Design, synthesis and biological evaluation of 4-aniline-thieno[2,3-d]pyrimidine derivatives as MNK1 inhibitors against renal cell carcinoma and nasopharyngeal carcinoma

Article abstract of DOI:10.1016/j.bmc.2019.04.022

MAP Kinase Interacting Serine/Threonine Kinase 1 (MNK1)play important roles in the signaling transduction of MAPK pathways. It is significantly overexpressed in renal clear cell carcinoma and head-neck squamous cell carcinoma tissues in both mRNA and prot

Full text of DOI:10.1016/j.bmc.2019.04.022

Accepted Manuscript
Design, synthesis and biological evaluation of 4- aniline-thieno[2,3-d]pyrimi-
dine derivatives as MNK1 inhibitors against renal cell carcinoma and nasophar-
yngeal carcinoma
Min Zhang, Li Jiang, Jia Tao, Zhaoping Pan, Mingyao He, Dongyuan Su, Gu
He, Qinglin Jiang
PII:
S0968-0896(19)30439-0
https://doi.org/10.1016/j.bmc.2019.04.022
BMC 14876
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
18 March 2019
10 April 2019
14 April 2019
Please cite this article as: Zhang, M., Jiang, L., Tao, J., Pan, Z., He, M., Su, D., He, G., Jiang, Q., Design, synthesis
and biological evaluation of 4- aniline-thieno[2,3-d]pyrimidine derivatives as MNK1 inhibitors against renal cell
carcinoma and nasopharyngeal carcinoma, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.04.022
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Design, synthesis and biological evaluation of 4-
aniline-thieno[2,3-d]pyrimidine derivatives as MNK1
inhibitors against renal cell carcinoma and
nasopharyngeal carcinoma
Min Zhang,†,1 Li Jiang,†,1 Jia Tao,1 Zhaoping Pan,2 Mingyao He,2 Dongyuan Su,3
Gu He2,* and Qinglin Jiang1,*
1 School of Pharmacy, Chengdu Medical College, Chengdu 610500, China.
2
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University and Collaborative Innovation Center for
Biotherapy, Chengdu 610041, China.
3
Department of Gastroenterology, Chongzhou People's Hospital, Chengdu 611230,
China
† These authors contributed equally.
* Corresponding authors: Prof. Qinglin Jiang: jql_cmc@163.com; Prof. Gu He:
hegu@scu.edu.cn

ABSTRACT: MAP Kinase Interacting Serine/Threonine Kinase 1 (MNK1) play
important roles in the signaling transduction of MAPK pathways. It is significantly
overexpressed in renal clear cell carcinoma and head-neck squamous cell carcinoma
tissues in both mRNA and protein levels. Based on the crystallographic structure of
MNK1 protein and binding modes analysis of known MNK inhibitors, we have
designed and synthesized a series of 4-aniline-thieno[2,3-d]pyrimidine derivatives as
potential MNK1 inhibitors. These synthetic compounds are tested in biochemical and
cell proliferation assays, and six of them display potent inhibitory capacity against
MNK1 kinase and cancer cell lines. Compound 12dj with strongest inhibitory
capacity is transferred to molecular mechanism studies, and the results indicated that
12dj remarkably suppresses the phosphorylation of EIF4E, a substrate of MNK1. And
the expression levels of MNK1, ERK1/2 and pERK1/2 are not affected by compound
12dj incubation in SUNE-1 and 786-O cells. In summary, our works suggested that
these novel 4-aniline-thieno[2,3-d]pyrimidine based MNK1 inhibitors might be
attractive lead compounds for targeted therapy of renal cell carcinoma and
nasopharyngeal carcinoma.
Keywords: MNK; thieno[2,3-d]pyrimidine; apoptosis; renal cell carcinoma;
nasopharyngeal carcinoma.

1. Introduction
The MAPK-interacting kinases (MNKs, including MNK1 and MNK2) is belong to
a serine/threonine protein kinases family that are phosphorylated and activated by
MAPK pathways such as ERK1/2 or p38 MAPK and downstream regulating
translation initiator complexes by phosphorylating the eukaryotic initiation factor 4E
(eIF4E).[1-6] The oncogenic roles of MNKs and eIF4E had been reported in several
types of malignances, including breast cancer, Castrate-resistant Prostate Cancer,
hepatocellular carcinoma and lymphoma, etc.[7-13] We analyzed the MNK1 mRNA
and protein expression profiles in the Cancer Genome Atlas (TCGA)[14, 15] and
ProteinAtlas database[16], respectively (Figure 1).
Figure 1. The mRNA and protein expression levels of MNK1 in KIRC and HNSC.
(A and B) The boxplot of MNK1 expression in KIRC and HNSC cohort retrieved
from TCGA database, respectively (*, p<0.05); (C and D) The survival curves of
KIRC and HNSC patients differed by the median expression levels of MNK1; (E and
F) The protein expression levels of MNK1 determined by IHC in KIRC and HNSC
tissues, retrieved from ProteinAtlas database.

The results suggest MNK1 is significantly overexpressed at the mRNA and protein
levels in patients with renal cell carcinoma (KIRC) or head-neck squamous cell
carcinoma (HNSC).[17] Moreover, the patient with higher MNK1 expression is
suffered the worse prognosis in KIRC and HNSC, respectively. In the past years,
there were several series of MNKs inhibitors reported with diverse chemical scaffold,
such as pyrazolo[3,4-d]pyrimidin derivative CGP57380[18-20] and dibenzofuran
derivatives cercosporamide (see Figure 2),[21-23] which displayed MNKs inhibitory
capacities at micromolar levels but also demonstrated strong inhibition on the
off-target kinases. The imidazopyridine based inhibitor eFT508 exerted nanomolar
inhibitory capacity on MNK1 and MNK2 without obvious effects on p38, JNK1,
ERK1/2 and Src, etc.[24] Moreover, a multi-kinase inhibitor merestinib, which had
entered to phase I clinical trials against leukemia and solid tumors, also displayed
potent inhibition activity to MNKs.[25-27] In addition, an indazol-6-yl-pyridinone
based inhibitor SEL-201 was reported inhibitory capacities on MNKs, the
proliferation and metastasis of melanoma both in vitro and in vivo.[28] Recently, Jin
et al. reported aseries of novel MNKs inhibitors with thieno[2,3-d]pyrimidine scaffold,
inhibiting the proliferation of cancer cells as well as the high-fat feed induced.[29]

Figure 2. Chemical structures of structurally diverse MNK inhibitors.
Based on our previous works on the pyrido-thieno[2,3-d]pyrimidine based orally
available kinase inhibitor with both inhibitory potency and good selectivity,[29] in the
current manuscript, we have designed and synthesized a panel of potential MNK1
inhibitors, which showed promising cytotoxicity and apoptosis-inducing capacity
against renal cell carcinoma and nasopharyngeal carcinoma cells. In particular,
compound 12dj exerts strongest MNK1 inhibitory capacity, and it is transferred to
molecular mechanism studies, and the results indicate that 12dj remarkably
suppresses the phosphorylation of EIF4E, a substrate of MNK1. And the expression
levels of MNK1, ERK1/2 and pERK1/2 are not affected by compound 12dj
incubation in SUNE-1 and 786-O cells. In brief, our works suggest that these novel
4-aniline-thieno[2,3-d]pyrimidine based MNK1 inhibitors might be attractive lead
compounds for targeted therapy of renal cell carcinoma and nasopharyngeal
carcinoma.

Figure 3. (A) Typical binding model of inhibitors to MNK1, DS12881479 was
utilized as a representative inhibitor; (B) Strategy for the design of novel MNK1
inhibitors.
2. Results and discussion
2.1 Design and synthesis of novel MNK1 inhibitors
The binding modes of MNK1 to its inhibitors were visualized in Figure 3A, by
using the co-crystalized structures of MNK1 and DS12881479 (PDB No. 5WVD).[30]
The ligand binding pocket of MNK1 was comprised of an inner hydrophobic pocket
and an outer hydrophilic region. Moreover, the hydrogen bonds between Leu127 and
inhibitors were crucial to maintain binding affinities (Figure 3A). After compared the

structures of known MNK1 inhibitors, we found that there were three main fragments:
a hydrophobic fragment to the inner hydrophobic pocket, the hydrogen
donor/acceptor to Leu127 residue and a hydrophilic or hydrophobic fragment to bind
the outer region. Therefore, a series of novel MNK1 inhibitors were designed based
on a pyridothieno[2,3-d]pyrimidine scaffold (Figure 3B).
Scheme 1. Synthesis of Compounds 6a-6f, 11a-11k, 12a-12v, 13aa-13dk.
Reagents and conditions: a.NCCH2CO2Et, S8, Et3N, EtOH, reflux, 12h; b.formamidine acetate,
DMF, 100℃,16h; c.SOCl2, reflux, 4h; d.NaH, 1,4-dioxane, 90℃, 2hours.
Reagents and conditions: a.NCCH2CO2Et, S8, Et3N, EtOH, reflux, 12h; b.formamidine acetate,
DMF, 100℃,16h; c.SOCl2, reflux, 4h; d.NaH, 1,4-dioxane, 90℃, 2hours.

Reagents and conditions: a. 9N HCl, ethylacetate, 25℃, 30 min and then Et3N, THF, rt,4hours.
The synthesis route of compounds 6a-6f, 11a-11k and 12aa-12fk were shown in
Scheme 1. In general, for the preparation of the 6a-11a and 12aa, we chose
cyclohexanone and N-Boc-4-piperidone as the starting materials respectively with
ethyl cyanoacetate and sulfur to obtain 2 and 8 in yield 76% and 87%, the compound
2 and 8 was respectively reacted with formamidine acetate in DMF for 12h at 100℃to
give 3 and 9 in yield 91% and 90%, the compound 3 and 9 was converted to 4 and 10
by treatment with SOCl2 in yield 70% and 73%, the compound 4 was reacted with 5a
in 2-propanol at 100℃ to give 6a in yield 73%, meanwhile the compound 9 was
reacted with 5a and NaH in anhydrous 1,4-dioxane to give 11a in yield 53%, Finally,
the benzyl group of 11a was removed by 9N HCl, the obtained compound was treated
with iodomethane in THF to give 12aa in yield 49%. The synthesized target
1
compounds and key intermediates were characterized by H NMR, 13C NMR and
HR-MS (high-resolution mass spectrometry), the detailed experimental procedures
and characterization were presented in experimental sections.

Table 1. Evaluations of compounds Compounds 6a-6f, 11a-11k and 12aa-12fk.
Cell Vialibity(%)b,c
MNK1
No.
R1
R2
R3
R4
IC50(μM)a
SUNE-1
786-O
6a
6b
6c
6d
6e
H
H
H
H
H
F
Cl
H
H
Cl
H
H
-
-
-
-
-
2.18
68.08
71.20
2.43
66.16
59.39
62.19
66.98
77.60
68.00
76.80
75.20
H
1.62
OCH3
CH3
1.70
1.79
1.51
2.19
2.17
2.24
1.95
2.50
1.81
1.62
1.72
2.16
1.34
1.70
2.04
6f
11a
11b
11c
11d
11e
11f
H
H
H
F
Br
H
-
52.52
71.42
73.99
65.80
71.35
76.39
63.28
44.92
50.60
53.94
38.77
57.85
65.18
64.00
76.00
79.20
77.60
79.20
82.40
71.20
62.40
55.20
59.20
53.60
60.80
72.00
-
H
Cl
H
H
-
H
Cl
H
-
H
OCH3
CH3
H
-
H
H
-
H
Br
F
-
11g
11h
11i
F
H
-
Br
F
H
F
-
H
Cl
F
-
11j
OCH3
Br
H
H
-
-
11k
12aa
H
CH3
H
F
CH3

12ab
12ac
12ad
12ae
12af
12ag
12ah
12ai
H
H
Cl
H
H
Cl
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Bn
1.87
1.82
2.29
2.22
1.69
1.09
1.47
1.85
0.93
1.65
2.08
2.29
1.54
1.92
2.11
1.69
1.10
1.12
1.31
1.00
66.91
53.65
65.97
69.22
55.07
35.66
38.81
47.79
32.47
45.83
58.84
64.75
48.16
58.76
67.33
42.86
31.61
39.86
45.72
33.50
72.00
59.20
71.20
86.40
61.60
56.00
54.40
66.40
57.60
57.60
64.00
68.00
66.40
64.80
76.80
65.60
48.00
56.00
60.80
45.60
H
OCH3
CH3
H
H
H
H
Br
F
F
H
Br
F
H
F
H
Cl
F
12aj
12ak
12ba
12bb
12bc
12bd
12be
12bf
12bg
12bh
12bi
12bj
OCH3
Br
H
H
H
CH3
H
F
H
Cl
H
Bn
H
H
Cl
H
Bn
H
OCH3
CH3
H
Bn
H
H
Bn
H
Br
F
Bn
F
H
Bn
Br
F
H
F
Bn
H
Cl
F
Bn
OCH3
H
Bn

12bk
12ca
12cb
12cc
12cd
12ce
12cf
12cg
12ch
12ci
Br
H
H
F
CH3
H
Bn
1.57
1.99
1.64
1.65
2.02
2.06
1.25
1.08
1.16
1.26
1.10
1.65
50.11
62.13
56.07
41.83
51.30
67.32
33.60
28.52
36.27
35.55
29.37
53.90
66.40
78.50
68.86
56.66
57.04
79.46
50.08
42.82
58.62
57.24
44.30
65.52
H
Cl
H
H
H
Cl
H
H
OCH3
CH3
H
H
H
H
Br
F
F
H
Br
F
H
F
H
Cl
F
12cj
12ck
OCH3
Br
H
H
CH3
12da
12db
12dc
12dd
12de
12df
H
H
H
H
H
H
F
Cl
H
H
1.39
1.40
1.22
2.14
1.41
1.07
48.46
47.66
30.54
56.43
49.14
30.24
62.77
66.93
50.83
69.14
63.31
50.59
H
Cl
H
OCH3
CH3
H
H
Br

12dg
12dh
12di
12dj
12dk
12ea
12eb
12ec
12ed
12ee
12ef
12eg
12eh
12ei
F
Br
F
H
H
F
F
0.83
1.00
1.15
0.65
1.89
2.02
1.74
1.50
1.88
2.40
1.73
1.10
1.28
1.54
28.96
34.02
31.44
21.20
55.52
50.40
53.30
46.80
59.85
68.00
54.00
31.05
36.10
40.42
39.97
53.61
54.75
36.96
72.41
68.32
69.84
55.04
72.68
76.00
68.80
44.84
56.88
61.94
H
Cl
F
OCH3
Br
H
H
H
CH3
H
F
H
Cl
H
H
H
Cl
H
H
OCH3
CH3
H
H
H
H
Br
F
F
H
Br
F
H
F
H
Cl

12ej
12ek
12fa
12fb
12fc
12fd
12fe
12ff
12fg
12fh
12fi
OCH3
Br
H
H
H
F
CH3
H
0.93
1.68
1.50
1.38
1.63
2.35
2.13
1.39
1.46
0.97
1.36
0.94
32.32
49.45
48.89
50.64
45.25
62.84
66.64
44.28
37.82
32.49
39.21
28.89
49.86
62.76
56.71
58.91
66.60
66.27
70.91
66.62
57.45
54.79
59.37
42.31
F
H
Cl
H
H
H
Cl
H
H
OCH3
CH3
H
H
H
H
Br
F
F
H
Br
F
H
F
H
Cl
F
12fj
12fk
OCH3
H
Br
-
H
-
CH3
-
1.56
2.74
47.70
57.48
60.56
61.35
CGP-5
7380
-
a IC50 values were determined from KinaseProfiler of Eurofins. The data represent the mean values of two
independent experiments.
b Cells were incubated to 1.0 μM corresponding compound for 24 hours.
b Each compound was tested in triplicate; the data are presented as the mean values.

Figure 4. (A) Cytotoxicity of compounds 6a-f, 11a-k, 12aa-fk against nasopharyngeal
carcinoma cell lines SUNE-1 based on the MTT method; (B) IC50 values of compound
12dj against a panel of renal cell carcinoma and nasopharyngeal carcinoma cell lines;

(C) and (D) Apoptosis in SUNE-1 cells treated with compound 12dj based on Annexin
V/PI dual staining. Values are mean ± SD (n = 3).
2.2 Novel MNK1 inhibitors possess good kinase inhibition and suppress cancer
cells proliferation
We have evaluated the MNK1 kinase inhibitory capacities of compounds 6a-6f,
11a-11k and 12aa-12fk by using a γ-[32P]-ATP competitive inhibition assay. As
positive control drugs, we used the known inhibitor CGP-57380. The IC50 values for
each compound were determined as the mean values of three duplicated experiments
(Table 1). The inhibition rates on cell viability of SUNE-1 and 786-O cells for each
compound at 1.0 μM were determined were also measured by MTT method (Figure 4A
and Table 1). Further structure-activity relationships investigation also revealed that the
R1 and R3 substitution of 4-aniline fragment could be replaced by 2,4-di-F (12dg),
2-Br-4-F (12dh), 2-F-4-Cl (12di), 2-OCH3-4-F (12dj) or 2-Br-4-CH3 (12dk) with
obviously increasing the MNK1 inhibitory potencies as well as cell proliferation
inhibition. For the R4 substitution, the alkyl or benzyl substituted compounds
12aa-12bk exhibited moderate MNK1 inhibitory capacities, and the aromatic sulfonyl
substituted compounds demonstarted better inhibitory potencies in both MNK1 and
cell
proliferation
assays.
When
the
R4
position
of
pyrido[4',3':4,5]thieno[2,3-d]pyrimidine ring substituted by a 4-methoxyphenyl
sulfonyl group, the resulting compound 12dj was almost four times stronger potent than
the lead molecule 6a. Compounds 12df-12dj worked better than the other compounds
by inhibiting the kinase activity of MNK1 as well as the proliferation of renal cell

carcinoma and nasopharyngeal carcinoma cell lines (Figure 4B). Among the more
active compounds 12df-12dj, compound 12dj with a halogen at the 4-F,2-OMe-phenyl
on the 4-position of thieno[2,3-d]pyrimidine scaffold demonstrated the greatest MNK1
inhibition and cytotoxicity. Compound 12dj inhibited both the MNK1 kinase and
cancer cell proliferation prior than known inhibitor CGP-57380. Moreover, compound
12dj displayed submicromolar inhibition IC50 values on MNK1 kinase and SUNE-1
cell viability assays. Therefore, compound 12dj was transferred to further molecular
mechanism studies.

Figure 5. (A) Comparison of the binding modes of compound 12dj (green) with the
experimental conformation of DS12991479 (red) bound to MNK1 kinase domain; (B)
Schematic depiction of the potential interaction modes of compound 12dj in the ATP
pocket of MNK1; (C) Western blotting analysis of MNK1, ERK1/2, eIF4E and their
phosphorylated proteins in SUNE-1 and 786-O cells after 24-h incubation of 1μM
CGP-57380, or 6h and 24-h incubation of 1μM compound 12dj, respectively.

2.3 Novel MNK1 inhibitor 12dj induces apoptosis without affects the expression
and phosphorylation of ERK1/2
The apoptotic cell death of SUNE-1 and 786-O cells induced by compound 12dj
incubation were determined by the flowcytometry analysis with an Annexin V/PI dual
staining kit (Figure 4C and 4D). According to Figure 4C-4D, the percentage of total
apoptotic cells in the 5μM 12dj treatment groups were raised from 5.7% to 61.1% in
SUNE-1 cell and 49.6% in 786-O cell, respectively. In addition, the early apoptotic
cells in 5μM 12dj treated groups were remarkably higher than in the 1μM 12dj
treated groups for both cells. For the late apoptotic cells, there were no significant
differences between the 1μM 12dj treated groups and 5μM 12dj treated groups.
Above results suggested that the cytotoxicity of compound 12dj possibly dependent
on the apoptotic cell death subroutine with a concentration-dependent manner.[31-33]
The potential binding modes of compound 12dj into the ATP pocket of MNK1
kinase domain was performed by CDOCKER molecular docking module in Accelrys
Discovery Studio 2.5 software (Figure 5A). By comparison to the co-crystallized
inhibitor DS12881470, compound 12dj formed similar hydrogen bonds with Leu127,
hydrophobic interaction to the inner residues such as Leu108, Phe124, Leu177 and
Phe192. In addition, the possible hydrogen bond between the 4-F,2-OMe-phenyl
fragment and Asp191, and the cation-π interaction between Lys126 and the
4-methoxybenzenesulfonamide fragment might enhanced the binding affinity of 12dj
to MNK1 kinase domain. The 2D-diagram of interactions between 12dj and
interactive residues were depicted into Figure 5B. To further verify the selectivity of

compound 12dj to the ERK-MNK-eIF4E signaling axis, we determined its influence
on the expression of ERK1/2, pERK1/2, MNK1 eIF4E and p-eIF4E in SUNE-1 and
786-O cells with the known MNK1 inhibitor CGP-57380 as a positive control (Figure
5C). As shown in Figure 5C, the 12dj or CGP-57380 incubation for 24 hours
selectively declined the phosphorylation of eIF4E in both cells, the phosphorylation of
ERK1/2 and expression of total ERK1/2, MNK1 and eIF4E were not affected.
Furthermore, the 12dj induced inhibition of eIF4E phosphorylation, a MNK1
substrate, by a time-dependent manner. These results suggested that compound 12dj
could competitively suppress the MNK1 without interfere the upstream ERK1/2
functions. In addition, the inhibitory capacity of eIF4E phosphorylation of 12dj in
SUNE-1 cells was obviously stronger than that of 786-O cells, which might explain
the cytotoxic difference in the renal cell carcinoma cells and nasopharyngeal
carcinoma cells after 12dj incubation.
3. Conclusions
In the current study, based on the rational drug design method, we have prepared, in
good yield, a series of 4- aniline-pyridothieno[2,3-d]pyrimidine derivatives bearing
various substituted groups. Structure-activity analysis of the prepared compounds
afforded 12dj, which showed strong ability to inhibit both MNK1 and a panel of
cancer cell lines. The compound 12dj also selectively suppressed the eIF4E
phosphorylation and induced apoptosis in both SUNE-1 and 786-O cells. Taken
together, these results suggested that these novel 4-aniline-thieno[2,3-d]pyrimidine
based MNK1 inhibitors might be attractive lead compounds for targeted cancer

therapy, especially for nasopharyngeal carcinoma.
4. Experimental Section
4.1 Chemistry
The general synthesis procedures of representative compounds and intermediates
were described bellowed, the detailed synthesis and characterization of all compounds,
and the spectra of 1H NMR, 13C NMR and HR-MS were provide in the supplementary
materials. [34-36]
General Procedures of Method for the Synthesis of 6a, 11a, 12aa, 12ca
Ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (2). To a
mixture of cyclohexanone(1)(9.80g, 100mmol) , ethyl cyanoacetate (11.32 g, 200.0
mmol), and sulfur (3.20 g, 100.0 mmol) in absolute ethanol (200 mL) was added
triethylamine (20mL) and refluxed for 12 h; the reaction mixture was concentrated
and the residue was partitioned between water and ethyl acetate. The organic layer
was separated, and concentrated, and the crude product was recrystallized with
1
ethanol (150ml), to give 2. Yield 76%, light yellow crystal. H NMR (400 MHz,
Chloroform-d) δ 7.20 (s, 2H), 4.14 (q, J = 7.2 Hz, 2H), 2.59 (t, J = 5.6 Hz, 2H), 2.41
(t, J = 5.6 Hz, 2H), 1.74 – 1.58 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H).
3,4,5,6,7,8-Hexahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-one(3). A mixture of (2)
(4.50g, 20.0mmol)and formamidine acetate (3.12g, 30.0mmol)was sterred in DMF for
12 h at 100℃. The reaction mixture was cooled, water was added, and the precipitate
formed was collected by filtration, and washed thoroughly with water to give 3, Yield,

91%. 1H NMR (400 MHz, Chloroform-d) δ 7.91(s,1H) , 3.02-2.99 (m,2H) , 2.79-2.76
(m, 2H) , 2.02-1.83 (m, 4H).
4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine(4).A mixture of (3)
(12.0g,58.2mmol), SOCl2 (100mL)and DMF(2ml) was refluxed for 4 h. The reaction
mixture was cooled in anice bath and then carefully neutralized by the addition of
aqueoussodium bicarbonate solution. The resulting mixture was extractedwith ethyl
acetate, the organic layer was separated, dried overMgSO4, and concentrated, and the
crude product was purified by silicagel column chromatography using a mixture of
Petroleum ether: Dichloromethane (5:1), to give 4, Yield, 70%. 1H NMR (400 MHz,
Chloroform-d) δ8.69 (s, 1H), 3.10-3.07(m, 2H), 2.88-2.86 (m, 2H), 1.92-1.89 (m,
4H).
N-(3-fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine
(6a)
A
mixture
of
3-fluoroaniline(5a)(333mg,3mmol)
and
4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (4) (449 mg, 2mmol)
and 2-propanol (15 mL) in sealed tube was sterred for 6 h at 100℃. The reaction
mixture was concentrated, and the residue was partitioned between water and
dichloromethane. The organic layer was washed with water, dried over MgSO4, and
concentrated. The crude product was purified by silica gel column chromatography
using a mixture of hexane: ethyl acetate (6:1) to give 6a.Yield 73%,White powder, m.
p. 114.5 – 116.3℃. 1H NMR (400 MHz, Chloroform-d) δ 8.52 (s, 1H), 7.75 (dt, J =
11.1, 2.3 Hz, 1H), 7.33 – 7.17 (m, 3H), 6.92 – 6.68 (m, 1H), 3.07 (tt, J = 6.0, 3.1 Hz,
2H), 2.87 (tt, J = 6.0, 3.1Hz, 2H), 2.11 – 1.86 (m, 4H). 13C NMR (100 MHz,

Chloroform-d) δ 166.52, 163.09(d, J= 244 Hz, 1C), 154.26, 152.34, 140.21(d, J= 11
Hz, 1C), 135.22, 130.00(d, J= 9 Hz, 1C), 124.50, 116.77 , 115.91 (d, J= 3 Hz, 1C),
110.32(d, J= 22 Hz, 1C), 108.24(d, J= 27 Hz, 1C), 26.53, 25.57, 22.56, 22.39 .
HRMS(ESI) calcd for C16H15FN3S+ [M+H]+ 300.0965, found 300.0966.
6-(tert-butyl) 3-ethyl
2-amino-4,7-dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxylate(8). Compound(8)
was synthesized from N-Boc-4-piperidone (7), in a manner similar to(2),after stirring
for 16 h at room temperature, the precipitatewas collected by filtration and washed
with ethanol to give8Yield 87%, white powder,1H NMR (400 MHz, Chloroform-d) δ
6.02 (s, 2H), 4.35 (s, 2H), 4.26 (q, J = 7.0 Hz, 2H), 3.61 (t, J = 5.7 Hz, 2H), 2.80 (t, J
= 5.7 Hz, 2H), 1.48 (s, 9H), 1.34 (t, J = 7.0 Hz, 3H).
tert-butyl
4-oxo-3,5,6,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidine-7(4H)-carboxyl
ate(9). Compound (9) was synthesized from compound(8) and formamidine acetate,
in a manner similar to (9),Yield, 90%,light yellowpowder,1H NMR (400 MHz,
Chloroform-d) δ 11.99 (s, 1H), 7.99 (s, 1H), 4.66 (s, 2H), 3.74 (t, J = 5.8 Hz, 2H),
3.13 (t, J = 5.8 Hz, 2H), 1.50 (s, 9H).
tert-butyl
4-chloro-5,8-dihydropyrido[4',3':4,5]thieno[2,3-d]pyrimidine-7(6H)-carboxylate(
10). Compound (10) was synthesized from compound(9) and SOCl2, in a manner

similar to (4),Yield, 73%,white powder,1H NMR (400 MHz, Chloroform-d) δ 8.77 (s,
1H), 4.74 (s, 2H), 3.80 (t, J = 5.8 Hz, 2H), 3.21 (t, J = 5.8 Hz, 2H), 1.51 (s, 9H).
tert-butyl4-((3-fluorophenyl)amino)-5,8-dihydropyrido[4',3':4,5]thieno[2,3-d]pyr
imidine-7(6H)-carboxylate (11a). To a suspension of 60% NaH(120mg, 3mmol) in
anhydrous 1,4-dioxane(10ml) was added a solution of 3-fluoroaniline(5a) (222mg,
2mmol) in anhydrous 1,4-dioxane(5ml), the reaction mixture was stirred for 20 min at
room temperature and then compound(10)(325mg, 1mmol) in 1,4-dioxane(5ml) was
added to the above reaction mixture, the mixture was sterredfor another 2 hours at
90℃,at the end of the reaction, the excessive NaH was quenched by H2O, the mixture
was concentrated and the residue was partitioned between water and ethyl acetate.
The organic layer was separated, and concentrated, and then the crude product was
purified by silica gel column chromatography using a mixture solvent of petroleum
ether: ethylacetate (15:1),to give 11a. Yield 53%,Light yellow powder, m. p. 164.4 –
165.0℃. 1H NMR (400 MHz, Chloroform-d) δ 8.55 (s, 1H), 7.73 (d, J = 11.0 Hz, 1H),
7.32 (td, J = 8.1, 6.3 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.03 (s, 1H), 6.83 (tdd, J = 8.2,
2.5, 1.0 Hz, 1H), 4.72 (s, 2H), 3.86 (t, J = 5.7 Hz, 2H), 3.14 (t, J = 5.7Hz, 2H), 1.51
13
(s, 9H). C NMR (100 MHz, Chloroform-d) δ 167.00 , 163.04 (d, J = 243 Hz, 1C),
154.64 , 154.33 , 152.81 , 139.85(d, J = 11 Hz, 1C), 130.07 (d, J = 9 Hz, 1C), 116.13 ,
116.07(d, J = 3 Hz, 1C), 110.65 (d, J = 21 Hz, 1C), 108.42 (d, J = 23 Hz, 1C), 80.83 ,
28.41(3C), 26.45. HRMS(ESI) calcd forC20H22FN4O2S+[M+H]+401.1442, found
401.1441.

N-(3-fluorophenyl)-7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyri
midin-4-amine (12aa). Compound 11a (800mg, 2.0mmol) was dissolved in 10ml
ethyl acetate and then 9N HCl (5ml) was added, the reaction mixture was stirred for
30 min at room temperature, the reaction solvent was neutralized by NaHCO3 to
pH=8, 50ml ethyl acetate and 30ml H2O was added, after stirred adequately, the
organic layer was separated, and concentrated, and then the crude product was
purified by silica gel column chromatography using a mixture solvent of
dichloromethane: methanol (20:1),to give white powder (540mg); Above white
powder (150mg, 0.5mmol) was dissolved in THF (10ml), iodomethane(107mg,
0.75mmol) and triethylamine(0.5ml) were added in the solvent.afterthe reaction
mixture was stirred for 1hour at room temperature, the solvent was concentrated, the
residueswas purified by silica gel column chromatography using a mixture solvent of
dichloromethane: methanol (40:1),to give(12aa)Yield 49%,White powder,m. p. 116.7
1
– 118.5℃. H NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 7.75 (dt, J = 11.1, 2.3
Hz, 1H), 7.31 (td, J = 8.1, 6.2 Hz, 1H), 7.26 – 7.22 (m, 1H), 7.08 (s, 1H), 6.82 (tdd, J
= 8.2, 2.5, 1.1 Hz, 1H), 3.72 (t, J = 1.9 Hz, 2H), 3.17 (tt, J = 5.7, 1.9 Hz, 2H), 2.90 (t,
13
J = 5.7 Hz, 2H), 2.54 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 166.85, 163.09
(d, J=243Hz, 1C), 154.65, 152.68, 140.04 (d, J=11Hz, 1C), 132.49, 130.06 (d, J=9Hz,
1C), 122.76, 116.18, 116.02 (d, J=3Hz, 1C), 110.54 (d, J=21Hz, 1C), 108.38 (d,
J=27Hz, 1C) , 54.03 , 51.66 , 45.33 , 26.75. HRMS(ESI) calcd for C16H16FN4S+
[M+H]+ 315.1074, found 315.1079.

N-(3-fluorophenyl)-7-((2-nitrophenyl)sulfonyl)-5,6,7,8-tetrahydropyrido[4',3':4,5
]thieno[2,3-d]pyrimidin-4-amine (12ca).Compound(12ca)was synthesized from
compound (11a) and 2-nitrobenzenesulfonyl chloride, in a manner similar to (12aa).
1
Yield 88%, White powder, m. p.212.2 – 212.8℃. H NMR (400 MHz, DMSO-d6) δ
8.49 (s, 1H), 8.40 (s, 1H), 8.15 (dd, J = 7.7, 1.5 Hz, 1H), 8.03 (dd, J = 7.8, 1.5 Hz,
1H), 7.95 – 7.86 (m, 2H), 7.63 (dt, J = 11.7, 2.3 Hz, 1H), 7.48 – 7.32 (m, 2H), 6.91
(td, J = 8.4, 2.5 Hz, 1H), 4.70 (s, 2H), 3.72 (t, J = 5.7 Hz, 2H), 3.34 (t, J = 5.7 Hz,
13
2H). C NMR (100 MHz, DMSO-d6) δ 167.16, 162.50 (d, J = 240 Hz, 1C), 155.14,
153.08, 148.09, 141.46(d, J = 11 Hz, 1C), 135.40, 133.11, 130.96, 130.73, 130.39 (d,
J = 11 Hz, 1C), 128.68, 125.73, 124.96, 118.02 (d, J =3 Hz, 1C), 116.82, 110.12 (d, J
= 21 Hz, 1C), 108.97 (d, J = 26 Hz, 1C), 45.29, 43.16, 26.30. HRMS(ESI) calcd for
+
C21H17FN5O4S2 [M+H]+ 486.0701, found 486.0698.
4.2 Molecular docking.
The molecular docking analysis was performed by the CDOCKER module in the
Accelrys Discovery Studio 2.5 software packages (BIOVIA, San Diego, CA, USA).
The initial co-crystallized structure of MNK1 (PDB No. 5WVD) was retrieved from
the Protein Data Bank (http://www.rcsb.org). All the computational parameters were
used default settings without individual descriptions.[37-39]
4.3 Kinase inhibition assay of MNK1.

The kinase inhibition assay of MNK1 was provided by Eurofins Co. Ltd. with the
KinaseProfiler services. The detailed experimental procedures had been described in
our previous reports.[39-41]
4.4 Cell viability and apoptosis assays.
The cell viability assays of screened compounds were performed in SUNE-1 and
786-O cells, respectively with a constant concentration of 1μM and 24-hour
incubation. The IC50 values of compound 12dj on a panel of renal cell carcinoma and
nasopharyngeal carcinoma cells were performed on a series of concentration gradient
with 8-10 points. The detailed experimental procedures had been described in our
previous reports. And the known MNK1 inhibitor CGP-57380 was utilized as a
positive control. Apoptosis assay of compound 12dj was carried out by a BD
FACSCalibur Flow Cytometer on SUNE-1 and 786-O cells, respectively. The detailed
experimental procedures had been described in our previous reports.[42-44]
4.5 Western blot analysis
The primary antibodies of ERK1/2, pERK1/2, MNK1, eIF4E and p-eIF4E were
btained from Cell Signaling Technology (Danvers, MA, USA). After treatment by the
procedures according to the figure legends, SUNE-1 or 786-O cells were harvested,
trypsin digested and then washed twice by cold PBS. After lysated by RIPA buffer
(Invitrogen, CA, USA) on ice, the cellular lysate was sonication denaturation and then
cold centrifuged at 10000+ g for 30 min. The supernatant was collected, quantified
the total protein concentration, and then separated by a SDS-PAGE electrophoresis,
electro-transferred into a PVDF membrane, incubated by the corresponding primary

and secondary antibodies, and then visualized by an ECL (enhanced
chemiluminescence) Kit.
Acknowledgements. We are grateful for financial support from the National
Natural Science Foundation of China (21772131), the Fundamental Research Funds
of Science & Technology Department of Sichuan Province (2017JY0123 and
19SYXHZ015), the Fundamental Research Funds for the Central Universities and
Distinguished Young Scholars of Sichuan University (2015SCU04A13), the China
Postdoctoral Science Foundation and the Open Research Fund from State Key
Laboratory of Breeding Base of Systematic Research, Development and Utilization of
1
13
Chinese Medicine Resources. The HNMR and CNMR spectrum were supplied as
Supporting Information. This material is available free of charge via the Internet.
Competing interests. The authors declare no competing interests.
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