Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.7b01714

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

Full text of DOI:10.1021/acs.jmedchem.7b01714

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Optimization of Selective Mitogen-activated Protein Kinase Interacting
Kinases 1 and 2 (MNK1/2) Inhibitors for the treatment of Blast Crisis Leukemia
Hai Yan Yang, Lohitha Rao Chennamaneni, Melvyn Wai Tuck Ho, Shi Hua Ang, Eldwin Sum Wai Tan,
Duraiswamy Athisayamani Jeyaraj, Yoon Sheng Yeap, Boping Liu, Esther HQ Ong, Joma Kanikadu Joy,
John Liang Kuan Wee, Perlyn Kwek, Priya Retna, Nurul Dinie, Thuy Thi Hanh Nguyen, Shi Jing Tai,
Vithya Manoharan, Vishal Pendharkar, Choon Bing Low, Yun Shan Chew, Susmitha Vuddagiri, Kanda
Sangthongpitag, Meng Ling Choong, May Ann Lee, Srinivasaraghavan Kannan, Chandra S. Verma,
Anders Poulsen, Sharon Lim, Charles Chuah, Tiong Sin Ong, Jeffrey Hill, Alex Matter, and Kassoum Nacro
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01714 • Publication Date (Web): 23 Apr 2018
Downloaded from http://pubs.acs.org on April 23, 2018
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Ong, Tiong; Duke-NUS Medical School; Department of Medicine, Duke
University Medical Center
Hill, Jeffrey; Experimental Therapeutics Centre, Medicinal Chemistry
Matter, Alex; Experimental Therapeutics Center, Agency of Science
Technology and Research
Nacro, Kassoum; Experimental Therapeutics Centre/ ASTAR, Medicinal
Chemistry
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Optimization of Selective Mitogenꢀactivated Protein Kinase Interacting
Kinases 1 and 2 (MNK1/2) Inhibitors for the treatment of Blast Crisis
Leukemia
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Haiyan Yang , Lohitha Rao Chennamaneni , Melvyn Way Tuck Ho , Shi Hua Ang , Eldwin Sum Wai
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Tan , Duraiswamy Athisayamani Jeyaraj , Yoon Sheng Yeap , Boping Liu , Esther HQ Ong , Joma
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Kanikadu Joy , John Liang Kuan Wee , Perlyn Kwek , Priya Retna , Nurul Dinie , Thuy Thi Hanh
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, Susmitha Vuddagiri , Kanda Sangthongpitag , Meng Ling Choong , May Ann Lee ,
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Anders Poulsen , Sharon Lim , Charles Chuah ,
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Tiong Sin Ong , Jeffrey Hill , Alex Matter Kassoum Nacro
,
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Experimental Therapeutics Centre (ETC), A*STAR, 31 Biopolis Way, Nanos #03ꢀ01, Singapore
138669
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Organic Chemistry, Institute of Chemical and Engineering Sciences (ICES), A*STAR, 8
Biomedical Grove, Neuros, #07ꢀ01, Singapore 138665
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Bioinformatics Institute (BII), A*STAR, 30 Biopolis Street, #07ꢀ01 Matrix, Singapore 138671
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School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,
Singapore 637551
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Department of Biological Sciences, National University of Singapore, 14 Science Drive 4,
Singapore 117543
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DukeꢀNUS Medical School, 8 College Road, Singapore, Singapore 169857
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Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
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*Corresponding Author: Kassoum Nacro, Ph.D., Experimental Therapeutics Centre, 31 Biopolis
Way, Nanos #03-01, Singapore 138667, Tel: +65 6407 0343, Fax: +65 6478 8768, email:
knacro@etc.a-star.edu.sg
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Keywords: MNK1/2 kinases, eIF4E, Blast crisis chronic myelogenous leukemia, leukemic stem
cells, selfꢀrenewal, tumor growth inhibition, MNK inhibitor, dasatinib, tyrosine kinase inhibitor,
imidazopyrazines, imidazopyridazines, imidazopyridines, structureꢀactivity relationship, lead
optimization.
ABSTRACT
Chronic Myeloid Leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a
constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of
leukemic stem cells (LSCs). Inhibition of BCRꢀABL1 kinase activity offers long term relief to CML
patients. However, for a proportion of them, BCRꢀABL1 inhibition will become ineffective at
treating the disease and CML will progress to blast crisis (BC) CML with poor prognosis. BCꢀCML
is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E)
which renders LSCs capable of proliferating via selfꢀrenewal, oblivious to BCRꢀABL1 inhibition. In
vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor
of MNK1/2 should reduce the level of phosphorylated eIF4E and reꢀsensitize LSCs to BCRꢀABL1
inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure activity
relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate,
compound 48 (ETCꢀ206), which in combination with dasatinib prevents BC CML LSC selfꢀrenewal
in vitro, and enhances dasatinib antitumor activity in vivo.
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Introduction
Messenger RNA (mRNA) translation is a highly regulated process pervasive to many biological
processes in which, eukaryotic translation initiation factor 4E (eIF4E), a cap dependent translation
factor plays a crucial role. eIF4E, one of the five MAP kinase interacting serine/threonine kinases 1
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and 2 (MNK1/2) substrates, is phosphorylated in vivo only by MNK1/2 on Ser209 and accordingly,
2
phosphoꢀeIF4E is not detected in Mnk1/2 knock out mice. Overexpression of phosphorylated eIF4E
3
ꢀ5
6
7ꢀ9
has been linked to inflammation, CNS disorders and malignancies such as glioblastoma, breast
1
0ꢀ12
13,14
15,16
cancer,
pancreatic ductal adenocarcinoma,
and leukemias.
Abnormally high mRNA
translation of oncogenic transcripts is thought to fuel the growth of cancers, including chronic
myeloid leukemia (CML), where tumorigenic pathways are activated through eIF4E overexpression
17ꢀ22
and/or phosphorylation.
Specifically, blast crisis chronic myeloid leukemia (BC CML) is a dire
stage of CML, where patients are no longer responsive to treatment with BCRꢀABL1 kinase
23
24,25
inhibitors such as imatinib, dasatinib or nilotinib,
that in most cases ensure long term survival to
1
6,17,26
patients in chronic phase (CP) CML. BC CML patients have high levels of nuclear βꢀcatenin,
27,28
MNK1/2, eIF4E and phosphorylated eIF4E
that are believed to promote not only resistance to
treatment with BCRꢀABL1 inhibitors, but also stimulate a proliferation of leukemic stems cells
1
6,29
30
28
(LSCs)
via selfꢀrenewal. Lim et al have shown that phosphoꢀmimetic eIF4E (eIF4Eꢀ209D)
elicits LSC selfꢀrenewal ability whereas the unꢀphosphorylatable eIF4E (eIF4Eꢀ209A) does not.
They have, along with others, also shown that inhibition of βꢀcatenin impairs the ability of primary
16,26
BC CML cells to selfꢀrenew to form colonies
and that the tumorigenic MNK/eIF4E/βꢀcatenin
2
8,31,32
axis plays an important role in BC CML as well as in other cancers.
33
The current best and only option to cure BC CML is an allogenic bone marrow transplantation,
33
(only beneficial to a small proportion of patients ) and consequently, longꢀterm treatment of patients
with BC CML remains an unmet medical need.
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Pharmacologic and knockꢀdown inhibition of eIF4E phosphorylation by MNK1/2 prevents βꢀcatenin
28,31,32
28
activation
and suppresses the ability of LSCs to selfꢀrenew, indicating that selective MNK1/2
kinase inhibition by a small molecule could be an avenue to treat patients with BC CML. In addition,
selective MNK1/2 kinase inhibition may be associated with minimal adverse effects as Mnk knockꢀ
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out mice are viable, healthy and have normal haematopoiesis.
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4,35
36ꢀ38
Several inhibitors of MNK1/2 kinases have recently been reviewed,
notably CGP57380
and
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cercosporamide. Both cercosporamide and CGP57380 lack kinase selectivity and the latter is a
weak MNK1/2 inhibitor with potency in the low micromolar range. Theodosia et al. have revealed
40,41
MNK inhibitors with antiꢀproliferative activity against AML cell lines.
Likewise, merestinib, a
nonꢀselective MEK inhibitor was also shown to inhibit MNK1/2 in AML progenitors with antiꢀ
4
2
leukemic effects. The effectiveness of pharmacologic coꢀinhibition of MNKs and mTORC1 against
43,44
45
BC CML cells was recently published.
Han et al. have disclosed potent MNK1/2 inhibitors but
revealed no disease relevant biological data. Recently, two MNK inhibitors BAY1143269 and
4
6,47
eFT508 have progressed to clinical trial. BAY1143269
a predominantly MNK1 inhibitor was
48
administered in a phase I clinical study to patients with advanced solid tumors. eFT508 a potent
and selective MNK1/2 inhibitor is in phase II for the treatment of lymphoma, colorectal cancer, triple
49ꢀ51
negative breast cancer and hepatocellular carcinoma.
To date there is little efficacy data and very few publications supporting the use of small molecule
MNK inhibitors to treat BC CML. We have recently published our discovery efforts to generate a
5
2
dual MNK and BCRꢀABL1 inhibitor for the treatment of BC CML with a single agent and we
report herein a complementary approach with the design, synthesis and structure activity
relationships of very selective MNK1/2 inhibitors. These compounds intentionally lack BCRꢀABL1
inhibitory activity, and therefore can potentially be combined with the appropriate tyrosine kinase
inhibitor for the treatment of BC CML patients. Starting from fragments, small molecule inhibitors
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of MNK1/2 were optimized and a nominated preclinical development candidate was tested in a K562
mouse xenograft model.
Chemistry
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The compounds described herein fall into 3 classes following the 3 central bicyclic rings explored,
imidazopyrazine, imidazopyridazine or imidazopyridine.
Imidazopyrazines
Compounds 4 and 5, as shown in Scheme 1, were prepared in 3 steps starting from commercially
available 6ꢀbromoimidazo[1,2ꢀa]pyrazine 1, which was submitted to a copper mediated amination
leading to 6ꢀamide imidazopyrazines 2a,b. The amide derivatives were brominated using NBS to
give 3ꢀbromoꢀ6ꢀamide imidazopyrazines 3a,b, which were subjected to a Suzuki cross coupling
reaction to afford 4 and 5 respectively. Compound 8 was prepared in two successive Suzuki coupling
steps, as depicted in Scheme 1, with 66% overall yield.
Scheme 1. Synthesis of Nꢀ(3ꢀ(4ꢀhydroxyphenyl)imidazo[1,2ꢀa]pyrazinyl)ꢀ6 amides 4 and 5, and 6-
a
phenyl 8
a
Reagents and conditions: (i) acetamide or benzamide, N,Nꢀdimethylethylenediamine, K CO , CuI,
2
3
Toluene, 90 °C, 12 h; (ii) NBS, CH
2 2 2 3 2
Cl , 1 h; (iii) (4ꢀhydroxyphenyl)boronic acid, Na CO , Pd(OAc) ,
P(PPh ) , 1,4ꢀdioxane, 90 °C, 12 h; (iv) (4ꢀhydroxyphenyl)boronic acid, Na CO , Pd(PPh ) , 1,4ꢀdioxane, 90
3
4
2
3
3 4
2 2 2 3 2
°C, 12 h; (v) phenylboronic acid, Pd(dppf) Cl , Cs CO , DMF, H O, 140 °C, 30 min.
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Imidazopyrazine analogs 19-33, shown in Scheme 2, were prepared in a similar manner from 1.
Beginning with a Suzuki coupling to introduce a paraꢀbenzoyl ester at the imidazopyrazine 6ꢀ
position to give 9, which was halogenated at Cꢀ3 with either NBS or NIS to afford compounds 10
and 11. The esters were hydrolyzed to carboxylic acids 12 and 13 that, in turn were converted to
amides 14-18. Arylation at Cꢀ3 using a second Suzuki cross coupling afforded 19-25 and 27-33.
Carboxylic acid 25 was converted into a protected hydroxamic acid intermediate, which was treated
with an aqueous solution of hydrochloric acid to afford 26.
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a
Scheme 2. Synthesis of imidazopyrazine analogs 19-33
a
Reagents and conditions: (i) (4ꢀ(ethoxycarbonyl)phenyl)boronic acid, Pd(dppf)
2 h; (ii) NBS, MeOH, CCl or NIS, DMF; (iii) LiOH, H O, THF, CH
DMF; (v) arylboronic acid, Pd(PPh , K PO , Na CO , H O, 1,4ꢀdioxane, 90 °C
EDCI•HCl, Oꢀ(tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)hydroxylamine, CH Cl , DMF, rt, 12 h; (vii) 1M aq. HCl, CH CN,
2
Cl
2
, Cs
2 3
CO , toluene, 90 °C,
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1
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2
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OH; (iv) R R NH, HATU, NMM,
3
)
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2
, 12 h; (vi) DIPEA, HOBt,
2
2
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MeOH, rt, 4 h.
Compounds 36 and 37 were synthesized in 3 steps comprising a Suzuki coupling, ester hydrolysis
and amide formation as depicted in Scheme 3.
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a
Scheme 3. Synthesis of imidazopyrazine analogs 36 and 37
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3 4 2 3 2
Reagents and conditions: (i) (4ꢀ(ethoxycarbonyl)phenyl)boronic acid, Pd(PPh ) , Na CO , H O, DMF, 90 °C,
1
3
2 h for 34a, 2 h for 34b; (ii) LiOH, THF, CH OH, rt, 5 h for 35a and 12 h for 35b; (iii) 1ꢀ(piperazinꢀ1ꢀ
yl)ethanꢀ1ꢀone or N,Nꢀdimethylpiperidinꢀ4ꢀamine, HATU, NMM, DMF, rt, 3 h.
Imidazopyridazines
As illustrated in Scheme 4, compounds 39 and 40 were both prepared in one step from 38.
Compound 42 was synthesized in two steps starting with a Suzuki cross coupling between 38 and 4ꢀ
boronobenzoic acid to give 41, which was converted to amide 42. Compound 45 was also prepared
in 2 steps from commercially available acid 43, which was converted to amide 44 followed by a
Suzuki cross coupling reaction to give 45.
a
Scheme 4. Synthesis of imidazopyridazine analogs 39, 40, 42 and 45
a
Reagents and conditions: (i) (4ꢀ(4ꢀmethylpiperazineꢀ1ꢀcarbonyl)phenyl)boronic acid, Pd(PPh
CO , 140 °C, microwave reactor, 30 min; or morpholino(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ
ꢀyl)phenyl)methanone, Pd(dppf) Cl , DMF, H O, Cs CO , microwave reactor, 140 °C, 30 min; (ii) 4ꢀ
boronobenzoic acid, Pd(dppf) Cl , DMF, H O; Cs CO , 90 °C, 18 h; (iii) tertꢀbutyl piperazineꢀ1ꢀcarboxylate,
3 4
) , Toluene,
EtOH, K
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HATU, NMM, DMF, rt, 18 h; (iv) TFA, CH
2 2
Cl , rt; (v) N,Nꢀdimethylpiperidinꢀ4ꢀamine, HATU, NMM,
DMF; (vi) (4ꢀcyanophenyl)boronic acid, K CO , Pd(PPh ) , 1,4ꢀdioxane, H O, 90 °C, 6 h.
2
3
3 4
2
Imidazopyridines
The preparation of 47 and 48 is depicted in Scheme 5.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 5. Synthesis of imidazopyridine 47 and 48
a
Reagents and conditions: (i) (4ꢀmethylpiperazinꢀ1ꢀyl)(4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀ
yl)phenyl)methanone, (AꢀPhos) PdCl , NaHCO , DMF, H O, 90 °C, 1 h; or morpholino(4ꢀ(4,4,5,5ꢀ
2
2
3
2
3 4 3 4 2
tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)phenyl)methanone, Pd(PPh ) , K PO , 1,4ꢀdioxane, H O, 90 °C, 19 h.
RESULTS AND DISCUSSION
5
3
Fragment inhibitors of MNK1 published by Oyarzabal et al. offered a diversity of scaffolds among
which, a few imidazopyridazine analogs displayed high ligand efficiencies ranging from 0.32ꢀ0.52
kcal/mol per heavy atom. We found the imidazopyridazine scaffold to be an attractive starting point
to generate novel inhibitors of MNK1/2 with drugꢀlike properties, suitable for proof of concept in
man. Accordingly, we gave it the highest priority.
Docking studies suggested that interactions with MNK1/2 could be improved by varying the bicyclic
core and by modifying the substituents at Cꢀ3 and at Cꢀ6, whose alterations would considerably
widened the SAR scope. Bicyclic cores were only selected, if modeling proposed a binding mode to
the kinases similar to that of the imidazopyridazine. All these taken together suggested a need for a
structureꢀactivityꢀrelationships (SAR) exploration of bicyclic analogs bearing substituents at both Cꢀ
3, Cꢀ6. Accordingly, our strategy was to divide this scaffold into 3 parts as outlined in Figure 1,
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2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
6
where each part was investigated and the best of each was combined to give an optimized MNK1/2
inhibitor.
Figure 1. SAR exploration strategy
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Modeling: We relied on molecular modeling to understand how our bicyclicꢀderived inhibitors
interact with MNK1/2 and to guide optimization of their potency. A common structural feature in
5
4
both MNK kinases is the DFD motif, in which an aspartic acid Asp193 and Asp228 for MNK1 and
MNK2 respectively, replaces the glycine of the DFG motif that is more common to kinases. A DFDꢀ
5
5
out structure of MNK1 (2HW6) is available while MNK2 has been crystalized in both the DFDꢀin
5
5
54
(
2HW7) and DFDꢀout (2AC3) conformations. To validate our modeling, we searched the PDB
for a coꢀcrystal structure with inhibitors having the same scaffold. We found a structure [4YMJ] of
TRK kinase in the DFGꢀout conformation with a 4ꢀ(imidazoꢀpyridazinꢀ3ꢀyl)ꢀbenzonitrile inhibitor.
This compound has the same core as some of our inhibitors and docking studies supported that it
bind similarly to the TRK and MNK kinases. Consequently, we used the MNK DFDꢀout
conformation in our modeling studies. The ATP binding site of MNK1 and MNK2 share high
5
6
5
7
sequence homology and modeling indicates that the compounds bind similarly to both kinases.
Specifically, the bicyclic core acceptor nitrogen (Nꢀ1) hydrogen bonds with the amide backbone of
the kinase hinge residues Leu127 in MNK1 and Met162 in MNK2. The Cꢀ3 substituents reside in the
hydrophobic pocket formed by residues Phe124, Val63, Leu108 and Cys190 in MNK1 and the
homologous residues Phe159, Val98, Leu143 and Cys225 in MNK2. The substituents at the core 6ꢀ
position occupy the phosphate binding pocket close to the ATP ribose binding pocket. A major part
of the inhibitor is buried inside the protein except for the substituents at the 6ꢀposition, which interact
with the glycineꢀrich Pꢀloop and the loop connecting the hinge region and helix αD of the Cꢀterminal
lobe. Compounds were docked into MNK1 and MNK2 using the automated docking program Glide
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2017ꢀ3 release using standard settings, and showed that top ranked poses had the same binding
mode as when manually modeled. The compounds described in this manuscript interact mostly with
the same amino acid residues in both MNK1 and MNK2. Furthermore, they inhibit both MNK1 and
MNK2 with similar potency (less than 2 fold difference in all cases). In light of the similarities of the
amino acids engaged in compound binding to MNK1/2 and the similarity between the inhibitory
activities of the compounds against both MNK1 and MNK2, we will only discuss the modeling of
inhibitors bound to MNK2.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
52
The inhibitory activity of all compounds against MNK1/2 was assessed in a biochemical assay with
ATP at Km concentration of 1.2 mM and 250 ꢀM for MNK1 and MNK2 assays respectively.
Compounds with a potency of 1 ꢀM or lower against either MNK1 or MNK2, were assayed in a
52
HeLa cell line for their ability to inhibit the phosphorylation of eIF4E by MNK1/2. This functional
assay relies on a sandwich immunoassay to quantify phosphorylated eIF4E. In both enzymatic and
cell based assays, each compound was tested in a dose dependent manner, in duplicate, using 8ꢀ
points from a 3ꢀfold serial dilution. IC50 values were determined by nonꢀlinear regression using
GraphPad prism and the values reported are the average of at least two experiments.
C-6 substituent variation. A quick probing of bicyclic scaffolds led to the imidazopyrazine
derivatives is shown in Table 1. The acetamide of 4 was replaced by a benzamide as in 5 which
resulted in potency loss. The inhibitory activity towards MNK1/2 was restored by reversing the
benzamide in 5 resulting in 19. The primary amide function was removed to yield compound 8,
which was slightly less potent than compound 19, suggesting that the amide of 19 contributes to
potency.
1
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1
1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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Table 1. SAR of MNK1 and MNK2 inhibitor analogs 4, 5, 8 and 19
IC50 (ꢀM)
a
a
b
#
4
R
1
MNK1
0.789
3.35
MNK2
0.515
1.08
HeLa
>2.5
na
AcNH
PhCONH
Ph
0
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9
0
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9
0
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9
0
1
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5
6
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9
0
1
2
3
4
5
6
7
8
9
0
5
8
0.528
0.310
0.415
0.193
0.819
1.08
19
4ꢀPhCONH
2
a
In vitro assay were compounds’ ability to inhibit MNK1 or MNK2 phosphorylation of a peptide
substrate is determined as IC50 values from the average of at least 2 measures. In an in vitro assay
using HeLa cell line where compounds ability to inhibit MNK1/2 phosphorylation of eIF4E on
Ser209 and IC50s were determined as the average of at least 2 measures. na: not available.
Figure 2. Compound 4 (Table 1) modeled into the MNK2 DFDꢀout Xꢀray structure (2AC3). The
binding site residues of MNK2 are shown in thin tube representation with grey carbon atoms. 4 is
shown in thick tube with green carbon. Hydrogen bonds between MNK2 and 4 are shown with
yellow dashed lines, πꢀπ interactions with magenta dashed lines and aromatic hydrogen bond with
blue dashed line.
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Modeling of 4 (Table 1) into the ATP site of MNK2 show that the imidazole Nꢀ1 hydrogen bond
acceptor forms a hydrogen bond to the kinase hinge (Figure 2), very similar to that established by the
Nꢀ1 of the adenine base of ATP. This is the backbone NH hydrogen bond donor of residues Leu127
in MNK1 and Met162 in MNK2. This hydrogen bond is stable during the Molecular Dynamics (MD)
simulations (Supporting Information, MD file compound 4). The 2ꢀposition of the imidazopyrazine
is pointing towards the oxygen of the backbone carbonyl of Glu160 and the 8ꢀposition is pointing
towards the oxygen of the backbone carbonyl of Met162.
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2
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6
0
1
2
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6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
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8
9
0
1
2
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9
0
1
2
3
4
5
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7
8
9
0
Interactions between the aromatic hydrogen at the 2ꢀ and 8ꢀpositions and the backbone carbonyls are
5
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expected to be favorable to inhibitor binding to the kinases. When either the 2ꢀ or 8ꢀposition is
substituted all activity is lost (Supporting Information Table S1).
The phenyl in the 3ꢀposition has extensive hydrophobic interactions with the kinase. It is sandwiched
between the gatekeeper residue Phe159 and the DFDꢀmotive residue Phe227 and engages in Van der
Waals contacts with the side chains of Val98, Ala111, Leu143, Cys225 and Leu212. The hydroxyl of
4 (Table 1) forms a stable hydrogen bond per MD simulations, with the backbone carbonyl of
Asp226 from the DFD motif. The acetamide is solvent exposed on one side and binds in a largely
hydrophobic area, where it interacts with the side chains of Leu90, Leu212 and Phe227. During the
MD simulations, the nitrogen or carbonyl of the amide (4, 5) periodically hydrogen bonds with the
backbone NH or sidechain of Ser166 either directly or water mediated. However these interactions
are not stable during the MD simulations and probably do not contribute much to the binding
affinity.
The substitution of the methyl of acetamide (4) for a phenyl (5) led to a drop in potency, possibly due
to a steric clash between the phenyl and Leu212 of the glycineꢀrich loop. Removing the amide linker
favors a better interaction between the phenyl directly connected to Cꢀ6 (8, 19) and residues Leu90,
Leu212 and Phe227. Furthermore, the backbone NH of Ser166 is pointing towards the face of the
phenyl resulting in an attractive electrostatic interaction between the negative π cloud of the phenyl
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2
2
2
2
2
2
2
2
3
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3
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3
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and the partial positive charge of the NH donor (Figure 3). These observations are in favor of the
improvement in potency observed when a phenyl occupies the Cꢀ6 position. The amide of 19
marginally improved potency relatively to 8, probably due to a hydrogen bond between the carbonyl
and the side chain hydroxyl of Ser166. Both compounds 8 and 19 inhibit eIF4E phosphorylation in
cells with similar potencies and 19, exhibited a good permeability and favorable in vitro ADME
properties as shown in Table 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Profile of Hit Compound 19
a
Average of at least two determinations except
a
Assay/Property
Value
330.35
2.03
b
MW. Distribution coefficient between 1ꢀ
molecular weight (g/mol)
octanol and aqueous phosphate buffer at pH 7.4.
b
LogD7.4)
c
Parallel Artificial Membrane Permeability
ꢀ6
c
PAMPA (10 cm/s)
22.7
d
Assay at pH 7.4. Half life in human and mouse
d
HLM/MLM T1/2 (min)
60/46
>20/>20
e
liver microsomes. IC50 for Cyp 3A4 and 2D6
e
Cyp 3A4/2D6 IC50 (ꢀM)
inhibition.
The primary amide of 19 was replaced by larger amides to yield Nꢀmethylꢀpiperazine benzamide 20
and morpholine benzamide 32 (Table 3), both equipotent with 19, but with improved permeability
and solubility. The inhibitory activities of 19, 20, 32 and 36 showed that the substituents represented
1
by the R equal to O, NCH , NAc (Table 3) had a moderate impact on inhibitory potency towards
3
MNK1/2. Modeling (Figure 3) shows that the phenyl at 6ꢀposition makes Van der Waals contacts
with the side chains of Leu90, Leu212, Ser166 βꢀcarbon and Gly165 αꢀcarbon. In addition, to the
favorable interaction between the NH of Ser166 and the phenyl at Cꢀ6 described above, the
piperazine substituent makes Van der Waals contacts with the methylenes of Glu92 and Glu209.
However, these residues are on the edge of the binding site and are solvated. Any interaction
between the piperazine basic nitrogen and either acid is not likely to contribute much to the binding
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affinity. The methyl piperazine benzamide 20 and morpholine benzamide 32 amides appear to be the
best overall Cꢀ6 substituents.
C-3 substituent variation. We next kept the methyl piperazine benzamide, our best solubilizing
group, constant at Cꢀ6, and explored derivatization at Cꢀ3. Moieties such as pyridine derivatives,
pyrimidine, bicyclic heterocycle, carbonyl bridged heterocycles failed to improve potencies relative
to a phenyl substituent (Supporting Information Table S2). These observations are in agreement with
modeling studies, that support the hydrophobic nature of the binding site occupied by the phenyl at
Cꢀ3. Consequently, our SAR investigations were then narrowed to only substituting the para position
of phenyl at Cꢀ3. Removing the hydroxyl (21) or moving it to the ortho (22) or meta (23) positions
resulted in a 5 to 30 fold loss in potency. The following para substituents illustrate how this part of
the binding pocket can accommodate both hydrophobic and hydrophilic groups. The OH was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3 3 2
replaced by CH (24), COOH (25), CONHOH (26), CN (27), Cl (28), CF (29), CHF (30) and
CONH (31) and led to derivatives that inhibit MNK1 and MNK2 with similar potencies as
2
compared to 20.
The hydroxamic acid derivative 26 showed a substantial improvement in potency relative to 20, but
26 as well as the primary amide (31) and acid (25), all had poor permeability and consequently
lacked cellꢀbased activity. The carboxylic acid was replaced by a tetrazole (Supporting Information
Table S3) to give an equipotent compound, but with a bad permeability and practically no cell
activity. The benzonitrile 27, with a LogD of 1.67 (Table 3) is less lipophilic than analogs of similar
potency (28, 29, 30) and displayed excellent solubility and permeability properties. Accordingly, the
benzonitrile group was selected as the preferred substituent at Cꢀ3. At this stage, the improved
compounds, exemplified by 27 and 33, exhibited an imidazopyrazine with a benzonitrile at Cꢀ3 and
methylpiperazine or morpholine benzamide at Cꢀ6.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. SAR of imidazopyrazine derivatives 20-33, 36, 37 and their physicochemical properties
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC50 (ꢀM)
PAMPA
Solubility
(ꢀg/mL)
114
#
R
1
R
2
LogD7.4
a
a
b
-6
MNK1
0.284
2.68
MNK2
HeLa
(10 cm/s)
20
NCH
NCH
NCH
NCH
NCH
NCH
NCH
NCH
NCH
NCH
NCH
NCH
O
3
3
3
3
3
3
3
3
3
3
3
3
4ꢀOH
H
0.140
1.43
0.189
1.92
na
2.25
na
34.7
na
21
22
23
24
na
2ꢀOH
3ꢀOH
9.50
5.17
na
na
na
1.30
0.742
0.410
0.355
0.031
0.502
0.217
0.389
0.336
0.187
0.113
0.454
0.160
0.143
1.33
1.21
>2.5
>2.5
0.440
0.759
1.99
1.63
>2.5
0.130
0.748
>2.5
0.640
2.31
na
33.2
na
116
4ꢀCH
4ꢀCO
3
0.600
0.700
0.060
0.499
0.331
0.574
0.369
0.294
0.125
0.570
0.224
0.238
na
25
2
H
ꢀ1.37
na
0.10
na
>22
26
27
28
29
4ꢀCONH(OH)
4ꢀCN
na
1.67
2.96
2.95
2.55
0.98
2.21
1.77
1.68
0.73
102
71.3
87.4
76.2
1.78
50.2
43.1
2.82
>125
118
4ꢀCl
4ꢀCF
3
90.4
66.9
126
30
4ꢀCHF
2
31
32
33
36
4ꢀCONH
4ꢀOH
2
11.5
11.1
144
O
4ꢀCN
NCOCH
3
4ꢀOH
37
CHN(CH
3
)
2
4ꢀCN
64.7
>45
a
b
In vitro IC against MNK1 and MNK2; Average of at least two determinations. eIF4E
50
phosphorylation inhibition measured using HeLa cell line, average of at least two determinations. na:
not available.
MNK2 Xꢀray structures were examined to understand why the para position of the Cꢀ3 phenyl
tolerates both hydrophobic and hydrophilic groups. Figure 2 represents 4 docked into MNK2 and
shows that the para position is surrounded by hydrophobic residues Val98, side chain methylenes of
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Lys113, Phe227 and Phe159. Consequently, compounds with small hydrophobic para substituents
like Me (24), Cl (28), CF
than compound 21 the unsubstituted analog. On the other hand, compounds with more polar
substituents such as 4ꢀOH (20), 4ꢀCO H (25), 4ꢀCONHOH (26), 4ꢀCN (27) and 4ꢀCONH (31) also
3 2
(29) and CHF (30) have more hydrophobic interactions with the kinase
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
have good inhibitory activity that could be the result of hydrogen bonding to the backbone carbonyl
of Asp226 for hydrogen bond donating substituents (Figure 2) or with the basic side chain of Lys113
for acidic or hydrogen bond accepting groups as shown in Figure 3.
Figure 3. Compound 27 modeled into MNK2 DFDꢀout Xꢀray structure (2AC3). The binding site
residues of MNK2 are shown in thin tube representation with grey carbon atoms. 27 is shown in
thick tube drawing with green carbons. Hydrogen bonds between MNK2 and 27 are shown with
yellow dashed lines, πꢀπ interactions with magenta dashed lines and aromatic hydrogen bond with
blue dashed line.
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
6
Modeling studies showed that the cyano group of 27 forms a hydrogen bond with Lys113 and during
an MD simulation (Supporting Information, MD file compound 27), this hydrogen bond was present
for 60% of the time. The increased potency of 26 could be the result of two hydrogen bond
interactions between the NH and OH of the hydroxamic acid group to Asp226 and Lys113,
respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Microsomal stability, Cyp inhibition, permeability and plasma protein binding properties of
selected compounds
a
b
d
HLM/MLM
T1/2 (min)
41/37
RLM/DLM
Cyp 3A4/2D6
IC50 (ꢀM)
>20/>20
>20/>20
>20/>20
>20/>20
>20/>20
5.6/na
Cacoꢀ2
Papp AꢀB
6.87
Cacoꢀ2
Efflux ratio
5.6
Human PPB
Mouse PPB
(%)
#
c
T1/2 (min)
(%)
98.4
na
2
7
3
7
9
na
na
87.1
na
3
3
3
>60/>60
>60/>60
17/23
17.9
1.73
na
1.14
7.57
na
na
na
14.6
4.84
99.9
99.9
na
95.9
94.5
na
40
53/>60
>60/42
47/>60
20/11
>60/>60
na
38.5
0.64
42
45
47
48
1.13
29.9
na
na
3.00
13.27
4.84
na
na
na
3.9/>20
>20/>20
5.43
99.7
90.33
84.2
48/56
>60/>60
30.50
1.42
99.7
a
b
HLM/MLM: metabolic stability measured in human and mouse liver microsomes. RLM/DLM:
c
ꢀ6
d
metabolic stability determined in rat and dog liver microsomes. Papp in 10 cm/s; PPB: plasma
protein binding; na: not available.
C-6 substituent variation. We reexamined the suitability of benzamide moieties at Cꢀ6 to verify that
the SAR established with the Cꢀ3 phenol derivatives holds true for the Cꢀ3 benzonitrile analogs.
Accordingly, two benzonitrile derivatives, morpholino (33) and N,Nꢀdimethylamino piperidinyl (37)
were generated. 27 and 33 are equipotent towards MNK1/2 inhibition, just as the phenolic
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derivatives 20 and 32 are. Analogs 27, 33 and 37, were all of similar potency towards MNK1
inhibition, but for MNK2, 37 is 3 and 4 fold more potent than 27 and 33 respectively.
Compounds 27, 33 and 37 were found to have favorable ADME properties as shown in Table 4.
They were administered orally to mice, and 27 with a bioavailability of 48%, was the fastest
absorbed, with a Cmax and AUC was 4ꢀ6 time higher than those of 33 and 37 (Supporting Information
Table S4). Compound 27 achieves an excellent selectivity and inhibits only 4 kinases at more than
50% out of 104 kinases when tested at a concentration of 10 ꢀM (Supporting Information Table S5).
Overall, 27, combines excellent physicochemical properties (Table 3) with good in vitro and in vivo
ADME properties, excellent PK properties and kinase selectivity profile. Thus, compound 27 was
selected as the lead compound for the lead optimization phase.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Multi-parameter Lead Optimization
Increasing potency: swapping cores. Alteration at Cꢀ3 and Cꢀ6 of the imidazopyrazine core
produced 27 with an inhibitory potency of 0.5 ꢀM against MNK1 and MNK2, in both enzymatic and
in cellular assays. At this stage, the core is the only part of the scaffold that was not modified and we
60ꢀ62
thought that its alterations could modulate potency and/or ADME properties as done by others.
Accordingly, in addition to imidazopyrazine, six bicyclic rings were investigated (Table 5 and
Supporting Information Table S6). Modeling studies have shown that their binding mode to MNK1/2
remain similar to that of imidazopyrazine analogs, and all derived analogs have in common the Nꢀ1
binding to hinge Ser166, the CN hydrogen bonding to Lys223, and exhibited previously described
hydrophopic interactions for phenyl groups at both Cꢀ3 and Cꢀ6.
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1
1
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1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Influence of the bicyclic core on potency: from imidazopyrazine to imidazopyridazine and
imidazopyridine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
IC50 (ꢀM)
PAMPA
Solubility
1
2
#
X
X
R
LogD7.4
MNK1a
MNK2a
b
-6
HeLa
(10 cm/s)
(ꢀg/mL)
c
2
7
N
CH
N
NCH
NCH
O
3
0.499
0.020
0.023
0.016
0.009
0.084
0.502
0.025
0.041
0.026
0.011
0.072
0.086
0.440
0.326
0.421
0.220
0.431
0.229
0.321
102
25.0
30.0
45.8
75.3
81.9
74.4
1.67
2.82
2.10
2.00
2.19
2.48
2.61
>125
86.0
1.80
115
3
9
CH
CH
CH
CH
CH
CH
3
4
0
2
N
4
N
NH
45
N
CHN(CH
3
)
2
148
4
7
8
CH
CH
NCH
O
3
137
4
0.064
55.4
a
b
In vitro IC50 against MNK1 and MNK2. eIF4E phosphorylation inhibition measured using HeLa
c
cell line. 27 was shown to illustrate the bicycle ring influence on potency in one table. IC5 are the
0s
average of at least two determinations.
Briefly, in comparison to the imidazopyrazine 27, [1,2ꢀa]pyrimidine and 1Hꢀimidazo[4,5ꢀc]pyridine
analog (Supporting Information Table S6) was less potent, and 1Hꢀbenzo[d]imidazole derivative
(Supporting Information Table S6) was equipotent to 27. In contrast, the pyrazolo[1,5ꢀa]pyrimidine
analog was 10 fold more potent than 27 (Supporting Information Table S6), and compounds from
pyrazolo[1,5ꢀa]pyrimidine series unveiled similar SAR, physicoꢀchemical and PK properties as
imidazopyridazine analogs. Therefore, we focused on the imidazopyrazine, imidazopyridazine and
imidazopyridine cores, and derived analogs as shown in Table 5.
Similarly, imidazopyridazine analogs 39, 40 and 42 are more potent than imidazopyrazine analogs 27
and 33. This trend is also observed when the imidazopyrazine core is replaced by an imidazopyridine
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with an improvement in potency, of 6ꢀ9 fold for MNK1 and of 5ꢀ7 fold for MNK2, seen when
comparing imidazopyrazines 27, 33 to imidazopyridines 47, 48.
In HeLa cells, inhibitory data of eIF4E phosphorylation shown in Table 5 are clustered between 0.22
and 0.44 ꢀM. This inhibition is often weaker by 3 to 47 fold in whole cell as compared to cell free
assays, probably due to permeability issues (39, 40, 42), nonspecific binding or a combination of
both. Highly permeable compounds (27, 47, 48) exhibit a smaller potency difference between
enzymatic and cellular data, and 27, the most permeable compound, is equally potent in both assays.
Compound 45 is 47ꢀfold less potent in cellular assays despite a good PAMPA permeability.
However, 45 has a high Cacoꢀ2 efflux ratio of 13 (Table 4) that could explain the loss of potency.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To rationalize the impact of N-7 on potency we examined the structural models which revealed that
the imidazopyrazine Nꢀ7 (Fig 2 and 3) is not involved in any hydrogen bond in any of the docked
complexes studied. On the contrary, it resides in a hydrophobic environment and has Van der Waals
interactions with Gly165 alpha carbon and Leu90 side chain as visualized in Figures 2 and 3. This
may account for part of the approximately 5 to 10 fold gain in potency observed when this Nꢀ7
63
nitrogen is replaced by a CH (compare 33 to 48). Indeed, our previous modeling studies have also
shown the important contribution of hydrophobic interactions to the potency of the compounds.
It may be surprising that compounds with an imidazopyridazine scaffold were also substantially
more potent than the imidazopyrazine analogs as both cores have two nitrogen atoms with a lone pair
that can be solvated. The N-5 of the imidazopyridazine is also surrounded by hydrophobic residues
(Leu90 and Leu212). We believe that the difference in exposure of the lone pairs plays a determining
role in the influence of the core on potency. In the imidazopyrazines the lone pair is shielded by the
Cꢀ6 phenyl where as in the imidazopyridazines the lone pair is shielded by both the Cꢀ3 and Cꢀ6
phenyls. This impacted both the interaction between the ligand and protein as well as the solvation
energy.
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1
2
2
2
2
2
2
2
2
2
2
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 4. A single water molecule was placed
in front of the imidazopyrazine N-7 (A) and
imidazopyridazine N-5 (B). The complexes
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were minimized using DFTꢀB3LYP 6ꢀ31G**.
DFT calculations indicate that the desolvation penalty for the imidazopyrazine N-7 may be much
higher than that of the imidazopyridazine N-5 and it costs 10.8 kJ/mol more to remove the water
from the imidazopyrazine complex in Figure 4.
In contrast to the benzamide minor influence on binding in imidazopyrazine series, the 4ꢀN,Nꢀ
dimethylamino piperidine 45 in the imidazopyridazine series, is substantially more potent than
piperazine and morpholine benzamide analogs 39 and 40 respectively.
The piperidineꢀdimethylamine (45) in Figure 5 is sandwiched between hydrophobic residues Gly91,
Leu168, Phe227 while the base may interact with the acids Glu92 and Glu209. Figure 5 shows the
interactions of the dimethylamino piperidine group 45 with Glu209 and Phe227 of MNK2. In the
MD simulations the basic nitrogen interacts with the side chain of Glu209 during most of the
simulation. However, when bound to MNK2, this part of the inhibitor is solvent exposed and solvent
effects are likely to shield the contributions of any electrostatic interactions and they may not
contribute much to the binding affinity.
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1
1
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2
2
2
2
2
2
2
2
2
3
3
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3
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3
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4
4
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5
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Compound 45 docked into MNK2 catalytic site. MNK2 is shown as an electrostatic
surface. The inhibitors are shown in stick with green carbon and a transparent green surface.
We speculate that the N,N-dimethylamino piperidine interacts better with Phe227 and Glu209 than
the Nꢀmethyl piperazine. During the MD simulations (Supporting Information, MD file compound
45), the interactions with these residues were stable for 45 while they were only observed during
50% of the simulations for 20.
ADME and PK
Modulation of ADME Properties: correcting issues related to N-alkylated benzamides.
A selection of compounds, shown in Table 4, were evaluated in vitro to assess their metabolic
stability, Cyp inhibition and protein binding properties. It appears that substituting the
imidazopyrazine core effectively led to more potent derivatives 39 and 47 (Table 5), but with an
increased metabolic liability (Table 4). Methylpiperazine 39 and 47 are unstable in human and mouse
liver microsomes, and this is most likely attributable to Nꢀdemethylation. To address that issue
morpholine 40, piperazine 42, and N,Nꢀdimethylamino piperidine 45 derivatives were generated and
were found to be metabolically stable. Unfortunately, both piperazine 42 and dimethylamino
piperidine 45 derivatives were subject to a high efflux ratio (Table 4) which might negatively impact
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2
2
2
2
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5
5
5
5
5
6
their PK properties. Morpholine derivatives 40 and 48 showed low metabolic liability in mouse, rat,
dog and human microsomes, low Cyp 3A4 and 2D6 inhibition and they exhibited a higher cell
permeability as per the Cacoꢀ2 assay (Table 4) compared to the basic piperazine and piperidine
analogs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Parmacokinetic Properties. In spite of the high efflux ratio of 45, it was orally administered to mice
at 5 mg/kg and gave a poor systemic exposure with a Cmax of 69 ng/mL and an AUC0ꢀinf of 347
ng.h/ml. In contrast, 40 and 48 exhibited higher exposure and substantially improved PK properties
relative to the lead 27 when comparing AUC and Cmax values (Table 6). Indeed, compared to 27,
compounds 40 and 48 exhibited several characteristics favorable to good exposure including lower
basicity, higher Cacoꢀ2 permeability and a lower Cacoꢀ2 efflux ratio.
Table 6. Mouse pharmacokinetics data for 40 and 48.
a
F (%) calculated after iv and PO
#
40
48
Dose (mg/kg)
Route of
1
5
1
5
administration at 1 mg/kg and 5 mg/kg
respectively of 40 and 48. Formulations,
PO: 0.5%MC + 0.1%TW80 + sterile MQ
water, iv: 10%NMP + 40%PEG300 +
sterile MQ water, 10%Cremophor +
10%DMA + sterile MQ water.
IV
PO
IV
PO
C (ng/mL)
0
2940
2379
Cmax (ng/mL)
4405
0.5
2481
0.5
Tmax (h)
T
1/2 (h)
2.32
0.16
6124
2.14
1.7
0.56
1761
1.77
CL* (L/h/kg)
AUC0ꢀt (last)
24093
5484
a
F (%)
79
64
Kinase selectivity. Compounds 40 and 48, were tested against a panel of 104 kinases together with
36ꢀ38
39
52, 64
representative MNK inhibitors such as CGP57380,
cercosporamide and ASTꢀ487.
These
known MNK inhibitors are often used in biological studies but their selectivity profiles were not
6
5
readily available. CGP57380 is a multikinase inhibitor as it was found to inhibit ABL1 (E255K),
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ABL1 (WT), MEK1, MEK2, MNK1, MNK2 and RET at more than 99% (Supporting Information,
Table S5).
Table 7. Kinase selectivity profile of 40 and 48 screened against a panel of 104 kinases at 1 ꢀM.
Compounds
TREEspots
40
48
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
2
Number of
Kinases
>
65%
90%
(
DRAK1; FLT3; KIT; MNK2;
(
MEK; MNK2)
PDGFRA; PDGFRB; PHKG2; SRPK3)
3
1
inhibited*
>
(FLT3; MNK2; PDGFRB)
(MNK2)
*Competition assay with a proprietary ligand to determine compounds binding affinity against a
panel of 104 kinases.
Table 7 shows that 40 and 48 are selective and specifically, imidazopyridine 48 inhibits only 2
kinases at ≥65%, 6 kinases fewer than imidazopyridazine 40. The bicyclic core of 48 had an
unexpected influence on its kinase selectivity and the difference in the torsion between the bicyclic
ring and the phenyl at Cꢀ3 could be a plausible explanation of why 48 is more selective than 40.
There is a 29° versus 42° torsion variation in DFTꢀB3LYP 6ꢀ31** minimized structures. The more
coplanar 40 probably has a shape that allows it to fit better to a greater number of kinases.
Cell-based anti-proliferative activity. The antiproliferative effects of 40 and 48 were assessed in
vitro, using CellTiterꢀGlo viability assay against 25 hematological cancer cell lines including the
2
8
K562 cell line that overexpresses eIF4E (K562 o/e eIF4E) (Supporting information, Table S7). The
34,43,45,66
IC50s were in general in the micromolar range similar to published data.
Compound 40
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
appears to be more potent than 48 with submicromolar IC50, especially against GKꢀ5, DOHH2, AHHꢀ
1 and P3HRꢀ1 cell lines. We speculate that the superior activity of 40 against these cell lines could be
the result of the combination of its potency and offꢀtarget inhibition of kinases such as PDGFRA and
FLT3, against which 40 gave an inhibitory potency of 0.25 and 0.88 ꢀM respectively. MNK1/2
inhibitors that show strong antiꢀproliferative effects in vitro have been known to often inhibit other
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
34,39
kinases.
In contrast, 40 and 48 alone or in combination with imatinib or dasatinib were both able to suppress
the ability of leukemic stem cells from BC CML patient to form colony (data to be published
elsewhere). This was previously described for nonꢀselective MNK1/2 inhibitors ASTꢀ487,
CGP57380 and cercosporamide and it was shown that imatinib and dasatinib alone do not inhibit the
28
selfꢀrenewal activity of LSCs.
Selection of compound 48 as a preclinical candidate. A comparison of the two optimized
compounds 40 and 48 shows that 40 is the most potent in the enzymatic assay, but that potency did
not carry over to eIF4E phosphorylation inhibition in HeLa cells, where 48 is slightly more potent.
Compound 48 has a greater solubility, permeability and an excellent kinase selectivity. In maximum
tolerated dose studies, oral administration of 48, showed no weight loss and no clinical adverse
effects in healthy mice up to the tested dose of 200 mg/kg.
To avoid any ambiguities in interpreting efficacy studies, a compound which inhibits fewer kinases
will be preferred for investigating the impact of the inhibition of MNK1/2 on biological processes. In
light of these data, compound 48 was ranked higher and was selected as the preclinical candidate.
In vivo efficacy. We have reported that dual specific MNK1/2 and BCRꢀABL1 inhibitors potently
inhibited the proliferation of five BC CML lines in vitro and these inhibitors elicited a dose
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dependent antiꢀtumor activity in an in vivo mouse xenograft model. As MNK1/2 and BCRꢀABL1
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inhibitory activities were combined into one molecule, it was challenging to distinguish the
contribution of MNK1/2 inhibition in the observed biological response. Now, we have shown that
selective MNK1/2 inhibitors 40 and 48 are much less antiꢀproliferative against the five BC CML cell
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lines (Supporting information Table S7). However, there are emerging data
that support that
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MNK1/2 inhibition effect in vivo could be greatly enhanced by the tumor microꢀenvironment or the
stroma without which, the amplitude of MNK1/2 inhibitors antiꢀproliferative effect on BC CML cell
lines in in vitro cellular assays could appear to be much weaker. Accordingly, compound 48 was
selected for further evaluation in vivo in a mouse xenograft model. The antitumor effect of
compound 48 was then assessed in a K562 e/o eIF4E mouse xenograft model after oral
administration at 25, 50 or 100 mg/kg alone or in combination with a 2.5 mg/kg fixed dose of
dasatinib throughout the study (Figure 6).
Figure 6A shows that the treatment was well tolerated and the weight loss observed with the vehicle
or compound 48 administered alone is due to the burden of the tumor growth on mice.
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6B
Figure 6. Antitumor efficacy of 48 after oral administration alone or in combination with dasatinib
to K562 o/e eIF4E SCID mouse xenograft model with eight mice per group. A: Mouse body weights
expressed as (mean ±SEM). B: Tumor growth rates expressed as volumes (mean ±SEM).
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Dasatinib at 2.5 mg/kg elicited a tumor growth inhibition (TGI) of 88% (Figure 6B) with one tumor
free animal. In contrast, 48 alone only yields a maximum TGI of 23% at the highest administered
dose of 100 mg/kg which did not impede tumor growth, and was similar to the nonꢀtreated animals.
Combining 48 with 2.5 mg/kg of dasatinib not only increased tumor growth inhibition in a dose
dependent manner but, more importantly led to 2, 5 and 8 out of 8 tumor free animals at 25, 50 and
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100 mg/kg respectively (Supporting Information, Table S8). The combination of 48 and dasatinib
inhibited tumor growth at all tested doses and no weight loss was recorded (Figure 6A). Both the
combination of 48 and dasatinib and on the other hand, the dual MNK1/2 and BCRꢀABL1
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inhibitors, prevented tumor growth in the same mouse xenograft model. The combination study
clearly showed, that a selective MNK1/2 inhibitor plays a role in TGI by enhancing the antiꢀtumor
effect of dasatinib.
Biomarker study. Western blot analysis of tumor samples excised from mice administered with 2.5
mg/kg of dasatinib and 100 mg/kg of 48 (Supporting information Figure S1), revealed, a maximum
reduction in phosphorylated CRKL, a BCRꢀABL1 substrate, from 4 to 8 hours postꢀdose. The
reduction of phosphorylated eIF4e was significant after 1 hour postꢀdose and was sustained at 4 and
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hours postꢀdose. These observations are consistent with the tumor growth inhibition induced by the
combination of 48 and dasatinib and shows that both MNK1/2 and BCRꢀABL1 kinases are inhibited
in vivo.
Conclusion
Starting from bicyclic fragment hits, selective and potent MNK1/2 inhibitors were designed and
synthesized. Substituents at both Cꢀ3 and Cꢀ6 and the bicyclic moiety were optimized. The
imidazopyridazine and imidazopyridine derivatives which required a nonꢀbasic benzamide for
optimal PK properties, also showed that these cores were equally effective at increasing potency for
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MNK1/2 inhibition and at modulating kinase selectivity profiles. Imidazopyridine derivative 48
displayed excellent kinase selectivity and significantly enhanced the antiꢀtumor activity of dasatinib
in a BC CML mouse xenograft model. This is in agreement with our previous antiꢀtumor data
obtained with single compound dual MNK/BCRꢀABL1 inhibitors for which, the impact of MNK
inhibition could not be clearly established. Biomarker studies show that the combination effectively
reduced the ability of both MNK1/2 and BCRꢀABL1 kinases to phosphorylate their substrates eIF4E
and CRKL respectively. A treatment of patients resistant to dasatinib with a combination of MNK1/2
inhibitor and dasatinib should overcome the MNKꢀeIF4Eꢀaxis mediated resistance and restore
sensitivity to dasatanib treatment. In addition, the combination might require a lower dose of
dasatinib to achieve efficacy, alleviating dasatinib toxicity. This combination therapy with MNK1/2
inhibitors could be extended not only to other BCRꢀABL1 inhibitors current or future, but also to
other therapies using kinase inhibitors that have become ineffective due to an overexpression of
phosphorylated eIF4E.
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Compound 48 is currently in phase 1 clinical trial where its benefit to BC CML patients treated with
dasatinib is being evaluated.
EXPERIMENTAL SECTION
Molecular Modeling.
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The apo DFGꢀout MNK1 and MNK2 Xꢀray structure PDB entries 2HW6 and 2AC3 respectively,
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were downloaded from the Protein Data Bank and prepared with the protein preparation wizard in
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Maestro 2017ꢀ3 release using standard settings. This included the addition of hydrogen atoms, bond
assignments, removal of water molecules further than 5Å from the ligand, protonation state
assignment, optimization of the hydrogen bond network and restrained minimization using the
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OPLS3 force field. The missing loops were added to the 2AC3 structure, these were adopted from a
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