Discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d[pyrimidine derivatives as potent Mnk inhibitors: Synthesis, structure-activity relationship analysis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2015.03.032

Phosphorylation of the eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases (Mnks) is essential for oncogenesis but unnecessary for normal development. Thus, pharmacological inhibition of Mnks may offer an effective and non-toxic anti-cancer therapeutic strategy. Herein, we report the discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimidine derivatives as potent Mnk inhibitors. Docking study of 7a in Mnk2 suggests that the compound is stabilised in the ATP binding site through multiple hydrogen bonds and hydrophobic interaction. Cellular mechanistic studies on MV-4-11 cells with leads 7a, 8e and 8f reveal that they are able to down-regulate the phosphorylated eIF4E, Mcl-1 and cyclin D1, and induce apoptosis.

Full text of DOI:10.1016/j.ejmech.2015.03.032

European Journal of Medicinal Chemistry 95 (2015) 116e126
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]
pyrimidine derivatives as potent Mnk inhibitors: synthesis,
structureeactivity relationship analysis and biological evaluation
Mingfeng Yu, Peng Li, Sunita K.C. Basnet, Malika Kumarasiri, Sarah Diab, Theodosia Teo,
Hugo Albrecht, Shudong Wang*
Centre for Drug Discovery and Development, Sansom Institute for Health Research and Center for Cancer Biology, School of Pharmacy and Medical Sciences,
University of South Australia, Adelaide, South Australia 5001, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Phosphorylation of the eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase
(MAPK)-interacting kinases (Mnks) is essential for oncogenesis but unnecessary for normal develop-
ment. Thus, pharmacological inhibition of Mnks may offer an effective and non-toxic anti-cancer ther-
apeutic strategy. Herein, we report the discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno
[2,3-d]pyrimidine derivatives as potent Mnk inhibitors. Docking study of 7a in Mnk2 suggests that the
compound is stabilised in the ATP binding site through multiple hydrogen bonds and hydrophobic
interaction. Cellular mechanistic studies on MV-4-11 cells with leads 7a, 8e and 8f reveal that they are
able to down-regulate the phosphorylated eIF4E, Mcl-1 and cyclin D1, and induce apoptosis.
© 2015 Elsevier Masson SAS. All rights reserved.
Received 27 August 2014
Received in revised form
15 October 2014
Accepted 13 March 2015
Available online 17 March 2015
Keywords:
eIF4E
Mnk
Inhibitor
Structureeactivity relationship
Thieno[2,3-d]pyrimidine derivatives
1. Introduction
biological studies provide a rationale for the potential application of
specific Mnk inhibition as an effective and non-toxic therapeutic
Dysregulation of protein synthesis is implicated in the pro-
gression 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 the
eukaryotic initiation factor 4E (eIF4E) [2]. This general translation
factor is regulated by mitogen-activated protein kinase (MAPK)-
interacting kinases (Mnks) through phosphorylation at Ser209 [3].
The phosphorylation process is necessary for oncogenic trans-
formation [4,5]. Perturbation of the process by mutating eIF4E at
Ser209 [6], silencing Mnk1 with small hairpin RNA (shRNA) [7] or
knocking out Mnk1/2 [7] attenuated or even prevented tumour
formation. Conversely, activation of Mnk1 promoted tumouri-
genesis in a manner similar to eIF4E [8]. In contrast with their
pivotal role in tumourigenesis, Mnks seem to be dispensable for
normal development [4,5]. For example, neither cap-dependent
translation nor global protein synthesis was influenced in Mnk-
deficient embryonic fibroblasts [9]. Taken together, these
strategy against cancer. Hence, there is a pressing demand in
discovering pharmacologically selective Mnk inhibitors.
Regrettably, little progress has been made in the discovery of
pharmacological Mnk inhibitors since the first isolation and iden-
tification of the two kinases in 1997 [3,10]. The known Mnk in-
hibitors
include
CGP052088
[11],
CGP57380
[12,13],
cercosporamide [14] and hypothemycin derivatives [15e17] (Fig. 1).
These small molecules inhibit Mnks at nanomolar to micromolar
concentrations. However, all of them target multiple protein ki-
nases. Earlier this year, a series of 5-(2-(phenylamino)pyrimidin-4-
yl)thiazol-2(3H)-one derivatives was reported as potent Mnk2 in-
hibitors with sub-micromolar affinity, but the kinase selectivity
remains unconquered [18].
The high structural similarity of the ATP binding pockets among
kinases renders selective Mnk inhibition by exogenous inhibitors
challenging. Previous crystallographic analyses of Mnks reveal two
distinct structural features: (i) a noncanonical DFD motif (vs. a
common DFG motif in other kinases) and (ii) three Mnk-specific
inserts (i.e., insertions I1, I2 and I3) at the catalytic domain
[19,20]. In the absence of a ligand, Mnks predominantly adopt an
* Corresponding author.
E-mail address: shudong.wang@unisa.edu.au (S. Wang).
http://dx.doi.org/10.1016/j.ejmech.2015.03.032
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
117
Fig. 1. Chemical structures of known Mnk inhibitors.
unusual DFD-out conformation, in which the Phe residue flips into
the ATP binding pocket, thus preventing the accessibility of ATP
[21]. Exploiting the above characteristics would assist in the
rational design of highly selective Mnk inhibitors. Nevertheless, the
lack of structural details on the ATP-bound Mnks has hampered the
development of specific Mnk inhibitors.
methoxyethyl)-5-methylpyridin-2(1H)-one 3b in excellent yields
(3a: 94% and 3b: 100%). Chlorination of methyl 5-methyl-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-6-carboxylate 4 with thionyl
chloride in the presence of N,N-dimethylformamide (DMF) yielded
methyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-carboxylate 5
in a yield of 89%. Each of amines 3a and 3b was individually coupled
with chloride 5 in the presence of p-toluenesulfonic acid under
microwave irradiation to give the corresponding methyl 4-((1-
alkyl-2-oxo-5-methyl-1,2-dihydropyridin-3-yl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylates 6a and 6b in good
yields (6a: 86% and 6b: 54%), which were subsequently subjected to
saponification in an alkaline methanolic solution to yield the car-
boxylic acids 7a and 7b respectively in excellent yields (7a: 100%
and 7b: 84%). Amination of carboxylic acids 7a and 7b with
appropriate amines was effected in anhydrous DMF in the presence
of the coupling reagent 1-[bis(dimethylamino)methylene]-1H-
In our earlier attempt to identify novel selective Mnk inhibitors,
the ChemBridge database consisting of approximately 572,000
molecules was pre-filtered according to molecular weights, and the
remaining 387,000 compounds with molecular weights ranging
from 300 to 450 were subjected to a comprehensive virtual
screening using both protein- and ligand-based approaches. In the
former method, the only available staurosporine-bound Mnk2
(D228G) crystal structure (PDB ID: 2HW7) was adopted to create a
binding site for the protein-ligand docking. In the latter approach,
CGP57380 and cercosporamide, two most extensively studied Mnk
inhibitors, were selected as query molecules for the ligand-based
alignment. Successful deployment of these two methods was fol-
lowed by structure-guided optimisation and the evaluation of Mnk
inhibitory activity of the remaining candidates using biochemical
assays to reveal 4-aminothieno[2,3-d]pyrimidine as a valuable
chemical scaffold for Mnk inhibitors [22]. Using analogue-based
drug design, we further explored the utility and versatility of this
scaffold. Herein, we describe the synthesis and biological evalua-
tion of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]py-
rimidine derivatives.
1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate
(HATU) and N,N-diisopropylethylamine (DIPEA) to give the corre-
sponding amides 8a-f in moderate to good yields (36e80%).
The above synthetic approach was successfully applied to the
preparation of methyl 4-((1-methyl-6-oxo-1,6-dihydropyridin-3-
yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate
(12)
and its derivatives (13 and 14) (Scheme 2). In brief, 5-nitropyridin-
2(1H)-one 9 sequentially underwent alkylation and reduction to
give 5-amino-1-methylpyridin-2(1H)-one 11. Due to its instability
in the air, amine 11 was directly used in the next synthetic step
without further purification and characterisation; p-toluene-
sulfonic acid-catalysed substitution of chloride 5 with amine 11 was
achieved by microwave irradiation to give the desired methyl 4-((1-
methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylate 12. Ester 12 was readily hydro-
lysed to afford carboxylic acid 13 in a yield of 88%, which was
subsequently aminated with ammonia to give amide 14 in 54%
yield.
2. Results and discussion
2.1. Chemistry
The synthetic route deployed to prepare methyl 4-((1-alkyl-2-
oxo-5-methyl-1,2-dihydropyridin-3-yl)amino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylates (6a and 6b) and their derivatives
(7a, 7b and 8aef) is delineated in Scheme 1. N-Alkylation of 5-
methyl-3-nitropyridin-2(1H)-one
bromo-2-methoxyethane in the presence of potassium carbonate
gave 1,5-dimethyl-3-nitropyridin-2(1H)-one 2a or 1-(2-
methoxyethyl)-5-methyl-3-nitropyridin-2(1H)-one 2b respec-
tively in good yields (2a: 95% and 2b: 60%), which underwent
palladium-catalysed hydrogenation to afford the corresponding 3-
amino-1,5-dimethylpyridin-2(1H)-one 3a or 3-amino-1-(2-
1
with iodomethane or 1-
2.2. Structureeactivity relationship analysis
Up-regulation of eIF4E is observed in acute myeloid leukaemia
(AML) [23] and targeting eIF4E by pharmacologic inhibition of its
activating kinases Mnks may provide a new approach for the
treatment of AML. A recent study demonstrated that the suppres-
sive effects of cercosporamide on AML correlated with its Mnk

118
M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
Scheme 1. Synthesis of methyl 4-((1-alkyl-2-oxo-5-methyl-1,2-dihydropyridin-3-yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylates (6aeb) and their derivatives (7aeb
and 8aef). Reagents and conditions: (a) appropriate alkyl halide, K2CO3, DMF, 0 ꢀC to rt for CH3I, 0 ꢀC to 70 ꢀC for BrCH2CH2OCH3, o/n, 2a: 95%, 2b: 60%; (b) H2, 10% Pd/C, CH3OH, rt, o/
n, 3a: 94%, 3b: 100%; (c) SOCl2, DMF, reflux, 2 h, 89%; (d) TsOH$H2O, 1,4-dioxane, microwave 200e300 W, 150 ꢀC, 30 min, 6a: 86%, 6b: 54%; (e) 2 M NaOH, CH3OH, reflux, 2 h, 7a:
100%, 7b: 84%; (f) appropriate amine, HATU, DIPEA, DMF, 0 ꢀC to rt, o/n, 8a: 80%, 8b: 36%; 8c: 66%, 8d: 58%; 8e: 37%, 8f: 66%.
Scheme 2. Synthesis of methyl 4-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (12) and its derivatives (13 and 14). Reagents
and conditions: (a) CH3I, K2CO3, DMF, 0 ꢀC to rt, o/n, 99%; (b) H2, 10% Pd/C, CH3OH, rt, o/n; (c) 5, TsOH$H2O, 1,4-dioxane, microwave 200e300 W, 150 ꢀC, 30 min, 68% over steps (b)
and (c); (d) 2 M NaOH, CH3OH, rt, 48 h, 88%; (e) NH3 (0.5 M in 1,4-dioxane), HATU, DIPEA, DMF, 0 ꢀC to rt, o/n, 54%.
inhibitory activity [24], supporting the potential application of
pharmacologic Mnk inhibitors in anti-leukaemia therapy. As one of
the most studied AML cell lines, MV-4-11 was selected for cell
viability assay and cellular mechanistic investigation (vide infra)
due to its increasing use for the assessment of anti-leukaemic ef-
fects of Mnk inhibition [18,24,25] as well as its sensitivity to Mnk
inhibitors [18,22].
Mnk inhibitory activity of compounds 6e8 and 12e14 was
evaluated using the ADP-Glo™ kinase assay [26], and the anti-
proliferative activity tested by a cell viability assay using resa-
zurin [18]. CGP57380, a known Mnk inhibitor, served as a positive
control for both enzymatic and cellular assays. The results obtained
from these biological assays are summarised in Table 1. All methyl
esters (R2 ¼ OCH3), i.e., 6a (R1 ¼ CH3 in general structure I), 6b
(R1 ¼ CH2CH2OCH3) and 12 (R1 ¼ CH3 in general structure II), are
not active against both Mnk1 and Mnk2 with Ki values being
inhibitory activity and cytotoxicity are consistent with the results
previously obtained for methyl 4-(phenylamino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylates [22]. Conversion of the methyl
ester moiety into the carboxylic acid group resulting in 7a
(R1 ¼ CH3, R2 ¼ OH in general structure I), 7b (R1 ¼ CH2CH2OCH3,
R2 ¼ OH) and 13 (R1 ¼ CH3, R2 ¼ OH in general structure II),
dramatically boosts the inhibitory activity against both Mnks,
implying the involvement of the carboxylic acid in the interaction
with the kinases. Carboxylic acids 7a, 7b and 13 inhibit Mnk1 and
Mnk2 with Ki values in the range of 2.06e4.82
0.24e0.90 M respectively, which are comparable and superior to
those of CGP57380 (Ki ¼ 1.01 M for Mnk1 vs. Ki ¼ 0.88 M for
Mnk2). Compound 7a possesses the highest selectivity towards
Mnk2 over Mnk1 by approximate 14-fold (Ki ¼ 4.82 M for Mnk1
vs. Ki ¼ 0.35 M for Mnk2), whereas compound 7b represents the
most potent dual Mnk inhibitor with Ki values of 2.06 M for Mnk1
and 0.24 M for Mnk2. Despite the significant improvement in Mnk
inhibitory activity, carboxylic acids 7a, 7b and 13 display lower or at
mM and
m
m
m
m
m
m
greater than 10
mM, and are moderately toxic against MV-4-11 cells
m
with GI50 values ranging from 13.44 to 47.82
mM. Both kinase

M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
119
Table 1
Summary of chemical structures and their biological activities.
Compound
R1
R2
Mnk inhibition Ki (
m
M)a
Anti-proliferation GI50
MV-4-11
(m
M)b
Mnk1
Mnk2
6a
6b
7a
7b
8a
8b
8c
8d
8e
8f
12
13
CH3
CH2CH2OCH3
CH3
CH2CH2OCH3
CH3
CH3
CH3
CH3
CH3
CH2CH2OCH3
CH3
CH3
CH3
OCH3
OCH3
OH
OH
NH2
NHCH3
NHCH2CH3
NHCH2CH2OCH3
>10
>10
4.82
2.06
>10
>10
>10
5.40
5.00
2.33
>10
2.15
4.51
1.01
>10
>10
0.35
0.24
>10
9.05
5.99
3.17
2.63
0.34
13.44 0.70
26.22 2.70
15.18 1.91
59.58 3.48
17.51 1.02
10.37 2.80
9.66 0.72
11.30 3.81
5.73 0.38
NHCH2CH2CH2OCH3
NH2
OCH3
OH
NH2
e
13.96 2.21
47.82 13.84
88.99 4.11
89.59 13.62
4.88 0.45
>10
0.90
1.60
0.88
14
CGP57380
e
a
Km values were used as ATP concentrations. Apparent inhibition constants (Ki) were calculated using IC50 values and appropriate Km (ATP) values for each kinase, and are
expressed as mean values derived from two replicates.
b
GI50 values were determined by 72 h resazurin assay, and are presented as mean standard deviation derived from at least two replicates.
the best similar cytotoxicity in comparison with the corresponding
methyl esters 6a, 6b and 12 (GI50 > 15 M for carboxylic acids vs.
GI50 > 13 M for methyl esters). Amination of carboxylic acids re-
However, it appears to be a discrepancy between Mnk inhibitory
potency and cellular anti-proliferative effects, and two plausible
explanations are: (i) in vitro inhibition of Mnks at low micromolar
or sub-micromolar concentrations may not necessarily cause cell
death during the 72-h incubation, and/or (ii) the tested compounds
may also target other kinase(s), both of which from a biochemical
standpoint warrant the further investigation of these Mnk in-
hibitors in kinase and cell panel screens. Thus, 7a, 7b and 8e were
tested against a panel of 9 kinases, including cyclin-dependent ki-
nases (i.e., CDK2/cyclinA and CDK9/cyclinT1), Mnk upstream acti-
vating kinases in Ras/Raf/MAPK and PI3K/Akt/mTOR pathways (i.e.,
m
m
duces or even abolishes Mnk inhibitory activity. Accordingly, car-
boxamides 8f (R1 ¼ CH2CH2OCH3, R2 ¼ NH2) and 14 (R1 ¼ CH3,
R2 ¼ NH2 in general structure II) show marginally weaker Mnk
inhibitory potency than do the corresponding carboxylic acids 7b
and 13, whereas carboxamide 8a (R1 ¼ CH3, R2 ¼ NH2 in general
structure I) loses inhibitory activity against both Mnks (Ki > 10 mM).
The distinct kinase inhibitory activities between 8a and each of 8f
and 14 imply that both the nature of R1 substituent and the to-
pology of the dihydropyridinone ring are important factors for the
affinity for Mnks. The anti-proliferative effects of 8a and 14 (with
MAPK1, mTOR, PKBa, SAPK2a and PI3K), as well as FLT3 and Pim-1.
The results obtained from this kinase panel screen are presented in
their respective GI50 values of 17.51 and 89.59
are comparable to those of the corresponding carboxylic acids 7a
and 13, whereas 8f shows significantly enhanced anti-
proliferative activity by comparison to its carboxylic acid counter-
part 7b (GI50 ¼ 59.58 M for 7b vs. GI50 ¼ 13.96 M for 8f). N-
m
M) on MV-4-11 cells
Fig. 2. Both 7a and 7b have no inhibitory activity against all kinases
tested at the concentration of 10 mM, confirming their selectivity for
Mnks and their potential use as pharmacologic tools for the
a
m
m
Alkylation of the carboxamide 8a restores Mnk inhibitory activity
and enhances cytotoxicity. Along with the increase in the alkyl
chain length, Mnk inhibitory activity improves with the selectivity
towards Mnk2. However, Mnk inhibitory activities of all carbox-
amides, i.e., 8a-e, decrease when compared to the parent carboxylic
acid 7a, suggesting a preferable role of the carboxylic acid in the
interaction with Mnks. All N-monoalkylated carboxamides, i.e., 8b-
f, demonstrate potent anti-proliferative activity with GI50 values
ranging from 5.73 to 11.30 mM. Compound 8e with the longest chain
(R1 ¼ CH3, R2 ¼ NHCH2CH2CH2OCH3) exhibits the highest toxicity
against MV-4-11 cells with a GI50 value of 5.73 mM, which is com-
parable with that of CGP57380 (GI50 ¼ 4.88
mM).
Taken together, low micromolar to sub-micromolar Mnk inhi-
bition has been achieved with the 4-(dihydropyridinon-3-yl)ami-
nothieno[2,3-d]pyrimidine framework. The selectivity of Mnk2
over Mnk1 can be optimised by (i) manipulating the topology and/
or N-substitution of the dihydropyridinone ring, and/or (ii) varying
the C-6 functionality at the thieno[2,3-d]pyrimidine moiety.
Fig. 2. Selectivity of kinase inhibition by compound 7a, 7b and 8e. Compounds were
tested at the concentration of 10
values.
mM. ATP concentrations used were within 15 mM of Km

120
M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
required.
2.4. Cellular mechanism of action
Carboxylic acid 7a is the most selective Mnk2 inhibitor whereas
N-(3-methoxypropyl) carboxamide 8e displays the highest cyto-
toxicity against MV-4-11 cells. Thus, these two molecules were
chosen for detailed cellular mechanistic investigation. Carbox-
amide 8f was also selected in virtue of its well-balanced biological
activity, i.e., high potency in both Mnk inhibition and anti-
proliferation. CGP57380 was employed as a positive control. Com-
pounds were tested at concentrations which are approximately
twice as much as their respective GI50 values (2 ꢁ GI50) with the
exception of 8e which was used at the concentration of approxi-
mately 4 ꢁ GI50 value, i.e., 7a and 8f at 30
mM, 8e at 20 mM and
CGP57380 at 10 M.
m
Cell death occurs through several pathways including apoptosis,
autophagocytosis and necrosis. To evaluate whether apoptosis
contributes to the anti-proliferative effects of 7a, 8e and 8f, annexin
V/propidium iodide (PI) staining of MV-4-11 cells was carried out
upon incubation with each compound for 24 h (Fig. 4A). In com-
parison with DMSO diluent, 7a, 8e and 8f induced more apoptotic
cells (annexin Vþ/PI- and annexin Vþ/PIþ) by 6e10%. CGP57380
was more effective in inducing apoptosis, resulting in an increment
of 13% apoptotic cells.
Fig. 3. Proposed binding mode of 7a to the ATP-binding pocket of Mnk2. 7a is engaged
in four hydrogen bonds and a p-p stacking interaction with the protein. The protein is
depicted as cyan ribbons with the hinge region in magenta. Nitrogen atoms are shown
in blue, sulphur atom in yellow, oxygen atoms in red, hydrogen atoms in grey, receptor
carbon atoms in green, and ligand carbon atoms in salmon. The glycine rich loop was
omitted for clarity. The binding pose was generated by induced fit docking of 7a to a
€
DFD-in conformation model of Mnk2 using Schrodinger (Glide version 6.1, Prime
€
version 3.4, Schrodinger, LLC, New York, NY, 2013). The figure was prepared using
Pymol version 1.5.
To determine whether the apoptosis observed with 7a, 8e or 8f
is caspase-dependent, MV-4-11 cells were treated with each com-
pound for 24 h, and the activity of caspases 3 and 7 was measured
(Fig. 4B). No significant changes in caspase 3/7 activity were
observed, which is in agreement with the previous results obtained
with 5-(2-(phenylamino)pyrimidin-4-yl)thiazol-2(3H)-one de-
rivatives [18].
To further assess whether the anti-proliferative effect of 7a, 8e
or 8f is a consequence of inhibition of cell cycle progression, MV-4-
11 cells were exposed to each compound for 24 h, and analysed by
flow cytometry. A similar cell cycle profile was observed for the
cells treated with 7a, 8e or 8f (Fig. 4C). Each compound caused a
marked increase in the population of G1-phase cells and a minor
accumulation of sub-G1-phase cells, which was companied by a
substantial loss in S- and G2/M-phase cells. These results are
consistent with previously reported observations on alternative
Mnk inhibitors [18,22].
To investigate cellular Mnk inhibitory potency of 7a, 8e or 8f,
MV-4-11 cells were incubated with each compound for 24 h, and
analysed by Western blot (Fig. 4D). Treatment with 7a, 8e or 8f
abolished the phosphorylation of eIF4E at Ser209, but barely
affected the levels of Mnk1 and total eIF4E, confirming their cellular
Mnk inhibitory activity. CGP57380 also reduced the level of phos-
phorylated eIF4E. 7a, 8e and 8f were capable of down-regulating
anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and
causing poly (ADP-ribose) polymerase (PARP) cleavage, both of
which are associated with the apoptosis observed in MV-4-11.
CGP57380 exerted similar effects on Mcl-1 and PARP. 7a, 8e and 8f
moderately to significantly blocked the expression of cyclin D1,
which well correlates with the observation that these molecules
induced G1 cell cycle arrest (vide supra).
evaluation of Mnk inhibition. Compound 8e is not active against
these kinases with the single exception of FLT3 where 31% inhibi-
tory activity was detected. Given that FLT3 was previously sug-
gested to be important for the proliferation and survival of AML
cells [27] and that MV-4-11 cells express FLT3 protein [28], dual
inhibitory activity of 8e against both Mnks and FLT3 may contribute
to its potent anti-proliferative effect.
2.3. Molecular basis for Mnk2 inhibitory activity
In order to understand the origins of the Mnk inhibitory potency
of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimi-
dine derivatives, 7a was docked to an in-house model of Mnk2 with
the DFD-in conformation [29] using the induced fit protocol
[30,31]. The docking was performed at the ATP binding pocket and
the best scoring model was selected following visual inspection of
the top scoring binding poses. Fig. 3 illustrates this plausible
binding mode. 7a is stabilised in the ATP binding pocket by four
hydrogen bonds: the compound interacts with Phe227 of the DFD
motif through the carbonyl oxygen atom of the dihydropyridinone
ring and the NH linker, whereas the N-1 of the pyrimidine moiety
and the carboxyl oxygen atom form two additional hydrogen bonds
with positive-charged side chains of Lys113 and Arg125 respec-
tively. Furthermore, the dihydropyridinone ring exhibits a
p-p
stacking interaction with the gatekeeper Phe159. Interestingly, no
hinge region interactions, which are common among most kinase
inhibitors, are observed. It appears that the salt bridge between the
carboxylate moiety of 7a and the guanidine side chain of Arg125
makes a significant contribution to the binding affinity. When the
carboxylate moiety is replaced by a methyl ester group (6a), the
interaction with Arg125 is severed, reducing the binding affinity.
On the other hand, removal of the negative charge from the
carboxylate moiety by the substitution with an amide group ap-
pears to disturb the binding mode in a subtle way. This substitution
adversely affects the binding affinity in the case of 8a while further
replacement of the N-methyl group with an N-methoxyethyl chain
(8f) may assist in recovering the binding affinity. To elucidate such
subtle interplays between substituents, more rigorous calculations,
potentially assisted by experimental structure determinations, are
3. Conclusions
A
series of 4-(dihydropyridinon-3-yl)amino-5-methylthieno
[2,3-d]pyrimidine derivatives was synthesised and their structure
activity relationships were investigated. Many compounds dis-
played potent Mnk inhibitory activity with the selectivity towards
Mnk2. The induced fit docking study with a DFD-in conformation
model of Mnk2 implies 7a is stabilised in the ATP binding pocket by

M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
121
Fig. 4. Cellular mechanism of action of 7a, 8e and 8f. (A) Induction of apoptosis by 7a, 8e, 8f or CGP57380 in MV-4-11 cells. Cells were exposed to each compound for 24 h, and
analysed by Annexin V/PI staining. The percentage of cells undergoing apoptosis is defined as the sum of early apoptotic (annexin Vþ/PI-) cell percentage and late apoptotic
(annexin Vþ/PIþ) cell percentage. (B) Relative caspase 3/7 activity in MV-4-11 cells after treatment with 7a, 8e, 8f or CGP57380 for 24 h. (C) Cell cycle analysis of MV-4-11 cells after
incubation with 7a, 8e, 8f or CGP57380 for 24 h. (D) Western blot analysis of MV-4-11 cells treated with 7a, 8e, 8f or CGP57380 for 24 h. b-Actin antibody was used as an internal
control. In all experiments, DMSO diluent was employed as a control, and compounds were tested at concentrations of their respective (2 ꢁ GI50) values with the exception of 8e
which was used at the concentration of approximately 4 ꢁ GI50 value, i.e., 7a and 8f at 30
mM, 8e at 20 mM and CGP57380 at 10 mM.
four hydrogen bonds and hydrophobic interaction. Cellular mech-
anistic studies on MV-4-11 cells with lead compounds 7a, 8e and 8f
reveal that they are able to block the phosphorylation of eIF4E via
targeting Mnks, down-regulate Mcl-1 and cause PARP cleavage.
This study suggests that exploration of the structural diversity in
the context of 4-aminothieno[2,3-d]pyrimidine would offer potent
and highly selective Mnk inhibitors.
298 K on a Bruker AVANCE III HD 500 spectrometer (1H at
500.16 MHz and 13C at 125.76 MHz; Faellanden, Switzerland), and
were analysed using Bruker Topspin 3.2 software. 1H and 13C NMR
spectra are referenced to 1H signals of residual nondeuterated
solvents and 13C signals of deuterated solvents respectively. 1H
NMR signals are reported with chemical shift values
d (ppm),
multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,
dd ¼ doublet of doublets, m ¼ multiplet and br ¼ broad), relative
integral, coupling constants J (Hz) and assignments. High resolution
mass spectra were recorded on an AB SCIEX TripleTOF 5600 mass
spectrometer (Concord, ON, Canada), and ionisation of all samples
was carried out using ESI. Melting points were determined using an
open capillary on a Stuart SMP10 melting point apparatus and are
uncorrected. The purity of compounds used for biological evalua-
tion was determined by analytic RP-HPLC which was carried out on
a Shimadzu Prominence UFLC system (UltraFast Liquid Chromato-
graph, Kyoto, Japan) equipped with a CBM-20A communications
bus module, a DGU-20A5R degassing unit, an LC-20AD liquid
chromatograph pump, an SIL-20AHT auto-sampler, an SPD-M20A
photo diode array detector, a CTO-20A column oven and a Phe-
nomenex Kinetex 5u C18 100A 250 mm ꢁ 4.60 mm column.
Method A (gradient 5%e95% CH3OH containing 0.1% formic acid
(FA) over 7 min at a flow rate of 1 mL/min, followed by 95% CH3OH
containing 0.1% FA over 13 min) and method B (gradient 5%e95%
CH3CN containing 0.1% FA over 7 min at a flow rate of 1 mL/min,
followed by 95% CH3CN containing 0.1% FA over 13 min) were used
4. Experimental
4.1. Chemistry
All reactions except microwave-assisted syntheses were carried
out with continuous magnetic stirring in ordinary glassware;
microwave-assisted syntheses were performed in a 10 mL Asynt
DrySyn insert (Isleham, UK) using a CEM Discover SP and Explorer
48/72/96 microwave system (Matthews, NC, USA) controlled by
Synergy software (Firmware version DSCA02.17). Heating of re-
actions was conducted with a DrySyn® single- or multi-position
heating block (Isleham, UK); cooling of reactions was achieved
using an ice or ice-salt bath. All reagents and solvents (including
anhydrous solvents) were purchased from SigmaeAldrich, Alfa
Aesar, Merck, GL Biochem, Combi-block or Ajax Finechem, and
were used as received. Methyl 5-methyl-4-oxo-3,4-dihydrothieno
[2,3-d]pyrimidine-6-carboxylate (4) was purchased from Le Sun
Pharmaceuticals (China). 1H and 13C NMR spectra were recorded at

122
M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
for analytic RP-HPLC. Data acquired from analytic RP-HPLC were
processed using LabSolutions Analysis Data System. Analytical TLC
was performed on Merck silica gel 60 F254 pre-coated aluminium
plates (0.2 mm) and visualised under UV light (254 nm). Column
chromatography was carried out using a fritted solid loader packed
(DCM:CH3OH ¼ 98:2) 0.49. m.p. 185e186 ꢀC. 1H NMR (CDCl3)
d 2.18
(s, 3H, pyridinone-CH3), 3.64 (s, 3H, NCH3), 7.53 (d, 1H, J 1.5,
pyridinone-H), 8.18 (d, 1H, J 2.5, pyridinone-H). 13C NMR (CDCl3)
d
16.9, 38.7, 112.8, 138.0, 140.6, 143.2, 154.3. HRMS (ESI-TOF) m/z
191.0404 [MþNa]þ; calcd. for C7H8N2NaOþ3 191.0427 [MþNa]þ.
with GRACE Davison DAVISIL® silica gel 60 Å (40e63
mm) on a
Biotage FlashMaster Personalþ flash chromatography system.
4.1.2. 1-(2-Methoxyethyl)-5-methyl-3-nitropyridin-2(1H)-one (2b)
5-Methyl-3-nitropyridin-2(1H)-one (1, 1.54 g, 9.99 mmol) and
1-bromo-2-methoxyethane (1.88 mL, 20.0 mmol) were reacted
using general synthetic procedure A. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
EtOAc) to give 2b as a yellow solid (1.27 g, 60%). RF (EtOAc) 0.52.
General synthetic procedure A: N-Alkylation of pyridin-2(1H)-ones
To a suspension of pyridin-2(1H)-one (1.0 equiv.) and K2CO3 (2.0
equiv.) in DMF (0.60 M in pyridin-2(1H)-one) at 0 ꢀC was added
alkyl halide (1.5e2.0 equiv.) dropwise. The reaction mixture was
allowed to warm up to room temperature (in the case of iodo-
methane) or heated at 70 ꢀC (in the case of 1-bromo-2-
methoxyethane), stirred overnight, concentrated under reduced
pressure. The residue was diluted with H2O (200 mL) and extracted
with DCM (6 ꢁ 100 mL). The organic extracts were combined and
concentrated under reduced pressure, and the residue was purified
by Biotage® FlashMaster Personalþ flash chromatography (silica
gel, DCM ramping to DCM:CH3OH ¼ 98:2 unless otherwise stated)
to give the desired N-alkylated pyridine-2(1H)-one.
m.p. 103e105 ꢀC. 1H NMR (CDCl3)
d 2.00 (s, 3H, pyridinone-CH3),
3.10 (s, 3H, OCH3), 3.48 (t, 2H, J 5.0, CH2CH2), 4.04 (t, 2H, J 5.0,
CH2CH2), 7.49 (d, 1H, J 1.5, pyridinone-H), 8.02 (d, 1H, J 2.5,
pyridinone-H). 13C NMR (CDCl3)
d 16.3, 50.0, 58.4, 68.9, 112.0, 137.0,
140.4, 144.3, 153.3. HRMS (ESI-TOF) m/z 213.0847 [MþH]þ; calcd.
for C9H13N2Oþ4 213.0870 [MþH]þ.
4.1.3. 3-Amino-1,5-dimethylpyridin-2(1H)-one (3a)
Nitro compound 2a (169 mg, 1.00 mmol) was reduced according
to general synthetic procedure B. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:CH3OH ¼ 98:2) to give 3a as an orange oil
General synthetic procedure B: reduction of nitro compounds to
amines
To a suspension of a nitro compound (1.0 equiv.) in CH3OH
(50 mM in nitro compound) was added 10% Pd/C (0.01 equiv.). The
reaction mixture was bubbled with H2 for 1 h at room temperature
and stirred under H2 overnight. The solids were filtered off and
washed with DCM (25 mL). The filtrate and DCM washing were
combined and concentrated under reduced pressure to give the
desired amine.
(131 mg, 94%). RF (DCM:CH3OH ¼ 98:2) 0.15. 1H NMR (CDCl3)
d 1.90
(s, 3H, pyridinone-CH3), 3.42 (s, 3H, NCH3), 4.15 (br s, 2H, NH2), 6.33
(d, 1H, J 2.0, pyridinone-H), 6.40 (s, 1H, pyridinone-H). 13C NMR
(CDCl3)
d 17.5, 37.1, 115.1, 115.7, 123.5, 136.8, 157.1. HRMS (ESI-TOF)
m/z 139.0876 [MþH]þ; calcd. for C7H11N2Oþ 139.0866 [MþH]þ.
4.1.4. 3-Amino-1-(2-methoxyethyl)-5-methylpyridin-2(1H)-one
(3b)
General synthetic procedure C: substitution of chloride with amines
To a suspension of chloride 5 (1.0 equiv.) and amine (1.0 equiv.)
in 1,4-dioxane (0.40 M in amine) was added TsOH$H2O (0.20 equiv).
The reaction mixture was heated at 150 ꢀC under microwave irra-
diation for 30 min, cooled down, and concentrated under reduced
pressure. The residue was purified by Biotage® FlashMaster Per-
sonalþ flash chromatography (silica gel, DCM ramping to
DCM:CH3OH ¼ 95:5 unless otherwise stated) to give the desired
secondary amine.
Nitro compound 2b (212 mg, 0.999 mmol) was reduced ac-
cording to general synthetic procedure B to give 3b as a brown oil
(182 mg, 100%). RF (DCM:CH3OH ¼ 9:1) 0.57. 1H NMR (CDCl3)
d 1.83
(s, 3H, pyridinone-CH3), 3.15 (s, 3H, OCH3), 3.52 (t, 2H, J 5.0,
CH2CH2), 3.93 (t, 2H, J 5.0, CH2CH2), 4.18 (br s, 2H, NH2), 6.26 (d,1H, J
1.5, pyridinone-H), 6.39 (s, 1H, pyridinone-H). 13C NMR (CDCl3)
d
17.4, 49.3, 58.6, 70.2, 115.0, 115.1, 123.5, 136.8, 156.5. HRMS (ESI-
TOF) m/z 205.0947 [MþNa]þ; calcd. for C9H14N2NaOþ2 205.0947
[MþNa]þ.
General synthetic procedure D: hydrolysis of esters
4.1.5. Methyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-
carboxylate (5)
To a suspension of methyl 5-methyl-4-oxo-3,4-dihydrothieno
[2,3-d]pyrimidine-6-carboxylate (4, 4.48 g, 20.0 mmol) in SOCl2
To a suspension of ester (1.0 equiv.) in CH3OH (50 mM in ester)
was added 2 M NaOH (7.5 equiv.). The reaction mixture was heated
at reflux for 2 h, cooled down to room temperature, and washed
with DCM (20 mL). The aqueous layer was acidified with 2 M HCl to
pH 3. The precipitate was filtered, washed with H2O (3 ꢁ 25 mL),
and dried under reduced pressure to give the desired carboxylic
acid.
(40.0 mL, 548 mmol) was added DMF (800 mL, 10.3 mmol). The
reaction mixture was heated at reflux under N2 for 2 h, cooled
down to room temperature, stirred overnight and concentrated
under reduced pressure. The residue was purified by Biotage®
FlashMaster Personalþ flash chromatography (silica gel, DCM
ramping to DCM:EtOAc ¼ 98:2) to give 5 as a white solid (4.30 g,
89%). RF (DCM:EtOAc ¼ 98:2) 0.57. m.p. 141e142 ꢀC. 1H NMR
General synthetic procedure E: amide bond formation
To a suspension of the carboxylic acid (1.0 equiv.) in anhydrous
DMF (3 mL) at 0 ꢀC were added DIPEA (3.0 equiv. with the single
exception of 10 equiv. in the case of methylamine hydrochloride)
and HATU (1.1 equiv.). The reaction mixture was stirred at 0 ꢀC for
10 min, and amine (5.0 equiv.) was added. The reaction mixture was
allowed to warm up to room temperature, stirred under N2 over-
night and filtered. The solids were washed with ice-cold DMF
(3 mL) and H2O (30 mL) to give the desired amide.
(CDCl3)
d 2.97 (s, 3H, thiophene-CH3), 3.91 (s, 3H, COOCH3), 8.79 (s,
1H, pyrimidine-H). 13C NMR (CDCl3)
d
15.8, 52.8, 127.9, 128.8, 139.2,
154.2, 157.3, 162.4, 169.1. HRMS (ESI-TOF) m/z 243.0004 [M(35Cl)þ
H]þ, 244.9975 [M(37Cl)þH]þ; calcd. for C9H8ClN2O2Sþ 242.9990
[M(35Cl)þH]þ, 244.9960 [M(37Cl)þH]þ.
4.1.6. Methyl 4-((1,5-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)
amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylate (6a)
Chloride 5 (485 mg, 2.00 mmol) and amine 3a (273 mg,
1.98 mmol) were subjected to general synthetic procedure C. The
residue was purified by Biotage® FlashMaster Personalþ flash
chromatography (silica gel, DCM ramping to DCM:CH3OH ¼ 99:1)
4.1.1. 1,5-Dimethyl-3-nitropyridin-2(1H)-one (2a)
5-Methyl-3-nitropyridin-2(1H)-one (1, 1.39 g, 9.02 mmol) and
iodomethane (840
mL, 13.5 mmol) were reacted using general
synthetic procedure A to give 2a as a yellow solid (1.44 g, 95%). RF

M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
123
to give 6a as a yellow solid (587 mg, 86%). RF (DCM:CH3OH ¼ 95:5)
0.45. m.p. 270e272 ꢀC. 1H NMR (CDCl3)
2.20 (s, 3H, pyridinone-
7.86 (br d, 2H, J 59.0, CONH2), 8.59 (d, 1H, J 1.5, pyridinone-H), 8.66
(s, 1H, pyrimidine-H), 9.23 (s, 1H, pyridinone-NH-pyrimidine). 13C
d
CH3), 3.21 (s, 3H, thiophene-CH3), 3.63 (s, 3H, NCH3), 3.93 (s, 3H,
NMR (DMSO-d6) d 15.2, 17.0, 36.5, 113.8, 117.5, 123.4, 128.1, 129.1,
COOCH3), 6.82 (s, 1H, pyridinone-H), 8.69 (s, 2H, pyridinone-H &
130.2,153.8,155.1,156.5,163.5,165.1 (one carbon signal overlapping
pyrimidine-H), 9.49 (br s, 1H, NH). 13C NMR (CDCl3)
d
15.9, 18.2,
or obscured). HRMS (ESI-TOF) m/z 352.0838 [MþNa]þ; calcd. for
37.9, 52.5, 116.3, 118.6, 123.7, 124.9, 127.5, 129.3, 138.3, 155.3, 156.3,
C
15H15N5NaO2Sþ 352.0839 [MþNa]þ. Anal. RP-HPLC Method A: tR
157.6, 163.3, 167.3. HRMS (ESI-TOF) m/z 345.1033 [MþH]þ; calcd. for
10.88 min, purity >98%; Method B: tR 8.77 min, purity >98%.
C
16H17N4O3Sþ 345.1016 [MþH]þ. Anal. RP-HPLC Method A: tR
12.76 min, purity >98%; Method B: tR 11.18 min, purity >97%.
4.1.11. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-
N,5-dimethylthieno[2,3-d]pyrimidine-6-carboxamide (8b)
4.1.7. Methyl 4-((1-(2-methoxyethyl)-5-methyl-2-oxo-1,2-
dihydropyridin-3-yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-
carboxylate (6b)
Carboxylic acid 7a (83.0 mg, 0.251 mmol) and methylamine
hydrochloride (84.0 mg, 1.24 mmol) were coupled using general
synthetic procedure E to give 8b as a beige solid (31.0 mg, 36%).
Chloride 5 (243 mg, 1.00 mmol) and amine 3b (182 mg,
0.999 mmol) were subjected to general synthetic procedure C to
give 6b as a yellow solid (210 mg, 54%). RF (DCM:CH3OH ¼ 95:5)
m.p. > 300 ꢀC. 1H NMR (DMSO-d6)
d 2.12 (d, 3H, J 0.5, pyridinone-
CH3), 2.80 (d, 3H, J 4.5, CONHCH3), 2.91 (s, 3H, thiophene-CH3), 3.54
(s, 3H, NCH3), 7.23 (dd, 1H, J 2.0 & 1.0, pyridinone-H), 8.42 (q, 1H, J
4.5, CONHCH3), 8.59 (d, 1H, J 2.0, pyridinone-H), 8.66 (s, 1H,
pyrimidine-H), 9.22 (s, 1H, pyridinone-NH-pyrimidine). 13C NMR
0.58. m.p. 216 ꢀC. 1H NMR (CDCl3)
d 2.18 (s, 3H, pyridinone-CH3),
3.18 (s, 3H, thiophene-CH3), 3.33 (s, 3H, CH2OCH3), 3.70 (t, 2H, J 4.5,
CH2CH2), 3.91 (s, 3H, COOCH3), 4.16 (t, 2H, J 4.5, CH2CH2), 6.84 (s,1H,
pyridinone-H), 8.65 (s, 1H, pyrimidine-H), 8.69 (s, 1H, pyridinone-
(DMSO-d6)
d 15.7, 17.5, 26.5, 37.2, 114.2, 117.7, 123.6, 128.3, 128.6,
128.9, 130.4, 154.3, 155.3, 156.6, 162.5, 165.2. HRMS (ESI-TOF) m/z
344.1167 [MþH]þ; calcd. for C16H18N5O2Sþ 344.1176 [MþH]þ. Anal.
RP-HPLC Method A: tR 11.15 min, purity >97%; Method B: tR
9.11 min, purity >97%.
H), 9.29 (s, 1H, NH). 13C NMR (CDCl3)
d 15.9, 18.3, 50.4, 52.5, 59.2,
70.4, 115.6, 118.5, 123.5, 125.2, 128.1, 129.1, 138.4, 155.3, 156.3, 157.2,
163.2, 167.4. HRMS (ESI-TOF) m/z 389.1267 [MþH]þ; calcd. for
C
18H21N4O4Sþ 389.1278 [MþH]þ. Anal. RP-HPLC Method A: tR
11.34 min, purity >99%; Method B: tR 9.97 min, purity >98%.
4.1.12. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-N-
ethyl-5-methylthieno[2,3-d]pyrimidine-6-carboxamide (8c)
Carboxylic acid 7a (83.0 mg, 0.251 mmol) and ethylamine (2.0 M
4.1.8. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylic acid (7a)
in THF, 625
procedure E to give 8c as a beige solid (59.0 mg, 66%). m.p.
284e286 ꢀC. 1H NMR (DMSO-d6)
1.14 (t, 3H, J 7.5, NHCH2CH3), 2.12
mL, 1.25 mmol) were coupled using general synthetic
Ester 6a (751 mg, 2.18 mmol) was hydrolysed using general
synthetic procedure D to give 7a as a greenish solid (720 mg, 100%).
d
m.p. > 300 ꢀC. 1H NMR (DMSO-d6)
d
2.12 (s, 3H, pyridinone-CH3),
(s, 3H, pyridinone-CH3), 2.90 (s, 3H, thiophene-CH3), 3.26e3.32 (m,
2H, NHCH2CH3), 3.54 (s, 3H, NCH3), 7.23 (d, 1H, J 1.0, pyridinone-H),
8.51 (t, 1H, J 5.5, NHCH2CH3), 8.59 (d, 1H, J 2.5, pyridinone-H), 8.66
(s, 1H, pyrimidine-H), 9.22 (s, 1H, pyridinone-NH-pyrimidine). 13C
3.09 (s, 3H, thiophene-CH3), 3.54 (s, 3H, NCH3), 7.23 (s, 1H,
pyridinone-H), 8.59 (d, 1H, J 2.0, pyridinone-H), 8.67 (s, 1H,
pyrimidine-H), 9.28 (s, 1H, NH) (one carboxylic acid proton signal
(COOH) not observed). 13C NMR (DMSO-d6)
d
15.0, 17.5, 37.2, 114.2,
NMR (DMSO-d6) d 14.7,15.8,17.6, 34.4, 37.2,114.3,117.7,123.6,128.3,
118.3, 123.7, 128.3, 128.7, 154.9, 155.6, 156.6, 163.8, 166.0 (two car-
bon signals overlapping or obscured). HRMS (ESI-TOF) m/z
331.0871 [MþH]þ; calcd. for C15H15N4O3Sþ 331.0859 [MþH]þ. Anal.
RP-HPLC Method A: tR 11.89 min, purity >98%; Method B: tR
9.56 min, purity >98%.
128.6, 129.3, 130.2, 154.3, 155.3, 156.7, 161.8, 165.2. HRMS (ESI-TOF)
m/z 358.1329 [MþH]þ; calcd. for C17H20N5O2Sþ 358.1332 [MþH]þ.
Anal. RP-HPLC Method A: tR 11.44 min, purity >98%; Method B: tR
9.57 min, purity >99%.
4.1.13. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-N-
(2-methoxyethyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide
(8d)
4.1.9. 4-((1-(2-Methoxyethyl)-5-methyl-2-oxo-1,2-dihydropyridin-
3-yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylic acid
(7b)
Carboxylic acid 7a (74 mg, 0.22 mmol) and 2-
Ester 6b (155 mg, 0.399 mmol) was hydrolysed using general
synthetic procedure D to give 7b as a greenish solid (125 mg, 84%).
methoxypropylamine (96
eral synthetic procedure E to give 8d as a pale yellow solid (50 mg,
58%). m.p. 245e246 ꢀC. 1H NMR (DMSO-d6)
2.12 (s, 3H, pyr-
mL, 1.1 mmol) were coupled using gen-
m.p. 295e296 ꢀC. 1H NMR (DMSO-d6)
d
2.11 (s, 3H, pyridinone-
d
CH3), 3.05 (s, 3H, thiophene-CH3), 3.26 (s, 3H, OCH3), 3.63 (t, 2H, J
5.5, CH2CH2), 4.13 (t, 2H, J 5.5, CH2CH2), 7.16 (s, 1H, pyridinone-H),
8.55 (d, 1H, J 1.5, pyridinone-H), 8.64 (s, 1H, pyrimidine-H), 9.20
(s, 1H, NH) (one carboxylic acid proton signal (COOH) not
idinone-CH3), 2.90 (s, 3H, thiophene-CH3), 3.30 (s, 3H, OCH3),
3.41e3.45 (m, 2H, CH2CH2), 3.47e3.50 (m, 2H, CH2CH2), 3.54 (s, 3H,
NCH3), 7.23 (d, 1H, J 1.0, pyridinone-H), 8.55 (t, 1H, J 5.5, CONHCH2),
8.59 (d, 1H, J 2.0, pyridinone-H), 8.66 (s, 1H, pyrimidine-H), 9.23 (s,
observed). 13C NMR (DMSO-d6)
d
15.1, 17.6, 49.0, 58.1, 69.4, 114.0,
1H, pyridinone-NH-pyrimidine). 13C NMR (DMSO-d6)
d 15.8, 17.5,
118.2, 124.1, 128.3, 128.7, 137.1, 155.0, 155.6, 156.3, 163.8, 166.1 (one
carbon signal overlapping or obscured). HRMS (ESI-TOF) m/z
375.1118 [MþH]þ; calcd. for C17H19N4O4Sþ 375.1122 [MþH]þ. Anal.
RP-HPLC Method A: tR 10.85 min, purity >99%; Method B: tR
8.62 min, purity >99%.
37.1, 58.0, 70.2, 114.2, 117.6, 123.5, 128.3, 128.6, 129.2, 130.3, 154.3,
155.2, 156.6, 162.1, 165.3 (one carbon signal overlapping or
obscured). HRMS (ESI-TOF) m/z 388.1446 [MþH]þ; calcd. for
C
18H22N5O3Sþ 388.1438 [MþH]þ. Anal. RP-HPLC Method A: tR
11.30 min, purity >99%; Method B: tR 9.30 min, purity >99%.
4.1.10. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxamide (8a)
4.1.14. 4-((1,5-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)amino)-N-
(3-methoxypropyl)-5-methylthieno[2,3-d]pyrimidine-6-
carboxamide (8e)
Carboxylic acid 7a (83.0 mg, 0.251 mmol) and NH3 (0.5 M in 1,4-
dioxane, 2.50 mL, 1.25 mmol) were coupled using general synthetic
procedure E to give 8a as an off-white solid (66.0 mg, 80%). m.p. >
Carboxylic acid 7a (132 mg, 0.400 mmol) and 3-
methoxypropylamine (204
general synthetic procedure E to give 8e as a pale yellow solid
(60.0 mg, 37%). m.p. 230e231 ꢀC. 1H NMR (DMSO-d6)
1.75e1.81
mL, 2.00 mmol) were coupled using
300 ꢀC. 1H NMR (DMSO-d6)
d
2.12 (s, 3H, pyridinone-CH3), 2.94 (s,
3H, thiophene-CH3), 3.54 (s, 3H, NCH3), 7.23 (s, 1H, pyridinone-H),
d

124
M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
(m, 2H, CH2CH2CH2), 2.12 (s, 3H, pyridinone-CH3), 2.90 (s, 3H,
thiophene-CH3), 3.26 (s, 3H, OCH3), 3.29e3.34 (m, 2H, NHCH2CH2),
3.40 (t, 2H, J 6.5, CH2CH2O), 3.53 (s, 3H, NCH3), 7.22 (d, 1H, J 1.0,
pyridinone-H), 8.49 (t, 1H, J 5.5, CONHCH2), 8.58 (d, 1H, J 2.0,
pyridinone-H), 8.65 (s, 1H, pyrimidine-H), 9.21 (s, 1H, pyridinone-
pyridinone-H), 7.86 (d, 1H, J 3.0, pyridinone-H), 8.40 (s, 1H, NH),
8.41 (s, 1H, pyrimidine-H) (one carboxylic acid proton signal
(COOH) not observed). 13C NMR (DMSO-d6)
d 15.5, 37.0, 117.3, 118.2,
118.3, 123.1, 135.8, 138.6, 140.9, 155.1, 158.0, 160.7, 163.9, 166.7.
HRMS (ESI-TOF) m/z 317.0684 [MþH]þ; calcd. for C14H13N4O3Sþ
317.0703 [MþH]þ. Anal. RP-HPLC Method A: tR 8.15 min, purity
>98%; Method B: tR 6.57 min, purity >99%.
NH-pyrimidine). 13C NMR (DMSO-d6)
d 15.8, 17.5, 29.0, 36.9, 37.1,
58.0, 69.7, 114.2, 117.6, 123.5, 128.3, 128.6, 129.2, 130.1, 154.3, 155.2,
156.6, 162.0, 165.2. HRMS (ESI-TOF) m/z 402.1577 [MþH]þ; calcd.
for C19H24N5O3Sþ 402.1594 [MþH]þ. Anal. RP-HPLC Method A: tR
11.56 min, purity >96%; Method B: tR 9.60 min, purity >96%.
4.1.19. 5-Methyl-4-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)
amino)thieno[2,3-d]pyrimidine-6-carboxamide (14)
Carboxylic acid 13 (111 mg, 0.351 mmol) and NH3 (0.5 M in 1,4-
dioxane, 3.50 mL, 1.75 mmol) were coupled using general synthetic
procedure E to give 14 as a white solid (60.0 mg, 54%). m.p. >
4.1.15. 4-((1-(2-Methoxyethyl)-5-methyl-2-oxo-1,2-
dihydropyridin-3-yl)amino)-5-methylthieno[2,3-d]pyrimidine-6-
carboxamide (8f)
300 ꢀC. 1H NMR (DMSO-d6)
d 2.85 (s, 3H, thiophene-CH3), 3.45 (s,
Carboxylic acid 7b (75 mg, 0.20 mmol) and NH3 (0.5 M in 1,4-
dioxane, 2.0 mL, 1.0 mmol) were coupled using general synthetic
procedure E to give 8f as a yellowish solid (49 mg, 66%). m.p.
3H, NCH3), 6.42 (d, 1H, J 9.5, pyridinone-H), 7.53 (dd, 1H, J 9.5 & 3.0,
pyridinone-H), 7.77 (br d, 2H, J 31.0, CONH2), 7.86 (d, 1H, J 2.5,
pyridinone-H), 8.31 (s, 1H, pyridinone-NH-pyrimidine), 8.38 (s, 1H,
263e264 ꢀC. 1H NMR (DMSO-d6)
d
2.12 (s, 3H, pyridinone-CH3),
pyrimidine-H). 13C NMR (DMSO-d6)
d 15.9, 36.9, 116.9, 118.2, 127.8,
2.93 (s, 3H, thiophene-CH3), 3.25 (s, 3H, OCH3), 3.64 (t, 2H, J 5.5,
CH2CH2), 4.14 (t, 2H, J 5.5, CH2CH2), 7.17 (s, 1H, pyridinone-H), 7.86
(br d, 2H, J 56.0, CONH2), 8.60 (d, 1H, J 2.0, pyridinone-H), 8.65 (s,
1H, pyrimidine-H), 9.20 (s, 1H, pyridinone-NH-pyrimidine). 13C
132.1, 135.7, 141.0, 154.3, 157.7, 160.6, 164.0, 165.7 (one carbon signal
overlapping or obscured). HRMS (ESI-TOF) m/z 316.0880 [MþH]þ;
calcd. for C14H14N5O2Sþ 316.0863 [MþH]þ. Anal. RP-HPLC Method
A: tR 8.65 min, purity >99%; Method B: tR 7.04 min, purity >99%.
NMR (DMSO-d6) d 15.3,17.1, 48.5, 57.7, 69.2,113.6,117.5,123.6,127.9,
128.1, 129.1, 130.2, 153.8, 155.1, 156.2, 163.5, 165.1. HRMS (ESI-TOF)
m/z 374.1263 [MþH]þ; calcd. for C17H20N5O3Sþ 374.1281 [MþH]þ.
Anal. RP-HPLC Method A: tR 10.04 min, purity >98%; Method B: tR
7.90 min, purity >97%.
4.2. Computational methods
An in-house Mnk2 DFD-in conformation that was modelled
using homology modelling and molecular dynamics simulations
was used for the docking [29]. The docking grid was generated to
€
4.1.16. 1-Methyl-5-nitropyridin-2(1H)-one (10)
encompass the ATP binding site. Schrodinger's induced fit docking
5-Nitropyridin-2(1H)-one (9, 700 mg, 5.00 mmol) and iodo-
methane (467 mL, 7.50 mmol) were reacted using general synthetic
protocol [30,31] was used to dock 7a, while allowing residues in a
5 Å radius to be flexible. The top 10 poses were visually inspected to
reveal that the three best scoring binding modes are highly similar.
Subsequently, the best scoring mode was selected to rationalise the
binding affinity of 7a for Mnk2.
procedure A to give 10 as a pale yellow solid (763 mg, 99%). RF
(DCM:CH3OH ¼ 98:2) 0.41. m.p. 173e174 ꢀC. 1H NMR (CDCl3)
d 3.65
(s, 3H, NCH3), 6.55 (d, 1H, J 10.0, pyridinone-H), 8.08 (dd, 1H, J 10.0
& 3.0, pyridinone-H), 8.64 (d, 1H, J 3.0, pyridinone-H). 13C NMR
(CDCl3)
d
38.9, 119.3, 130.6, 133.4, 140.2, 162.0. HRMS (ESI-TOF) m/z
4.3. Biology
155.0453 [MþH]þ; calcd. for C6H7N2Oþ3 155.0451 [MþH]þ.
4.3.1. ADP-Glo
™ kinase assay
4.1.17. Methyl 5-methyl-4-((1-methyl-6-oxo-1,6-dihydropyridin-3-
yl)amino)thieno[2,3-d]pyrimidine-6-carboxylate (12)
The luminescent ADP-GloTM assay kits were purchased from
Promega (Madison, WI, USA). Serial dilution of compounds 6e8
and 10e12 with a dilution factor of 1:3 was carried out to give 8
Nitro compound 10 (308 mg, 2.00 mmol) was reduced according
to general synthetic procedure B to give 11 as a dark green semi-
solid. The semi-solid and chloride 5 (242 mg, 0.997 mmol) were
subjected to general synthetic procedure C to give 12 as a yellow
solid (225 mg, 68%). RF (DCM:CH3OH ¼ 95:5) 0.29. m.p. 260 ꢀC. 1H
concentrations ranging from 10 mM to 4.5 nM with 0.5% DMSO.
Each compound was added to the kinase reaction containing
1 ꢁ kinase reaction buffer, 0.1 mg/mL BSA, Milli-Q H2O, eIF4E
peptide substrate (custom synthesis, Resolving Images, Thomas-
town, Vic) and Mnk (Merck Millipore, Kilsyth, Vic) in a total assay
NMR (CDCl3) d 3.09 (s, 3H, thiophene-CH3), 3.60 (s, 3H, NCH3), 3.94
(s, 3H, COOCH3), 6.65 (d, 1H, J 9.5, pyridinone-H), 7.02 (br s, 1H, NH),
7.37 (dd,1H, J 9.5 & 3.0, pyridinone-H), 7.89 (d,1H, J 3.0, pyridinone-
volume of 15
initiated by addition of ATP, incubated at 30 ꢀC for 45 min and
stopped with ADP Glo™ reagent (15 L), followed by incubation in
the dark at room temperature for 40 min. Kinase detection reagent
(30 L) was added. The mixture was further incubated for 40 min
before reading in an EnVision multi-label plate reader (Perki-
nElmer, Beaconsfield, Buckinghamshire, UK). Positive and negative
controls were performed with 0.5% DMSO in the absence and
presence of Mnk respectively.
mL and incubated for 10 min. The kinase reaction was
H), 8.55 (s, 1H, pyrimidine-H). 13C NMR (CDCl3)
d
16.0, 38.1, 52.6,
m
117.4, 118.1, 121.0, 124.1, 133.8, 137.0, 138.4, 155.5, 157.8, 161.7, 163.2,
167.9. HRMS (ESI-TOF) m/z 331.0840 [MþH]þ; calcd. for
m
C
15H15N4O3Sþ 331.0859 [MþH]þ. Anal. RP-HPLC Method A: tR
7.54 min, purity >96%; Method B: tR 9.11 min, purity >96%.
4.1.18. 5-Methyl-4-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)
amino)thieno[2,3-d]pyrimidine-6-carboxylic acid (13)
To a suspension of 12 (225 mg, 0.681 mmol) in CH3OH (15 mL)
was added 2 M NaOH (4.50 mL, 9.00 mmol). The reaction mixture
was stirred at room temperature for 48 h and filtered, the solids
were washed with CH3OH (5 mL). The filtrate and washing were
combined, acidified with 2 M HCl to pH 3. The precipitate was
filtered, washed with H2O (3 ꢁ 25 mL), and dried under reduced
pressure to give 13 as a white solid (190 mg, 88%). m.p. > 300 ꢀC. 1H
4.3.2. Kinase panel assay
Percentages of residual kinase activity upon treatment with 7a,
7b or 8e at the concentration of 10
Millipore KinaseProfiler services, and ATP concentrations used
were within 15 M of Km values.
mM were determined using the
m
4.3.3. Cell culture
NMR (DMSO-d6)
6.43 (d, 1H, J 9.5, pyridinone-H), 7.54 (dd, 1H, J 9.5 & 3.0,
d
2.99 (s, 3H, thiophene-CH3), 3.45 (s, 3H, NCH3),
MV-4-11 cells were provided by Professor Richard D'Andrea
(University of South Australia) and maintained in Roswell Park

M. Yu et al. / European Journal of Medicinal Chemistry 95 (2015) 116e126
125
Memorial Institute medium (RPMI)-1640 with 10% fetal bovine
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Cell cycle analysis and apoptosis detection were performed with
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were seeded at 8 ꢁ 104 and incubated at 37 ꢀC, 5% CO2 overnight.
After treatment with each compound for 24 h, cell pellets were
collected and centrifuged (300 ꢁ g, 5 min). For cell cycle analysis,
cell pellets were fixed with 70% ethanol on ice for 15 min and
collected again. The collected pellets were incubated with propi-
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(Beckman Coulter, Brea, CA, USA) within 1 h after staining. Data
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4.3.7. Western blots
Western blotting was performed as described previously
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Mcl-1, cyclin D1, PARP, cleaved PARP and b-actin. Both anti-mouse
and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase-
conjugated antibodies (Dako, Glostrap, Denmark) were used as
secondary antibodies. Enhanced Chemiluminescence (ECL) re-
agents (GE Life Sciences) were used for Western blotting detection.
Acknowledgements
This work is supported by funding from Australia Government
National Health and Medical Research Council (project grant
1050825) and South Australian Health and Medical Research
Institute, Beat Cancer Project Principal Cancer Research Fellowship
to S.W.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.032.

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