An integrated approach for discovery of highly potent and selective Mnk inhibitors: Screening, synthesis and SAR analysis

Article abstract of DOI:10.1016/j.ejmech.2015.09.008

Deregulation of protein synthesis is a common event in cancer. As MAPK-interacting kinases (Mnks) play critical roles in regulation of protein synthesis, they have emerged as novel anti-cancer targets. Mnks phosphorylate eukaryotic initiation factor 4E (eIF4E) and promote eIF4E-mediated oncogenic activity. Given that the kinase activity of Mnks is essential for oncogenesis but is dispensable for normal development, the discovery of potent and selective pharmacological Mnk inhibitors provides pharmacological target validation and offers a new strategy for cancer treatment. Herein, comprehensive in silico screening approaches were deployed, and three thieno[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine derivatives were identified as hit compounds. Further chemical modification of thieno[2,3-d]pyrimidine derivative 3 has given rise to a series of highly potent Mnk2 inhibitors that could be potential leads for the treatment of acute myeloid leukemia.

Full text of DOI:10.1016/j.ejmech.2015.09.008

European Journal of Medicinal Chemistry 103 (2015) 539e550
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
An integrated approach for discovery of highly potent and selective
Mnk inhibitors: Screening, synthesis and SAR analysis
Theodosia Teo, Yuchao Yang, Mingfeng Yu, Sunita K.C. Basnet, Todd Gillam, Jinqiang Hou,
Raffaella M. Schmid, Malika Kumarasiri, Sarah Diab, Hugo Albrecht, Matthew J. Sykes,
Shudong Wang*
Center for Drug Discovery and Development, Sansom Institute for Health Research, Center for Cancer Biology, and 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:
Deregulation of protein synthesis is a common event in cancer. As MAPK-interacting kinases (Mnks) play
critical roles in regulation of protein synthesis, they have emerged as novel anti-cancer targets. Mnks
phosphorylate eukaryotic initiation factor 4E (eIF4E) and promote eIF4E-mediated oncogenic activity.
Given that the kinase activity of Mnks is essential for oncogenesis but is dispensable for normal
development, the discovery of potent and selective pharmacological Mnk inhibitors provides pharma-
cological target validation and offers a new strategy for cancer treatment. Herein, comprehensive in silico
screening approaches were deployed, and three thieno[2,3-d]pyrimidine and pyrazolo[3,4-d]pyrimidine
derivatives were identified as hit compounds. Further chemical modification of thieno[2,3-d]pyrimidine
derivative 3 has given rise to a series of highly potent Mnk2 inhibitors that could be potential leads for
the treatment of acute myeloid leukemia.
Received 11 September 2014
Received in revised form
7 October 2014
Accepted 5 September 2015
Available online 9 September 2015
Keywords:
CGP57380
Eukaryotic initiation factor 4E
Mnk inhibitors
© 2015 Elsevier Masson SAS. All rights reserved.
Structure-activity relationship
Thieno[2,3-d]pyrimidine
Virtual screening
1. Introduction
for the transformation activity of eIF4E. The tumorigenic effect of
Mnks has been further confirmed by Mnk1/2 double knockout
Dysregulation of cellular translational machinery is a hallmark
of cancer [1e3]. Translational initiation is a key rate-limiting step
for protein synthesis and is involved in the assembly of eukaryotic
initiation factor 4F (eIF4F), including eIF4A RNA helicase, eIF4G
scaffold protein and eIF4E cap-binding factor. Binding of eIF4E to
eIF4G assists in the recruitment of mRNA on the 40S subunit [4].
Given that the rate of translation is controlled by the availability of
eIF4E in cells, eIF4E is thus considered as the key regulator for cap-
dependent mRNA translation [5,6].
The oncogenic role of eIF4E has been confirmed with a mouse
lymphoma model [7,8]. Activation of eIF4E via phosphorylation by
the human mitogen-activated protein kinase (MAPK)-interacting
kinases (Mnks) [9] repressed apoptosis and engendered tumor
formation. Consistently, mice in which Ser209 of eIF4E was
mutated to Ala were resistant to Ptenꢀ/ꢀ-induced prostate cancer
[10], implying the importance of Mnk-mediated phosphorylation
Ptenꢀ/ꢀ mice, which were found to be resistant to lymphogenesis
when compared to the parental Ptenꢀ/ꢀ mice [11]. Importantly,
while the activity of Mnks is necessary for eIF4E-mediated
tumorigenesis, it is dispensable for normal tissue development
[12]. Thus, inhibiting Mnks offers an exciting opportunity for
effectively treating cancer with low toxicity.
Despite the increasing understanding of Mnk-related cancer
biology, little progress has been made with the discovery of phar-
macological Mnk inhibitors. CGP57380, a known inhibitor of Mnks,
showed low micromolar cytotoxicity against various cancer cells [3]
while targeting many other kinases with low micromolar IC50
values [13]. Cercosporamide, a natural product isolated from Cer-
cosporidium henningsii, was initially identified as a broad-spectrum
anti-fungal agent [14]. It was only recently discovered that cerco-
sporamide is also a potent Mnk1/2 inhibitor with nanomolar IC50
values [15]. Cercosporamide was the first Mnk inhibitor to show
in vivo anti-tumor efficacy in human xenograft tumor models, and
it effectively blocked eIF4E phosphorylation at Ser209, suppressed
cancer cell proliferation and led to induction of apoptosis. More
* Corresponding author.
E-mail address: shudong.wang@unisa.edu.au (S. Wang).
recently, we have identified
a series of 5-(2-(phenylamino)
http://dx.doi.org/10.1016/j.ejmech.2015.09.008
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

540
T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
pyrimidin-4-yl)thiazole-2(3H)-one derivatives as potent Mnk2 in-
hibitors using an analog-based drug design approach [16]. Two
compounds were tested against a panel of cancer cell lines, and the
results revealed the cell-specific effect of Mnk inhibition. Our study
also suggested that Mnk2 inhibitors were capable of reducing the
level of anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1), and
of inducing apoptosis in MV4-11 acute myeloid leukemia (AML)
cells. However, all known Mnk inhibitors target other kinases
[13,15], rendering the discovery of potent and selective Mnk in-
hibitors challenging. Achieving mono- and dual-specific Mnk in-
hibition is essential to understand the biological relevance and
clinical impacts of individual Mnk as most biological studies re-
ported previously were based on the non-selective molecules
CGP57380 and cercosporamide.
Structure-based screening has become a prevalent approach in
the discovery of small-molecule therapeutics after the successful
identification of inhibitors for a number of targets, including
FK506 binding proteins, trihydroxynaphthalene reductase, serine
protease factor Xa, and v-raf murine sarcoma viral oncogene ho-
molog B1 [17,47e50]. The uniqueness of Mnks is highlighted by
two structural elements in the C-terminal lobe, i.e. a DFD-motif
that replaces the common DFG-motif in other kinases, and three
insertions that are exclusively found in Mnks [5,18,19]. Further-
more, this DFD-motif adopts an unusual DFD-out conformation,
in which the Phe residue points into the ATP-binding pocket.
Such an auto-inhibitory conformation prevents the accessibility of
ATP to the binding pocket, and exposes an additional hydrophobic
site [20].
via the Erk pathway. Mnk2 does not respond to the stimuli-induced
p38 activation, and therefore may have a different role in inducing
cytokine production and regulating cytokine responses in com-
parison with Mnk1 [25]. As no specific Mnk1 or Mnk2 inhibitors
exists, our first-in-class selective inhibitors of Mnk1/2 will be
powerful pharmacological tools in distinguishing Mnk1/2-related
biology, pharmacology and drug discovery.
2. Results and discussion
2.1. Validation of computational approaches
To assess the reproducibility of bioactive conformations by the
3D conformation generator OMEGA, the co-crystallized staur-
osporine in the Mnk2 protein structure (PDB ID code: 2HW7) was
selected as the starting point. The co-crystallized conformation was
extracted and aligned with the conformations generated by
OMEGA using vROCS. A good correlation was observed between the
highest scoring OMEGA generated conformation and the co-
crystallized staurosporine conformation (Fig. 1A), as indicated by
a TanimotoCombo score of 1.47. All output conformations produced
by OMEGA were of interest nonetheless, as it is known that the
There are currently three crystal structures of Mnk2, including
apo (PDB ID code: 2AC3) and D228G mutant (PDB ID code: 2AC5)
forms as well as Mnk2D228G in complex with staurosporine (PDB
ID code: 2HW7) in the Protein Data Bank (PDB). In contrast, only
one apo Mnk1 crystal structure (PDB ID code: 2HW6) is available.
Staurosporine is a highly potent multi-kinase inhibitor widely used
as a chemical probe in diverse protein kinase studies [20]. Closer
investigation of the Mnk2D228G-staurosporine complex using
docking methods showed that the rigid planar polycyclic ring
system of staurosporine lies in a flat orientation in the ATP-binding
pocket and the lactam ring forms a total of two hydrogen bonds
with the Glu160 and Met162 residues in the hinge region [5]. Such
an observation is consistent with the results obtained from a pre-
vious study, in which different docking methods were used to
replicate the data [21]. Notably, the gatekeeper residue Phe159 in
Mnk2 may assist in the formation of favorable hydrophobic in-
teractions with the polycyclic ring system of staurosporine,
possibly stabilizing the overall protein-ligand complex [3]. Given
that the only crystal structure of Mnk1 adopts the DFD-out
conformation, attempts to dock small-molecule Mnk inhibitors
into Mnk1 have been hindered. In contrast, crystallographic anal-
ysis of the Mnk2D228G-staurosporine complex, where Mnk2 exists
in both DFG/D-in and -out conformations, provides a potentially
valuable source of structural information for the design of selective
Mnk2 inhibitors.
The current study aimed to discover Mnk inhibitors using pro-
tein structure-guided and ligand-based screening approaches.
Based on chemical structural features of hits identified in silico, a
series of hit-derived compounds was designed, synthesized and
evaluated using human AML cell lines. A number of compounds
exhibited improved efficacy and selectivity towards Mnk2. In-
house established Mnk1 kinase assays also showed that some of
the compounds are potent Mnk1 inhibitors. Clearly, there are
distinct roles in the regulation by MAPKs and in basal activity be-
tween Mnk1 and Mnk2, despite their high structural similarity
[9,22e24]. Mnk1 is activated by Erk and p38 kinases in response to
mitogens and stress, respectively, but Mnk2 is only phosphorylated
Fig. 1. Validation of computational approaches. (A) Overlay of two staurosporine
poses obtained from the co-crystallized structure with Mnk2 (PDB ID code: 2HW7)
(green sticks) and OMEGA-constructed method (black sticks), respectively. The figure
was generated using VIDA v4.0.3. (B) Docking of the co-crystallized staurosporine pose
(green sticks) and the OMEGA-constructed pose (black sticks) against the Mnk2 re-
ceptor (PDB ID code: 2HW7). Mnk2 is shown as cyan ribbons. The Phe gatekeeper,
hinge residues and DFD motif are in capped sticks. Hydrogen bonds are shown as black
dashed lines.
pep stacking interactions between the aromatic rings are shown as red
dashed lines. The figure was generated using Pymol v1.6.

T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
541
ability to generate an ensemble of conformations encapsulating the
bioactive conformation impacts the identification of the correct
binding poses [26]. Therefore, the maximum number of output
conformations was varied (50, 100 and 200) and applied to the
molecules of a previously built in-house control database, including
the three known Mnk inhibitors (see Experimental Section 4.1.1).
An exploratory docking of these generated conformer sets against
the Mnk2 receptor using the X-ray crystal structure (PDB ID code:
2HW7) revealed that the ranking of the active molecules were
either unchanged by the introduction of a larger number of con-
formations or degraded (Table S1). Hence, for computational effi-
ciency, a maximum number of 50 conformers was used for the
subsequent screening.
The ability of OEDocking [27] to identify accurate binding poses
was assessed by two approaches. The first method assessed its
ability to reproduce the co-crystallized staurosporine pose (Fig. 1B).
The root mean square deviation (rmsd) between the co-crystallized
and docked staurosporine poses was only 0.45 Å, providing initial
evidence of the applicability of OEDocking in this study. Therefore,
the parameter sets used in the initial conformer generation and
docking were employed for the Mnk2 screening that followed. In
the second approach, the docking results generated by the Mnk2
receptor against the control database were re-assessed to deter-
mine the threshold point (in terms of docking score) for retrieving
potential Mnk2 hits. Out of the 38 molecules in this database, 7
molecules were identified as actives. Not unexpectedly,
CGP052088, a known Mnk inhibitor derived from staurosporine
[28], was the top ranked molecule with a docking score of ꢀ14.09
(Table S1). This was followed by one of the most active compounds
among the hits (JQ12445675), accounting for a docking score
of ꢀ12.67. Despite the broad distribution of the actives throughout
the ranking list, all of the 7 actives were retrieved from the database
with a docking score of ꢀ6.50 or less. Hence, a threshold score
of ꢀ6.50 was set to identify potential Mnk2 hits.
Collectively, these stringent screening criteria successfully filtered
out ~97% of molecules, leaving a set of 40 structurally diverse
compounds. The drug-like properties and commercial availability
of these compounds were considered and 13 candidates were
selected and purchased for biological evaluation (Table S2).
2.3. Assessment of Mnk2 inhibitors
The selected 13 candidates were evaluated at concentrations of
1 and 10 mM by employing an Mnk2 biochemical assay (Fig. 2B).
CGP57380 was included as a positive control. Candidates that
exhibited > 50% inhibitory activity in the assay were further sub-
jected to IC50 determination against Mnk1 and Mnk2. CDK2/cyclin
A plays a regulatory role during the G1 to S phase transition in cell
cycle [30], and inhibition of CDK2 leads to cell arrest in G1/S phase
[31]. Inhibition of Mnk results in down-regulation of cell cycle
regulatory proteins including cyclin D causing G1 cell arrest
[3,51e53]. In our recent study, CDK9 inhibition led to the down-
regulation of Mnk and the consequent reduction of eIF4E phos-
phorylation in ovarian cancer cells [32]. To ensure that the bio-
logical effects observed are exerted through Mnk inhibition and are
not consequences of inhibiting CDK2 and CDK9, the assessment
against both CDKs was also carried out for the initial selectivity
profile. This procedure eliminated 10 of the 13 hits, yielding a vir-
tual screening hit rate of 23%. The structures and biological activ-
ities of the remaining three hit compounds are summarized in
Fig. 2B. Hits 1 and 3 (ZINC00132478 and ZINC00098359, respec-
tively) are thieno[2,3-d]pyrimidine derivatives while hit
2
(ZINC00450245) shares a pyrazolo[3,4-d]pyrimidine scaffold with
CGP57380. Hit 3 demonstrated potent Mnk2 inhibitory activity
with a Ki value of 0.620
tency (Ki ¼ 3.430 M). Both thieno[2,3-d]pyrimidine derivatives
demonstrated excellent selectivity for Mnk2 over CDK2/A and
CDK9/T1 (Ki > 10 M). Hit 2 was also found to be a potent Mnk2
inhibitor (Ki ¼ 0.523 M), being comparable to CGP57380; however
it also targets CDK2/A and CDK9/T1 (Ki ¼ 3.046 and 7.873 M,
respectively). All hits displayed cytotoxicity with GI50 values
mM, whereas hit 1 showed a modest po-
m
m
m
2.2. Virtual screening approaches
m
Fig. 2A depicts the workflow of the virtual screening, including
database preparation and filtration followed by the combinatorial
in silico screening. Initially, the ChemBridge database consisting of
572 K compounds was pre-filtered and the resulting 387 K com-
pounds were converted to 3D conformers using OMEGA (see
Experimental Section 4.1.1). The original ionization states of the
compounds were retained and the new 3D database generated was
subjected to two different approaches to maximize the virtual
screening hit rate.
The first approach involved docking of the 3D database
against the active site of Mnk2. The 1000 top-scoring compounds
were selected for inspection. The ensuing molecules were
analyzed for their ability to form interactions with the protein
residues known to contribute significantly to Mnk2 binding, in
the context of the following criteria: possession of (1) at least one
hydrogen bond with the hinge residues (i.e. Glu160, Lys161 and
ranging between 4.95 and 8.81
leukemic cell lines.
mM against MV4-11 and MOLM-13
The importance of adopting the ligand-based alignment method
is highlighted when all three hits were shown to be retrieved from
this method in the virtual screening study (Table S2). Fig. 2C depicts
the molecular shape and chemical feature matches between
CGP57380 and hit 3 in a similarity assessment using the Tanimo-
toCombo metric. As indicated by the partial charge density, hit 3
shows chemical features similar to CGP57380. On the basis of the
docking study (Fig. 2D), the presence of at least one hydrogen bond
with the hinge residue(s) of Mnk2 and the adoption of an appro-
priate geometry for the
pep stacking interaction with the gate-
keeper Phe159 (distance of ꢁ 4 Å), are two key parameters as the
preliminary filter for selecting hits, which is consistent with our
previous proposed models for the optimal Mnk2 ligand binding
[3,5]. As this study aimed to identify selective Mnk inhibitors, the
thienopyrimidine scaffold, which has a distinctive core structure
relative to the known Mnk2 inhibitors, was pursued further for
structural optimization.
Met162), (2) a central aromatic moiety that forms
pep stacking
interactions with the gatekeeper Phe159 within a distance of 4 Å
or less, and (3) one hydrophobic group pointing towards the
hydrophobic region 1 (HR1; formed by residue Phe159) or hy-
drophobic region 2 (HR2; formed by residues Met162, Gly164,
Gly165 and Leu212) [5].
2.4. Hit optimization, assay validation and structure-activity
relationship analysis
In the second approach, the same 3D database was submitted
for ligand-based alignment. CGP57380 and cercosporamide, rep-
resenting two distinct chemical scaffolds, were selected as queries.
The top 200 compounds from each alignment set, ranked according
to the TanimotoCombo score [29], were retrieved and re-docked to
the Mnk2 receptor to meet the aforementioned criteria.
To facilitate chemical modifications on the thienopyrimidine
core, further exploration of the binding modes of CGP57380 or
cercosporamide against Mnk2 was undertaken. Close inspection of
the docking models showed that cercosporamide protrudes its
acetyl group into a relatively acidic pocket (adjacent to the specific

Fig. 2. Cascade screening for Mnk inhibitors. (A) Computational steps including database filtration, conformer generation, docking-based design and ligand-based study were performed using Openeye Scientific Software. SBVS,
structure-based virtual screening; LBVS, ligand-based virtual screening. See also Tables S1-2. (B) Chemical structures of the final three hits and their virtual screening and biological evaluation results. Hits 1e3 were purchased from
Hit2Lead online chemical store that reported to have a compound purity of ꢂ 95% by NMR and LC-MS. vROCS alignment result when CGP57380 was used as a query. Number of hydrogen bond interactions with the Mnk2 hinge residues
(i.e. Glu160, Lys161 and Met162) in the docking analysis using VIDA. Apparent inhibition constants (Ki) were calculated from IC50 values and the appropriate Km (ATP) values for each kinase. The assays were tested by Millipore
KinaseProfiler™ services. Cytotoxicity by 72 h resazurin assays. The data given are mean values derived from at least two replicates SD. (C) vROCS alignment of hit 3 (orange sticks) shown in the partial charge mesh surface when
CGP57380 (green sticks) was used as a query molecule. (D) Proposed binding modes of CGP57380 (green sticks) and hit 3 (orange sticks) against the Mnk2 receptor (PDB ID code: 2HW7). Mnk2 is shown as cyan ribbons. The Phe
gatekeeper, hinge residues and DFD motif are in capped sticks. Hydrogen bonds are shown as black dashed lines. The figure was generated using Pymol v1.6.

T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
543
DFG/D-motif, Fig. 3A) formed by residues Glu92, Glu209, Asn210
and Ile211, which has not been found in the Mnk2 complex with
CGP57380 or hit 3. Given that cercosporamide is the most potent
Mnk2 inhibitor [15] among the previously identified compounds,
exploitation of this region with an appropriate functionality may
confer an increase in potency towards Mnk2.
employed as model inhibitors for the in-house assays and the
published IC50 values from radio-active kinase assays [13,15] were
used as standard references in the current study (Table 1). A 2-fold
inhibitory preference of Mnk1 over Mnk2 was previously reported
for CGP57380 (IC50 values ¼ 0.87 and 1.60
mM for Mnk1 and Mnk2,
respectively) [13]. Our data, however, revealed no significant dif-
ference in relative potency against the two kinases (Ki ¼ 1.010 and
Analysis of the docking mode of hit 3 in Mnk2 showed that an
additional substitution at the ortho-position of the aniline ring, in
the context of the thienopyrimidine core, might be able to target
the pocket to enhance the binding affinity (Fig. 3B). The synthesis of
analogs of hit 3, i.e. 4-((4-fluorophenyl)amino)-5-methylthieno[2,
3-d]pyrimidine-6-carboxylate derivatives, was hence pursued and
is outlined in Scheme 1. Compounds 5aec, 6aec and 7aee were
assessed for kinase inhibitory potency and selectivity against Mnk1,
Mnk2, CDK2/A and CDK9/T1 (Table 1). Our previous work has
shown that Mnk2 inhibitors demonstrated tumor cell growth in-
hibition in a cell-type specific manner, being particularly cytotoxic
against MV4-11 cells [16]. Thus, MV4-11, together with MOLM-13
(which are also acute monoblastic leukemia cells, i.e. M5 subtype,
expressing Fms-like tyrosine kinase 3-internal tandem duplication
(FLT3-ITD) mutations as MV4-11) [33], were chosen for the anti-
proliferative assessment of the compounds.
Profiling of Mnk inhibition involved the two in-house assays, i.e.
an initial rapid screen using immobilized metal ion affinity particle
(IMAP) technology [34] followed by a doseeresponse analysis
adopting the ADP-GloTM luminescent kinase assay [35]. Validation
of the assays was performed by optimizing the concentrations of
Mnk1 and Mnk2, eIF4E-derived substrates and ATP (Figure S2). Two
well-defined Mnk inhibitors, CGP57380 and cercosporamide, were
0.877 mM for Mnk1 and Mnk2, respectively). In contrast, cerco-
sporamide was found to be 6-fold more potent against Mnk2 than
Mnk1 (Ki ¼ 0.507 and 0.079 M for Mnk1 and Mnk2, respectively)
m
in our study, which is in close correlation with the data obtained
from the literature [15]. Hence, the established Mnk1 and Mnk2
assays were used to analyze newly synthesized compounds.
A change of substituent on the C6-position of thienopyrimidine
in hit 3, from an ethyl ester to a methyl ester (5a, R1 ¼ H, R2 ¼ OMe),
was found to abolish Mnk2 inhibitory activity, and the same holds
true for the cytotoxicity. Additionally, no Mnk1 inhibitory activity
was detected in 5a. Further introduction of a methyl group at the
ortho position of aniline (5b, R1 ¼ Me, R2 ¼ OMe) shows no
improvement in either kinase activity or cellular potency. Intro-
ducing an o-isopropoxyl group gives rise to 5c (R1 ¼ OCHMe2,
R2 ¼ OMe) with slightly restored Mnk2 activity and cytotoxicity. No
inhibition of Mnk1, CDK2/A and CDK9/T1 was observed.
In contrast, conversion of the methyl ester moiety of 5a into the
carboxylic acid group (6a, R1 ¼ H, R2 ¼ OH) was found to dramat-
ically enhance Mnk2 inhibitory activity, which is more than 3-fold
greater in comparison with hit 3, but has a detrimental effect on
cellular activity. However, in comparison with 6a, the methyl de-
rivative 6b (R1 ¼ Me, R2 ¼ OH) displays a reduced activity against
Fig. 3. Stereoview of structures of inhibitors in ATP-binding site of Mnk2. (A) CGP57380 (black sticks) and hit 3 (green sticks) possess a fluoroaniline moiety, in which the
aromatic ring is likely to be stabilized by the Phe159 gatekeeper. A pocket (adjacent to the DFG/D-motif, red arrow) formed by residues Glu92, Glu209, Asn210 and Ile211, ac-
commodates the hydrophobic moiety on cercosporamide (yellow sticks), highlighting the importance of assessing to such a pocket for modulating potency. (B) A proposed binding
mode of hit 3 against Mnk2 after the introduction of an isopropoxyl group at o-position of fluoroaniline (pink sticks). The compound protrudes the isopropoxyl group into the
alternative pocket, in in a way similar to that of the cercosporamide (yellow sticks). Docking analysis also revealed a favored C6-carboxamide in the hydrophobic region 2 (formed by
residues Met162, Gly164, Gly165 and Leu212). The binding modes were generated by docking the multiple poses to the Mnk2 X-ray structure (PDB ID code: 2HW7) using OEDocking
and the pose with best scoring function is presented. The figure is generated using Pymol v1.6.

544
T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
Scheme 1. Synthesis of methyl 4-((4-fluorophenyl)amino)-5-methylthieno[2,3-d]pyrimidine-6-carboxylates (5aec) and their derivatives (6aec and 7aee). Reagents and
conditions: (a) SOCl2, DMF, reflux, 2 h, 80%; (b) H2, 10% Pd/C, MeOH, rt, 2e3 h, 4a: 100%, 4b: 100%, 4c: 92%; (c) TsOH,H2O, 1,4-dioxane, microwave, 150 ꢃC, 0.5 h, 5a: 18%, 5b: 32%, 5c:
79%; (d) For 5a and 5b, NaOH, MeOH/THF, 70 ꢃC, 4 h, 6a: 87%, 6b: 91%; for 5c, LiOH, MeOH/THF/H2O, rt, o/n, 6c: 67%; (e) appropriate amine, HATU, DIPEA, DMF, rt, o/n, 7a: 86%, 7b:
63%, 7c: 34%, 7d: 51%, 7e: 48%.
Table 1
Summary of structure and biological activity.
c
Code
Structure
R1
Kinase inhibition Ki (
m
M)
72 h GI50
MV4-11
(m
M) SD
MOLM-13
R2
Mnk2a
Mnk1a
CDK2Ab
CDK9T1b
CGP57380
Cercosporamide
e
e
0.877
0.079
>10
1.010
0.507
>10
>10
>10
>10
e
>10
e
6.89 0.16
e
8.81 0.80
e
e
e
5a
5b
5c
6a
6b
6c
7a
7b
7c
7d
7e
H
Me
OCHMe2
H
Me
OCHMe2
OCHMe2
OCHMe2
OCHMe2
OCHMe2
OCHMe2
OMe
OMe
OMe
OH
OH
OH
NH2
NHMe
e
e
78.6 8.01
88.1 6.94
9.38 0.10
78.3 2.34
89.4 9.58
7.30 0.75
6.08 1.99
5.29 1.89
8.84 1.58
7.44 0.77
15.3 2.62
68.3 5.15
93.1 9.81
4.61 0.36
79.3 0.08
>100
5.65 0.84
6.12 0.44
5.76 1.05
8.65 1.77
7.19 0.08
50.4 7.40
>10
e
e
4.050
0.164
0.500
0.068
0.110
0.018
0.018
0.031
0.164
>10
e
>10
e
1.900
>10
e
e
0.186
1.020
0.106
0.933
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
NHEt
NHCH2CH2OMe
NHCH2CH2CH2OMe
0.320
aThe kinase assays were developed and conducted in-house. See also Fig. S2. bThe assays were tested by Merck Millipore. a,bThe ATP concentrations used in these assays were
within 15
M of Km. Apparent inhibition constants (Ki) were calculated from IC50 values and the appropriate Km (ATP) values for each kinase. cAnti-proliferative activity by 72 h
m
resazurin assays. Data shown are the mean values derived from at least two replicates SD.
Mnk2, but is comparable to hit 3. This compound demonstrates an
excellent selectivity for Mnk2 over Mnk1, but has a reduced cellular
potency. These results imply that the carboxylic acid may facilitate
the interaction with Mnks but give rise to a poor cell-based activity
plausibly due to poor permeability. Thus, replacement of the
methyl ester of 5c with the carboxylic acid in 6c (R1 ¼ OCHMe2,
R2 ¼ OH) results in a > 50-fold increase in Mnk inhibitory activity.
Of note, 6c has a Mnk2 activity very similar to that of cercospor-
amide, along with a 2-fold improvement in Mnk1 activity. These
findings indicate a balance between the two key substituents R1
and R2 need to be achieved for optimal activity. Once again, no CDK
inhibition is seen, indicating the anti-proliferative effects exhibited
by 6c on AML cells are derived from the inhibition of Mnks.
Since both satisfactory Mnk inhibitory activity and cytotoxicity
were achieved by compound 6c, the proposed Mnk2-6c binding
model was analyzed. It reveals a favored C6-carboxamide in the
HR2 (formed by residues Met162, Gly164, Gly165 and Leu212,
Fig. 3B), suggesting optimization at the C6-position of 6c may result
in further enhancement of activity against Mnk2. Hence, analogs
7a-e were synthesized by amide formation between 6c and
selected amines. As expected, compound 7a (R1 ¼ OCHMe2,
R2 ¼ NH2) shows no improvement in both enzymatic and cellular
activity compared to 6c. A slight increase in the hydrophobicity of
7a, as shown by compound 7b bearing an N-methyl C6-
carboxamide, improves the affinity for both Mnk1 and Mnk2 by
~6-fold. Introduction of a bulkier alkyl substituent into 7a, i.e. 7c
(R2 ¼ Et) and 7d (R2 ¼ CH2CH2OMe) retains the Mnk2 activity but
reduces Mnk1 inhibition by at least 9-fold. However, in the case of
7e (R2 ¼ CH2CH2CH2OMe), Mnk2 inhibitory activity decreases
whereas the affinity for Mnk1 is restored. In general, Mnk1 activity
decreases when the substituent at C6-position becomes bulkier.
This trend may be attributed to the structural observation that
Mnk1 comprises a more compact ATP-binding pocket when
compared with Mnk2 [5]. Taken together, these data suggest the

T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
545
structural modification at the C6-position plays a pivotal role in
4.1.1. 3D database preparation
governing the kinase activities, particularly for Mnk2.
Molecules including the three known Mnk inhibitors (i.e.
CGP052088, CGP57380 and cercosporamide) and the previously in-
house built database containing a total of 35 Mnk2 actives and
inactives was prepared manually using the drawing tool PICTO and
stored as a control database.
The ChemBridge database with over 572,000 2D purchasable
compounds (March 2012), was obtained from the ZINC library. As
the database contains only atom connectivity with no 3D structural
information, it was converted to 1D notation using Babel. A data
reduction tool, FILTER, was employed to eliminate compounds with
molecular weight (MW) outside the range of 300e450 Da. This MW
range is generally considered drug-like and thus was adopted in the
filtering process. The filtered compounds comprising 387,000
compounds were saved as a screening database.
A maximum of 50 conformers for each molecule in these data-
bases was generated by applying emaxconfs flag through OMEGA.
Additionally, flags estrictstereo and ecanonOrder were disabled to
prevent the failure of conformer generation for molecules with
specified stereo centers and to deactivate the automatic reordering
of conformers to a canonical atom and bond order, respectively.
None of compounds 7aee are active against CDK2/A and
CDK9/T1 (Ki
>
10
mM) and show cellular cytotoxicity
(GI50 ¼ 5.29e8.84
m
M) with the exception of 7e which exhibits
lower anti-proliferative activity. As suggested by our previous
studies with CDK2 and CDK9 inhibitors [16,36], two hydrogen bond
interactions of a given inhibitor with the hinge residues Leu83 of
CDK2 (Cys106 in CDK9) followed by a
pep interaction with the
gatekeeper Phe80 (Phe103 in CDK9) are required for activity. In
contrast, our docking studies of 6c and 7d in CDK2 or CDK9 failed to
generate any appreciable binding poses (Figure S3). The 1S and 6C-
carbonyl of thienopyrimidine seem unable to make any appropriate
interaction with the hinge residues.
It is also worth noting that a recent patent application has
described a series of thienopyrimidine-based compounds as highly
potent Mnk inhibitors [37], which serves as further validation of
our discovery strategy. Despite the similar chemical scaffold to the
patented structure, the current work has showcased an integrated
computational and experimental approach to identify the most
potent compound series to date.
4.1.2. Database alignment against selected query molecules
3. Conclusions
Ligand-based approaches adopt the approach that compounds
bearing structural similarity to a query molecule with known bio-
logical activity are likely to exert similar properties. The ROCS
overlay method assigns a score based on a the concept of Gaussian
volume overlap, which undertakes a rapid assessment of the
centre-of-mass of the query and database molecules, overlays their
principal components of inertia and hence maximizes the shared
volume overlap [29]. ROCS was used to investigate such similarities
between known Mnk2 inhibitors and the developed screening
database. Both CGP57380 and cercosporamide were adopted as
query molecules to account for the diversity of inhibitors involved.
In order to utilize the most energetically favorable conformation,
OMEGA was applied to generate one conformer for each query. The
color force field dropdown in vROCS was adjusted to the Implic-
itMillsDean option as it is the default setup that involves a pKa
model assuming pH ¼ 7. This automatically assigns appropriate
physiologically relevant ionization states for each aligned molecule.
Assessment of the conformers was computed by TanimotoCombo
(shape and color (i.e. chemical features include anions, cations,
hydrogen bond donors and acceptors, hydrophobes and rings)
combination) as it has been found as the most useful metric in a
number of other studies [44,45]. A total combo score of 2 indicates
an identical match of both shape and color between a given
molecule and a query. Each resulting alignment was analyzed by
the inbuilt visualization tool, VIDA.
This report has described the discovery, optimization and
characterization of a series of thienopyrimidine-based Mnk in-
hibitors that possess high potency and selectivity. The in silico vir-
tual screening that used an integrated approach of ligand-based
and structure-based has led to identification of Mnk2 hits. Further
structural optimization of hit 3 by adding an isopropoxyl group at
the ortho-position of 4-fluoroaniline to occupy a pocket adjacent to
the DFG/D-motif of Mnk2 and by modifying the length of C6-
carboxamide to reside in the HR2 region has yielded a series of
compounds that inhibit Mnk2 at low nanomolar concentrations
and exhibit good anti-leukemic activity in MV4-11 and MOLM-13
cells. In particular, compounds 7b, 7c and 7d have similar high
potency against Mnk2, which could be attributed to their bulkiness
of C6-carboxamide substituents. Compound 7b is the most potent
derivative with Ki values of 106 nM and 18 nM against Mnk1 and
Mnk2, respectively. In-house Mnk kinase assays also identified
compound 7d as a Mnk2-selective inhibitor that could be adopted
as a powerful pharmacological tool to elucidate the Mnk-eIF4E-
mediated tumorigenic mechanism. The molecular basis of activity
and/or selectivity of compounds 7b and 7d against Mnks remain
unknown due to the lack of an active Mnk1 and Mnk2 structure to
facilitate such an investigation. In conclusion, this study suggests
the potential utility of the thienopyrimidine derivatives as anti-
leukemic agents.
4.1.3. Database screening against Mnk2 protein kinase
The crystal structure of Mnk2 in complex with staurosporine
(PDB ID code: 2HW7) was used for docking purposes, as the pres-
ence of a bound ligand in the crystal structure facilitates the
docking program in defining the target active site. The docking
experiments were performed using the OEDocking module with its
complementary tools including MAKE RECEPTOR and FRED.
Initially, the Mnk2 receptor binding site was defined by forming an
enclosed box through molecular cavity detection in MAKE RECEP-
TOR. The active site shape was characterized by inner and outer
contours of a shape potential. Every single heavy atom of a docked
pose is required to fit within the shape of the outer contour and at
least one heavy atom lies within the volume of the inner contour.
Keeping the default settings, the defined receptor was ready for
docking experiments [27]. The multi-conformer databases were
respectively docked into the established receptor using the FRED
4. Experimental section
4.1. Experimental procedures for computational-aided virtual
screening
Openeye Scientific Software was utilized with Microsoft Win-
dow XP on a 2.66 GHz Intel Core2 processor system with 3.21 GB
RAM for all virtual screening applications. Visual Interface to Drug
design Application v4.0.3 (VIDA) [38] was used as the core graphical
platform to access most of the Openeye modules; PICTO v4.0.3 [38],
MAKE RECEPTOR v3.0.0 [39], Fast Rigid Exhaustive Docking (FRED)
v3.0.0 [39] and vROCS v3.1.2 [40]. Other modules including Babel
v3.3 [41], FILTER v2.1.1 [42] and OMEGA v2.4.6 [43] are driven by
command line prompts.

546
T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
command-line prompt. The number of poses to be generated for
each docked molecule was adjusted to 50 to allow a broad analysis
of the docked poses in the active site. Each docking pose was
evaluated by the default scoring function, Chemgauss4, and the
screened candidates were ranked accordingly.
using either sodium hydroxide or lithium hydroxide in a mixture of
tetrahydrofuran/methanol/water to give the corresponding car-
boxylic acid 6a, 6b or 6c in good to excellent yields (67e91%).
Amidation of carboxylic acid 6c with appropriate amines resulted
in amides 7a-e.
4.2. Synthesis of chemical compounds
4.2.1. Methyl 4-chloro-5-methylthieno[2,3-d]pyrimidine-6-
carboxylate (2)
Chemical reagents and solvents were purchased from com-
mercial sources, and were used as received unless otherwise
specified. All reactions except microwave-assisted synthesis were
carried out with continuous magnetic stirring in ordinary glass-
ware; microwave-assisted synthesis was performed in an Asynt
DrySyn insert (10 mL) (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).
Reactions were monitored using thin layer chromatography (TLC),
which was performed on Merck silica gel 60 F254 pre-coated
aluminum plates (0.2 mm), and was visualized under UV light
(254 nm). Flash column chromatography was carried out using
either pre-packed silica gel cartridges KP-SIL (ca. 0.05 mm) on
Biotage® Isolera™ Four automated flash chromatography or fritted
solid loaders packed with Scharlau® silica gel (0.04e0.06 mm) on
Biotage® FlashMaster Personalþ flash chromatography. High reso-
lution mass spectra were recorded using an AB SCIEX TripleTOF®
5600 mass spectrometer, and ionization of all samples was carried
out using ESI. 1H NMR and 13C NMR spectra were obtained at 298 K
using a Bruker AVANCE III HD spectrometer at 500.16 and
125.76 MHz, respectively, and were analyzed using a Bruker
Topspin 3.2 program. 1H and 13C NMR spectra are referenced to 1H
signals of residual non deuterated solvents and 13C signals of the
deuterated solvents, respectively. 1H NMR signals are reported with
To a solution of methyl 5-methyl-4-oxo-3,4-dihydrothieno[2,3-
d]pyrimidine-6-carboxylate (1, 2.00 g, 8.92 mmol) in SOCl2
(20.0 mL, 276 mmol) was added DMF (400 mL, 5.19 mmol). The
reaction mixture was heated at reflux under N2 for 2 h and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 99:1) to give 2 as a white solid
(1.73 g, 80%). RF (DCM:EtOAc ¼ 98:2) 0.57. m.p.141e142 ꢃC. 1H NMR
(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) m/z 243.0032 [M(35Cl)þH]þ,
245.0000 [M(37Cl)þH]þ; calcd. for C9H8ClN2O2Sþ 242.9990
[M(35Cl)þH]þ, 244.9960 [M(37Cl)þH]þ.
4.2.2. 4-Fluoroaniline (4a)
To a solution of 4-fluoro-1-nitrobenzene (3a, 212 mL, 2.00 mmol)
in MeOH (10 mL) was added 10% Pd/C (21 mg, 0.020 mmol). The
reaction mixture was stirred and bubbled with H2 at room tem-
perature for 3 h. 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 4a as a clear yellow oil
(222 mg, 100%). HRMS (ESI) m/z 112.0541 [MþH]þ; calcd. for
C6H7FNþ 112.0558 [MþH]þ.
chemical shift values
d
(ppm), multiplicity (s ¼ singlet, d ¼ doublet,
4.2.3. 4-Fluoro-2-methylaniline (4b)
t ¼ triplet, q ¼ quartet, dd ¼ doublet of doublets, td ¼ triplet of
doublets, m ¼ multiplet and br ¼ broad), relative integral, coupling
constants J (Hz) and assignments. The purity of compounds used for
biological evaluations was determined to be greater than 95% using
Shimadzu Prominence UltraFast Liquid Chromatography (UFLC)
system (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-
To a solution of 4-fluoro-2-methyl-1-nitrobenzene (3b, 243 mL,
2.00 mmol) in MeOH (10 mL) was added 10% Pd/C (21 mg,
0.020 mmol). The reaction mixture was stirred and bubbled with H2
at room temperature for 2 h. The solids were filtered off and
washed with DCM (25 mL). The filtrate and DCM washing were
combined and concentrated under reduced pressure. The residue
was purified by flash column chromatography (silica gel, P.E.
ramping to PE:EtOAc ¼ 4:1) to give 4b as a pink oil (250 mg, 100%).
RF (EtOAc:PE ¼ 1:4) 0.18. 1H NMR (CDCl3)
d 2.03 (s, 3H, CH3), 4.65 (s,
nomenex Kinetex 5
mM C18 100 Å 250 mm ꢄ 4.60 mm column.
2H, NH2), 6.56 (dd, 1H, J 5.0 & 8.5, benzene-H), 6.70 (td, 1H, J 3.0 &
9.0, benzene-H), 6.77 (dd, 1H, J 3.0 & 9.5, benzene-H). HRMS (ESI)
m/z 126.0778 [MþH]þ; calcd. for C7H9FNþ 126.0714 [MþH]þ.
Method A (gradient 5%e95% MeOH containing 0.1% formic acid
over 7 min at a flow rate of 1 mL/min, followed by 95% MeOH
containing 0.1% formic acid over 13 min) and method B (gradient
5%e95% MeCN containing 0.1% formic acid over 7 min at a flow rate
of 1 mL/min, followed by 95% MeCN containing 0.1% formic acid
over 13 min) were used for analytic RP-HPLC. Melting points were
determined using an open capillary on a Stuart SMP10 melting
point apparatus, and are uncorrected.
The synthesis of analogs of hit 3, i.e. 4-((4-fluorophenyl)amino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxylate derivatives is
outlined in Scheme 1. Chlorination of methyl 5-methyl-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidine-6-carboxylate
chloride in the presence of N,N-dimethylformamide yielded methyl
4-chloro-5-methylthieno[2,3-d]pyrimidine-6-carboxylate in
yield of 80%. Treatment of 4-fluoro-1-nitrobenzene 3a, 3b or 3c
with hydrogen gas in the presence of 10% Pd/C gave the corre-
sponding amine 4a, 4b or 4c in excellent yields (92e100%). The
microwave-assisted coupling of amine 4a, 4b or 4c to chloride 2
was carried out in 1,4-dioxane in the presence of p-toluenesulfonic
acid to give the corresponding methyl 4-((4-fluorophenyl)amino)-
5-methylthieno[2,3-d]pyrimidine-6-carboxylate 5a, 5b or 5c. The
subsequent saponification of ester 5a, 5b or 5c was accomplished
4.2.4. 4-Fluoro-2-isopropoxyaniline (4c)
To
a solution of 4-fluoro-2-isopropxy-1-nitrobenzene (3c,
199 mg, 0.999 mmol) in anhydrous EtOH (10 mL) was added 10%
Pd/C (11 mg, 0.010 mmol). The reaction mixture was stirred and
bubbled with H2 at room temperature for 2 h. The solids were
filtered off and washed with DCM (25 mL). The filtrate and DCM
washing were combined and concentrated under reduced pressure.
The residue was purified by Biotage® Isolera™ Four automated
flash chromatography (Biotage® SNAP cartridge KP-Sil 10 g, hexane
ramping to EtOAc:hexane ¼ 1:4) to give 4c as a colorless oil
1
with thionyl
2
a
(155 mg, 92%). RF (PE:EtOAc ¼ 9:1) 0.40. 1H NMR (CDCl3)
d 1.35 (d,
6H, J 6.0, 2 ꢄ CH3), 3.72 (s, 2H, NH2), 4.44e4.52 (m, 1H, OCH(CH3)2),
6.49 (td, 1H, J 8.5 & 2.5, benzene-H), 6.57 (dd, 1H, J 10.5 & 3.0,
benzene-H), 6.63 (dd, 1H, J 8.5 & 5.5, benzene-H). 13C NMR (CDCl3)
d
22.1, 71.0, 101.5 (d, JC-F 25.9), 106.5 (d, JC-F 22.0), 115.1 (d, JC-F 9.1),
132.9 (d, JC-F 2.5), 146.0 (d, JC-F 9.6), 156.3 (d, JC-F 234.3) (one carbon
signal overlapping or obscured). HRMS (ESI) m/z 170.1144 [MþH]þ;
calcd. for C9H13FNOþ 170.0976 [MþH]þ. The spectroscopic data
were in agreement with those in the literature [46].

T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
547
4.2.5. Methyl 4-((4-fluorophenyl)amino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylate (5a)
solution (1 mL, 1 mmol) were added MeOH (5 mL) and THF (5 mL).
The reaction mixture was stirred at 70 ꢃC for 4 h and quenched with
1 M aqueous HCl solution (1 mL, 1 mmol). The organic solvents
were removed under reduced pressure, and the precipitate was
filtered off, washed with H2O (3 ꢄ 10 mL) and dried in vacuo to give
6a as a white solid (74 mg, 87%). RF (EtOAc:PE ¼ 1:1) 0.05. m.p.
To a solution of 4a (222 mg, 2.00 mmol) in 1,4-dioxane (3.5 mL)
were added 2 (485 mg, 2.00 mmol) and TsOH$H2O (77 mg,
0.40 mmol). The reaction mixture was heated at 150 ꢃC under
microwave irradiation for 0.5 h and concentrated under reduced
pressure. The residue was purified by flash column chromatog-
raphy (silica gel, P.E. ramping to PE:EtOAc ¼ 7:3) and recrystallized
(hexane:acetone ¼ 1:5) to give 5a as greenish needles (114 mg,
18%). RF (EtOAc:PE ¼ 1:1) 0.61. m.p. 191e193 ꢃC. 1H NMR (DMSO-
293e295 ꢃC. 1H NMR (DMSO-d6)
d
3.03 (s, 3H, CH3), 7.22 (t, 2H, J 8.5,
benzene-H), 7.62 (dd, 2H, J 3.5 & 8.5, benzene-H), 8.45 (s, 1H,
pyridine-H), 8.63 (s, 1H, NH). 13C NMR (DMSO-d6)
15.5,115.2 (d, JC-
d
F 22.5), 117.8, 123.6, 125.8 (d, JC-F 7.5), 134.9, 138.8, 155.0 (d, JC-F 15.0),
157.3, 158.1, 160.1, 164.0, 166.9 (one carbon signal overlapping or
obscured). HRMS (ESI) m/z 304.0660 [MþH]þ; calcd. for
d6)
H), 7.58e7.62 (m, 3H, benzene-H & NH), 8.54 (s, 1H, pyrimidine-H).
13C NMR (DMSO-d6)
16.0, 52.6, 116.1 (d, JC-F 22.6), 117.5, 124.2,
d 3.13 (s, 3H, CH3), 3.93 (s, 3H, OCH3), 7.14 (t, 2H, J 8.5, benzene-
d
C
14H11FN3O2Sþ 304.0551 [MþH]þ. Anal. RP-HPLC Method A: tR
124.9 (d, JC-F 13.1), 133.2 (d, JC-F 2.9), 137.4, 154.5, 157.5, 159.4, 161.3,
163.0 (two carbon signals overlapping or obscured). HRMS (ESI) m/
z 318.0694 [MþH]þ; calcd. for C15H13FN3O2Sþ 318.0708 [MþH]þ.
Anal. RP-HPLC Method A: tR 11.38 min, purity >95%; Method B: tR
12.48 min, purity >95%.
10.38 min, purity >97%; Method B: tR 8.62 min, purity >99%.
4.2.9. 4-((4-Fluoro-2-methylphenyl)amino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid (6b)
To a solution of 5b (164 mg, 0.50 mmol) in 1 M aqueous NaOH
solution (1 mL, 1 mmol) were added MeOH (5 mL) and THF (5 mL).
The reaction mixture was stirred at 70 ꢃC for 4 h and quenched with
1 M aqueous HCl solution (1 mL, 1 mmol). The organic solvents
were removed under reduced pressure, and the precipitate was
filtered off, washed with water (3 ꢄ 10 mL) and dried in vacuo to
give 6b as a white solid (145 mg, 91%). RF (EtOAc:PE ¼ 1:1) 0.03.
4.2.6. Methyl 4-((4-fluoro-2-methylphenyl)amino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylate (5b)
To a solution of 4b (250 mg, 2.00 mmol) in 1,4-dioxane (3.5 mL)
were added 2 (485 mg, 2.00 mmol) and TsOH$H2O (76 mg,
0.40 mmol). The reaction mixture was heated at 150 ꢃC under
microwave irradiation for 0.5 h and concentrated under reduced
pressure. The residue was purified by flash column chromatog-
raphy (silica gel, P.E. ramping to PE:EtOAc ¼ 4:1) and recrystallized
(hexane:acetone ¼ 1:5) to give 5b as greenish needles (209 mg,
32%). RF (EtOAc:PE ¼ 1:1) 0.54. m.p. 243e245 ꢃC. 1H NMR (CDCl3)
m.p. 294e296 ꢃC. 1H NMR (DMSO-d6)
d 2.18 (s, 3H, CH3), 3.03 (s, 3H,
CH3), 7.07 (dt, 1H, J 3.0 & 8.5, benzene-H), 7.17 (dd, 1H, J 3.0 & 9.5,
benzene-H), 7.40 (dd, 1H, J 5.5 & 8.5, benzene-H), 8.33 (s, 1H,
pyrimidine-H), 8.48 (s, 1H, NH). 13C NMR (DMSO-d6)
d 15.7, 18.2,
112.8 (d, JC-F 21.9), 116.7 (d, JC-F 22.1), 123.0, 129.4 (d, JC-F 8.6), 133.4
(d, JC-F 2.5), 137.8 (d, JC-F 8.3), 139.0, 155.3, 157.9, 159.4, 161.3, 164.0,
166.8. HRMS (ESI) m/z 318.0830 [MþH]þ; calcd. for C15H13FN3O2Sþ
318.0708 [MþH]þ. Anal. RP-HPLC Method A: tR 10.28 min, purity
>97%; Method B: tR 8.59 min, purity >99%.
d
2.31 (s, 3H, CH3), 3.13 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 7.00e7.04
(m, 2H, benzene-H), 7.34 (s, 1H, NH), 7.61 (dd, 1H, J 5.0 & 8.0,
benzene-H), 8.52 (s, 1H, pyrimidine-H). 13C NMR (CDCl3)
16.1, 18.7,
d
52.6, 113.8 (d, JC-F 22.4), 117.5, 117.7 (d, JC-F 22.5), 124.1, 127.8 (d, JC-F
22.5), 131.4 (d, JC-F 2.8), 135.4 (d, JC-F 8.3), 137.5, 154.7, 158.1, 160.1,
162.1, 163.0. HRMS (ESI) m/z 332.1376; calcd. for C16H15FN3O2Sþ
332.0864 [MþH]þ. Anal. RP-HPLC Method A: tR 11.00 min, purity
>99%; Method B: tR 10.04 min, purity >98%.
4.2.10. 4-((4-Fluoro-2-isopropoxyphenyl)amino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxylic acid (6c)
To a solution of 5c (200 mg, 0.533 mmol) in a mixture of THF/
MeOH/H2O (1:1:1, 60 mL) was added LiOH (382 mg, 16.0 mmol).
The reaction mixture was stirred at room temperature overnight
and washed with DCM (20 mL). The aqueous layer was heated at
50 ꢃC for 10 min and acidified to pH 3 with 2 M HCl. The precipitate
was filtered, washed with H2O (3 ꢄ 25 mL) and dried under
reduced pressure to give 6c as a white solid (128 mg, 67%). RF
4.2.7. Methyl 4-((4-fluoro-2-isopropoxyphenyl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxylate (5c)
To a solution of 4c (153 mg, 0.904 mmol) in 1,4-dioxane (3 mL)
were added 2 (219 mg, 0.902 mmol) and TsOH,H2O (35 mg,
0.18 mmol). The reaction mixture was heated at 150 ꢃC under mi-
crowave irradiation for 0.5 h and concentrated under reduced
pressure. The residue was dissolved in DCM (25 mL) and washed
with H2O (4 ꢄ 25 mL). The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 97:3) to give 5c as a white solid
(268 mg, 79%). RF (DCM:EtOAc ¼ 98:2) 0.33. m.p. 182e183 ꢃC. 1H
(DCM:MeOH ¼ 9:1) 0.28. m.p. > 300 ꢃC. 1H NMR (DMSO-d6)
d 1.32
(d, 6H, J 6.0, OCH(CH3)2), 3.07 (s, 3H, thiophene-CH3), 4.75e4.80 (m,
1H, OCH(CH3)2), 6.80 (td, 1H, J 8.5 & 2.5, benzene-H), 7.07 (dd, 1H, J
11.0 & 2.5, benzene-H), 8.47 (s, 1H, NH), 8.54 (s, 1H, pyrimidine-H),
8.56 (dd, 1H, J 9.0 & 6.5, benzene-H) (one carboxylic acid proton
signal not observed). 13C NMR (DMSO-d6)
d 15.3, 21.7, 71.5, 101.1 (d,
JC-F 26.8), 106.1 (d, JC-F 21.6), 117.8, 121.9 (d, JC-F 9.4), 123.9, 125.0 (d,
JC-F 2.6), 137.5, 148.3 (d, JC-F 10.6), 155.1, 156.1, 158.6 (d, JC-F 239.0),
163.9, 166.3 (one carbon signal overlapping or obscured). HRMS
(ESI) m/z 362.1500 [MþH]þ; calcd. for C17H17FN3O3Sþ 362.0969
[MþH]þ. Anal. RP-HPLC Method A: tR 16.95 min, purity >97%;
Method B: tR 12.48 min, purity >97%.
NMR (CDCl3) d 1.43 (d, 6H, J 6.0, OCH(CH3)2), 3.12 (s, 3H, thiophene-
CH3), 3.91 (s, 3H, COOCH3), 4.61e4.69 (m, 1H, OCH(CH3)2), 6.67 (dd,
1H, J 10.0 & 2.5, benzene-H), 6.71 (td, 1H, J 9.0 & 2.5, benzene-H),
8.44 (s, 1H, NH), 8.59 (s, 1H, pyrimidine-H), 8.75 (dd, 1H, J 9.0 &
6.5, benzene-H). 13C NMR (CDCl3)
d 15.8, 22.2, 52.5, 71.8, 100.4 (d, JC-
F 26.9), 106.7 (d, JC-F 21.6), 118.0, 121.7 (d, JC-F 9.0), 123.4, 124.8 (d, JC-F
3.0), 137.6, 147.6 (d, JC-F 9.8), 154.9, 156.3, 159.1 (d, JC-F 241.5), 163.2,
166.2 (one carbon signal overlapping or obscured). HRMS (ESI) m/z
376.1207 [MþH]þ; calcd. for C18H19FN3O3Sþ 376.1126 [MþH]þ.
Anal. RP-HPLC Method A: tR 13.98 min, purity >98%; Method B: tR
13.19 min, purity >97%.
4.2.11. 4-((4-Fluoro-2-isopropoxyphenyl)amino)-5-methylthieno
[2,3-d]pyrimidine-6-carboxamide (7a)
To a solution of 6c (160 mg, 0.442 mmol) in DMF (3 mL) were
added DIPEA (160
0.500 mmol). The reaction mixture was stirred on an ice bath for
30 min. NH3 (7 M in MeOH, 220 L, 1.54 mmol) was added, and the
mL, 0.919 mmol) and HATU (190 mg,
m
4.2.8. 4-((4-Fluorophenyl)amino)-5-methylthieno[2,3-d]
pyrimidine-6-carboxylic acid (6a)
reaction mixture was stirred at room temperature overnight and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
To a solution of 5a (89 mg, 0.28 mmol) in 1 M aqueous NaOH

548
T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
DCM ramping to DCM:EtOAc ¼ 4:1) to give 7a as a white solid
(138 mg, 86%). RF (MeOH:EtOAc ¼ 1:5) 0.43. m.p. 159e160 ꢃC. 1H
4.2.14. 4-((4-Fluoro-2-isopropoxyphenyl)amino)-N-(2-
methoxyethyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide
(7d)
NMR (DMSO-d6)
d 1.33 (d, 6H, J 6.0, OCH(CH3)2), 2.95 (s, 3H, thio-
phene-CH3), 4.78e4.83 (m, 1H, OCH(CH3)2), 6.83 (td, 1H, J 8.5 & 2.5,
benzene-H), 7.10 (dd, 1H, J 11.0 & 3.0, benzene-H), 7.83 (br d, 2H, J
56.0, NH2), 8.43 (s, 1H, pyrimidineeNHebenzene), 8.54 (s, 1H,
pyrimidine-H), 8.58 (dd, 1H, J 9.0 & 6.5, benzene-H).13C NMR
To a solution of 6c (200 mg, 0.553 mmol) in DMF (3 mL) were
added DIPEA (193
The reaction mixture was stirred on an ice bath for 30 min. 2-
Methoxypropylamine (54.0 L, 0.609 mmol) was added, and the
mL, 1.11 mmol) and HATU (316 mg, 0.830 mmol).
m
(DMSO-d6)
d
15.8, 22.7, 71.4, 101.1 (d, JC-F 26.8), 106.0 (d, JC-F 21.7),
reaction mixture was stirred at room temperature overnight and
concentrated under reduced pressure. The residue was dissolved in
DCM (20 mL), washed with saturated NaHCO3 (3 ꢄ 20 mL), 10%
citric acid (3 ꢄ 20 mL), H2O (3 ꢄ 20 mL) and brine (3 ꢄ 20 mL), and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 4:1) to give 7d as a white solid
(118 mg, 51%). RF (DCM:EtOAc ¼ 7:3) 0.54. m.p. 186e187 ꢃC. 1H
117.3, 121.8 (d, JC-F 9.3), 125.1 (d, JC-F 2.7), 128.6, 131.1, 148.2 (d, JC-F
10.2), 154.3, 155.7, 158.5 (d, JC-F 238.9), 163.9, 165.3 (one carbon
signal overlapping or obscured). HRMS (ESI) m/z 361.1135 [MþH]þ;
calcd. for C17H18FN4O2Sþ 361.1129 [MþH]þ. Anal. RP-HPLC Method
A: tR 15.88 min, purity >98%; Method B: tR 11.63 min, purity >95%.
4.2.12. 4-((4-Fluoro-2-isopropoxyphenyl)amino)-N,5-
dimethylthieno[2,3-d]pyrimidine-6-carboxamide (7b)
To a solution of 6c (55 mg, 0.15 mmol) in DMF (3 mL) were
NMR (CDCl3) d 1.43 (d, 6H, J 6.0, OCH(CH3)2), 3.06 (s, 3H, thiophene-
CH3), 3.41 (s, 3H, OCH3), 3.56e3.60 (m, 2H, CH2CH2O), 3.63e3.67
(m, 2H, CH2CH2O), 4.64e4.69 (m, 1H, OCH(CH3)2), 6.34 (br s, 1H,
CONH), 6.69 (dd, 1H, J 10.0 & 3.0, benzene-H), 6.73 (td, 1H, J 8.5 &
2.5, benzene-H), 8.47 (br s, 1H, pyrimidineeNHebenzene), 8.61 (s,
1H, pyrimidine-H), 8.77 (dd, 1H, J 9.0 & 6.5, benzene-H). 13C NMR
added DIPEA (40 mL, 0.23 mmol) and HATU (80 mg, 0.21 mmol). The
reaction mixture was stirred on an ice bath for 30 min. Methyl-
amine hydrochloride (80 mg, 1.2 mmol) was added, and the reac-
tion mixture was stirred at room temperature overnight, and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 4:1) to give 7b as a white solid
(35 mg, 63%). RF (DCM:EtOAc ¼ 4:1) 0.44. m.p. 223e224 ꢃC. 1H
(CDCl3) d 16.1, 22.2, 40.1, 59.1, 70.3, 71.9, 100.5 (d, JC-F 27.0), 106.8 (d,
JC-F 21.6), 118.3, 121.7 (d, JC-F 9.0), 124.9, 127.3, 132.9, 147.7 (d, JC-F
9.5), 154.2, 156.2, 159.1 (d, JC-F 241.5), 162.9 (two carbon signals
overlapping or obscured). HRMS (ESI) m/z 419.1394 [MþH]þ; calcd.
for C20H24FN4O3Sþ 419.1548 [MþH]þ. Anal. RP-HPLC Method A: tR
16.01 min, purity >96%; Method B: tR 11.98 min, purity >96%.
NMR (DMSO-d6)
d 1.33 (d, 6H, J 6.0, OCH(CH3)2), 2.79 (d, 3H, J 4.5,
NHCH3), 2.92 (s, 3H, thiophene-CH3), 4.77e4.83 (m, 1H,
OCH(CH3)2), 6.83 (td,1H, J 11.5 & 8.5, benzene-H), 7.10 (dd,1H, J 10.5
& 2.5, benzene-H), 8.40 (q, 1H, J 4.5, NHCH3), 8.42 (s, 1H, pyr-
imidineeNHebenzene), 8.54 (s, 1H, pyrimidine-H), 8.58 (dd, 1H, J
4.2.15. 4-((4-Fluoro-2-isopropoxyphenyl)amino)-N-(3-
methoxypropyl)-5-methylthieno[2,3-d]pyrimidine-6-carboxamide
(7e)
9.0 & 6.5, benzene-H). 13C NMR (DMSO-d6)
d 15.8, 21.7, 26.5, 71.4,
101.1 (d, JC-F 3.2), 106.0 (d, JC-F 21.7), 117.2, 122.8 (d, JC-F 9.2), 125.1 (d,
JC-F 2.7), 128.4, 130.6, 148.2 (d, JC-F 10.5), 154.3, 155.7, 158.5 (d, JC-F
238.7), 162.6, 165.2 (one carbon signal overlapping or obscured).
HRMS (ESI) m/z 375.1525 [MþH]þ; calcd. for C18H20FN4O2Sþ
375.1286 [MþH]þ. Anal. RP-HPLC Method A: tR 16.06 min, purity
>95%; Method B: tR 12.00 min, purity >96%.
To a solution of 6c (65 mg, 0.18 mmol) in DMF (3 mL) were
added DIPEA (58
reaction mixture was stirred on an ice bath for 30 min. 3-
Methoxypropylamine (37 L, 0.36 mmol) was added, and the re-
mL, 0.33 mmol) and HATU (75 mg, 0.20 mmol). The
m
action mixture was stirred at room temperature overnight and
concentrated under reduced pressure. The residue was dissolved in
DCM (20 mL), washed with saturated NaHCO3 (3 ꢄ 20 mL), 10%
citric acid (3 ꢄ 20 mL), H2O (3 ꢄ 20 mL) and brine (3 ꢄ 20 mL), and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 1:1) to give 7e as a white solid
(37 mg, 48%). RF (DCM:EtOAc ¼ 7:3) 0.32. m.p. 170e171 ꢃC. 1H NMR
4.2.13. N-Ethyl-4-((4-fluoro-2-isopropoxyphenyl)amino)-5-
methylthieno[2,3-d]pyrimidine-6-carboxamide (7c)
To a solution of 6c (50 mg, 0.14 mmol) in DMF (3 mL) were
added DIPEA (40 mL, 0.23 mmol) and HATU (80 mg, 0.21 mmol). The
reaction mixture was stirred on an ice bath for 30 min. Ethylamine
(2.0 M in THF, 1.0 mL, 2.0 mmol) was added, and the reaction
mixture was stirred at room temperature overnight and concen-
trated under reduced pressure. The residue was dissolved in DCM
(20 mL), washed with saturated NaHCO3 (3 ꢄ 20 mL), 10% citric acid
(3 ꢄ 20 mL), H2O (3 ꢄ 20 mL) and brine (3 ꢄ 20 mL), and
concentrated under reduced pressure. The residue was purified by
Biotage® FlashMaster Personalþ flash chromatography (silica gel,
DCM ramping to DCM:EtOAc ¼ 4:1) to give 7c as a white solid
(18 mg, 34%). RF (DCM:EtOAc ¼ 4:1) 0.29. m.p. 205e206 ꢃC. 1H
(CDCl3)
d 1.43 (d, 6H, J 6.0, OCH(CH3)2), 1.88e1.93 (m, 2H,
CH2CH2CH2), 3.08 (s, 3H, thiophene-CH3), 3.41 (s, 3H, OCH3),
3.56e3.60 (m, 4H, CH2CH2CH2), 4.64e4.70 (m, 1H, OCH(CH3)2), 6.70
(dd, 1H, J 10.0 & 2.5, benzene-H), 6.74 (td, 1H, J 8.5 & 2.5, benzene-
H), 6.98 (br s, 1H, CONH), 8.59 (s, 1H, pyrimidineeNHebenzene),
8.61 (s, 1H, pyrimidine-H), 8.75 (dd, 1H, J 9.0 & 6.0, benzene-H). 13C
NMR (CDCl3)
d 16.0, 22.2, 28.7, 39.8, 59.2, 71.9, 72.7, 100.5 (d, JC-F
27.0), 106.8 (d, JC-F 21.6), 118.4, 121.7 (d, JC-F 9.0), 124.9, 127.9, 132.6,
147.7 (d, JC-F 9.7), 154.0,156.2, 159.1 (d, JC-F 254.2), 162.5 (two carbon
signals overlapping or obscured). HRMS (ESI) m/z 433.1538
[MþH]þ; calcd. for C21H26FN4O3Sþ 433.1704 [MþH]þ. Anal. RP-
HPLC Method A: tR 16.25 min, purity >98%; Method B: tR 12.25 min,
purity >98%.
NMR (CDCl3)
d 1.28 (t, 3H, J 7.0, NHCH2CH3), 1.42 (d, 6H, J 6.0,
OCH(CH3)2), 3.05 (s, 3H, thiophene-CH3), 3.46e3.53 (m, 2H,
NHCH2CH3), 4.63e4.69 (m, 1H, OCH(CH3)2), 6.02 (br s, 1H,
NHCH2CH3), 6.68 (dd, 1H, J 10.5 & 2.5, benzene-H), 6.72 (td, 1H, J 9.0
& 6.0, benzene-H), 8.42 (s, 1H, pyrimidineeNHebenzene), 8.58 (s,
1H, pyrimidine-H), 8.77 (dd, 1H, J 9.0 & 6.5, benzene-H). 13C NMR
4.3. Chemical and proteins
(CDCl3) d 15.0, 16.1, 22.2, 29.8, 35.4, 71.8, 100.4 (d, JC-F 27.5), 106.7 (d,
JC-F 21.3), 118.3, 121.5 (d, JC-F 8.8), 125.0 (d, JC-F 3.8), 127.1, 132.8, 147.6
(d, JC-F 10.0), 154.5, 156.2, 159.0 (d, JC-F 241.3), 162.8 (one carbon
signal overlapping or obscured). HRMS (ESI) m/z 389.1443 [MþH]þ;
The Mnk inhibitor N3-(4-fluorophenyl)-1H-pyrazolo-[3,4-d]py-
rimidine-3,4-diamine (CGP57380) and cercosporamide were pur-
chased from Sigma Aldrich and BioAustralis, respectively, and used
as standard references or controls. All the compounds used were
dissolved in dimethylsulfoxide (DMSO) at a stock concentration of
10 mM, and stored at ꢀ20 ꢃC in small aliquots. eIF4E-derived
calcd. for
C
19H22FN4O2Sþ 389.1442 [MþH]þ. Anal. RP-HPLC
Method A: tR 16.20 min, purity >97%; Method B: tR 12.29 min,
purity >97%.

T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
549
peptides: TAMRA-DTATKSGSTTKNR, TAMRA-DTATKSG(phospho)
Acknowledgment
STTKNR and DTATKSGSTTKNR, were custom synthesized from
Mimotopes Pty. Ltd. (Melbourne, Australia). Proteins Mnk1a and
Mnk2a were purchased from Life Technologies and Merck Milli-
pore, respectively.
This work is supported by funding from Australia Government
National Health and Medical Research Council (project grant
1050825 to S.W.), and South Australian Health and Medical
Research Institute, Beat Cancer Project Principal Cancer Research
Fellowship, to S.W. We thank Dr Frankie Lam for assistance in assay
development. We are grateful to Professor Richard D'Andrea (Uni-
versity of South Australia) for the generous supply of the AML cell
lines.
4.4. Cell culture
Both MOLM-13 and MV4-11 cells were kindly provided by Prof.
Richard D'Andrea (University of South Australia, Australia) and
were maintained in Roswell Park Memorial Institute (RPMI)-1640
with 10% fetal bovine serum (FBS).
Appendix A. Supplementary data
4.5. Kinase activity
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.09.008.
The Millipore KinaseProfilerTM services were used to measure
inhibition of Mnk2 (applied on virtual screening hits only), CDK2/
cyclin A and CDK9/cyclin T1 with a radiometric assay format. The
ATP concentrations were adjusted to Km(app) for each kinase and
half-maximal inhibition (IC50) values were calculated from 10-
point doseeresponse curves and apparent inhibition constants
(Ki) were calculated from the IC50 values and appropriate Km (ATP)
values for the kinases in question using the Cheng-Prusoff equation
[54].
References
[1] M.J. Clemens, U.A. Bommer, Translational control: the cancer connection, Int. J.
Biochem. Cell Biol. 31 (1999) 1e23.
[2] Y. Mamane, E. Petroulakis, L. Rong, K. Yoshida, L.W. Ler, N. Sonenberg, eIF4E-
from translation to transformation, Oncogene 23 (2004) 3172e3179.
[3] S. Diab, M. Kumarasiri, M. Yu, T. Teo, C. Proud, R. Milne, S. Wang, MAP kinase-
interacting kinases-emerging targets against cancer, Chem. Biol. 21 (2014)
441e452.
[4] R.E. Rhoads, eIF4E: new family members, new binding partners, new roles,
J. Biol. Chem. 284 (2009) 16711e16715.
[5] J. Hou, F. Lam, C. Proud, S. Wang, Targeting Mnks for cancer therapy, Onco-
target 3 (2012) 118e131.
[6] R. Duncan, S.C. Milburn, J.W. Hershey, Regulated phosphorylation and low
abundance of HeLa cell initiation factor eIF-4F suggest a role in translational
control. Heat shock effects on eIF-4F, J. Biol. Chem. 262 (1987) 380e388.
[7] H.G. Wendel, R.L.A. Silva, A. Malina, J.R. Mills, H. Zhu, T. Ueda, R. Watanabe-
Fukunaga, R. Fukunaga, J. Teruya-Feldstein, J. Pelletier, Dissecting eIF4E action
in tumorigenesis, Genes Dev. 21 (2007) 3232e3237.
[8] H.G. Wendel, E. De Stanchina, J.S. Fridman, A. Malina, S. Ray, S. Kogan,
C. Cordon-Cardo, J. Pelletier, S.W. Lowe, Survival signalling by Akt and eIF4E in
oncogenesis and cancer therapy, Nature 428 (2004) 332e337.
[9] A.J. Waskiewicz, A. Flynn, C.G. Proud, J.A. Cooper, Mitogen-activated protein
kinases activate the serine/threonine kinases Mnk1 and Mnk2, EMBO J. 16
(1997) 1909e1920.
[10] L. Furic, L. Rong, O. Larsson, I.H. Koumakpayi, K. Yoshida, A. Brueschke,
E. Petroulakis, N. Robichaud, M. Pollak, L.A. Gaboury, eIF4E phosphorylation
promotes tumorigenesis and is associated with prostate cancer progression,
Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 14134e14139.
[11] T. Ueda, M. Sasaki, A.J. Elia, I.I.C. Chio, K. Hamada, R. Fukunaga, T.W. Mak,
Combined deficiency for MAP kinase-interacting kinase 1 and 2 (Mnk1 and
Mnk2) delays tumor development, Proc. Natl. Acad. Sci. U. S. A. 107 (2010)
13984e13990.
4.5.1. IMAP
™
TR-FRET progressive binding system
M in 0.5% DMSO) were added to the
The inhibitors (1 and 10
m
kinase reaction containing 1 ꢄ IMAP reaction buffer with Tween-
20, dithiothreitol (DTT), dH2O, TAMRA-labeled eIF4E peptide sub-
strate and Mnk kinases (a total assay volume 20 ml) and incubated
for 10 min. The reactions were sequentially started by adding ATP.
The kinase reaction was run for 90 min at 30 ꢃC and stopped with
60
ml progressive binding solution (30% binding buffer A, 70%
binding buffer B, progressive binding reagent (1:600), Tb-donor
(1:400)), followed by incubation in the dark for 5 h. After the in-
cubation, the plate was read in EnVision multi-label plate reader
(PerkinElmer, Beaconsfield, Buckinghamshire, UK). Excitation
wavelength was set to 330 nm (Tb excitation) and emission was
measured at 545 nm (Tb emission) and 572 nm (TR-FRET emission).
Emission was measured in a time-resolved manner with a delay of
200 msec. Positive and negative controls were also included with
and without Mnk kinases, respectively, in 0.5% DMSO.
[12] T. Ueda, R. Watanabe-Fukunaga, H. Fukuyama, S. Nagata, R. Fukunaga, Mnk2
and Mnk1 are essential for constitutive and inducible phosphorylation of
eukaryotic initiation factor 4E but not for cell growth or development, Mol.
Cell. Biol. 24 (2004) 6539e6549.
4.5.2. ADP-GloTM kinase assay
Serial dilution of the inhibitor with a dilution factor of 1:3 was
[13] J. Bain, L. Plater, M. Elliott, N. Shpiro, C.J. Hastie, H. McLauchlan, I. Klevernic,
J.S.C. Arthur, D.R. Alessi, P. Cohen, The selectivity of protein kinase inhibitors:
a further update, Biochem. J. 408 (2007) 297e315.
carried out for 8 different concentrations, ranging from 10 mM to
4.5 nM, in 0.5% DMSO. The inhibitors were added to the kinase
reaction containing 1 ꢄ kinase reaction buffer, 1 mM bovine
serum albumin (BSA), dH2O, eIF4E peptide substrate and Mnk
[14] A. Sussman, et al., Discovery of cercosporamide, a known antifungal natural
product, as
a selective Pkc1 kinase inhibitor through high-throughput
screening, Eukaryot. Cell 3 (2004) 932e943.
kinases (a total assay volume of 15
The reactions were sequentially started by adding ATP. The kinase
reaction was run for 45 min at 30 ꢃC and stopped with 15
l ADP
Glo reagent, followed by incubation in the dark for 40 min. After
the incubation, 30 l of detection reagent was added and further
ml) and incubated for 10 min.
[15] B.W. Konicek, et al., Therapeutic inhibition of MAP kinase interacting kinase
blocks eukaryotic initiation factor 4E phosphorylation and suppresses
outgrowth of experimental lung metastases, Cancer Res. 71 (2011)
1849e1857.
[16] S. Diab, T. Teo, M. Kumarasiri, P. Li, M. Yu, F. Lam, S.K. Basnet, M.J. Sykes,
H. Albrecht, R. Milne, S. Wang, Discovery of 5-(2-(phenylamino)pyrimidin-4-
yl)thiazol-2(3H)-one derivatives as potent Mnk2 inhibitors: synthesis, SAR
analysis and biological evaluation, ChemMedChem 9 (2014) 962e972.
[17] F.M. Balis, Evolution of anticancer drug discovery and the role of cell-based
screening, J. Natl. Cancer Inst. 94 (2002) 78e79.
[18] R. Jauch, S. Jakel, C. Netter, K. Schreiter, B. Aicher, H. Jackle, M.C. Wahl, Crystal
structures of the Mnk2 kinase domain reveal an inhibitory conformation and a
zinc binding site, Structure 13 (2005) 1559e1568.
[19] J. Hou, T. Teo, M.J. Sykes, S. Wang, Insights into the importance of DFD-motif
and insertion I1 in stabilizing the DFD-out conformation of Mnk2 kinase, ACS
Med. Chem. Lett. 4 (2013) 736e741.
m
m
incubated for 40 min before read in EnVision multi-label plate
reader (PerkinElmer). Positive and negative controls were also
included with and without Mnk kinases, respectively, in 0.5%
DMSO.
4.6. Cell viability assay
Resazurin (Sigma Aldrich) assays were performed in MOLM-13
and MV4-11 cells as previously reported [16]. Compound concen-
trations required to inhibit 50% of cell growth (GI50) were calcu-
lated using non-linear regression analysis.
[20] R. Jauch, M.K. Cho, S. Jakel, C. Netter, K. Schreiter, B. Aicher, M. Zweckstetter,
H. Jackle, M.C. Wahl, Mitogen-activated protein kinases interacting kinases
are autoinhibited by a reprogrammed activation segment, EMBO J. 25 (2006)
4020e4032.

550
T. Teo et al. / European Journal of Medicinal Chemistry 103 (2015) 539e550
[21] J. Oyarzabal, N. Zarich, M.I. Albarran, I. Palacios, M. Urbano-Cuadrado,
G. Mateos, I. Reymundo, O. Rabal, A. Salgado, A. Corrionero, J.s. Fominaya,
J. Pastor, J.R. Bischoff, Discovery of mitogen-activated protein kinase-
interacting kinase 1 inhibitors by a comprehensive fragment-oriented vir-
tual screening approach, J. Med. Chem. 53 (2010) 6618e6628.
[22] K. Slentz-Kesler, J.T. Moore, M. Lombard, J. Zhang, R. Hollingsworth,
M.P. Weiner, Identification of the human Mnk2 gene (MKNK2) through pro-
tein interaction with estrogen receptor beta, Genomics 69 (2000) 63e71.
[23] G.C. Scheper, N.A. Morrice, M. Kleijn, C.G. Proud, The mitogen-activated pro-
tein kinase signal-integrating kinase Mnk2 is a eukaryotic initiation factor 4E
kinase with high levels of basal activity in mammalian cells, Mol. Cell. Biol. 21
(2001) 743e754.
[24] J.L. Parra, M. Buxade, C.G. Proud, Features of the catalytic domains and C
termini of the MAPK signal-integrating kinases Mnk1 and Mnk2 determine
their differing activities and regulatory properties, J. Biol. Chem. 280 (2005)
37623e37633.
[25] S. Joshi, L.C. Platanias, Mnk kinases in cytokine signaling and regulation of
cytokine responses, Biomol. Concepts 3 (2012) 127e139.
[26] J. Bostrom, J.R. Greenwood, J. Gottfries, Assessing the performance of OMEGA
with respect to retrieving bioactive conformations, J. Mol. Graph. Model 21
(2003) 449e462.
[27] M. McGann, FRED pose pediction and virtual screening accuracy, J. Chem. Inf.
Model 51 (2011) 578e596.
[28] C. Tschopp, U. Knauf, M. Brauchle, M. Zurini, P. Ramage, D. Glueck, L. New,
J. Han, H. Gram, Phosphorylation of eIF-4E on Ser 209 in response to mito-
genic and inflammatory stimuli is faithfully detected by specific antibodies,
Mol. Cell Biol. Res. Commun. 3 (2000) 205e211.
[29] J.A. Grant, M.A. Gallardo, B.T. Pickup, A fast method of molecular shape
comparison: a simple application of a Gaussian description of molecular
shape, J. Comput. Chem. 17 (1996) 1653e1666.
relationship, and anticancer activities, J. Med. Chem. 56 (2013) 640e659.
[37] T. Lehmann-Lintz, J. Kley, N. Redemann, A. Sauer, L. Thomas, D. Wiedenmayer,
M. Austen, J. Danilewicz, M. Schneider, K. Schreiter, P. Black, W. Blackaby, and
I. Linney, WO 2011/104337 A1, 2011.
[38] VIDA, Version 4.0.3, Openeye Scientific Software, Sante Fe, NM, 2010.
[39] OEDocking, Version 3.0.0, Openeye Scientific Software, Sante Fe, NM, 2012.
[40] ROCS, Version 3.1.2, Openeye Scientific Software, Sante Fe, NM, 2011.
[41] Babel, Version 3.3, Openeye Scientific Software, Sante Fe, NM, 2007.
[42] FILTER, Version 2.1.1, Openeye Scientific Software, Sante Fe, NM, 2010.
[43] OMEGA, Version 2.4.6, Openeye Scientific Software, Sante Fe, NM, 2012.
[44] G.B. McGaughey, R.P. Sheridan, C.I. Bayly, J.C. Culberson, C. Kreatsoulas,
S. Lindsley, V. Maiorov, J.F. Truchon, W.D. Cornell, Comparison of topological,
shape, and docking methods in virtual screening, J. Chem. Inf. Model 47
(2007) 1504e1519.
[45] M.J. Sykes, R.A. McKinnon, J.O. Miners, Prediction of metabolism by cyto-
chrome P450 2C9: alignment and docking studies of a validated database of
substrates, J. Med. Chem. 51 (2008) 780e791.
[46] A.B. Reitz, E.W. Baxter, E.E. Codd, C.B. Davis, A.D. Jordan, B.E. Maryanoff,
C.A. Maryanoff, M.E. McDonnell, E.T. Powell, M.J. Renzi, M.R. Schott, M.K. Scott,
R.P. Shank, J.L. Vaught, Orally active benzamide antipsychotic agents with
affinity for dopamine D2, serotonin 5-HT1A, and adrenergic alpha1 receptors,
J. Med. Chem. 41 (1998) 1997e2009.
[47] P. Burkhard, U. Hommel, M. Sanner, M.D. Walkinshaw, The discovery of ste-
roids and other novel FKBP inhibitors using a molecular docking program, J.
Mol. Biol 287 (1999) 853e858.
[48] D.B. Jordan, G.S. Basarab, D.I. Liao, W.M. Johnson, K.N. Winzenberg,
D.A. Winkler, Structure-based design of inhibitors of the rice blast fungal
enzyme trihydroxynaphthalene reductase, J. Mol. Graph. Model 19 (2001)
434e447, 470-431.
[49] J.W. Liebeschuetz, S.D. Jones, P.J. Morgan, C.W. Murray, A.D. Rimmer,
J.M. Roscoe, B. Waszkowycz, P.M. Welsh, W.A. Wylie, S.C. Young, H. Martin,
J. Mahler, L. Brady, K. Wilkinson, PRO_SELECT: combining structure-based
drug design and array-based chemistry for rapid lead discovery. 2. The
development of a series of highly potent and selective factor Xa inhibitors, J.
Med. Chem 45 (2002) 1221e1232.
[50] H. Park, H. Choi, S. Hong, S. Hong, Identification of novel BRAF kinase in-
hibitors with structure-based virtual screening, Bioorg. Med. Chem. Lett 21
(2011) 5753e5756.
[30] S. Lapenna, A. Giordano, Cell cycle kinases as therapeutic targets for cancer,
Nat. Rev. Drug Discov. 8 (2009) 547e566.
[31] V. Dulic, W.K. Kaufmann, S.J. Wilson, T.D. Tlsty, E. Lees, J.W. Harper,
S.J. Elledge, S.I. Reed, p53-dependent inhibition of cyclin-dependent kinase
activities in human fibroblasts during radiation-induced G1 arrest, Cell 76
(1994) 1013e1023.
[32] F. Lam, A.Y. Abbas, H. Shao, T. Teo, J. Adams, P. Li, T.D. Bradshaw, P.M. Fischer,
E. Walsby, C. Pepper, Y. Chen, J. Ding, S. Wang, Targeting RNA transcription
and translation in ovarian cancer cells with pharmacological inhibitor CDKI-
73, Oncotarget 5 (2014) 7691e7704.
[33] H. Quentmeier, J. Reinhardt, M. Zaborski, H.G. Drexler, FLT3 mutations in acute
myeloid leukemia cell lines, Leukemia 17 (2003) 120e124.
[51] T. Teo, M. Yu, Y. Yang, T. Gillam, F. Lam, M.J. Sykes, S. Wang, Pharmacologic co-
inhibition of Mnks and mTORC1 synergistically suppresses proliferation and
perturbs cell cycle progression in blast crisis-chronic myeloid leukemia cells,
Cancer Lett 357 (2015) 612e623.
[34] E.R. Sharlow, S. Leimgruber, A. Yellow-Duke, R. Barrett, Q.J. Wang, J.S. Lazo,
Development, validation and implementation of immobilized metal affinity
for phosphochemicals (IMAP)-based high-throughput screening assays for
low-molecular-weight compound libraries, Nat. Protoc. 3 (2008) 1350e1363.
[35] M. Koresawa, T. Okabe, High-throughput screening with quantitation of ATP
consumption: a universal non-radioisotope, homogeneous assay for protein
kinase, Assay. Drug Dev. Technol. 2 (2004) 153e160.
[36] H. Shao, S. Shi, S. Huang, A.J. Hole, A.Y. Abbas, S. Baumli, X. Liu, F. Lam,
D.W. Foley, P.M. Fischer, M. Noble, J.A. Endicott, C. Pepper, S. Wang,
Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active
CDK9 inhibitors: synthesis, X-ray crystal structures, structure-activity
[52] M. Yu, P. Li, S.K.C. Basnet, M. Kumarasiri, S. Diab, T. Teo, H. Albrecht, S. Wang,
Discovery of 4-(dihydropyridinon-3-yl)amino-5-methylthieno[2,3-d]pyrimi-
dine derivatives as potent Mnk inhibitors: synthesis, structure-activity rela-
tionship analysis and biological evaluation, Eur. J. Med. Chem 95 (2015)
116e126.
[53] T. Teo, F. Lam, M. Yu, Y. Yang, S.K. Basnet, H. Albrecht, M.J. Sykes, S. Wang,
Pharmacologic inhibition of MNKs in acute myeloid leukemia, Mol. Pharmacol
88 (2015) 380e389.
[54] Y. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K1) and
the concentration of inhibitor which causes 50 per cent inhibition (I50) of an
enzymatic reaction, Biochem. Pharmacol 22 (1973) 3099e3108.