5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine zipper kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.12.051

Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes and displays increased levels of expression in numerous cancer classes (colon, breast, brain, ovary, prostate and lung). Although no selective MELK inhibitors ha

Full text of DOI:10.1016/j.bmcl.2018.12.051

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Accepted Manuscript
5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leu-
cine zipper kinase inhibitors
Nicolas Boutard, Aleksandra Sabiniarz, Klaudia Czerwińska, Małgorzata
Jarosz, Anna Cierpich, Ewa Kolasińska, Katarzyna Wiklik, Karolina Gluza,
Claude Commandeur, Anna Buda, Agata Stasiowska, Aneta Bobowska,
Mariusz Galek, Charles-Henry Fabritius, Marta Bugaj, Edyta Palacz, Andrzej
Mazan, Adrian Zarębski, Karolina Krawczyńska, Małgorzata Żurawska,
Przemysław Zawadzki, Mariusz Milik, Paulina Węgrzyn, Monika Dobrzańska,
Krzysztof Brzózka, Piotr Kowalczyk
PII:
S0960-894X(18)30999-5
https://doi.org/10.1016/j.bmcl.2018.12.051
BMCL 26220
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 November 2018
17 December 2018
21 December 2018
Please cite this article as: Boutard, N., Sabiniarz, A., Czerwińska, K., Jarosz, M., Cierpich, A., Kolasińska, E.,
Wiklik, K., Gluza, K., Commandeur, C., Buda, A., Stasiowska, A., Bobowska, A., Galek, M., Fabritius, C-H., Bugaj,
M., Palacz, E., Mazan, A., Zarębski, A., Krawczyńska, K., Żurawska, M., Zawadzki, P., Milik, M., Węgrzyn, P.,
Dobrzańska, M., Brzózka, K., Kowalczyk, P., 5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal
embryonic leucine zipper kinase inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/
10.1016/j.bmcl.2018.12.051
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maternal embryonic leucine zipper kinase inhibitors
Nicolas Boutard, Aleksandra Sabiniarz, Klaudia Czerwińska, Małgorzata Jarosz, Anna Cierpich, Ewa
Kolasińska, Katarzyna Wiklik, Karolina Gluza, Claude Commandeur, Anna Buda, Agata Stasiowska, Aneta
Bobowska, Mariusz Galek, Charles-Henry Fabritius, Marta Bugaj, Edyta Palacz, Andrzej Mazan, Adrian
Zarębski, Karolina Krawczyńska, Małgorzata Żurawska, Przemysław Zawadzki, Mariusz Milik, Paulina
Węgrzyn, Monika Dobrzańska, Krzysztof Brzózka and Piotr Kowalczyk*

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Bioorganic & Medicinal Chemistry Letters
5-Keto-3-cyano-2,4-diaminothiophenes as selective maternal embryonic leucine
zipper kinase inhibitors
Nicolas Boutarda, Aleksandra Sabiniarza, Klaudia Czerwińskaa, Małgorzata Jarosza, Anna Cierpicha, Ewa
Kolasińska, Katarzyna Wiklika, Karolina Gluzaa, Claude Commandeura, Anna Budaa, Agata Stasiowskaa,
Aneta Bobowskaa, Mariusz Galeka, Charles-Henry Fabritiusa, Marta Bugaja, Edyta Palacza, Andrzej
Mazan,a Adrian Zarębskia, Karolina Krawczyńskaa, Małgorzata Żurawskaa, Przemysław Zawadzkia,
a
a
a
a
a

Mariusz Milik , Paulina Węgrzyn , Monika Dobrzańska , Krzysztof Brzózka , and Piotr Kowalczyk
a Selvita S.A., Bobrzyńskiego, 14, 30-338 Kraków, Poland
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Maternal embryonic leucine zipper kinase (MELK) is involved in several key cellular processes
and displays increased levels of expression in numerous cancer classes (colon, breast, brain,
ovary, prostate and lung). Although no selective MELK inhibitors have yet been approved,
increasing evidence suggest that inhibition of MELK would constitute a promising approach for
cancer therapy. A weak high-throughput screening hits (17, IC50 ≈ 5 M) with lead-like
properties was optimized for MELK inhibition. The early identification of a plausible binding
mode by molecular modeling offered guidance in the choice of modifications towards compound
52 which displayed a 98 nM IC50. A good selectivity profile was achieved for a representative
member of the series (29) in a 486 protein kinase panel. Future elaboration of 52 has the
potential to deliver compounds for further development with chemotherapeutic aims.
Keywords:
Maternal embryonic leucine zipper kinase (MELK)
Kinase
5-Keto-3-cyano-2,4-diaminothiophene
MELK inhibitor
Cancer
Maternal embryonic leucine zipper kinase (MELK) is an
atypical member of the AMPK family of serine–threonine
kinases and its role has been extensively studied in the last two
decades. It has been implicated in various cellular processes
including stem cell renewal,1 cell cycle progression,2,3
cytokinesis,4,5 mRNA splicing6 and apoptosis.7 MELK expression
and activity are cell cycle dependent. Expression peaks during
the G2/M phase and MELK activity, correlated with its
phosphorylation by the MPF (Maturation-Promoting Factor) and
MAPK1 (Mitogen-Activated Protein Kinase) mitotic kinases,
reaches a maximum during the M phase.8
demonstrated in numerous experimental systems that reduction
of MELK level (using si/shRNA) leaded to decreased viability of
kidney, pancreas, breast and colon cancer cell lines and inhibited
colony formation as well as tumor growth in vivo.11,7 Similarly, in
astrocytoma cell lines, Ras and Akt overexpressing astrocytes
and primary cultures from human glioblastoma and
medulloblastoma, MELK knockdown resulted in decreased
proliferation, anchorage-independent growth and survival in
vitro.9,12,16 Consistent with previous studies, shRNA-mediated
MELK knock-down in gastric cancer cells affected proliferation
both in vitro and in xenograft model.17 It was also shown that
MELK played an important role in the acquisition of resistance to
radiotherapy and chemotherapy by tumors. In glioblastoma,
radiation causes an increase in the level of MELK kinase in vitro
and in vivo,18,19 and a simultaneous inhibition of its activity with
small molecule inhibitors induces apoptosis.20 Similar
MELK was found to be overexpressed in many tumor types,
including colon, breast, brain, ovary, prostate and lung.1,9–11,7
MELK overexpression in tumors strongly correlates with poor
prognosis in glioblastoma12 and breast cancer.13 MELK has also
been identified as one of the genes for which overexpression is
characteristic of triple negative breast cancer.14 High expression
in cancer cells, while germ-line knock-out mice are viable
without any noticeable phenotypes, indicated MELK as a
promising therapeutic target for multiple cancers.11,15,8 It has been
observations have also been made in the case of colon cancer
cells treated with radio- or chemotherapy.21
———
 Corresponding author. Tel.: +48 12 297 47 00; e-mail: piotr.kowalczyk@selvita.com

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These reports clearly suggest pharmacological inhibition of
the MELK kinase activity to be an attractive therapeutic approach
in a wide range of solid tumors.
Figure 1. Representative known MELK inhibitors.20,22–28
Several inhibitors of the MELK kinase activity have been
described (compounds 1-9, Figure 1). The most extensively
studied compound is the potent OTS167 1 (IC50 = 0.41 nM). A
wide range of biological activities attributed to 1 have been
reported in the literature, including inhibition of breast cancer
cells mammosphere formation and abrogation of tumor growth in
breast, lung, prostate, and pancreas cancer cells xenograft
experiments in mice.29 OTS167 is currently being assessed in
several clinical trials in patients with acute and chronic myeloid
leukemia (AML, CML), acute lymphoblastic leukemia (ALL),
and triple negative breast cancer (TNBC).30 It is unclear however
whether all of these effects are attributable solely to its activity
on MELK as recent reports revealed OTS167’s broad multikinase
inhibition,31,32 and its preserved anti-proliferative activity in
several MELK-knockout cancer cell lines.33,34 Compounds 2-7
display nanomolar activity on MELK and inhibit the proliferation
of several cancer cell lines.20,22–26,35 As in the case of OTS167, X-
Ray crystallography or docking studies indicate they bind to the
“hinge” region in the “DFG-in” MELK active conformation (type
I inhibitors). On the other hand, compounds 8-9 are considered as
type II inhibitors, binding to the “DFG-out”, MELK inactive
conformation.27,28 In spite of a decent number of disclosed
inhibitors, the development of compounds with distinctive
MELK selectivities is still needed for the further characterization
of its biological functions and therapeutic potential. We present
herein our initial efforts in the identification of MELK inhibitors
based on the 5-keto-3-cyano-2,4-diaminothiophene scaffold
which may prove useful for the study of this target.
Figure 2. Reported kinase activity of 5-keto-3-cyano-2,4-diaminothiophenes.
The close succession of hydrogen bond donors (NH2) and
acceptors (CN, C=O) in the scaffold, as in compounds 2 and 5-8
(Figure 1, hinge binding atoms highlighted in blue), hinted at a
hinge backbone binding mode. Molecular modeling studies using
MELK crystallized with compound 2 (PDB 4BKY) supported
hypothesis that 17 binds as a type I inhibitor (Figure 3).
A high-throughput screening campaign of the Selvita
compound collection identified 16 and 17 (Table 1) as moderate
MELK inhibitor hits (≈ 5 M IC50). Literature search revealed
that among the diverse activities attributed to compounds built
around a 5-keto-3-cyano-2,4-diaminothiophene scaffold,36–45 only
weak kinase inhibition (2-digits M IC50) had been reported on
CK246 (compound 10, Figure 2) and p38 MAP Kinase
(compound 11).47 A 2005 patent claims activity of compounds 13
and 14 on TrkB, KIT, GSK-3, GSK-3 and MAPKAP, yet
without reporting IC50 nor % inhibition.48
Figure 3. Predicted binding mode for 17 in the MELK ATP binding site
(PDB code 4BKY used as model protein for docking, hydrogen bonds in
green, hydrophobic contacts in mauve, salt-bridge in orange, binding site
solvent accessible surface colored by hydrophobicity).
In this binding mode, the C=O and NH2 groups would form
hydrogen bonds with backbone atoms of the hinge residue
Cys89, and their suitable parallel orientation would be stabilized
by intramolecular hydrogen bonding. Additional interactions of
the iPrNH group with carboxylate of Glu93 would provide a
supplementary anchoring point in the active site. The phenyl
substituent would be buried in the lipophilic back pocket of the