Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family

Article abstract of DOI:10.1016/j.bmc.2020.115724

We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family of serine/threonine kinases. Here we described the optimisation of those compounds to improve their physicochemical and ADME properties as well as reducing their off-targets activities against other kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies, advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in a concentration dependent manner.

Full text of DOI:10.1016/j.bmc.2020.115724

Journal Pre-proofs
Aminothiazolones as potent, selective and cell active inhibitors of the PIM
kinase family
Camilo E. Quevedo, Carole J. R. Bataille, Simon Byrne, Matthew Durbin, Jon
Elkins, Abigail Guillermo, Alan M. Jones, Stefan Knapp, Anna Nadali,
Roderick G. Walker, Isabel Wilkinson, Graham M. Wynne, Stephen G.
Davies, Angela J. Russell
PII:
S0968-0896(20)30554-X
https://doi.org/10.1016/j.bmc.2020.115724
BMC 115724
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 June 2020
14 August 2020
17 August 2020
Please cite this article as: C.E. Quevedo, C. J. R. Bataille, S. Byrne, M. Durbin, J. Elkins, A. Guillermo, A.M.
Jones, S. Knapp, A. Nadali, R.G. Walker, I. Wilkinson, G.M. Wynne, S.G. Davies, A.J. Russell,
Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family, Bioorganic &
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115724
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Aminothiazolones as potent, selective and cell active inhibitors of the PIM kinase family
Camilo E. Quevedo,a Carole J. R. Bataille,a Simon Byrne,a Matthew Durbin,a Jon Elkins,b Abigail
Guillermo,c Alan M. Jones,a Stefan Knapp,b Anna Nadali,c Roderick G. Walker,c Isabel Wilkinson,a
Graham M. Wynne,a Stephen G. Davies,a Angela J. Russell*a,c
aDepartment of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road,
Oxford OX1 3TA, U.K.
bStructural Genomics Consortium, University of Oxford, Old Road Campus Research Building,
Roosevelt Drive, Oxford U.K.
cDepartment of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, U.K.
*Corresponding author: angela.russell@chem.ox.ac.uk
Abstract
We have previously reported the discovery of a series of rhodanine-based inhibitors of the PIM family
of serine/threonine kinases. Here we described the optimisation of those compounds to improve their
physicochemical and ADME properties as well as reducing their off-targets activities against other
kinases. Through molecular modeling and systematic structure activity relationship (SAR) studies,
advanced molecules with high inhibitory potency, reduced off-target activity and minimal efflux were
identified as new pan-PIM inhibitors. One example of an early lead, OX01401, was found to inhibit
PIMs with nanomolar potency (15 nM for PIM1), inhibit proliferation of two PIM-expressing leukaemic
cancer cell lines, MV4-11 and K562, and to reduce intracellular phosphorylation of a PIM substrate in
a concentration dependent manner.
Introduction
The mammalian Pim family of serine/threonine-specific kinases were originally identified as proviral
integration sites for Moloney murine leukemia virus in mouse models in the 1980s1. The human family
consists of Pim-1, Pim-2 and Pim-3, which are oncogenes, and whose overexpression has been shown
to promote proliferation and survival of cancer cells.2 Pim kinases have been linked to the development
of a range of human leukaemias and lymphomas as well as solid cancers3 and have become highly
1

attractive as therapeutic targets4-6. As they are highly homologous proteins, whose functions and
expression patterns are partially overlapping;7-8 simultaneous targeting of all Pim isoforms using pan-
PIM inhibitors has proven to be advantageous in treating cancers. It is noteworthy that inhibition of all
Pim kinases would not induce undesirable side-effects for patients, as mice deficient in the three Pim
genes are viable and fertile with a normal life span, only showing a slight deficiency in their growth
responses.8 We have recently reported the identification and preliminary optimization of pan-PIM
inhibitors using computational chemistry and in silico modelling, whose structural make-up is
exemplified by compound OX0642; their potent PIM inhibition being demonstrated using both in vitro
enzyme and cell-based assays.
Novel ATP competitive PIM inhibitors such as SGI-1776, AZ1208, LGH4479-18 which have advanced
to clinical trials were compared to our initial lead, compound OX0642. Our comparison revealed that
the high potency of OX0642 and favourable selectivity made it a good starting point for further
development.
O
NH
N
H2N
O
F
F
F
NH
NH
S
N
N
N
S
S
N
H
N
H2N
N
H
N
N
F3C
F3CO
O
O
SGI-1776
OX00642
LGH447
off-target effect:
PKN1, PKCT, GSK3-b
AZD1208
off-target effect:
FLT3, BAD, hERG
Mcl-1; GSK3-b
off-target effect:
BAD
off-target effect:
GSK3-b
(IC50 of 0.4 nM, 5 nM, and
1.9 nM for Pim1, Pim2, and
Pim3 in cell-free assays,
respectively)
(IC50 of 6 pM, 18 pM, and 9
pM for Pim1, Pim2, and
Pim3 in cell-free assays,
respectively)
(IC50 of 70 nM for Pim1)
(IC50 of 7 nM for Pim1 with IC50 in
a cell-free assay, 50- and 10-fold
selective versus Pim2 and Pim3)
Figure 1: Comparison OX0642 with reported PIM inhibitors.
We decided to use compound OX0642 as a benchmark and optimise it further in order to obtain a lead
compound for potential in vivo investigations. One of the major hurdles is the poor solubility of
compound OX0642 (kinetic solubility <5 μM). From our previous work, we determined that changes
on the left-hand side of OX0642 (aromatic moiety) were well tolerated. Other areas where
improvements could be implemented were also identified; the right hand-side of compound OX0642
appeared to present a good starting point for the development of a new series of compounds aimed at
2

increasing the solubility and simultaneously maintaining or improving other physicochemical
properties of the series.
This paper describes efforts towards optimisation of benchmark compound OX0642 into a novel series
of pan-PIM inhibitors, using computational chemistry, in silico modelling, protein crystallography and
SAR- analysis.
Result and discussion
In our previous studies we had demonstrated that the C=S functionality was responsible for metabolic
instability seen in the rhodanine-based first generation compounds: comparison of the metabolic
stability of OX0642 and OX0586 using a mouse liver microsome (MLM) stability assay (Figure 2)
demonstrated that the iminothiazolone OX0642 was substantially more stable that its rhodanine
counterpart OX0586.19
NH
NH
S
S
S
S
S
NH
N
N
F3C
F3C
O
O
OX0642
MLM t1/2 > 100 min
OX0586
MLM t1/2 = 49 min
Figure 2: MLM in min; MLM t1/2: half-life measured in mouse liver microsomes.
The iminothiazolone derivative OX0642 still only showed moderate solubility and it was decided to
investigate how this could be improved. It was envisaged that introduction of a substituent on the imino
group would allow the straightforward addition of hydrophilic moieties.
To assess the feasibility of our strategy and to determine whether the proposed structural variations
would be likely to reduce binding affinity of the inhibitors to the target enzyme, docking experiments
were performed, using a published crystal structure, to assess the possibilities of expanding our scaffold
via the NextR outside the thiazolidinone (Figure 3, PDB 2C3I).
Next
R
S
S
NH
N
F3C
O
3

Figure 3: Representative docking picture of OX0642 in the PIM1 active site. (E) Representative
docking picture of OX0642 in the PIM1 active site; x = H2O, PDB 2C3I.
Analysis of the docking results suggested that the preferred conformation of compound OX0642 bound
to with PIM1 has Next orientated towards the solvent-exposed surface. The rest of the molecule would
then be predicted to be placed in a lipophilic sandwich where no H-bond interactions are observed,
following this model. The aryl group on this molecule in this conformation was also predicted to be
close to the solvent-exposed surface and could be used to incorporate additional polar substituents if
required. Although the interactions of the iminothiazolidinedione seem to be mainly with a water
molecule placed in a pocket adjacent to Lys-67, the introduction of extra functionalities on the Next
should be tolerated. Therefore, it was decided to start a new SAR investigation for identification of new
analogues with potential PIM inhibitory capabilities.
S
HN RB
S
S
S
S
S
S
RA
RA
RA
iii, iv
i
ii
NH
N
O
Br
O
O
N
N
N
N
O
Scheme 1: General synthesis of 2-thioxothiazolidin-4-ones.
Reagents and conditions: (i) RAPhB(OH)2, Pd(PPh3)4, Na2CO3, EtOH/DME (1:1), 100 °C, 24 h. (ii) cat.
piperidine, rhodanine, EtOH, 70 °C, 16 h; (iii) MeI, DIPEA, MeOH, r.t., 16h; (iv) RBNH2, DIPEA,
EtOH, 140 °C, 20 min, MW.
Aminothiazolones were readily available from the commercially available 2-bromothiazole-4-
carbaldehyde in four high yielding steps (Scheme 1). In the course of this SAR study, an alternative
4

furan core was also utilised for the investigation of the different possible NHRB analogues. Because of
its comparable potency to the thiazole core and its commercial availability, the 5-aryl furan building
block (Scheme 2) was selected for our initial investigations. The furan, as a bioisostere, was confirmed
to be a good substitute for the thiazole as judged by the temperature shift (Tm) and IC50 assays (Table
1). While we did not progress the furan-containing molecules themselves, as they were generally found
to exhibit less potency than their thiazole counterparts,20 this allowed the preparation and analysis of a
large array of right hand-side N-substitutions in an efficient manner, with a three-step synthesis from
the commercially available aldehyde, 5-(3-chlorophenyl)furan-2-carbaldehyde (Scheme 2). The N-
substituted derivatives giving the most promising results were then incorporated into the corresponding
thiazoles.
S
HN RB
Cl
Cl
Cl
S
S
i
ii, iii
NH
N
O
O
O
O
O
O
Scheme 2: General synthesis of furan derivatives.
Reagents and conditions: (i) cat. piperidine, rhodanine, EtOH, 70 °C, 16 h; (ii) MeI, DIPEA, MeOH,
r.t., 16h; (iii) RBNH2, DIPEA, EtOH, 140 °C, 20 min, MW.
A range of furans and thiazole analogues were synthesised using a diverse set of amines and the requisite
thiazolidinones as shown in Table 1 (Figure 4). In our previous SAR studies on the related thiazolidine
series we had shown that a range of ortho, meta or para substituted electron withdrawing and electron
donating groups could be tolerated on the phenyl substituent.21 We therefore selected a small
representative set of variants of RA for this study. They were evaluated using a differential scanning
fluorimetry (DSF) assay against the recombinant PIM1 enzyme.21 The screen was performed at 10 µM
and a minimum threshold thermal shift (Tm) was defined as >3 °C. A coupled kinetic assay was also
used as a secondary orthogonal assay to determine IC50 values of the hit compounds against PIM1 and
rank their activity.22 Kinetic solubility and metabolic stability in mouse liver microsomes (MLMs) were
also assessed. Any compounds with favourable profile and enzymatic inhibitory activity were then
5

23
evaluated for antiproliferative activity against MV4-11 cells (human acute monocytic leukemia line),
24
which has been reported to be PIM sensitive.
An initial set of alkyl, aryl amines, and amino acids residues were first synthesised. Simple alkyl amines,
such as i-Pr, t-Bu, cyclopropyl, methylcyclopropyl, furan and THF (OX0693, OX0865, OX0764,
OX0758, OX1126, OX1287) showed similar ΔTm results to compound OX0642, but there was a drop
in IC50 in the enzymatic assay, with the most potent example, OX1126, being around three fold less
active (171±21 nM vs 70 ± 2 nM). It was then decided to investigate aromatic amines; the fluorobenzyl
derivative showed a definite drop in activity in the thermal stability shift assay (OX0767, ΔTm = 1.8 °C),
whereas amino phenols (OX0932, OX0763, OX0867) all had activities very close to the parent
compound OX0642 (in thermal stability shift and enzymatic assays), but there was, unfortunately, no
real improvement with regards to the solubility (OX01173, kinetic solubility 3.75 µM). Amino acids
were also prepared in an attempt to add a solubilising group (OX0786, OX0759), but they led to a
significant drop in activity in the thermal stability assay, which was confirmed by enzymatic assay
(OX0786, IC50 = 1990±14).
HN RB
HN RB
S
A
N
S
S
RA
RA
N
O
B
O
N
O
Figure 4: General structures of the furan and thiazole derivatives.
MV4-11
IC50 (µM )
ΔTm
(°C)
PIM1 IC50
(nM)
KinSol
(µM)
MLM
t1/2 (min)
ID
core
RA
RB
OX0642
A
p-CF3
H
9.4± 0.1
6.4±0.4
5.8±0.4
70 ± 2
>10
<5
nd
nd
>100
OX0865
OX0764
B
B
m-Cl t-Bu
m-Cl
nd
nd
nd
141±79
nd
nd
OX0758
B
A
m-Cl
4.6±0.4
4.3± 0.1
nd
nd
nd
nd
nd
nd
nd
OX01182
p-CF3
>10000
6

p-
OX01126
OX01287
A
B
4.2± 0.1
4.1±1.0
171±21
537a
nd
nd
nd
nd
nd
nd
OCF3
O
m-Cl
OH
OX0932
OX0763
OX0867
B
B
B
m-Cl
m-Cl
m-Cl
7.7±2.2
5.7±0.6
6.5±0.1
66.3±13.4
33.2±11
483±40
nd
nd
nd
nd
nd
nd
nd
nd
nd
OH
OH
OH
F
OX0767
OX0933
OX1001
B
B
A
m-Cl
m-Cl
p-CF3
1.8±0.1
7.4±2.8
6.8± 2.8
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
OH
OH
60.2±5.3
53.7±4.2
p-
OH
OX1173
A
7.8 ± 0.3 50.4±10.0
nd
3.75
222
OCF3
N
OX0756
OX0931
B
B
m-Cl
m-Cl
4.2±1.0
4.1±1.6
nd
nd
nd
nd
nd
nd
nd
O
103±26
OX0786
OX0759
B
B
m-Cl
m-Cl
1.4±0.1
2.1±0.4
199±1
nd
nd
nd
nd
nd
nd
nd
CO2H
CO2H
Table 1: Physical properties of molecules with various substitutions on the RA and RB positions and
a
biological activities against PIM1. ΔTm in °C; IC50 in nM, n = 2 unless otherwise stated. n = 1, R2 >
0.90, R2 values listed in SI; MLM t1/2: half-life measured in mouse liver microsomes in min; KinSol:
kinetic solubility in µM; MV4-11 IC50 in µM; nd: not determined.
A new range of amines was therefore prepared aiming to gain potential new extra interactions and
therefore increase the solubility without losing activity (Table 2). In the previously reported PIM
inhibitor SGI-1776, it was shown that the addition of a larger amine group led to a general improvement
of physical properties and stability as well as activity. It included basic tertiary amines, and straight as
7

well as branched and cyclic amino alcohols with stereo centres. Non-aromatic cyclic alcohols
(OX01174, OX1285, OX1384) did not lead to any improvement of solubility or activity (with the best
result obtained with OX1174, IC50 = 97 nM, kinetic solubility = 3.75 µM) compared to compound
OX0642 or the amino phenols. Linear amino alcohols (OX0927, OX0864) showed only moderate
activity (OX0864, IC50 = 522 nM), which was improved with the branched derivatives (OX1286,
OX1388, OX 1385, OX 1392) exhibiting a range of IC50 values (from 309 to 22 nM) in a similar range
to compound OX0642. Solubility was improved slightly, with the best result obtained with OX1385
(kinetic solubility 11.5 µM), which was still substantially lower than needed for a potential hit. From
the second set of amines, basic tertiary amine functionalities emerged as the structural motif which
conferred the highest potency. p-Methylpiperidinylmethanamine containing compound showed a
dramatic improvement in all assays as well as in solubility (OX0757, OX0919, OX1070, OX1383,
OX0993), with the best example OX0993 exhibiting high potency (IC50 = 10.8 nM) and much improved
solubility (65 µM). The position of the piperidine was deemed to be important as the m-
methylpiperidinylmethanamine was 20 times less potent than the corresponding para analogue
(OX0917 vs OX0757, IC50 = 809 vs 37 nM). The thiazole derivatives in general appeared to have a
better profile than their furan counterparts.
MV4-11
IC50
PIM1
IC50
ΔTm
KinSol MLM
ID
core RA
RB
(µM )
t1/2 (min)
(°C)
(nM)
(µM )
OX1174
OX1285
A
B
p-OCF3 (rac)
5.9± 0
96.7±4.0 nd
3.75
nd
781
nd
OH
m-Cl (rac)
5.2±0.9
301±12
20
HO
HO
m-NMeSO2Me
OX1384
A
0.3± 1.7
510a
nd
nd
nd
nd
(rac)
OH
OX0927
OX0864
B
B
m-Cl
5.3±1.4
6.1±0.1
65±19
nd
nd
nd
nd
m-Cl
389±218 nd
OH
8

OX1286
OX1388
OX1646
OX1385
B
A
A
A
m-Cl
5.7±0.1
4.0± 1
nd
140±37
309a
11
3.75
<2.9
3.75
11.5
nd
OH
OH
OH
OH
p-OCF3
8.8
nd
>120
>120*
5.68
m-OCF3
m-NMeSO2Me
94±12
172±9
3.8± 1.5
4.5
m-
OX1392
OX1175
OX0917
A
A
B
7.4± 0.1
7.9± 0.1
22.1a
3.6
nd
4.9
2
7.56
>120
nd
OH
NHSONMe2
p-OCF3
m-Cl
65±21
NMe2
N
nd
859±108 nd
nd
N
N
N
N
N
OX0757
OX0919
OX1070
OX1383
OX0993
B
A
A
A
A
m-Cl
8.9±0.1
37±4
nd
65
>120
>120
nd
p-CF3
11.1± 1.6 20±13
2.2
nd
65
p-NHSO2Me
m-NMeSO2Me
o-OH
9.3± 0.2
7.1± 0.9
96±59
23.6a
nd
1-3.5
>100
65
27.5
127
11.2± 0.6 10.8±6.7 0.8
Table 2: Physical properties of molecules with various substitutions on the RA and RB positions and
biological activities against PIM1. ΔTm in °C; IC50 in nM, n = 2 unless otherwise stated. an = 1, R2 >
0.90, R2 values listed in SI; MLM t1/2: half-life measured in mouse liver microsomes in min; KinSol:
kinetic solubility in µM; MV4-11 IC50 in µM; nd: not determined.
A third set of amines, based on the thiazole core, was then prepared, taking into account the information
collected from the two first sets (Table 3). The branched amino-alcohols (OX1392, OX1399, OX1400,
OX1389, OX1390, OX1398) gave a range of IC50 values, with the most potent compound OX1400
also showing moderate solubility (12.7 µM). The methyl piperidine derivatives (OX1453, OX1579,
9

OX0999) showed the best results with potent inhibitory activity and acceptable solubility for OX1579
and OX0999.
PIM1
IC50
(nM)
MV4-11
IC50
(µM )
MLM
ΔTm
(°C)
KinSol
(µM)
ID
core RA
RB
t1/2 (min)
m-
OX1392
A
7.4±0.1 22.1a
3.6
4.9
7.6
OH
NHSONMe2
OX1399
OX1400
OX1389
OX1390
A
A
A
A
o-OH
6.4±0.1 14.9 a
6.3±0.1 18.2 a
4.4±0.1 114 a
2.8±0.1 222 a
0.55 (1.3) 10.5
48.5*
28.9*
>120
>120
OH
OH
OH
OH
o-OH
1
12.7
<6.5
2
p-OCF3
p-OCF3
17
9.1
m-
OX1398
OX1453
A
A
4.9±0.1 263 a
2.1
nd
nd
OH
NHSONMe2
p-OCF3
nd
nd
28.2±8.1 8.2
4.9±2.8 11.2
<2.9
>120*
N
HO
N
OX1579
OX0999
A
A
o-OH
65
65
>120*
>120
HO
N
p-OCF3
10.2±0.1 22.4±7.6 1.7
Table 3: Physical properties of molecules with various substitutions on the RA and RB positions and
biological activities against PIM1, *MLM S9.1 ΔTm in °C; IC50 in nM, n = 2 unless otherwise stated. an
= 1, R2 > 0.90, R2 values listed in SI; MLM t1/2: half-life measured in mouse liver microsomes in min;
KinSol: kinetic solubility in µM; MV4-11 IC50 in µM; nd: not determined.
1 MLM S9 was run on those compounds based on the number of conjugable groups present in the molecule.
10

The p-methylpiperidinylmethanamine amine within OX0999 was initially selected based on the
alignment studies performed with aminothiazolone OX0999 and SGI-1776 using Forge (Cresset™,
UK) (Figure 5). In these studies, the best alignment was observed where both compounds have the
methylpiperidinylmethanamine substituent oriented in the same direction. In addition, docking studies
with OX0999 indicated that the direction of the amine was towards the solvent-exposed surface and in
contact with amino acid residues outside the active site.
A
B
Figure 5: A) : (A) Overlay of 1776 (grey) with OX0999 (pink) in the PIM1 active site. B) Docking
studies with OX0999 and the 2C3I crystal structure.
Our in silico hypothesis was confirmed when the crystal structure of Pim1 complexed with OX0999
was solved to a resolution of 2.1 Å (PDB ID 6QXK). In this crystal structure, the compound bound to
the protein in a similar manner to that predicted from the docking studies with compound OX0642
(Figure 5). Although no H-bond interactions were observed between the thiazole ring and the protein,
the crystal structure shows clearly that this heterocyclic core is positioned next to the kinase hinge
region and stabilised by lipophilic interactions. The pose of the compound inside the crystal allows the
left and right hand-side to also be well accommodated within the active site and also stabilised by
lipophilic interactions. This is consistent with the observation that a range of ortho, meta or para
substituents on the phenyl ring (RA) were found to show activity in the enzymic assay. As expected, the
addition of the amine moiety moved the thiazolidinone motif towards the solvent surface via
interactions with the protein. Four different H-bond interactions were identified for the binding of
OX0999. Three of these were to molecules of water; one was observed with the carbonyl of the
thiazolidinone head, another one with the NH outside the thiazolidine ring and the last one with the
11

NMe of the piperidine ring. The sole H-bond directly to the protein was between Lys-67 and the nitrogen
within the thiazolidine ring.
B
A
C
C
Figure 6: Co-crystal structure of OX0999 bound to PIM1. (A) Overview of OX0999 in the ATP
binding site of PIM1. (B) Showing the interactions of Phe49 with the piperidine and phenyl rings of
OX0999 and Leu44 with the phenyl ring of OX0999. (C) Showing a 2Fo-Fc electron density map
around OX0999 and the water molecules involved in hydrogen bonding with OX0999. (D) Zoomed-in
view to show the hydrogen bonding network involved in the binding of OX0999.
Following the encouraging cell activity and crystallography results, OX0999 was selected for more
detailed ADME evaluation. Results with OX0999 showed good kinetic solubility, a good stability
profile in microsomes and hepatocytes and no CYP inhibition against 5 CYPs (CYP1A, CYP2C9,
CYP2C19, CYP2D6, CYP3A4). Low permeability with high levels of efflux ratio was observed, but
despite that, OX0999 showed moderate to good activity against different cancer cell lines including
PC3 and MV4-11 cells (IC50 3.1 and 1.7 M respectively). Therefore, in light of the results from various
12

in vitro ADME evaluation experiments OX0999 was identified as a compound with a good balance of
properties (Figure 7).
IC50: 22.4 nM
KinSol: 65 M
MLM t1/2: >120 min
HLM t1/2:>120 min
HN
S
N
S
F3CO
Murine Hepatocytes t1/2: 52.3 min
Caco-2 ER: 26.9 (AB: 0.21, BA: 5.57)
No inhibition against 5 CYP P450s
No PPAR activation
N
N
O
OX0999
PC3 Cell IC50: 3.1 M
MV4-11 Cell IC50: 1.7 M
PPB 50% plasma fu: 0.015 (98.5% bound)
Figure 7: Physicochemical and ADME properties of lead compound OX0999
Rhodanine and closely related structures have been described as promiscuous and pan-assay
interference compounds (PAINS) in the past decade and their use in medicinal chemistry projects can
lead to misleading false positive results. There has been in the past years a strong trend to avoid all non-
specific or promiscuous compounds in drug discovery campaigns and many papers advocate the use of
substructure filters to recognize PAINS and exclude them from the starting point of the SAR process.25
It is a controversial issue however, as a number of currently marketed drugs would have not been found
if these type of filters had been applied dogmatically. The drug discovery community is currently
reviewing how these methodologies are used, with the knowledge that some compounds, which flag as
PAINS may be acceptable to pursue, so long as their activity is carefully assessed, and an appropriate
set of orthogonal screening assays are used.26 We have previously reported that structurally-related
compounds bearing a rhodanine moiety have a genuine PIM inhibitory effect, but keeping this in mind,
OX0999 was sent to be profiled via a kinase selectivity panel in order to assess if any unwanted off-
targets.
The screening panel of 22 kinases chosen for the analysis of OX0999 included a representative array
of diverse kinases from the human kinase phylogenetic tree, as well as ones that had shown off-target
activity with other PIM inhibitors (see figure 1), and the 3 PIM isozymes. No significant inhibition of
any of the chosen kinases (apart from PIM1) at 10 µM compound concentration was observed (Table
4). It was decided to generate IC50 for some of kinases for which 80% activity were found. Those IC50
13

confirmed that the inhibition of OX999 was mainly focussed on the PIMs isozymes. OX0999 also
showed selectivity for PIMs over FLT3 (fms-like tyrosine kinase 3), as opposed to SGI-1776, which
is a known PIM and FLT3 inhibitor.27 FLT3 is over-expressed in AML (acute myeloid leukemia) cells
often due to FLT3-ITD (internal tandem duplication) mutation, and PIM1 is transcriptionally
upregulated downstream of FLT3, phosphorylates and stabilizes FLT3, promoting FLT3 signalling in
cells with FLT3-ITD.28 It has been reported that a combination of PIM kinase and FLT3 inhibitors
enhances apoptosis and decreases growth of FLT3-ITD AML cell lines and primary patient cells in
vitro and in vivo, when compared to treatment with FLT3 or PIM inhibitors alone.29-30 Therefore
OX0999 would be useful to understand the roles of PIM1 and FLT3 in this oncogenic signalling
pathway.
OX0999 IC50 (nM)
BTK(h)
94
74
CDK1/cyclinB(h)
CHK1(h)
CK2(h)
79
76
>10000
8950
cSRC(h)
101
29
DAPK1(h)
DYRK2(h)
FLT3(h)
GSK3β(h)
Haspin(h)
IR(h)
81
137
147
98
102
131
98
JAK2(h)
JNK2α2(h)
14

KDR(h)
Pim-1(h)
Pim-2(h)
Pim-3(h)
104
1
9
nd
nd
86
75
<25%
25-80%
PKBα(h)
Plk1(h)
111
118
100
>80%
SAPK2a(h)
SIK(h)
105
30
TrkA(h)
7118
Table 4: Kinase Selectivity panel evaluation of OX0999 at 10 µM.
Encouraged by the kinase selectivity profile OX0999, it was decided to run cell mechanistic assays in
order to assess OX0999 potential target engagement in cancer cells; 4E-BP1 was selected as a potential
biomarker. Translation repressor protein 4E-BP1 (also known as PHAS-1) inhibits cap-dependent
translation by binding to the translation initiation factor eIF4E; its hyperphosphorylation has been
reported to disrupt this interaction and activate cap-dependent translation,31 with multiple 4E-BP1
residues being phosphorylated in vivo.32 Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase
regulate 4E-BP1 activity,33-34 and while phosphorylation by FRAP/mTOR at Thr37 and Thr46 does not
prevent the binding of 4E-BP1 to eIF4E, it is thought to prime 4E-BP1 for subsequent phosphorylation
at Ser65 and Thr70.35 As discussed previously, PIMs are important in the upregulation of proteins
involved in cell cycle regulation and cell survival via cap-dependent translation in cancer.36 Pim-2
phosphorylates tuberous sclerosis complex-2 (TSC2) to derepress mammalian target of rapamycin
complex-1 (mTORC1). mTORC1 then phosphorylates EIF4E-binding protein-1 (4EBP1) and
ribosomal protein S6 kinase (S6K).37 Western blot experiments carried out using the MV4-11 cell line
15

showed some evidence of reduction of phosphorylated 4EBP1 protein and total 4EBP1 at 10 M after
incubation with OX0999 consistent with cellular IC50 , albeit the effects were modest (Figure 8).2
OX0999
DMSO
1
3
10 (µM)
26 kDa
15 kDa
P-4E-BP1 (Thr 70)
4E-BP1
Actin
Figure 8: Western blot experiments using OX0999 on MV4-11.
Having identified a compound with suitable potency and adequate kinase selectivity, analysis of its
potential off target toxicity against relevant targets was next evaluated. OX0999 was examined in a
CEREP Express profile at 10 M concentration against 54 receptors, ion channels and amine
transporters to identify off-target activity. This assay revealed that OX0999 inhibited a considerable
number of enzymes above 50% (13 were inhibited above 85% and an additional 12 above 50%, see SI),
making it an undesirable compound to progress. In parallel to this assay, we also assessed the activity
of our series of compounds against hERG. A selection of compounds, including OX0999 were
evaluated using a Cloe® Screen hERG Channel Inhibition assay (carried out by Cyprotex plc, see SI for
further information, Table 5).3 OX0999 was found to inhibit hERG with an IC50 of 4.9 M, making it
a moderately compound against hERG; results from the other compounds were more encouraging
2
Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb detects endogenous levels of 4E-BP1 only when
phosphorylated at Thr37 and/or Thr46.
3
Cloe® is a registered trademark of Cyprotex plc
16

however, since none of them were inhibiting hERG at a relevant concentration. Previous studies on
SGI-1776 identified that the N-methylamino piperidine was responsible for the hERG inhibition.38
Consistent with these findings, compound OX0998, the imino analogue of OX0999 did not show any
hERG inhibition; also, when an alcohol group was added to the N-methylamino piperidine (OX1453)
and thereby reducing the basicity of the N-Me piperidine, the compound exhibited considerably less
activity in hERG than OX0999.
Another property of the compounds, which seemed to be associated with the N-methyl piperidine head
group was found to be high efflux; this was identified when representative compounds were evaluated
in Caco-2 permeability assays as shown in Table 5. The results from these assays showed both OX0999
(Caco-2 A-B = 0.21, and CaCo-2 B-A = 5.55) and OX1453 (Caco-2 A-B = 0.11, and CaCo-2 B-A =
10.5) gave high efflux ratios.
N
NHR
S
N
S
N
N
arom
H
N
N
N
F3CO
O
OX0998-1453
SGI-1776, 2
ID
SGI-1776
OX0999
OX0998
OX1453
N
N
amine
H
N
HO
IC50 (nM)
Caco-2 ER
hERG IC50
(µM)
46±12
4.0
22±8
26.9
39.7±4.9
1.5
28.2±8.1
99.3
1.0
4.9
>25
>25
Table 5: ADME properties of various amine-substituted analogues and biological activities against
PIM1. IC50 in nM, n = 2. hERG IC50 in µM.
It was therefore deemed necessary to change the N-methyl piperidine functionality in order to improve
compound profile and identify a new and better lead compound. A selection of compounds previously
17

synthesised that were structurally similar to OX0999, but devoid of the N-methyl piperidine group, and
possessed promising profiles were assessed for activity against a subset of off-target receptor (Table
XX) . The aim was to establish whether binding promiscuity identified with OX0999 was likely to be
a series or compound specific effect. Two promising aminothiazolones were examined against the top
10 off-target receptors bound by OX0999. The results from the CEREP data show a much cleaner
profile for OX1453 but particularly for OX1399. The data from the toxicity panel proved that the
relative promiscuity of OX0999 was a compound and not a series specific effect, and may well also be
related to the presence of the N-methylpiperidine group.
OH
HN
N
HN
N
OH
N
HN
N
S
N
S
N
S
N
S
S
S
F3CO
F3CO
OH
N
O
O
O
OX0999
OX1399
OX1453
OX0999 OX1453 OX1399
alpha 2 (non-selective)
CCK1 (CCKA) (h)
D1 (h)
67
88
86
67
72
84
68
62
84
99
3
84
90
97
96
94
98
94
71
87
95
21
65
2
H1 (h)
M3 (h)
-5
63
75
25
7
>80%
NK2 (h)
50-80%
<50%
5-HT1A (h)
Na+ channel (site 2)
Percentage of radio ligand
displacement at 10 µM
norepinephrine transporter (h)
dopamine transporter (h)
61
Table 6: Comparative CEREP Express profile of OX0999, OX1453 and OX1399
18

With these data in mind, our efforts were next focused on molecules containing branched or cyclic
amino alcohols derived from OX1453 and OX1399 without the presence of a basic N-Me (Figure 9).
The new set of compounds were all derived from the same synthetic precursor, bearing a rhodanine
moiety that can be easily functionalised via treatment with MeI to produce the corresponding SMe
followed by its substitution with the amine of choice.
HO
OH
HN
N
HN
N
HN
N
S
N
S
N
HN
N
S
N
S
S
S
N
S
R
R
R
S
OH
R
N
N
O
O
O
O
Figure 9: Evolution of the N-substituted aminothiazolone series.
The data obtained from the permeability assays and the hERG assay for the new derivatives showed
improved results over those for OX0999. While addition of a hydroxyl group reduced the hERG activity
(as observed in OX1453), complete removal of the basic N-Me resulted in a better hERG profile and
simultaneously much lower efflux ratios. The favourable properties (activity, solubility and metabolic
stability) associated with OX0999 were also observed in the new analogues (Table 7). Thiazole-
containing compounds like OX1399 with a phenol left hand side showed good overall metabolic
properties such as no hERG inhibition, no efflux, good solubility and good cell potency.
PIM1
KinSol
MLM
t1/2 (min)
>120
Caco-2
ER
MV4-11
ID
RA
RB
IC50 (µM )
IC50 (nM)
(µM )
N
OX0993
o-OH
10.8± 6.7 0.81
10.8±6.7
18.7
(>120)*
OX1399
OX1401
o-OH
o-OH
15.1a
15.1a
0.42
0.50
10.5
4.2a
48.5*
33.5*
0.4
0.3
OH
OH
19

OX1484
o-OH
OH 13.5± 2.9 2.6
3.8a
38.9*
0.4
OX1483
OX1485
OX1486
o-OH, 3-F
o-OH, 5-F
o-OH, 4-F
40± 7
13.1
65 a
44.4*
67.2*
45.6*
nd
0.8
nd
OH
OH
6.9± 0.5
147±6
3.9
nd
<20 a
65 a
OH
OH
OX1580
OX1645
o-OH, 3-F
5.2±0.7
97±8
2.7
0.3
<6.5
<2.4
22.1*
84.2*
nd
nd
o-OH,
OH
m-OCF3
Table 7: Physical properties of various substitutions on the RA and RB positions and biological activities
against PIM1, *MLM S9.4 IC50 in nM, n = 2 unless otherwise stated. an = 1, R2 > 0.90, R2 values listed
in SI; MLM t1/2: half-life measured in mouse liver microsomes in min; KinSol: kinetic solubility in µM;
MV4-11 IC50 in µM; nd: not determined.
Among this series of compounds, two examples OX1401 and OX1399, present balanced overall
properties with reduced off-target toxicity and minimal efflux making them very suitable candidates for
further optimisation toward the identification of new pan-PIM inhibitors for further optimisation (Figure
10).
4 MLM S9 was run on those compounds based on the amounts of conjugable groups present in the molecule.
20

OH
HN
N
OH
S
N
HN
N
S
N
S
S
OH
OH
O
O
OX1399
OX1401
IC50: 15.1 nM
KinSol: 10.5 M
MLM S9 t1/2: 48.5 min
Caco-2 ER: 0.31 (AB: 19.5, BA: 6.16)
IC50: 15.1 nM
KinSol: 4.2 M
MLM S9 t1/2: 33.5 min
Caco-2 ER: 0.40 (AB: 12.7, BA: 5.03)
PC3 Cell IC50: 1.1 M
MV4-11 Cell IC50: 0.4 M
cell biomarker: <1-3uM p4EBP1
PC3 Cell IC50: 1.2 M
MV4-11 Cell IC50: 0.5 M
cell biomarker: 1-5 uM p4EBP1 and 1-5 uM c-Myc
hERG >25 uM
P450 >25 uM
Figure 10: Potential lead compounds for further evaluation
Cell-based assays were also carried out with OX1401 and OX01399 to assess its potential target
engagement in cancer cells. We used the same assay described earlier in the assessment of OX0999,
using 4E-BP1. Western blot experiments carried out using the MV4-11 cell line showed some evidence
of reduction of phosphorylated 4EBP1 protein and total 4EBP1 at 10 M after incubation with OX1401;
this change was also dose dependent and consistent with cellular IC50 (Figure 11).
OX0999
DMSO 1
OX1401
1
3
10
3
10
(µM)
26 kDa
15 kDa
P-4E-BP1 (Thr 70)
4E-BP1
Actin
Figure 11: Western blot experiments using OX1401 on MV4-11.
21

Docking experiments were performed with OX1041 and OX1399 using the crystal structure obtained
with OX0999; similar docking poses to the observed mode of binding for OX0999 were observed
(Figure 12). The amino moieties are orientated in the same direction and occupy the same regions of
the protein. In the case of the phenol rings, even though the orientation of the phenol varies within the
two structures, they were predicted to fill the same area of the protein. The H-bond interactions observed
from the right hand-side of OX1399 are identical to those in OX0999. Interactions were also identified
with two molecules of water and two amino acid residues (Lys-67 and Asp-186), as seen in OX0999
crystal structure. In the case of OX1401 in addition to those described for OX1399 an extra H-bond
interaction with Asn-172 was also predicted via the primary alcohol on the right-hand side of the
molecule.
B
A
C
D
Figure 12: A) Surface representation from a docking pose of OX1399 with OX0999 crystal. B) Cartoon
representation from a docking pose of OX1399 with OX0999 crystal. C) Surface representation from a
docking pose of OX1401 with OX0999 crystal. D) Cartoon representation from a docking pose of
OX1401 with OX0999.
22

The results obtained from the docking experiments highlight the similarities between the three
compounds in their expected binding mode with Pim1 and present OX1399 and OX1401 as potential
attractive lead molecules upon which to continue structure activity studies and optimise further towards
compounds with properties suitable for in vivo evaluation.
Conclusions
In this paper, we described the optimisation of a promising pan-PIM inhibitor, compound 1, improving
physical properties (metabolic stability, kinetic solubility) as well as its enzymatic and cell activity
using a systematic, SAR study, supported by structure based design attributes. During the early phase
of our optimisation work, the first leading molecule with an appropriate ADME profile was found to
exhibit high levels of efflux, high levels of hERG activity and substantial off-target toxicity in a CEREP
Express assay, precluding its further progression.. A structure-property analysis revealed that the N-
substituent was likely responsible for both the hERG activity and the poor permeability. Building on
this analysis, another round of focused SAR studies was undertaken to identify alternative compounds
with a similar activity, physicochemical and ADME profile, but reduced off-target activity and an
appropriate profile that could be taken to in vivo animal studies.
Acknowledgments
This work was supported by Cancer Research UK through a Small Molecule Cancer Drug Discovery
Award (C17468/A9332, CEQ, SB, MD, AMJ, AN, and RGW). We would also like to thank Research
Councils United Kingdom for a fellowship (AJR). SK and JE are grateful for support by the SGC, a
registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer
Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada,
Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co.,
Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo
Research Foundation-FAPESP, Takeda, and Wellcome Trust (092809/Z/10/Z).
23

Competing interests
The authors declare that they have no competing interests.
References
1.
Theo Cuypers, H.; Selten, G.; Quint, W.; Zijlstra, M.; Maandag, E. R.; Boelens, W.; van
Wezenbeek, P.; Melief, C.; Berns, A., Murine leukemia virus-induced T-cell lymphomagenesis:
Integration of proviruses in a distinct chromosomal region. Cell 1984, 37 (1), 141-150.
2.
Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J., PIM serine/threonine
kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica-
the Hematology Journal 2010, 95 (6), 1004-1015.
3.
Amson, R.; Sigaux, F.; Przedborski, S.; Flandrin, G.; Givol, D.; Telerman, A., THE HUMAN
PROTOONCOGENE PRODUCT P33PIM IS EXPRESSED DURING FETAL HEMATOPOIESIS
AND IN DIVERSE LEUKEMIAS. Proceedings of the National Academy of Sciences of the United
States of America 1989, 86 (22), 8857-8861.
4.
in the Design and Evaluation of Pim Inhibitors. Current Medicinal Chemistry 2010, 17 (34), 4114-4133.
5. Morwick, T., Pim kinase inhibitors: a survey of the patent literature. Expert Opinion on
Therapeutic Patents 2010, 20 (2), 193-212.
6. Arunesh, G. M.; Shanthi, E.; Krishna, M. H.; Kumar, J. S.; Viswanadhan, V. N., Small molecule
Anizon, F.; Shtil, A. A.; Danilenko, V. N.; Moreau, P., Fighting Tumor Cell Survival: Advances
inhibitors of PIM1 kinase: July 2009 to February 2013 patent update. Expert Opinion on Therapeutic
Patents 2014, 24 (1), 5-17.
7.
Eichmann, A.; Yuan, L.; Breant, C.; Alitalo, K.; Koskinen, P. J., Developmental expression of
Pim kinases suggests functions also outside of the hematopoietic system. Oncogene 2000, 19 (9), 1215-
1224.
24

8.
Mikkers, H.; Nawijn, M.; Allen, J.; Brouwers, C.; Verhoeven, E.; Jonkers, J.; Berns, A., Mice
deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth
factors. Molecular and Cellular Biology 2004, 24 (13), 6104-6115.
9.
Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z.-S.; Ambudkar, S. V.; Baer, M. R.,
The Pim kinase inhibitor SGI-1776 decreases cell surface expression of P-glycoprotein (ABCB1) and
breast cancer resistance protein (ABCG2) and drug transport by Pim-1-dependent and -independent
mechanisms. Biochemical Pharmacology 2013, 85 (4), 514-524.
10.
Kirschner, A. N.; Wang, J.; van der Meer, R.; Anderson, P. D.; Franco-Coronel, O. E.; Kushner,
M. H.; Everett, J. H.; Hameed, O.; Keeton, E. K.; Ahdesmaki, M.; Grosskurth, S. E.; Huszar, D.;
Abdulkadir, S. A., PIM Kinase Inhibitor AZD1208 for Treatment of MYC-Driven Prostate Cancer.
Jnci-Journal of the National Cancer Institute 2015, 107 (2).
11.
Haddach, M.; Michaux, J.; Schwaebe, M. K.; Pierre, F.; O'Brien, S. E.; Borsan, C.; Tran, J.;
Raffaele, N.; Ravula, S.; Drygin, D.; Siddiqui-Jain, A.; Darjania, L.; Stansfield, R.; Proffitt, C.;
Macalino, D.; Streiner, N.; Bliesath, J.; Omori, M.; Whitten, J. P.; Anderes, K.; Rice, W. G.; Ryckman,
D. M., Discovery of CX-6258. A Potent, Selective, and Orally Efficacious pan-Pim Kinases Inhibitor.
Acs Medicinal Chemistry Letters 2012, 3 (2), 135-139.
12.
Burger, M. T.; Han, W.; Lan, J.; Nishiguchi, G.; Bellamacina, C.; Lindval, M.; Atallah, G.;
Ding, Y.; Mathur, M.; McBride, C.; Beans, E. L.; Muller, K.; Tamez, V.; Zhang, Y.; Huh, K.; Feucht,
P.; Zavorotinskaya, T.; Dai, Y.; Holash, J.; Castillo, J.; Langowski, J.; Wang, Y.; Chen, M. Y.; Garcia,
P. D., Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase
Inhibitors. Acs Medicinal Chemistry Letters 2013, 4 (12), 1193-1197.
13.
Keeton, E. K.; McEachern, K.; Dillman, K. S.; Palakurthi, S.; Cao, Y.; Grondine, M. R.; Kaur,
S.; Wang, S.; Chen, Y.; Wu, A.; Shen, M.; Gibbons, F. D.; Lamb, M. L.; Zheng, X.; Stone, R. M.;
DeAngelo, D. J.; Platanias, L. C.; Dakin, L. A.; Chen, H.; Lyne, P. D.; Huszar, D., AZD1208, a potent
and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid
leukemia. Blood 2014, 123 (6), 905-913.
14.
Raab, M. S.; Ocio, E. M.; Thomas, S. K.; Guenther, A.; Goh, Y.-T.; Lebovic, D.; Jakubowiak,
A.; Song, D.; Xiang, F.; Patel, A.; Vanasse, K. G.; Kumar, S., Phase 1 Study Update of the Novel Pan-
25

Pim Kinase Inhibitor LGH447 in Patients with Relapsed/ Refractory Multiple Myeloma. Blood 2014,
124 (21).
15.
Flanders, Y.; Dumas, S.; Caserta, J.; Nicewonger, R.; Baldino, M.; Lee, C.-S.; Baldino, C. M.,
A versatile synthesis of novel pan-PIM kinase inhibitors with initial SAR study. Tetrahedron Letters
2015, 56 (23), 3186-3190.
16.
Harada, M.; Benito, J.; Yamamoto, S.; Kaur, S.; Arslan, D.; Ramirez, S.; Jacamo, R.; Platanias,
L.; Matsushita, H.; Fujimura, T.; Kazuno, S.; Kojima, K.; Tabe, Y.; Konopleva, M., The novel
combination of dual mTOR inhibitor AZD2014 and pan-PIM inhibitor AZD1208 inhibits growth in
acute myeloid leukemia via HSF pathway suppression. Oncotarget 2015, 6 (35), 37930-47.
17.
Liu, Z.; He, W.; Gao, J.; Luo, J.; Huang, X.; Gao, C., Computational prediction and
experimental validation of a novel synthesized pan-PIM inhibitor PI003 and its apoptosis-inducing
mechanisms in cervical cancer. Oncotarget 2015, 6 (10), 8019-8035.
18.
Wang, H.-L.; Cee, V. J.; Chavez, F., Jr.; Lanman, B. A.; Reed, A. B.; Wu, B.; Guerrero, N.;
Lipford, J. R.; Sastri, C.; Winston, J.; Andrews, K. L.; Huang, X.; Lee, M. R.; Mohr, C.; Xu, Y.; Zhou,
Y.; Tasker, A. S., The discovery of novel 3-(pyrazin-2-yl)-1H-indazoles as potent pan-Pim kinase
inhibitors. Bioorganic & Medicinal Chemistry Letters 2015, 25 (4), 834-840.
19.
Bataille, C. J. R.; Brennan, M. B.; Byrne, S.; Davies, S. G.; Durbin, M.; Fedorov, O.; Huber,
K. V. M.; Jones, A. M.; Knapp, S.; Liu, G.; Nadali, A.; Quevedo, C. E.; Russell, A. J.; Walker, R. G.;
Westwood, R.; Wynne, G. M., Thiazolidine derivatives as potent and selective inhibitors of the PIM
kinase family. Bioorg Med Chem 2017, 25 (9), 2657-2665.
20.
ring. Chem Res Toxicol 2013, 26 (1), 6-25.
21. Niesen, F. H.; Berglund, H.; Vedadi, M., The use of differential scanning fluorimetry to detect
ligand interactions that promote protein stability. Nat. Protoc. 2007, 2 (9), 2212-2221.
22. Kiianitsa, K.; Solinger, J. A.; Heyer, W.-D., NADH-coupled microplate photometric assay for
Peterson, L. A., Reactive metabolites in the biotransformation of molecules containing a furan
kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal. Biochem. 2003,
321 (2), 266-271.
26

23.
through Pim-1. Br. J. Haematol. 2006, 134 (5), 500-509.
24. Meeker, T. C.; Loeb, J.; Ayres, M.; Sellers, W., The human Pim-1 gene is selectively
Kim, K.-T.; Levis, M.; Small, D., Constitutively activated FLT3 phosphorylates BAD partially
transcribed in different hemato-lymphoid cell lines in spite of a G+C-rich housekeeping promoter. Mol.
Cell. Biol. 1990, 10 (4), 1680-8.
25.
Baell, J.; Walters, M. A., Chemical con artists foil drug discovery. Nature 2014, 513 (7519),
481-483.
26.
Senger, M. R.; Fraga, C. A. M.; Dantas, R. F.; Silva, F. P., Filtering promiscuous compounds
in early drug discovery: is it a good idea? Drug Discovery Today 2016, 21 (6), 868-872.
27.
Hospital, M.-A.; Green, A. S.; Lacombe, C.; Mayeux, P.; Bouscary, D.; Tamburini, J., The
FLT3 and Pim kinases inhibitor SGI-1776 preferentially target FLT3-ITD AML cells. Blood 2012, 119
(7), 1791-1792.
28.
Natarajan, K.; Xie, Y.; Burcu, M.; Linn, D. E.; Qiu, Y.; Baer, M. R., Pim-1 Kinase
Phosphorylates and Stabilizes 130 kDa FLT3 and Promotes Aberrant STAT5 Signaling in Acute
Myeloid Leukemia with FLT3 Internal Tandem Duplication. PLoS ONE 2013, 8 (9), e74653.
29.
Kim, K.-T.; Baird, K.; Ahn, J.-Y.; Meltzer, P.; Lilly, M.; Levis, M.; Small, D., Pim-1 is up-
regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood 2005,
105 (4), 1759-1767.
30.
Patrick R. Baldwin, S. K., Karthika Natarajan, Rossana Trotta, Adriana Tron, Dennis Huszar,
Concurrent Inhibition of Pim-1 and
Eduardo Davila, P, Danilo Perrotti, and Maria R. Baer, ,
FLT3 Kinases in FLT3-ITD Acute Myeloid Leukemia Post-Translationally Downregulates the Anti-
Apoptotic Protein Mcl-1 through Downregulation of the Mcl-1 Deubiquitinase USP9X 2016.
31.
N., Insulin-dependent stimulation of protein synthesis by phosphorylation
of a regulator of 5'-cap function. Nature 1994, 371 (6500), 762-767.
Fadden, P.; Haystead, T. A. J.; Lawrence, J. C., Identification of phosphorylation sites in the
Pause, A.; Belsham, G. J.; Gingras, A.-C.; Donze, O.; Lin, T.-A.; Lawrence, J. C.; Sonenberg,
32.
translational regulator, PHAS-I, that are controlled by insulin and rapamycin in rat adipocytes. Journal
of Biological Chemistry 1997, 272 (15), 10240-10247.
27

33.
Gingras, A.-C.; Kennedy, S. G.; O’Leary, M. A.; Sonenberg, N.; Hay, N., 4E-BP1, a repressor
of mRNA translation, is phosphorylated and inactivated by the Akt(PKB) signalingꢀpathway. Genes &
Development 1998, 12 (4), 502-513.
34.
Brunn, G. J.; Hudson, C. C.; Sekulić, A.; Williams, J. M.; Hosoi, H.; Houghton, P. J.; Lawrence,
J. C.; Abraham, R. T., Phosphorylation of the Translational Repressor PHAS-I by the Mammalian
Target of Rapamycin. Science 1997, 277 (5322), 99-101.
35.
Gingras, A.-C.; Gygi, S. P.; Raught, B.; Polakiewicz, R. D.; Abraham, R. T.; Hoekstra, M. F.;
Aebersold, R.; Sonenberg, N., Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism.
Genes & Development 1999, 13 (11), 1422-1437.
36.
multiple myeloma. Blood Cancer Journal 2015, 5, e325.
37. Lu, J.; Zavorotinskaya, T.; Dai, Y.; Niu, X.-H.; Castillo, J.; Sim, J.; Yu, J.; Wang, Y.;
Keane, N. A.; Reidy, M.; Natoni, A.; Raab, M. S.; O'Dwyer, M., Targeting the Pim kinases in
Langowski, J. L.; Holash, J.; Shannon, K.; Garcia, P. D., Pim2 is required for maintaining multiple
myeloma cell growth through modulating TSC2 phosphorylation. Blood 2013, 122 (9), 1610-1620.
38.
Foulks, J. M.; Carpenter, K. J.; Luo, B.; Xu, Y.; Senina, A.; Nix, R.; Chan, A.; Clifford, A.;
Wilkes, M.; Vollmer, D.; Brenning, B.; Merx, S.; Lai, S.; McCullar, M. V.; Ho, K.-K.; Albertson, D.
J.; Call, L. T.; Bearss, J. J.; Tripp, S.; Liu, T.; Stephens, B. J.; Mollard, A.; Warner, S. L.; Bearss, D. J.;
Kanner, S. B., A Small-Molecule Inhibitor of PIM Kinases as a Potential Treatment for Urothelial
Carcinomas()(). Neoplasia (New York, N.Y.) 2014, 16 (5), 403-412.
28

29