Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells

Article abstract of DOI:10.1021/acs.jmedchem.5b01712

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

Full text of DOI:10.1021/acs.jmedchem.5b01712

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Article
Structure-Activity Relationship Studies of Mitogen Activated
Protein Kinase Interacting Kinase (MNK) 1 & 2 and BCR-
ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells
Joseph Cherian, Kassoum Nacro, Zhi Ying Poh, Samantha Guo, Duraiswamy Athisayamani
Jeyaraj, Yun Xuan Wong, Melvyn Ho, Hai Yan Yang, Joma Kanikadu Joy, Zekui Perlyn
Kwek, Boping Liu, John Liang Kuan Wee , Esther HQ Ong, Meng Ling Choong, Anders
Poulsen, May Ann Lee, Vishal Pendharkar, Li Jun Ding, Vithya Manoharan, Yun Shan
Chew, Kanda Sangthongpitag, Sharon Lim, S. Tiong Ong, Jeffrey Hill, and Thomas H. Keller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01712 • Publication Date (Web): 24 Mar 2016
Downloaded from http://pubs.acs.org on March 25, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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StructureꢀActivity Relationship Studies of Mitogen
Activated Protein Kinase Interacting Kinase (MNK)
1 & 2 and BCRꢀABL1 Inhibitors Targeting Chronic
Myeloid Leukemic Cells
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,ǁ
ǁ
ǁ
ǁ,§
ǁ,ǂ
Joseph Cherian,* Kassoum Nacro, Zhi Ying Poh, Samantha Guo, Duraiswamy A. Jeyaraj,
ǁ
ǁ,¶
ǁ
ǁ
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Yun Xuan Wong, Melvyn Ho, Hai Yan Yang, Joma Kanikadu Joy, Zekui Perlyn Kwek, Boping
ǁ
ǁ
ǁ
ǁ
ǁ
Liu, John Liang Kuan Wee, Esther HQ Ong, Meng Ling Choong, Anders Poulsen, May Ann
ǁ
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ǁ
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Lee, Vishal Pendharkar, Li Jun Ding, Vithya Manoharan, Yun Shan Chew, Kanda
ǁ
ɸ
ɸ
ǁ
ǁ
Sangthongpitag, Sharon Lim, S. Tiong Ong, Jeffrey Hill, Thomas H. Keller
ǁ
Experimental Therapeutics Centre, 13 Biopolis Way, Nanos, Singapore 13866;
ɸ
DukeꢀNUS Graduate Medical School, 8 College Road, Singapore.
KEYWORDS: Blast Crisis, Chronic Myeloid Leukemia, Leukemic stem cells, selfꢀrenewal,
MNK kinase, BCRꢀABL1.
ABSTRACT
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Clinically used BCRꢀABL1 inhibitors for the treatment of chronic myeloid leukemia (CML)
do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 & 2 inhibitors
prevent phosphorylation of eIF4E and eliminate the selfꢀrenewal capacity of LSCs. Herein, we
describe the identification of novel dual MNK1 & 2 and BCRꢀABL1 inhibitors, starting from the
known kinase inhibitor 2. Initial SAR studies resulted in compound 27 with loss of BCRꢀABL1
inhibition. Further modification led to orally bioavailable dual MNK1 & 2 and BCRꢀABL1
inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level
of phosphorylated eIF4E in the tumor tissues. Kinase selectivity of these compounds is also
presented.
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INTRODUCTION
Serine/threonine kinases MNK1 and MNK2 phosphorylate eukaryotic translation initiation
1,2
factor 4E (eIF4E) at Ser209 and regulate the translation initiation. Overexpression of eIF4E
and its phosphorylated form have been shown to be responsible for abnormal growth and
3
4
5
proliferation of breast, prostate, head and neck cancer cells as well as blast crisis chronic
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myeloid leukemia (BC CML) cells. It has been shown that in BC CML, overexpression of
eIF4E and its phosphorylated form gives selfꢀrenewal capacity to blast crisis granulocyte
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macrophage progenitors (GMPs), allowing them to function as leukemic stem cells (LSCs).
4
, 7
While phosphorylation of eIF4E is necessary for oncogenic transformation, the kinase activity
1
of MNK1 & 2 seems dispensable for normal development making MNK1 & 2 kinases attractive
anticancer targets.
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Inhibition of MNK1 and MNK2 using 1 (CGP57380) has been shown to be effective in
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preventing the phosphorylation of eIF4E as well as selfꢀrenewal of LSCs in vitro and in vivo.
However, the antiproliferative effect of 1 on BC CML cells was observed at doses higher than 10
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µM, probably due to its weak BCRꢀABL1 inhibition. While clinical BCRꢀABL1 inhibitors
exhibit very good antiproliferative activity against actively dividing progenitors, they do not
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,10ꢀ14
eliminate
LSCs.
4ꢀ[(4ꢀMethylpiperazinꢀ1ꢀyl)methyl]ꢀNꢀ(4ꢀmethylꢀ3ꢀ{[4ꢀ(pyridinꢀ3ꢀ
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yl)pyrimidinꢀ2ꢀyl]amino}phenyl)benzamide (imatinib) and Nꢀ(2ꢀchloroꢀ6ꢀmethylphenyl)ꢀ2ꢀ[[6ꢀ
[4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazinyl]ꢀ2ꢀmethylꢀ4ꢀpyrimidinyl]amino]ꢀ5ꢀthiazole
carboxamide
1
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(
dasatinib) do not inhibit MNK1 & 2 enzymes and had little effect in reducing the serial
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replating efficiency of LSCs at clinically relevant concentrations. Combination of BCRꢀABL1
inhibitor 4ꢀmethylꢀNꢀ[3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ꢀ 5ꢀ(trifluoromethyl)phenyl]ꢀ3ꢀ [(4ꢀpyridinꢀ
17
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ꢀylpyrimidinꢀ2ꢀyl) amino]benzamide (nilotinib) and JAK1/2 inhibitor (3R)ꢀ3ꢀcyclopentylꢀ3ꢀ
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[
4ꢀ(7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl)pyrazolꢀ1ꢀyl]propanenitrile (ruxolitinib) was successful in
preventing the selfꢀrenewal capacity of LSCs in vitro and in vivo, with some degree of toxicity to
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normal stem cells. As MNK1 & 2 inhibitors were able to prevent selfꢀrenewal of LSCs, we
envisaged that a single agent inhibitor of both BCRꢀABL1 and MNK1 & 2 may be able to cause
BCꢀCML cell death through the inhibition of BCRꢀABL and block the selfꢀrenewal capacity of
LSCs by inhibiting phosphorylation of eIF4E. Such an agent should be able to distinguish
between normal stem cells and LSCs to minimize toxicity.
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In addition to 1, other known MNK inhibitors include: 2 (ASTꢀ487) , 3 (staurosporine)
2
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and 4 (cercosporamide) (Figure 1) while a few other inhibitors have also appeared in the recent
9
,23,24
literature.
2 and 4 have been shown to inhibit the phosphorylation of
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Figure 1: Examples of MNK1 and MNK2 inhibitors known in the literature
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eIF4E and prevent selfꢀrenewal capacity of LSCs. However, 2 is a multiꢀkinase inhibitor and
has been shown to influence selfꢀrenewal capacity of hematopoietic stem cells (HSCs), thus
making it toxic towards normal HSCs. We used 2 as a starting point for the identification of
novel inhibitors of MNK1, MNK2 and BCRꢀABL1 that are nonꢀtoxic to HSCs.
CHEMISTRY:
2
5
2 was synthesized as previously reported. Scheme 1 describes synthesis of 9 from 5.
Reduction of the methyl ester group of 5 was carried out using lithium aluminium hydride to
furnish 6. Subsequent treatment of the primary alcohol 6 with MsCl/Et N followed by NaCN in
3
DMSO and hydrolysis of the resulting nitrile intermediate furnished the required carboxylic acid
25
7
. Amide coupling of 7 with the previously reported aniline 8 using HBTU/Et N gave 9.
3
Scheme 1: Synthesis of compound 9.
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a) LiAlH , THF, 0 Cꢀ rt, 4 h; b) MsCl, Et N, CH Cl ; 0 C – rt, 16 h; c) NaCN, DMSO, 90
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C, 14 h; d) 6N HCl, 90 C, 16 h; e) 8, HBTU, Et N, DMF, 16 h, rt.
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Scheme 2: Synthesis of compounds 15ꢀ17.
o
a) 2,4ꢀdichloropyridine, K CO , DMF, 90 C, 16 h; b) LiOH, THFꢀwater, rt, 14 h; c) aq.
MeNH , THF, 80 C, 15 h; d) Pd/C/H , rt; e) 14, HATU, Et N, DMF, rt; f) MeNH , THF, 80 C,
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Scheme 2 depicts synthesis of 15 ꢀ17. Methyl 2ꢀ(4ꢀhydroxyphenyl)acetate 10 was converted to
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1 by reacting with 2,4ꢀdichloropyridine in presence of K CO in DMF followed by ester
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hydrolysis using LiOH. In a similar manner, 12 was synthesized from 10 using 4,6ꢀ
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dichloropyrimidine. 11 was converted to 13 by treatment with aq. MeNH in THF. Coupling of
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Coupling of the acid 13 and previously reported aniline 14 using HATU/Et N gave 16.
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coupling with aniline 14 under previously mentioned conditions resulted in 17.
Converting 19 to the corresponding acid chloride and subsequent reaction with 14 and 18 gave
intermediates 24 and 25 (Scheme 3). Coupling of anilines 14 and 18 with commercially available
carboxylic acids 20ꢀ23 resulted in compounds 26ꢀ29. Intermediate 24 was subjected to Suzuki
coupling with (6ꢀacetamidopyridinꢀ3ꢀyl)boronic acid to provide compound 30. 25 was converted
to 31 by Suzuki coupling reaction with pyridineꢀ3ꢀboronic acid and subsequent Nꢀboc
deprotection.
Scheme 3: Synthesis of compounds 26ꢀ31.
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pyridineꢀ3ꢀboronic acid for 31, dioxaneꢀwater, 100 C; d) TFA, CH Cl .
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4. Suzuki coupling of 34 with pyridineꢀ3ꢀboronic acid resulted in compound 35.
Scheme 4: Synthesis of compound 35.
a) 33, NaBH CN, AcOH, MeOHꢀTHF, rt, 16 h; b) 19, HATU, DIPEA, THF, rt, 2 h; c)
Pd (dba) , PCy , K PO , Pyridineꢀ3ꢀboronic acid, dioxaneꢀwater, 100 C, 16 h.
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Arbuzove reaction of 36 with triethylphosphite and subsequent WittigꢀHorner reaction of the
resulting phosphonate with Nꢀethylpiperidinꢀ4ꢀone gave 37 (Scheme 5). Hydrogenation and
subsequent coupling with 2ꢀ(4ꢀ(pyridinꢀ3ꢀyl)phenoxy)acetic acid yielded 38.
Scheme 5: Synthesis of compound 38.
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Pt/C, EtOH, 5 h; d) 2ꢀ(4ꢀ(pyridinꢀ3ꢀyl)phenoxy)acetic acid, HATU, DIPEA, DMF, 16 h, rt.
Suzuki coupling reaction between 39 and cyclopropylboronic acid, reduction of the nitro group
using NiCl .6H O/NaBH in MeOH followed by HATU/DIPEA mediated coupling with 2ꢀ(4ꢀ
2
2
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(pyridinꢀ3ꢀyl)phenoxy)acetic acid gave 40 (Scheme 6).
Scheme 6: Synthesis of compound 40.
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a) Cyclopropylboronic acid, Pd(dppf)Cl .CH Cl dioxane, aq. K CO , 100 C, 16 h; b)
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NiCl .6H O, NaBH , MeOH, 0 Cꢀrt, 4 h; c) 2ꢀ(4ꢀ(pyridinꢀ3ꢀyl)phenoxy)acetic acid, HATU,
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DIPEA, THF, rt, 6 h.
27
Suzuki coupling of 41 with pyridineꢀ3ꢀboronic acid and subsequent coupling of the resulting
carboxylic acid intermediate with aniline 14 under HATU/Et N/DMF condition gave 42
3
(Scheme 7).
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Scheme 7: Synthesis of compound 42.
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Et N, DMF, rt, 1.5 h.
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Intermediates 46 and 47 were obtained by coupling of anilines 14 and 18 with 44. Suzuki
coupling of 46 and 47 with corresponding boronic acid or boronate esters (pyridineꢀ3ꢀboronic
acid, (6ꢀacetamidopyridinꢀ3ꢀyl)boronic acid, 55 and 56) and subsequent NꢀBoc deprotection
using TFA/CH Cl provided compounds 48ꢀ53 (Scheme 8). In a similar manner, 43 was
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converted to 54.
Scheme 8: Synthesis of compounds 48ꢀ54.
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a) HATU, Et N, THF, rt; b) corresponding boronic acid or ester derivative (pyridineꢀ3ꢀboronic
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acid, (6ꢀacetamidopyridinꢀ3ꢀyl)boronic acid, 55, 56), Pd(dppfCl ).CH Cl K CO , dioxaneꢀ
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RESULTS AND DISCUSSION:
Compound 2 inhibits both MNK1 and MNK2 with IC ’s of 1.76 µM and 20 nM, respectively
5
0
2
1
(Table 1). In addition to MNK1 and MNK2, 2 inhibits several other kinases. Our first objective
was to improve the kinase selectivity profile of 2 while maintaining inhibition of MNK1 and
MNK2. For this purpose, a brief SAR analysis was conducted. Modification of the urea linker to
the corresponding amide derivatives 9 and 15 showed that this is tolerated in 15, while 9 lost
MNK1 inhibition and is >30 fold weaker on MNK2. Conversion of Nꢀmethylaminopyrimidine
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Table 1. SAR of MNK1 and MNK2 inhibitors
IC (µM)*
50
ID
Structure
ABL1 MNK1 MNK2
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2
0.58
>10
>10
1.76
>10
3.69
0.020
0.760
0.022
9
15
O
N
O
1
1
6
7
N
N
0.58
3.88
0.37
0.54
0.008
0.010
F3C
N
H
NHMe
2
7
>10
>10
0.57
0.35
0.014
0.011
48
*
In vitro IC against ABL1, MNK1 and MNK2; n > 2.
50
ring of 15 to Nꢀmethylpyridineꢀ2ꢀamine derivative 16 led to significant improvement in MNK1
inhibition. We also noticed that the Nꢀmethylamino group was not required for the inhibition of
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2
2
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2
2
2
2
2
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5
5
6
MNK1 and 2 as indicated by IC values of 17. Removal of biaryl ether oxygen and modification
5
0
of phenylacetamide linker in 17 to a phenoxyacetamide for patentable structural novelty resulted
in compound 27 which exhibited IC s of 577 nM and 14 nM against MNK1 & 2. The inhibitory
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
9
0
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2
3
4
5
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7
8
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0
1
2
3
4
5
6
7
8
9
0
profile of the corresponding amine analog 48 was similar to 27. It may be noted that ABL1
inhibition was lost during these modifications. Apart from 16 with a donorꢀacceptor motif in the
form of ꢀNHꢀmethylaminopyridine, none of the other compounds were able to show
submicromolar inhibition of ABL1.
Any change to the position of nitrogen in the biaryl ring was not tolerated, as seen from the
enzymatic activities of compounds 26 and 28 (Table 2).
Table 2. SAR of MNK1 and MNK2 inhibitors
IC (µM)*
5
0
ID
Ar1
R
H
H
H
Ar
ABL1 MNK1 MNK2
2
2
2
7
6
8
>10
>10
>10
0.57
>10
>10
0.014
>10
1.91
3
5
H
>10
4.51
0.082
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38
40
H
>10
>10
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
>10
>10
>10
>10
0.35
>10
0.13
0.012
>10
3
4
1
2
Me
*
In vitro IC against ABL1, MNK1 and MNK2; n > 2.
50
Presence of basic amines as in the piperazine motif was essential for MNK1 & 2 inhibition as
shown by compounds 35 and 38. While compound 35 showed weak MNK1 inhibition and more
than 5 fold loss of MNK2 potency, 38 was inactive against both enzymes. We found that
removal of Nꢀethyl group did not affect potencies against either enzyme. Replacement of –CF3
with a cyclopropyl group resulted in loss of MNK1 potency with 10ꢀfold weaker MNK2
inhibition. Introduction of a methyl group on the methylene carbon as in compound 42 also was
not tolerated for MNK inhibition. Against ABL1, none of these compounds were active.
TM
The kinase selectivity of 27 was evaluated at 1 µM using the KINOMEscan approach
(Ambit biosciences) against a panel of 104 kinases (Supporting Information Table S1). Profiling
of compound 27 at 1 µM resulted in 90% inhibition of 11 kinases and showed a better selectivity
index in comparison to 2.
Molecular modeling of 27 with MNK2 and ABL1 enzyme models suggested the following
interactions between the compound and protein (Figure 2): i) the protonated piperazine nitrogen
may form a hydrogen bond in MNK2 with the carbonyl backbone of Ala202 and His203 (in
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5
5
5
6
ABL1, the residues are Ile360 and His361); ii) ꢀCF group binds in a hydrophobic pocket in
3
MNK2, formed by residues Leu133, Cys136, Val142, Leu196, Ile224 and Cys225 (in ABL1,
Met290, Ile293, Leu298, Leu354, Val379 and Ala380); iii) nitrogen on the pyridine ring may
form a hydrogen bond with the backbone nitrogen of MNK2 kinase hinge residue Met162
(Met318 in ABL1); iv) phenyl ring in the 5ꢀposition of the pyridine is sandwiched between the
MNK2 gatekeeper Phe159 and Phe227 (in ABL1, gatekeeper Thr315 and Phe382); v) hydrogen
bonding interaction between amide linker and the MNK2 backbone nitrogen of Glu129
(backbone nitrogen of Glu286 in ABL1). From these studies, we hypothesized that we could gain
additional interaction with ABL1 protein by introducing substituents in the hinge binding region,
and these modifications were suggested to be tolerated by both MNK1 and 2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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8
9
0
1
2
3
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9
0
1
2
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9
0
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27
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2: A) Compound 27 (stick representation; carbon in green, nitrogen in blue and oxygen
in red) docked into the ATPꢀbinding domain of: A) MNK2 (PDB entry 2AC3); B) ABL1 (PDB
entry 3OXZ). The kinase domain is depicted as a cartoon with carbon atoms in gray, nitrogen in
blue and oxygen in red.
Introduction of acetamide (30) resulted in 10ꢀfold weaker inhibition of MNK1 while activity
against MNK2 was maintained. Most importantly, 30 inhibited not only wild type ABL1, but
also its T315I mutant with IC s 0.39 and 0.17 µM, respectively. The corresponding reverse
5
0
amide derivative 29 exhibited complete loss of ABL1 and MNK1 inhibition with weak MNK2
inhibition. Compound 49, the amine analog of 30, showed similar activity profile as its ether
analog. Removal of the NꢀEt group had little effect on its enzymatic and antiproliferative activity
as shown by the data of compound 50. Isopropyl amide analog 51 showed >10 µM inhibition of
ABL1 with significant loss of MNK2 inhibition. Compound 52 with a cyclopropanamide moiety
retained ABL1 and its T315I mutant inhibition while a 10ꢀfold and 4ꢀfold improvement was
obtained in the inhibition of MNK1 & 2, respectively. This compound had good antiproliferative
activity with a GI of 80 nM against K562 cells overexpressing eIF4E. Reꢀintroducing the NꢀEt
5
0
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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
moiety retained ABL1 and MNK2 inhibition while suffering a 5ꢀfold loss over MNK1 inhibition
with a GI of 51 nM in K562 cells overexpressing eIF4E as shown by compound 53. Compound
50
54 with a fluorine on the phenyl ring improved ABL1 inhibition and MNK1 inhibition to 10 nM
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
and 200 nM, respectively while retaining ABL1 T315I and MNK2 inhibition. 54 also exhibited
very good antiproliferative activity as shown by its GI50 against K562 cells overexpressing
eIF4E. In addition to the antiproliferative activity, these compounds also showed reduction in the
phosphorylation of eIF4E at Ser209 in HeLa cells, with 50, 53 and 54 as the most potent
compounds in this assay.
Table 3. SAR of dual MNK1 & 2 and Abl inhibitors
GI50
IC (µM)^
5
0
(µM)
Compꢀ
ound
R1
X
R
R2
K562
o/e
eIF4E
ABL1 ABL1
T315I native
MNK1 MNK2 HeLa*
#
27
29
30
49
50
51
52
53
Et
Et
Et
Et
H
O
O
H
H
H
H
H
H
H
ND
ND
>10
>10
0.57
>10
0.014
6.86
0.098
ND
>10
ꢀCONHMe
ꢀNHAc
ꢀNHAc
ꢀNHAc
ND
O
0.17
0.09
0.24
>10
0.39
0.38
0.88
>10
5.68
5.98
3.92
7.70
0.51
2.52
0.033
0.044
0.032
0.13
0.68
0.32
0.20
0.86
0.32
0.18
0.10
0.34
0.40
3.30
0.08
0.051
NH
NH
NH
i
H
ꢀNHCO Pr
H
NH H ꢀNHCOCyc 0.10
NH H ꢀNHCOCyc 0.05
0.21
0.089
0.008
0.016
Et
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5
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7
8
9
5
4
Me NH F ꢀNHCOCyc 0.02
0.01
0.20
0.010
0.28
0.012
^
In vitro IC against ABL1, MNK1 and MNK2; n > 2. *concentration of the compound required
5
0
#
for 50% reduction in the phosphorylation of eIF4E in HeLa cells; concentration of the
compound required for 50% reduction in the growth of K562 cells overexpressing eIF4E. The
compounds were tested for dose response in triplicates. The dose response curve was done twice.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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30
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35
36
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Docking studies suggested additional Hꢀbonding interaction between the amide ꢀNH and
backbone carbonyl of the ABL1 hinge residue Met318 while the amide carbonyl may hydrogen
bond to the –OH of the glycineꢀrich loop residue Tyr253 (Figure 3). While the reason behind the
enhancement in the inhibition when a cyclopropyl group is introduced is not clear, additional
interaction of this group with the neighboring residues Phe317, Thr319 and Leu248 in ABL1
may be a plausible explanation. The equivalent residues in MNK2 are Lys161, Arg163 and
Leu90 (Figure 3).
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1
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5
5
5
6
0
1
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9
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9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3: Compound 53 (tube representation; carbon in green, nitrogen in blue and oxygen in
red) docked into the ATPꢀbinding domain of: A) ABL1 (PDB entry 3OXZ); B) MNK2 (PDB
entry 2AC3). The kinase domain is depicted as a cartoon with selected residues in thin tube with
carbon atoms in gray, nitrogen in blue and oxygen in red.
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48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4: Inhibition of phosphorylation of eIF4E and Crkl by 53 and 54 in K562 cells
overexpressing eIF4E. A) Total eIF4E and phosphoꢀeIF4E (Ser209) were detected using antiꢀ
eIF4E and antiꢀphosphoꢀeIF4E (Ser209) antibody; B) Crkl and phosphorꢀCrkl (Tyr207) were
detected using antiꢀCrkl and antiꢀphosphoꢀCrkl (Tyr207) antibody.
Figure 4 shows the ability of 53 and 54 to inhibit phosphorylation of both eIF4E (substrate of
MNK1 & 2) and Crkl (substrate of ABL1) in K562 cells overexpressing eIF4E. Compound 54
inhibited phosphorylation of eIF4E at all tested concentrations with a dose dependent reduction
of phosphorꢀCrkl, while 53 showed a dose dependent reduction of both phosphoꢀeIF4E and
phosphoꢀCrkl. This is in agreement with the biochemical assay data with 54 being more potent
inhibitor of both MNK1 & 2 and ABL1 than 53.
Compounds 53 and 54 were tested in a panel of CML cell lines (Table 4). 54 showed potent
antiꢀproliferative activity ranging from 1 nM to 13 nM while 53 was comparatively less potent
with GI values of 15 nM to 58 nM across the 5 different CML cell lines. In comparison,
50
imatinib exhibited values from 60 nM to 400 nM.
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5
6
7
8
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 4. Antiproliferative effect of 53 and 54 across a panel of CML cells
#
GI 48 h (µM)
50
Compound
K562 BVꢀ173 EMꢀ2 KCLꢀ22
JURLꢀMK1
0.030
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
5
5
3
4
0.058
0.012
0.200
0.030
0.001
0.10
0.015
0.001
0.26
0.050
0.013
0.43
0.001
Imatinib
0.06
#
The compounds were tested for dose response in triplicate. The dose response curve was done
twice.
TM
Selectivity screen of 53 and 54 in the kinase panel (104 kinases, Ambit KINOMEscan )
showed that at 1 µM, these compounds inhibited 90% activity of 12 and 15 kinases, respectively
(supporting information Table S1). Biochemical IC s were determined for those kinases against
50
which both compounds showed >99% inhibition (table 5). Among these, RET was most potently
inhibited by 53 and 54 followed by FLT3 and VEGFR2 with IC s < 24 nM while showing
50
comparatively weaker inhibition of KIT, PDGFRα, PDGFRβ and FGFR2.
Table 5: IC of 53 and 54 against selected kinases.
5
0
Kinase
IC (µM)*
50
53
54
FLT3
0.020 0.020
VEGFR2 0.024 0.021
RET
0.007 0.001
0.888 1.509
0.930 0.272
cKIT
FGFR2
PDGFRα 0.672 1.280
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PDGFRβ 1.083 1.055
*
Inhibition of kinase activity, n=2.
In general, thermodynamic solubility of compounds in the series was low at pH 7, with 53 and
4 exhibiting 127.5 and 182.7 µg/mL, respectively at pH 4 (Table 6). Both these compounds did
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
not inhibit CYP3A4 and CYP2D6. Compounds 53 and 54 also showed high plasma protein
binding (Table 6). In vivo pharmacokinetic studies of 53 and 54 showed oral bioavailability of 80
and 31 %, respectively (Table 7).
Table 6. In vitro PK parameters and thermodynamic solubility of selected compounds
Thermodynamic
solubility (µg/mL)
CYP
IC5
0
PAMPA Human
(µM)
ꢀ6
Compound
(10
PPB
(%)
cm/s)
pH 7
pH 4
3A4 2D6
49
50
52
53
54
3.2
2.4
1.1
>20 26.2
>20 1.13
>20 5.53
ND
0.6
ND
ND
0.1
3.8
ND
ND
131.5
109.1
127.5
182.7
ND
>30 >30 34.53
>30 >30 30.61
99.92
99.78
ND – Not Determined.
Table 7: In vivo PK properties of 53 and 54 in CDꢀ1 mice
a
b
iv
po 50 mg/kg
Compound
F (%)
Cl T1/2
(L/h/kg) (h)
Cmax AUC
(ng/ml) (ng.h/ml)
5
3
4
2.7
3
2.14
2.2
2490
1212
14337
5128
80
31
5
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2
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2
2
2
2
2
2
2
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3
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4
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4
4
4
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5
5
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5
5
5
5
5
5
5
6
a
formulated in 50% PEG+sterile MQ water as clear solution. 53 and 54 were dosed at 5 and 10
b
mg/kg respectively; formulated in 0.5% methylcellulose + 0.1% Tween 80 + sterile MQ water
as suspension. See supporting information table S4 for full pharmacokinetic parameters.
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
In an in vivo xenograft model employing K562 cells overexpressing eIF4E in NODꢀSCID mice,
both 53 and 54 inhibited tumor growth in a doseꢀdependent manner when dosed orally, QD for
1
4 days (Figure 5). Complete tumor regression was observed at 100 mg/kg for 54 while 53
showed dose dependent tumor growth inhibition at 100 and 200 mg/kg. Importantly, mice given
3 and 54 at these doses did not exhibit toxicity. Imatinib at 200 mg/kg QD resulted in 57%
5
tumor growth inhibition. We also monitored phosphorylation of eIF4E in the tumor tissues after
compound administration on day 14 (Figure 6). For both 53 and 54 at 50 mg/kg, maximum
inhibition of phosphorylation of eIF4E in the tumor was seen after 4 h of drug administration.
Thus, inhibition of BCRꢀABL1 kinase activity resulted in tumor size shrinkage while its MNK1
and MNK2 inhibition prevented phosphorylation of eIF4E in vivo. A 10ꢀfold better inhibition of
MNK1 by 54 is reflected in greater inhibition of phosphorylation of eIF4E in tumor tissues in
comparison to 53. The difference in tumor growth inhibition of these two compounds in the
mouse xenograft model could be attributed to enhanced Abl inhibition of 54 over 53.
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9
2000
1750
1500
Vehicle
5
5
5
3 - 100 mg/kg
3 - 200 mg/kg
4 - 50 mg/kg
10
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49
50
51
52
53
54
55
56
57
58
59
60
54 - 100 mg/kg
Imatinib 200 mg/kg
1250
1000
750
500
250
0
0
2
4
6
8
10
12
14
16
Days
Figure 5: Mouse efficacy study of 53 and 54 in a K562 o/e eIF4E xenograft model. Compounds
3, 54 and imatinib were administered by oral gavage, single dose per day for 14 days.
5
Compound 53 showed a dose dependent tumor growth inhibition at 100 and 200 mg/kg while 54
showed complete tumor regression at 100 mg/kg.
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5
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9
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2
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9
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9
0
Figure 6: Inhibition of phosphorylation of eIF4E by 53 and 54 in tumor tissue samples after 14
day QD study. Maximum inhibition was observed at 4 hours after compound administration.
The effect of these compounds on the selfꢀrenewal capacity of hematopoietic stem cells and
+
patient derived BC CML samples (primary CD34 cells) ex vivo was further evaluated (Sharon
2
8,29
Lim et al, unpublished results).
Results of these experiments and detailed biological
characterization of these compounds will be published in due course.
Conclusion
We have identified novel inhibitors of MNK1, MNK2 and BCRꢀABL1 which are orally
available and capable of effective target engagement in vivo. While BCRꢀABL1 inhibition brings
potent antiproliferative activity against CML cells, MNK1 & 2 inhibition prevents eIF4E
6
phosphorylation which is known to prevent selfꢀrenewal capacity of LSCs. As current CML
therapy using tyrosine kinase inhibitors do not eliminate LSCs, dual AblꢀMNK1 & 2 inhibitors
such as 53 and 54 might be beneficial in treating patients with CML and may warrant further
studies to evaluate their usefulness as clinical agents.
Experimental Section:
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Molecular modeling: The MNK2 Xꢀray structure PDB entry 2AC3 and ABL1 Xꢀray
3
1
structure PDB entry 3OXZ was downloaded from the Protein Data Bank [www.pdb.org] and
prepared with the protein preparation wizard in Maestro 9.3 (Schrödinger, LLC, New York, NY,
USA, 2012) using standard settings. This included the addition of hydrogen atoms, bond
assignments, removal of water molecules further than 7Å from the ligand, protonation state
assignment, optimization of the hydrogen bond network and restrained minimization using the
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3
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OPLS2005 force field. The prepared ABL1 structure was used as is where as a model was built
of the MNK2 activation loop using the prepared ABL1 structure as a template. The Mnk2
structure was superimposed on ABL1 using the protein structural alignment tool in Maestro. The
activation loop residues 226ꢀ231 in MNK2 were manually modeled to have the same backbone
torsions as found in the ABL1 structure. The sidechains of these residues was modeled as
homologous ABL1 residues except Asp228 which is a glycine in ABL1. The Asp228 sidechain
was positioned to form a salt bridge to Lys113.
The inhibitors were build using Maestro and minimized to conversion using the OPLS2005
33
force field and GB/SA continuum solvation method as implemented in Macromodel 9.9
(Schrödinger, LLC, New York, NY, USA, 2012). The inhibitors were then manually docked into
the ABL1 and MNK2 binding site using the ABL1 coꢀcrystalized ligand ponatinib as a template
for the bioactive conformation and binding orientation. The inhibitorꢀprotein complex was
finally minimized using Macromodel. All residues more than 7Å from the ligand were
constrained before the complex was subjected to 500 steps of PolakꢀRibiereꢀConjugateꢀ
3
4
Gradient minimization using the OPLS2005 force field and GB/SA continuum solvation
3
3
method. The PDB files of compounds 27 and 53 modeled into MNK2 are available in
Supporting Materials.
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General Procedures. All reagents were purchased from commercial sources and used as
received. Reaction progress was monitored by TLC using Merck silica gel 60 F254 on glass
plates with detection by UV at 254 nM. LC−MS analysis was carried out with a Shimadzu LCꢀ
0
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20AD and LCMSꢀ2020. The column used was a Phenomenex Kinetex 2.6 ꢁm, 50 × 2.10 mm).
1
3
1
13
Proton and C nuclear magnetic resonance ( H and C NMR) spectra were obtained using a
Bruker Ultrashield 400 PLUS/R system, operating at 400 MHz and 100 MHz respectively. All
resonance bands were referenced to tetramethylsilane (internal standard). Splitting patterns are
indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. The compound
purities were >95%, determined by Varian Prostar HPLC instrument (column: Phenomenex
kinetex 2.6 ꢁm XBꢀC18A 100A, 50 x 4.6 mm; solvent A: H O/0.1% HCO H; solvent B:
2
2
MeCN/0.1% HCO H; run time 10.5 min.; time/%B: 0.0/10, 2.0/10, 7.5/90, 9.0/90,10.0/10,
2
10.5/10; flow rate: 2 mL/min.; wavelength: 254 nm). Melting points were determined in Pyrex
capillary tubes using a StuartnSMP30 melting point apparatus. Purification of final compounds
and intermediates was carried out via normal or reverse phase chromatography. Normalꢀphase
chromatography was performed on an ISCO CombiFlash system with either an ISCO RediSep or
a Biotage silica gel disposable column eluted with EtOAc/hexanes or methanol/CH Cl . Reverseꢀ
2
2
phase chromatography was performed on a Shimadzu preparative system on a Phenomenex Luna
or Sunfire C18 preparative column with appropriate gradients of MeCN/H O/0.1%
2
trifluoroacetic acid (TFA) or methanol/H O/0.1% TFA as eluent at a flow rate of 40 mL/min.
2
(4ꢀ((4ꢀEthylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)methanol (6): To a stirred
suspension of LiAlH (0.17 g, 4.53 mmol) in THF (25 mL) was added 5 (1.0 g, 3.02 mmol) in
4
o
THF (5 mL) at 0 C. After 4 h at room temperature, the reaction mixture was quenched (sat. aq.
o
Na SO ) at 0 C, filtered through celite pad and concentrated. The residue was partitioned
2
4
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between EtOAc and water. The organic layer was separated, dried (anh. Na SO ), evaporated
2
4
and the residue purified by silica gel column chromatography (100–200 mesh) using 30% EtOAc
1
in hexane as eluent to afford 6 (0.65 g, 71%). HNMR (CDCl ): δ 7.78ꢀ7.76 (d, J = 8.0 Hz, 1H),
3
10
11
12
13
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7
.63 (s, 1H), 7.52ꢀ7.50 (d, J = 8.0 Hz, 1H), 4.73 (s, 2H), 3.65 (s, 2H), 2.51ꢀ2.39 (m, 10H), 1.10ꢀ
+
1.06 (t, J = 7.2 Hz, 3H). LCMS (ESI) m/z 303.2 (M+H) .
2ꢀ(4ꢀ((4ꢀEthylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)acetic acid (7): To a
solution of 6 (0.65 g, 2.15 mmol) in CH Cl (10 mL) was added Et N (0.66 g, 6.6 mmol) and
2
2
3
MsCl (0.41 g, 3.63 mmol) at 0 °C. After stirring at room temperature for 16 h, the reaction
mixture was diluted with CH Cl and washed with water. The organic layer was dried,
2
2
evaporated and the crude residue (0.5 g, 1.56 mmol) redissolved in DMSO (5 mL). NaCN (0.15
g, 3.12 mmol) in water (0.5 mL) was added and the reaction mixture was heated at 90 °C for 14
o
h. The reaction mixture was cooled to 0 C, water (15 mL) was added and extracted with EtOAc
(3x5 mL). The organic layer was washed with water (1x5 mL), brine (1x5 mL), dried over
anhydrous Na SO and evaporated. The residue was purified by column chromatography over
2
4
silica gel (100–200 mesh) using a solvent gradient of 1% MeOH in CH Cl as eluent to afford
2
2
1
0.14 g of 2ꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)acetonitrile [ H NMR:
(DMSOꢀd ): δ 1.08ꢀ1.12 (t, J = 7.0 Hz, 3H), 2.48ꢀ3.33 (m, 10H), 3.66 (s, 2H), 3.78 (s, 2H), 7.61ꢀ
6
7
.59 (d, J = 8.0 Hz, 1H), 7.67 (s, 1H), 7.78 (d, J = 8.0 Hz, 1H)] which was treated with 6N HCl
o
(5 mL) at 90 C for 16 h. The reaction mixture was evaporated, the residue dissolved in methanol
(1 mL) and triturated with Et O at 0 °C. The solid formed was filtered and dried to afford (0.10
2
1
g, 14 % over 3 steps) of 7 as hydrochloride salt. H NMR (DMSOꢀd ): δ 7.83ꢀ7.85 (d, J = 8.0
6
Hz, 1H), 7.57 (s, 1H), 7.49ꢀ7.51 (d, J = 8.0 Hz, 1H), 3.89 (s, 2H), 3.73 (s, 2H), 3.67ꢀ3.49 (m,
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H), 3.17ꢀ3.08 (m, 6H), 2.68ꢀ2.60 (m, 2H), 1.25ꢀ1.23 (t, J = 6.0 Hz, 3H). LCMS (ESI) m/z 331.2
+
(M+H) .
2ꢀ(4ꢀ((4ꢀEthylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀNꢀ(4ꢀ((6ꢀ(methylamino)ꢀ
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pyrimidinꢀ4ꢀyl)oxy)phenyl)acetamide (9): 7 (0.1 g, 0.30 mmol) and 8 (0.06 g, 0.30 mmol)
were dissolved in 3 mL of anhydrous DMF. HBTU (0.138 g, 0.36 mmol) and Et N (0.10 mL,
3
0
.72 mmol) were subsequently added and the resulting reaction mixture was stirred for 16 h,
diluted with water (30 mL) and extracted with EtOAc (3x15 mL). The combined organic layers
were washed with brine, dried over sodium sulfate and evaporated under reduced pressure. The
1
residue was purified by preparative HPLC to afford 9 (10.6 mg, 6 % yield) as a solid. HNMR
(DMSOꢀd ): δ 10.27 (s, 1H), 8.11 (s, 1H), 7.69ꢀ7.71 (m, 1H), 7.67 (s, 1H), 7.58ꢀ7.62 (m, 3H),
6
7.24ꢀ7.25 (m, 1H), 7.05ꢀ7.08 (m, 2H), 5.71 (s, 1H), 3.74 (s, 2H), 3.57 (s, 2H), 2.75 (s, 3H), 2.29ꢀ
1
3
2.38 (m, 10H), 0.96ꢀ0.99 (t, J = 7.2 Hz, 3H); C NMR (DMSOꢀd ): δ 168.4, 165.1, 148.1, 135.9,
6
1
35.5, 135.1, 133.0, 130.5, 127.1ꢀ 126.2 (q), 125.7, 123.0, 121.6, 120.2, 57.4, 52.7, 52.2, 51.4,
+
42.2, 27.3, 11.8. LCMS (ESI) m/z 529 (M+H) . HPLC purity: 99.3%; retention time: 3.85 min.;
o
Melting point: 147.9 C.
2ꢀ(4ꢀ((2ꢀChloropyridinꢀ4ꢀyl)oxy)phenyl)acetic acid (11): To a solution of 10 (2.45 g, 14.7
mmol) and 2,4ꢀdichloropyridine (2.18 g, 14.7 mmol) in 8 mL of DMF was added K CO (3.05 g,
2
3
o
22.1 mmol). After 16 h at 90 C, the reaction mixture was cooled to room temperature, diluted
with water (80 mL) and extracted with EtOAc (3x25 mL). The combined organic layer was
washed with brine, dried over sodium sulfate and evaporated under reduced pressure. The
residue was purified by flash column chromatography eluting with 1:4 EtOAc:hexane to yield
1
1
.68 g of methyl 2ꢀ(4ꢀ((2ꢀchloropyridinꢀ4ꢀyl)oxy)phenyl)acetate as a pale yellow oil [ HNMR
(CDCl ): δ 8.22 (s, 1H), 7.35ꢀ7.37 (d, J = 8.0 Hz, 1H), 7.12ꢀ7.14 (d, J = 8.8 Hz, 2H), 7.05ꢀ7.07
3
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(
d, J = 8.0 Hz, 1H), 6.81ꢀ6.83 (d, J = 8.8 Hz, 2H), 3.69 (s, 3H), 3.55 (s, 2H); LCMS (ESI) m/z
+
2
78 (M+H) ] which was dissolved in THF containing water (25 mL, 1:1) and treated with LiOH
(0.220 mg, 9.09 mmol). After 14 h, the reaction mixture was acidified with aq. citric acid. The
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solid formed was filtered, washed with water and dried in vacuuo to afford 1.59 g (40 % over
1
two steps) of 11. H NMR (CDCl ): δ 8.23 (d, J = 5.7 Hz, 1H), 7.39ꢀ7.37 (m, 2H), 7.08ꢀ7.06 (m,
3
+
2
H), 6.84ꢀ6.83 (m, 1H), 6.81ꢀ6.79 (m, 1H), 3.71 (s, 2H); LCMS (ESI) m/z 264.1 (M+H) .
2ꢀ(4ꢀ((2ꢀ(Methylamino)pyridinꢀ4ꢀyl)oxy)phenyl)acetic acid (13): A solution of 12 (200 mg,
0
.75 mmol) in THF (15 mL) was treated with aq.MeNH (15 mL) at 80 °C. After 15 h, the
2
reaction mixture was cooled to room temperature, evaporated and the residue purified by
1
preparative HPLC to afford 101 mg (54%) of 13 as a white solid. H NMR (DMSOꢀd ): δ 12.40
6
(brs, 1H), 8.31 (brs, 1H), 7.90ꢀ7.92 (d, J = 8.0 Hz, 1H), 7.40ꢀ7.43 (d, J = 8.5 Hz, 2H), 7.20ꢀ7.17
(d, J = 8.5 Hz, 2H), 6.58 (m, 1H), 6.10 (s, 1H), 3.64 (s, 2H), 2.83ꢀ2.85 (m, 3H); LCMS (ESI) m/z
+
2
59.2 (M+H) .
Nꢀ(4ꢀ((4ꢀEthylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)phenyl)ꢀ2ꢀ(4ꢀ((6ꢀ(methylamino)ꢀ
pyrimidinꢀ4ꢀyl)oxy)phenyl)acetamide (15): To a solution of 12 (0.10 g, 0.38 mmol) and 14
0.11 g, 0.38 mmol) in DMF (4 mL) of was added HATU (174 mg, 0.45 mmol) and Et N (0.06
(
3
mL, 0.45 mmol). After stirring at room temperature for 3 h, the reaction mixture was diluted with
water and extracted with EtOAc (3x10 mL). The combined organic extracts were washed with
brine, dried over sodium sulfate and evaporated under reduced pressure and the residue purified
by flash column chromatography, eluting with MeOH:CH Cl (1:20) to afford 151 mg of 2ꢀ(4ꢀ
2
2
((6ꢀchloropyrimidinꢀ4ꢀyl)oxy)phenyl)ꢀNꢀ(4ꢀ((4ꢀethylpiperazinꢀ1ꢀyl)methyl)ꢀ3ꢀ(trifluoromethyl)ꢀ
+
phenyl)acetamide [LCMS (ESI) m/z 534 (M+H) ] which was dissolved in THF and treated with
o
MeNH (3 mL of 2.0 M solution in THF) in a sealed tube. After heating at 80 C for 16 h, the
2
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