-204.1°C. IR (KBr, cm-1 ν): 3435, 3379, 2923, 2852, 1620, 1525, 1450,
1
1317, 1283, 822, LC-MS m/z: 347.3 [M+H]+, 369.3 [M+Na]+, 345.2 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.39 (s, 1H, 6-OH), 8.45 (d, 1H, J=8.5 Hz, 2-CONH-), 8.24 (s, 1H, 7-CONH2), 7.97 (s,
1H, 7-CONH2), 6.37 (s, 1H, 5-H), 3.89 (s, 3H, 4-OCH3), 3.75 (m, 1H), 2.58 (s, 3H, 3-CH3), 1.81 (d, 2H,
J= 1.8 Hz), 1.74 (d, 2H, J= 1.7 Hz), 1.33 (m, 6H).
Preparation
of
6-hydroxy-4-methoxy-3-methyl-2-(morpholine-4-carbonyl)benzofuran-7-
carboxamide (5g)
Yield 44.0%. M.p.: 171.3-172.7°C. IR (KBr, cm-1 ν): 3460, 3326, 3205, 2920, 2851, 1632, 1517,
1
1431, 1329, 1281, 826. LC-MS m/z: 335.5 [M+H]+, 357.5 [M+Na]+, 333.2 [M-H]-. H-NMR (400
MHz, DMSO-d6) δ: 13.90 (s, 1H, 6-OH), 8.27 (s, 1H, 7-CONH2), 7.38 (s, 1H, 7-CONH2), 6.42 (s, 1H,
5-H), 3.92 (s, 3H, 4-OCH3), 3.64 (m, 4H), 3.60 (m, 4H), 2.39 (s, 3H, 3-CH3). 13C-NMR (100 MHz,
DMSO-d6) δ: 170.3, 165.1, 159.8, 159.5, 152.5, 142.0, 120.6, 110.6, 96.0, 93.9, 66.6, 56.7, 10.8.
Preparation
of
6-hydroxy-4-methoxy-3-methyl-2-(piperidine-1-carbonyl)benzofuran-7-
carboxamide (5h)
Yield 43.0%. M.p.: 190.4-192.3°C. IR (KBr, cm-1 ν): 3477, 3328, 2924, 2852, 1613, 1516, 1445,
1327, 1269, 822. LC-MS m/z: 333.2 [M+H]+, 355.2 [M+Na]+, 371.2 [M+K]+, 331.0 [M-H]-. 1H-NMR
(400 MHz, DMSO-d6) δ: 13.87 (s, 1H, 6-OH), 8.30 (s, 1H, 7-CONH2), 7.33 (s, 1H, 7-CONH2), 6.41 (s,
1H), 3.92 (s, 3H), 3.52 (br, 4H), 2.36 (s, 3H), 1.57 (m, 4H), 1.24 (br, 2H). 13C-NMR (100 MHz,
DMSO-d6) δ: 169.9, 164.5, 159.1, 159.0, 151.9, 142.1, 119.1, 110.1, 95.4, 93.4, 56.2, 24.1, 10.3.
Preparation of 2-(4-ethylpiperazine-1-carbonyl)-6-hydroxy-4-methoxy-3-methylbenzofuran-7-
carboxamide (5i)
Yield 83.0%. M.p.: 204.1-205.6°C. IR (KBr, cm-1 ν): 3421, 3204, 2922, 2851, 1622, 1515, 1457,
1
1326, 1207, 824. LC-MS m/z: 362.2 [M+H]+, 384.2 [M+Na]+, 400 [M+K]+, 360.0 [M-H]-. H-NMR
(400 MHz, DMSO-d6) δ: 13.88 (s, 1H, 6-OH), 8.28 (s, 1H, 7-CONH2), 7.33 (s, 1H, 7-CONH2), 6.41 (s,
1H), 3.92 (s, 3H), 3.57 (br, 4H), 2.42 (br, 4H), 2.37 (s, 3H), 1.23 (s, 2H), 1.02 (t, 3H, J= 1.0 Hz).

Preparation of 6-hydroxy-4-methoxy-3-methyl-2-(4-phenylpiperazine-1-carbonyl)benzofuran-7-
carboxamide (5j)
Yield 68.0%. M.p.: 246.4-248.2°C. IR (KBr, cm-1 ν): 3470, 3337, 3186, 2923, 2852, 1634, 1515,
1463, 1387, 1211, 823. LC-MS m/z: 410.3 [M+H]+, 432.2 [M+Na]+, 448.2 [M+K]+, 408.0 [M-H]-. 1H-
NMR (400 MHz, DMSO-d6) δ: 13.90 (s, 1H, 6-OH), 8.27 (s, 1H, 7-CONH2), 7.44 (s, 1H, 7-CONH2),
7.24 (t, 2H, J= 7.2 Hz), 6.97 (d, 2H, J= 7.0 Hz), 6.82 (t, 1H, J= 6.8 Hz), 6.41 (s, 1H), 3.92 (s, 3H), 3.73
(br, 4H), 3.22 (br, 4H), 2.41 (s, 3H).
Preparation
of
2-(4-(4-fluorophenyl)piperazine-1-carbonyl)-6-hydroxy-4-methoxy-3-
methylbenzofuran-7-carboxamide (5k)
Yield 32.0 %. M.p.:249.4-250.9°C. IR (KBr, cm-1 ν): 3477, 2921, 2852, 1628, 1509, 1460, 1326,
1209, 752. LC-MS m/z: 450.2 [M+Na]+, 426.0 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 13.90 (s, 1H,
6-OH), 8.28 (br, 1H, 7-CONH2), 7.41 (br, 1H, 7-CONH2), 7.08 (m, 2H), 7.00 (m, 2H), 6.43 (s, 1H),
3.93 (s, 3H), 3.73 (br, 4H), 3.17 (br, 4H), 2.41 (s, 3H).
Preparation of 6-hydroxy-4-methoxy-3-methyl-2-(4-(3-(trifluoromethyl)phenyl)piperazine-1-
carbonyl)benzofuran-7-carboxamide (5l)
Yield 61.0 %. M.p.: 228.6-230.1°C. IR (KBr, cm-1 ν): 3475, 3329, 3186, 2922, 2864, 1631, 1496,
1
1443, 1324, 1211, 884, 790. LC-MS m/z: 474.3 [M+H]+, 496.3 [M+Na]+, 512.2 [M+K]+. H-NMR
(400 MHz, DMSO-d6) δ: 13.91 (s, 1H, 6-OH), 8.28 (br, 1H, 7-CONH2), 7.45 (t, 1H, J= 8.0 Hz), 7.42
(br, 1H, 7-CONH2), 7.25 (d, 1H, J= 8.6 Hz), 7.22 (s, 1H), 7.11 (d, 1H, J= 7.2 Hz), 6.43 (s, 1H), 3.93 (s,
3H), 3.74 (br, 4H), 3.35 (br, 4H), 2.42 (s, 3H). 13C-NMR (100 MHz, DMSO-d6) δ: 170.3, 165.2, 159.8,
159.6, 152.5, 152.4, 142.1, 130.5, 126.2, 123.5, 119.5, 115.5, 111.9, 110.7, 96.0, 93.9, 56.7, 48.3, 48.2,
10.9.
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-phenylbenzofuran-2,7-dicarboxamide (5m)
Yield 46.0%. M.p.: 286.8-287.7°C. IR (KBr, cm-1 ν): 3431, 3357, 3200, 2922, 2852, 1622, 1521,
1443, 1316, 1207, 754, 723. LC-MS m/z: 341.2 [M+H]+, 363.2 [M+Na]+, 339.0 [M-H]-. 1H-NMR (400
MHz, DMSO-d6) δ: 14.50 (s, 1H, 6-OH), 10.35 (s, 1H), 8.32 (s, 1H, 7-CONH2), 8.13 (s, 1H, 7-
CONH2), 7.66 (d, 2H, J= 8.0 Hz), 7.37 (t, 2H, J= 8.0 Hz), 7.14 (t, 1H, J=8.0 Hz), 6.41 (s, 1H), 3.92 (s,
3H), 2.65 (s, 3H).
Preparation
of
N2-(4-fluoro-3-(trifluoromethyl)phenyl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (5n)

Yield 49.0%. M.p.: 271.6-272.5°C. IR (KBr, cm-1 ν): 3430, 2921, 2852, 1622, 1531, 1452, 1322,
1
1210, 830, 619. LC-MS m/z: 443.2 [M+H]+, 465.2 [M+Na]+, 441.1 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.56 (s, 1H, 6-OH), 10.63 (s, 1H), 8.37 (s, 1H, 7-CONH2), 8.26 (d, 1H, J= 4.0 Hz), 8.07
(s, 1H, 7-CONH2), 8.02 (dd, 1H, J= 2.2 Hz, J= 8.8 Hz), 7.70 (d, 1H, J= 8.7 Hz), 6.40 (s, 1H), 3.91 (s,
3H), 2.64 (s, 3H).
Preparation
of
6-hydroxy-4-methoxy-3-methyl-N2-(4-methyl-3-(trifluoromethyl)phenyl)
benzofuran-2,7-dicarboxamide (5o)
Yield 57.0%. M.p.: 294.3-296.1°C. IR (KBr, cm-1 ν): 3457, 3358, 2922, 2852, 1623, 1505, 1451,
1
1320, 1211, 824, 699. LC-MS m/z: 445.2 [M+Na]+, 421.0 [M-H]-. H-NMR (400 MHz, DMSO-d6) δ:
14.57 (s, 1H, 6-OH), 10.53 (s, 1H), 8.38 (s, 1H, 7-CONH2), 8.12 (s, 1H, 7-CONH2), 8.07 (d, 1H, J= 4.0
Hz), 7.89 (dd, 1H, J= 2.2 Hz, J= 8.8 Hz), 7.45 (d, 1H, J= 8.7 Hz), 6.44 (s, 1H), 3.94 (s, 3H), 2.67 (s,
3H), 2.43 (s, 3H).
Preparation of N2-benzyl-6-hydroxy-4-methoxy-3-methylbenzofuran-2,7-dicarboxamide (5p)
Yield 52.0%. M.p.: 294.0-295.7°C. IR (KBr, cm-1 ν): 3427, 3332, 2923, 2852, 1622, 1534, 1450,
1329, 1212, 822, 748. LC-MS m/z: 355.2 [M+H]+, 377.2 [M+Na]+, 353.2 [M-H]-.
Preparation of N2-benzyl-6-hydroxy-4-methoxy-N2,3-dimethylbenzofuran-2,7-dicarboxamide (5q)
Yield 34.0%. M.p.: 231.3-232.8°C. IR (KBr, cm-1 ν): 3471, 3321, 3164, 2925, 2852, 1636, 1516,
1446, 1324, 1210, 735, 712. LC-MS m/z: 369.3 [M+H]+, 391.3 [M+Na]+, 367.2 [M-H]-. 1H-NMR (400
MHz, DMSO-d6) δ: 13.90 (s, 1H), 8.20 (br, 1H), 7.995 (br, 1H), 7.37 (m, 3H), 7.30 (m, 2H), 6.39 (s,
1H), 4.64 (s, 2H), 3.90 (s, 3H), 2.98 (br, 3H), 2.41 (s, 3H). 13C-NMR (100 MHz, DMSO-d6) δ: 170.3,
165.2, 159.5, 152.4, 142.5, 137.5, 129.1, 127.9, 120.9, 110.7, 96.0, 93.9, 56.7, 10.9.
Preparation
of
N2-(3-fluorobenzyl)-6-hydroxy-4-methoxy-3-methylbenzofuran-2,7-
dicarboxamide (5r)
Yield 28.0%. M.p.: 266.3-267.2°C. IR (KBr, cm-1 ν): 3430, 3291, 2922, 2851, 1621, 1533, 1452,
1327, 1212, 821, 786. LC-MS m/z: 373.2 [M+H]+, 395.2 [M+Na]+, 371.1 [M-H]-. 1H-NMR (400 MHz,
DMSO-d6) δ: 14.51 (s, 1H, 6-OH), 9.33 (t, 1H, J= 6.0 Hz), 8.28 (s, 1H, 7-CONH2), 7.91 (s, 1H, 7-
CONH2), 7.37 (q, 1H, J= 7.9 Hz), 7.14 (m, 2H), 7.07 (m, 1H), 6.39 (s, 1H), 4.49 (d, 2H, J= 6.0 Hz),
3.90 (s, 3H), 2.61 (s, 3H).
Preparation of tert-butyl 4-(7-carbamoyl-4,6-dimethoxy-3-methylbenzofuran-2-carboxamido)
piperidine-1-carboxylate (6a)

To a solution of 4 (2.79 g, 10 mmol) in DMSO, HATU (5.2 g, 13 mmol) and DIEA (3 mL, 19
mmol) were added at 25°C, then 1-Boc-4-aminopiperidine (2.0 g, 10 mmol) was added. After thin-
layer chromatograph (TLC) indicating the reaction completed, poured the solution to 150 mL ice-water
and stirred for 30 min, the precipitate was filtered and dried to afford 6a as white solid in 65.0% yield.
LC-MS m/z: 460.1 [M-H]-.
Preparation of tert-butyl 4-((7-carbamoyl-4,6-dimethoxy-3-methylbenzofuran-2-carboxamido)
methyl)piperidine-1-carboxylate (6b)
To a solution of 4 (2.79 g, 10 mmol) in DMSO, HATU (5.2 g, 13 mmol) and DIEA (3 mL, 19
mmol) were added at 25°C, then 1-Boc-4-(aminomethyl)piperidine (2.14 g, 10 mmpl) was added. After
thin-layer chromatograph (TLC) indicating the reaction completed, poured the solution to 150 mL ice-
water and stirred for 30 min, the precipitate was filtered and dried to afford 6a as white solid in 58.0%
yield. LC-MS m/z: 474.2 [M-H]-.
Preparation of 4,6-dimethoxy-3-methyl-N2-(piperidin-4-yl)benzofuran-2,7-dicarboxamide (7a)
To a solution of 6a (5 mmol) in dry DCM, saturated HCl/ ethyl acetate solution (10 mL) was
added and stirred for 1 h at room temperature. The precipitate was filtered and dried to afford 7a as
white solid in 92.0% yield. LC-MS m/z: 362.3 [M+H]+, 384.2 [M+Na]+.
Preparation of 4,6-dimethoxy-3-methyl-N2-(piperidin-4-ylmethyl)benzofuran-2,7-dicarboxamide
(7b)
To a solution of 6b (5 mmol) in dry DCM, saturated HCl/ ethyl acetate solution (10 mL) was
added and stirred for 1 h at room temperature. The precipitate was filtered and dried to afford 7b as
white solid in 88.0% yield. LC-MS m/z: 376.3 [M+H]+, 398.2 [M+Na]+.
General procedure for the synthesis of 8a-8p
To a solution of 7a/7b (1 mmol) in DMF, K2CO3 (0.828 g, 6 mmol) was added and the solution
was stirred for 0.5 h at 80°C, then corresponding substituted benzyl chloride (benzyl bromide) or
substituted benzoyl chloride was added and stirred for additional 5-6 h. After thin-layer chromatograph
(TLC) indicating the reaction completed, poured the solution to 150 mL ice-water and stirred for 30
min, the precipitate was filtered as yellowish white solid, washed the precipitate with diethyl ether.
Dissolving these solid(0.5 mmol) in dry DCM under nitrogen atmosphere, BBr3 (1 mmol) was added
drop-wise under nitrogen atmosphere at -40°C and stirred for 4 h, then warmed up to room temperature
and stirred for additional 2 h. The reaction was quenched with water and the DCM was removed by

evaporators, then sodium bicarbonate solution was added to the solution to adjust pH to 7. The
precipitate was filtered and recrystallized with ethyl alcohol, purified by column chromatography on
silica (DCM: methyl alcohol = 20:1 ) to give compounds 8a-8p as off-white solid.
Preparation
of
6-hydroxy-4-methoxy-3-methyl-N2-(1-(pyridin-4-ylmethyl)piperidin-4-yl)
benzofuran-2,7-dicarboxamide (8a)
Yield 11.0%. M.p.: 283.4-284.7°C. IR (KBr, cm-1 ν): 3428, 3270, 3185, 2923, 2852, 1661, 1508,
1
1451, 1347, 1289. LC-MS m/z: 439.3 [M+H]+, 461.3 [M+Na]+, 437.1 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.44 (s, 1H, 6-OH), 8.51 (d, 2H, J= 5.3 Hz), 8.48 (d, 1H, J= 8.0 Hz), 8.29 (s, 1H), 7.97
(s, 1H), 7.33 (d, J= 5.2 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.80 (m, 1H), 3.53 (s, 2H), 2.83 (d,
2H, J= 10.3 Hz), 2.60 (s, 3H, 3-Me), 2.08 (t, 2H, J= 11.1 Hz), 1.80 (d, 2H, J= 9.8 Hz), 1.68 (q, 2H, J=
11.8 Hz).
Preparation of N2-(1-((6-chloropyrimidin-4-yl)methyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-
methylbenzofuran-2,7-dicarboxamide (8b)
Yield 23.0%. M.p.: 310.2-311.6°C. IR (KBr, cm-1 ν): 3464, 3323, 3191, 2921, 2852, 1652, 1520,
1
1493, 1340, 1300，LC-MS m/z: 460.1 [M+H]+, 482.1 [M+Na]+, 457.9 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.42 (s, 1H, 6-OH), 8.42 (d, 1H, J= 8.0 Hz), 8.36 (s, 1H), 8.27 (s, 1H), 7.88 (s, 1H),
7.05 (s, 1H), 6.39 (s, 1H), 4.16 (m, 1H), 3.91 (s, 3H, 4-OMe), 3.08 (t, 2H, J= 12.6 Hz), 2.61 (s, 3H, 3-
Me), 1.89 (d, 2H, J= 9.8 Hz), 1.52 (m, 2H), 1.23 (m, 2H).
Preparation of N2-(1-(2-fluorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl-benzofuran-
2,7-dicarboxamide (8c)
Yield 43.0%. M.p.: 251.2-252.9°C. IR (KBr, cm-1 ν): 3441, 2922, 2851, 1656, 1524, 1490, 1322,
1
1291, 749.1. LC-MS m/z: 456.2 [M+H]+, 478.2 [M+Na]+,454.0 [M-H]-. H-NMR (400 MHz, DMSO-
d6) δ: 14.43 (s, 1H, 6-OH), 8.45 (d, 1H, J= 7.4 Hz), 8.27 (s, 1H), 7.95 (s, 1H), 7.41 (m, 1H), 7.32 (m,
1H), 7.18 (m, 2H), 6.39 (s, 1H), 3.91 (s, 3H, 4-OMe), 3.79 (br, 1H), 3.54 (s, 2H), 2.86 (d, 2H, J= 9.1
Hz), 2.60 (s, 3H, 3-Me), 2.07 (t, 2H, J= 9.6 Hz), 1.79 (d, 2H, J= 10.2 Hz), 1.63 (m, 2H).
Preparation of N2-(1-(4-fluorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl-benzofuran-
2,7-dicarboxamide (8d)
1
Yield 30.0%. LC-MS m/z: 456.2 [M+H]+, 478.2 [M+Na]+, 454.0 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.43 (s, 1H, 6-OH), 8.46 (d, 1H, J= 6.5 Hz), 8.28 (s, 1H), 7.96 (s, 1H), 7.34 (br, 2H),

7.15 (m, 2H), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.80 (br, 1H), 3.46 (s, 2H), 2.82 (br, 2H), 2.60 (s,
3H, 3-Me), 2.02 (m, 2H), 1.78 (m, 2H), 1.65 (m, 2H).
Preparation of N2-((1-(4-fluorobenzyl)piperidin-4-yl)methyl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8e)
Yield 21.0%. M.p.: 189.1-191.0°C. IR (KBr, cm-1 ν): 3432, 2921, 2851, 1621, 1512, 1451, 1329,
1286, 823. LC-MS m/z: 470.7 [M+H]+, 468.4 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 14.51 (s, 1H,
6-OH), 8.82 (t, 1H, J= 4.5 Hz), 8.30 (s, 1H), 7.90 (s, 1H), 7.56 (br, 2H), 7.30 (br, 2H), 6.40 (s, 1H),
4.08 (q, 1H, J= 5.0 Hz), 3.91 (s, 3H, 4-OMe), 3.37 (br, 2H), 3.17 (m, 4H), 2.92 (br, 2H), 2.61 (s, 3H, 3-
Me), 1.83 (br, 2H), 1.44 (br, 2H).
Preparation of N2-((1-(4-fluorobenzoyl)piperidin-4-yl)methyl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8f)
Yield 27.0%. M.p.: 173.6-174.4°C. IR (KBr, cm-1 ν): 3434, 2922, 2852, 1621, 1512, 1450, 1330,
1
1284, 878.3, 846.5, 823.6. LC-MS m/z: 484.3 [M+H]+, 506.2 [M+Na]+, 482.0 [M-H]-. H-NMR (400
MHz, DMSO-d6) δ: 14.49 (s, 1H, 6-OH), 8.81 (t, 1H, J= 5.5 Hz), 8.29 (s, 1H), 7.92 (s, 1H), 7.44 (t, 2H,
J= 8.2 Hz), 7.27 (t, 2H, J= 8.7 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.19 (s, 2H), 3.01 (m, 1H),
2.61 (s, 3H), 1.60-1.86 (m, 4H), 1.18 (m, 4H).
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-(1-(4-methylbenzyl)piperidin-4-yl)benzofuran
-2,7-dicarboxamide (8g)
Yield 26.0%. M.p.: 242.7-243.7°C. IR (KBr, cm-1 ν): 3425, 2921, 2851, 1623, 1515, 1451, 1328,
1263，822.9. LC-MS m/z: 452.3 [M+H]+, 474.3 [M+Na]+, 450.1 [M-H]-.
Preparation of N2-(1-(4-chlorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl-benzofuran-
2,7-dicarboxamide (8h)
Yield 32.0%. M.p.: 245.4-246.9°C. IR (KBr, cm-1 ν): 3474, 2922, 2851, 1657, 1523, 1491, 1323,
1290, 822. LC-MS m/z: 472.2 [M+H]+, 494.2 [M+Na]+, 470.2 [M-H]-. 1H-NMR (400 MHz, DMSO-d6)
δ: 14.43 (s, 1H, 6-OH), 8.45 (d, 1H, J= 7.9 Hz), 8.28 (s, 1H), 7.96 (s, 1H), 7.38 (d, 2H, J= 7.4 Hz), 7.33
(d, 2H, J= 7.4 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.80 (br, 1H), 3.47 (s, 2H), 2.82 (d, 2H, J=
9.1 Hz), 2.60 (s, 3H, 3-Me), 2.03 (t, 2H, J= 10.2 Hz), 1.77 (m, 2H), 1.63 (m, 2H).
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-(1-(4-(trifluoromethyl)benzyl)piperidin-4-
yl)benzofuran-2,7-dicarboxamide (8i)

Yield 21.0%. M.p.: 231.5-232.2°C. IR (KBr, cm-1 ν): 3422, 2922, 2852, 1625, 1513, 1450, 1327,
1292, 821. LC-MS m/z: 506.4 [M+H]+, 528.3 [M+Na]+, 504.3 [M-H]-. 1H-NMR (400 MHz, DMSO-d6)
δ: 14.43 (s, 1H, 6-OH), 8.46 (d, 1H, J= 8.0 Hz), 8.28 (s, 1H), 7.96 (s, 1H), 7.69 (d, 2H, J= 8.0 Hz), 7.54
(d, 2H, J= 8.0 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.81 (br, 1H), 3.58 (s, 1H), 2.83 (d, 2H, J=
11.2 Hz), 2.60 (s, 3H, 3-Me), 2.07 (t, 2H, J= 11.3 Hz), 1.80 (d, 2H, J= 11.1 Hz), 1.61-1.70 (m, 2H).
13C-NMR (100 MHz, DMSO-d6) δ: 170.3, 166.2, 159.8, 158.8, 152.2, 141.8, 129.8, 125.6, 122.9, 111.4,
96.2, 93.7, 56.6, 52.8, 46.5, 31.8, 11.1.
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-(1-(4-(trifluoromethyl)benzoyl)piperidin-4-
yl)benzofuran-2,7-dicarboxamide (8j)
Yield 34.0%. M.p.: 215.7-216.2°C. IR (KBr, cm-1 ν): 3449, 2923, 2852, 1658, 1519, 1452, 1324,
1
1276, 820.1, 848. LC-MS m/z: 520.1 [M+H]+, 542.2 [M+Na]+, 518.0 [M-H]-. H-NMR (400 MHz,
DMSO-d6) δ: 14.15 (s, 1H, 6-OH), 8.52 (d, 1H, J= 7.8 Hz), 8.31 (s, 1H), 7.94 (s, 1H), 7.84 (d, 2H, J=
8.0 Hz), 7.60 (d, 2H, J= 8.0 Hz), 6.40 (s, 1H, 5-H), 4.52 (m, 1H), 4.12 (m, 1H), 3.91 (s, 3H, 4-OMe),
3.53 (m, 1H), 2.94 (m, 1H), 2.61 (s, 3H, 3-Me), 1.95 (m, 1H), 1.81 (m, 1H), 1.50-1.60 (m, 2H).
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-(1-(3-(trifluoromethyl)benzyl)piperidin-4-
yl)benzofuran-2,7-dicarboxamide (8k)
Yield 28.0%. M.p.: 219.4-220.7°C. IR (KBr, cm-1 ν): 3437, 2922, 2851, 1620, 1524, 1452, 1329,
1291, 757. LC-MS m/z: 506.2 [M+H]+, 504.0 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 14.45 (s, 1H,
6-OH), 8.46 (d, 1H, J= 7.3 Hz), 8.30 (s, 1H), 7.96 (s, 1H), 7.60-7.67 (m, 4H, phenyl H), 6.40 (s, 1H, 5-
H), 3.92 (s, 3H, 4-OMe), 3.83 (m, 1H), 3.60 (s, 2H), 2.84 (d, 2H, J= 10.0 Hz), 2.61 (s, 3H, 3-Me), 2.08
(t, 2H, J= 12.0 Hz), 1.79 (m, 2H), 1.66 (m, 2H).
Preparation of 6-hydroxy-4-methoxy-3-methyl-N2-((1-(3-(trifluoromethyl)benzyl)piperidin-4-
yl)methyl)benzofuran-2,7-dicarboxamide (8l)
Yield 28.0%. M.p.: 181.5-183.4°C. IR (KBr, cm-1 ν): 3426, 2923, 2852, 1621, 1514, 1451, 1329,
1286, 877, 821. LC-MS m/z: 520.6 [M+H]+, 518.4 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 14.50 (s,
1H, 6-OH), 9.43 (br, 1H, NH), 8.82 (s, 1H, NH2), 8.30 (s, 1H, NH2), 7.70-7.81 (m, 4H, phenyl H), 6.40
(s, 1H), 4.40 (br, 1H), 3.91 (s, 3H, 4-OMe), 3.38 (m, 2H), 3.18 (s, 2H), 2.96 (m, 2H), 2.61 (s, 3H, 3-
Me), 1.84 (br, 4H), 1.44 (br, 2H).
Preparation
of
N2-(1-(2,6-dichlorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8m)

Yield 28.0%. M.p.: 224.6-225.3°C. IR (KBr, cm-1 ν): 3463, 2921, 2851, 2764, 1660, 1526, 1323,
1286, 928. LC-MS m/z: 506.3 [M+H]+, 528.3 [M+Na]+, 504.2 [M-H]-. 1H-NMR (400 MHz, DMSO-d6)
δ: 14.43 (s, 1H, 6-OH), 8.39 (d, 1H, J= 8.0 Hz), 8.25 (s, 1H), 7.92 (s, 1H), 7.46 (d, 2H, 8.0 Hz), 7.33 (t,
1H, J= 7.6 Hz), 6.39 (s, 1H, 5-OH), 3.91 (s, 3H, 4-OMe), 3.82 (m, 1H), 3.70 (s, 2H), 2.83 (d, 2H, J=
11.3 Hz), 2.60 (s, 3H, 3-Me), 2.26 (t, 2H, J= 10.5 Hz), 1.76 (d, 2H, J= 10.0 Hz), 1.55-1.63 (m, 2H).
13C-NMR (100 MHz, DMSO-d6) δ: 170.6, 166.2, 159.7, 158.7, 152.2, 141.8, 136.6, 134.5, 130.2, 129.0,
122.9, 111.4, 96.2, 93.7, 56.6, 56.4, 52.7, 46.3, 31.9, 11.1.
Preparation
of
N2-(1-(2,4-dichlorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8n)
Yield 40.0%. M.p.: 236.8-237.4°C. IR (KBr, cm-1 ν): 3472, 2922, 2852, 1625, 1525, 1448, 1325,
1292, 822. LC-MS m/z: 506.3 [M+H]+, 528.3 [M+Na]+, 504.2 [M-H]-. 1H-NMR (400 MHz, DMSO-d6)
δ: 14.43 (s, 1H, 6-OH), 8.44 (d, 1H, J= 8.0 Hz), 8.28 (s, 1H), 7.95 (s, 1H), 7.59 (d, 1H, J= 1.4 Hz), 7.50
(d, 1H, J= 8.4 Hz), 7.43 (dd, 1H, J= 1.4 Hz, 8.4 Hz), 6.39 (s,1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.83 (m,
1H), 3.56 (s, 2H), 2.84 (d, 2H, J= 10.7 Hz), 2.60 (s, 3H, 3-Me), 2.15 (t, 2H, J= 10.9 Hz), 1.79 (d, 2H,
J= 9.9 Hz), 1.64-1.69 (m, 2H). 13C-NMR (100 MHz, DMSO-d6) δ: 170.3, 166.2, 159.8, 158.8, 152.2,
141.9, 134.6, 132.3, 129.1, 127.7, 122.9, 111.4, 96.2, 93.7, 58.5, 56.6, 52.8, 46.5, 31.9, 11.1.
Preparation
of
N2-(1-(2,3-dichlorobenzyl)piperidin-4-yl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8o)
Yield 34.0%. M.p.: 159.6-160.4°C. IR (KBr, cm-1 ν): 3471, 2923, 2852, 1655, 1523, 1450, 1325,
1292, 823.1. LC-MS m/z: 506.3 [M+H]+, 528.3 [M+Na]+. 1H-NMR (400 MHz, DMSO-d6) δ: 14.43 (s,
1H, 6-OH), 8.46 (d, 1H, J= 8.0 Hz), 8.29 (s, 1H), 7.96 (s, 1H), 7.56 (d, 1H, J= 7.4 Hz), 7.49 (d, 1H, J=
7.4 Hz), 7.38 (t, 1H, J= 7.8 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.84 (m, 1H), 3.66 (s, 2H),
2.90 (d, 2H, J= 9.8 Hz), 2.60 (s, 3H, 3-Me), 2.23 (m, 2H), 1.82 (d, 2H, J= 10.1 Hz), 1.68 (q, 2H, J=
10.7 Hz). 13C-NMR (100 MHz, DMSO-d6) δ: 170.3, 166.2, 159.8, 158.8, 152.2, 141.9, 132.3, 129.6,
128.4, 122.9, 111.4, 96.2, 93.7, 59.6, 56.7, 52.8, 46.4, 31.7, 11.1.
Preparation of N2-((1-(3,4-dichlorobenzyl)piperidin-4-yl)methyl)-6-hydroxy-4-methoxy-3-methyl
benzofuran-2,7-dicarboxamide (8p)
Yield 41.0%. M.p.: 244.6-245.5°C. IR (KBr, cm-1 ν): 3417, 3271, 2922, 2851, 1656, 1530, 1452,
1339, 1288, 823. LC-MS m/z: 520.6 [M+H]+, 518.3 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 14.49
(s, 1H, 6-OH), 8.77 (t, 1H, J= 5.5 Hz), 8.27 (s, 1H), 7.92 (s, 1H), 7.56 (t, 2H, J= 10.4 Hz), 7.29 (d, 1H,

J= 7.8 Hz), 6.39 (s, 1H, 5-H), 3.91 (s, 3H, 4-OMe), 3.45 (s, 2H), 3.16 (t, 2H, J= 5.7 Hz), 2.78 (d, 2H,
J= 8.8 Hz), 2.60 (s, 3H, 3-Me), 1.93 (br, 2H), 1.65 (d, 2H, J= 10.9 Hz), 1.56 (m, 1H), 1.23 (m, 2H).
Preparation
of
4,6-dimethoxy-3-methyl-N2-(1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)
benzofuran-2,7-dicarboxamide (17a)
17a was synthesized by the method shown in Scheme 2 as white solid. Yield 51.0%. M.p.: 185.1-
1
186.5°C, LC-MS: 520.3 [M+H]+, 542.3 [M+Na]+. H-NMR (400 MHz, DMSO-d6) δ: 7.75 (d, 1H, J=
8.4 Hz), 7.66 (brs, 1H), 7.56-7.63 (m, 4H), 7.38 (brs, 1H), 6.59 (s, 1H, 5-H), 3.95 (s, 3H), 3.90 (s, 3H),
3.77 (m, 1H), 3.58 (s, 2H), 2.79 (d, 2H, J= 11.1 Hz), 2.57 (s, 3H, 3-Me), 2.08 (t, 2H, J= 11.5 Hz), 1.76
(m, 2H), 1.63 (m, 2H).
Preparation
of
4,6-dihydroxy-3-methyl-N2-(1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)
benzofuran-2,7-dicarboxamide (17b)
17b was synthesized by the method shown in Scheme 3 as white solid with a yield of 4.0%. M.p.:
219.6-221.3°C, LC-MS: 492.2 [M+H]+, 490.0 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 14.22 (s, 1H,
6-OH), 11.13 (s, 1H, 4-OH), 8.67 (d, 1H, J= 7.4 Hz), 8.22 (s, 1H, CONH2), 7.98 (s, 1H, CONH2), 7.86
(m, 3H), 7.75 (t, 1H, J= 7.8 Hz), 6.18 (s, 1H, 5-H), 4.43 (d, 2H, J= 4.4 Hz), 3.97 (m, 1H), 3.43 (d, 2H,
J= 11.5 Hz), 3.13 (m, 2H), 2.60 (s, 3H, 3-Me), 2.07 (d, 2H, J= 13.6 Hz), 1.87 (m, 2H).
Preparation
of
4,6-dimethoxy-3-methyl-N-(1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)
benzofuran-2-carboxamide (17c)
17c was synthesized by the method shown in Scheme 4 as white solid with a yield of 40.0%. M.p.:
1
145.0-146.7°C, LC-MS: 477.2 [M+H]+. H-NMR (400 MHz, DMSO-d6) δ: 8.10 (t, 1H, J= 7.4 Hz),
7.55-7.66 (m, 4H), 6.68 (d, 1H, J= 7.1 Hz), 6.41 (d, 1H, J= 1.6 Hz), 3.96 (d, 3H, J= 7.3 Hz), 3.84 (d,
3H, J= 19.0 Hz), 3.73-3.80 (m, 1H), 3.57 (m, 2H), 2.81 (d, 2H, J= 8.6 Hz), 2.59 (s, 3H, 3-Me), 2.06 (t,
2H, J= 10.7 Hz), 1.62-1.78 (m, 4H).
Preparation of 6-methoxy-3-methyl-N-(1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)benzofuran-2-
carboxamide (17d)
17d was synthesized by the method shown in Scheme 5 as white solid with a yield of 35.0%. M.p.:
210.7-212.4°C, LC-MS: 447.1 [M+H]+, 444.8 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 8.20 (d, 1H,
J= 8.2 Hz), 7.58-7.66 (m, 5H), 7.10 (d, 1H, J= 2.0 Hz, 4-H), 6.96 (dd, 1H, J= 2.2 Hz, 8.6 Hz, 5-H),
3.83 (s, 3H, 6-OMe), 3.79 (m, 1H), 3.57 (s, 2H), 2.81 (d, 2H, J= 10.6 Hz), 2.48 (s, 3H, 3-Me), 2.06 (t,
2H, J= 12.0 Hz), 1.60-1.78 (m, 4H).

Preparation of 6-hydroxy-3-methyl-N-(1-(3-(trifluoromethyl)benzyl)piperidin-4-yl)benzofuran -
2-carboxamide (17e)
17e was synthesized by the method shown in Scheme 5 as white solid with a yield of 20.0%. M.p.:
114.4-116.2°C, LC-MS: 433.1 [M+H]+, 430.9 [M-H]-. 1H-NMR (400 MHz, DMSO-d6) δ: 9.87 (s, 1H,
6-OH), 8.17 (d, 1H, J= 8.2 Hz), 7.56-7.66 (m, 4H), 7.48 (d, 1H, J= 8.4 Hz), 6.89 (d, 1H, J= 1.7 Hz, 4-
H), 6.80 (dd, 1H, J= 1.7 Hz, 8.4 Hz), 3.72-3.82 (m, 1H), 3.57 (s, 2H), 2.81 (d, 2H, J= 11.3 Hz), 2.46 (s,
3H, 3-Me), 2.06 (t, 2H, J= 10.7 Hz), 1.60-1.78 (m, 4H).
4.2 Biological assay
4.2.1. Mnks kinase assay
CGP-57380 was obtained from Selleck.cn as positive control in the biological evaluation assay,
Ser/Thr KinEase assay kit (CisBio) was used to determine the Mnk1/2 kinases inhibition of the
designed compounds using CGP-57380 as positive control. 0.99 ng of Mnk1 or Mnk2 was incubated
with different concentrations of test compounds in an 10 µL reaction mixture (1 µM substrate S3, 392
µM ATP, 10 mM MgCl2, 1 mM Dithiothreitol and 1× KinEASE enzymatic buffer) for 60 min at room
temperature. Reactions were terminated through adding 5 µL SA-XL665 and 5 µL STK Ab detection
reagents following the kit protocol. The ratio between the HTRF signals of 615 and 665 nm recorded
with an Infinite® F500 microplate reader (Tecan, Switzerland) and IC50 values were calculated from
the inhibition curves.
4.2.2. In Vitro Anti-proliferative Assay
The tested cells were plated in 96-well microtiter plates with a density of 2,000 cells/well and
incubated in a humidified atmosphere with 5% CO2 at 37 °C for 24 h. The cells were then exposed to
tested compounds of different concentrations in triplicate. After incubated for 96 h, 50 μL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide MTT solution (2 mg/mL) was added to each
well and incubated for an additional 4 h. Then the suspernatant was discarded and the formazan was
dissolved in 200 μL of dimethyl sulfoxide (DMSO). The plate was oscillated subtly at room
temperature for 10 minutes before the absorbance was measured at 570 nm by a microplate reader. Cell
inhibitory ratio was calculated with the following formula:
Inhibitory Rate (%) = [(Acontrol-Atreated)/Acontrol]×100%

The IC50 was determined as the concentration that caused half inhibition of cell proliferation. All
experiments were performed three times independently, and the results were reported presented as
mean.
4.2.3. Western blot analysis
The human colon cancer cell line HCT-116 cells were incubated in presence of the test compound
8k (in 0.5% DMSO) for 24 h, harvested, and rinsed with ice-cold PBS. Total protein extracts (50 μg)
were prepared by lysing cells in RIPA buffer [50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl
sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF),
100 μM leupeptin, and 2 μg/mL aprotinin (pH 8.0)]. Samples were separated on an 8% or 12% SDS-
polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were stained with 0.2%
Ponceau S red to assure equal protein loading and transfer. After blocking with 5% nonfat milk, the
membranes were incubated with a specific antibody of eIF4E, p-eIF4E and GAPDH overnight at 4°C.
Immunocomplexes were visualized using enhanced chemiluminescence western blotting detection
reagents (Amersham Biosciences, England, and UK). Protein quantitation was determined by the
Bradford protein binding assay.
4.3. Molecular modeling
Discovery studio 3.0 was used to perform in silico docking. The X-ray crystal structure of Mnk2
kinase in complex with staurosporine was retrieved from Protein Data Bank (PDB code 2HW7). All
calculations and manipulations were performed with libdock modules in the Discovery studio software
package. All water molecules were removed and hydrogen was added. Applying the default parameters,
the best docking result was selected to analysis based on the favorable binding affinity rank in kcal/mol
(docking score).

Acknowledgement
This work was supported by the National Natural Science Foundation of China (Grant No. 81773578).