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

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

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

Camilo E. Quevedo,^aCarole J. R. Bataille,^aSimon Byrne,^aMatthew Durbin,^aJon Elkins,^bAbigail

Guillermo,^cAlan M. Jones,^aStefan Knapp,^bAnna Nadali,^cRoderick G. Walker,^cIsabel Wilkinson,^a

Graham M. Wynne,^aStephen G. Davies,^aAngela 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.²Pim kinases have been linked to the development

of a range of human leukaemias and lymphomas as well as solid cancers³and have become highly

1

attractive as therapeutic targets^4-6. As they are highly homologous proteins, whose functions and

expression patterns are partially overlapping;^7-8simultaneous 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.⁸We 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, LGH447^9-18which 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

NH

S

N

S

N

H

N

H₂N

N

H

N

F₃C

F₃CO

O

SGI-1776

OX00642

LGH447

off-target effect:

PKN1, PKC_T, GSK3-b

AZD1208

off-target effect:

FLT3, BAD, hERG

Mcl-1; GSK3-b

off-target effect:

BAD

off-target effect:

GSK3-b

(IC₅₀of 0.4 nM, 5 nM, and

1.9 nM for Pim1, Pim2, and

Pim3 in cell-free assays,

respectively)

(IC₅₀of 6 pM, 18 pM, and 9

pM for Pim1, Pim2, and

Pim3 in cell-free assays,

respectively)

(IC₅₀of 70 nM for Pim1)

(IC₅₀of 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

S

NH

N

F₃C

O

OX0642

MLM t_1/2> 100 min

OX0586

MLM t_1/2= 49 min

Figure 2: MLM in min; MLM t_1/2: 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 N_extR outside the thiazolidinone (Figure 3, PDB 2C3I).

N_ext

R

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 N_extorientated 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 N_ext

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 R_B

S

R_A

iii, iv

i

ii

NH

N

O

Br

O

N

O

Scheme 1: General synthesis of 2-thioxothiazolidin-4-ones.

Reagents and conditions: (i) R_APhB(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) R_BNH₂, 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 NHR_Banalogues. 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 (T_m) and IC₅₀assays (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,²⁰this 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 R_B

Cl

S

i

ii, iii

NH

N

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) R_BNH₂, 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.²¹We therefore selected a small

representative set of variants of R_Afor this study. They were evaluated using a differential scanning

fluorimetry (DSF) assay against the recombinant PIM1 enzyme.²¹The 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 IC₅₀values of the hit compounds against PIM1 and

rank their activity.²²Kinetic 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 IC₅₀in 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 R_B

S

A

N

S

R_A

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

t_1/2(min)

ID

core

R_A

R_B

OX0642

A

p-CF₃

H

9.4± 0.1

6.4±0.4

5.8±0.4

70 ± 2

>10

<5

nd

>100

OX0865

OX0764

B

m-Cl t-Bu

m-Cl

nd

141±79

nd

OX0758

B

A

m-Cl

4.6±0.4

4.3± 0.1

nd

OX01182

p-CF₃

>10000

6

p-

OX01126

OX01287

A

B

4.2± 0.1

4.1±1.0

171±21

537^a

nd

OCF₃

O

m-Cl

OH

OX0932

OX0763

OX0867

B

m-Cl

7.7±2.2

5.7±0.6

6.5±0.1

66.3±13.4

33.2±11

483±40

nd

OH

F

OX0767

OX0933

OX1001

B

A

m-Cl

p-CF₃

1.8±0.1

7.4±2.8

6.8± 2.8

nd

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

m-Cl

4.2±1.0

4.1±1.6

nd

O

103±26

OX0786

OX0759

B

m-Cl

1.4±0.1

2.1±0.4

199±1

nd

CO₂H

Table 1: Physical properties of molecules with various substitutions on the R_Aand R_Bpositions and

a

biological activities against PIM1. ΔT_min °C; IC₅₀in nM, n = 2 unless otherwise stated. n = 1, R²>

0.90, R²values listed in SI; MLM t_1/2: half-life measured in mouse liver microsomes in min; KinSol:

kinetic solubility in µM; MV4-11 IC₅₀in 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 IC₅₀values (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 R_A

R_B

(µM )

t_1/2(min)

(°C)

(nM)

(µM )

OX1174

OX1285

A

B

p-OCF_{3 (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

m-NMeSO₂Me

OX1384

A

0.3± 1.7

510^a

nd

(rac)

OH

OX0927

OX0864

B

m-Cl

5.3±1.4

6.1±0.1

65±19

nd

m-Cl

389±218 nd

OH

8

OX1286

OX1388

OX1646

OX1385

B

A

m-Cl

5.7±0.1

4.0± 1

nd

140±37

309^a

11

3.75

<2.9

3.75

11.5

nd

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

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

OX0757

OX0919

OX1070

OX1383

OX0993

B

A

m-Cl

8.9±0.1

37±4

nd

65

>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

23.6^a

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 R_Aand R_Bpositions and

biological activities against PIM1. ΔT_min °C; IC₅₀in nM, n = 2 unless otherwise stated. ^an = 1, R²>

0.90, R²values listed in SI; MLM t_1/2: half-life measured in mouse liver microsomes in min; KinSol:

kinetic solubility in µM; MV4-11 IC₅₀in 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 IC₅₀values, 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 R_A

R_B

t_1/2(min)

m-

OX1392

A

7.4±0.1 22.1^a

3.6

4.9

7.6

OH

NHSONMe₂

OX1399

OX1400

OX1389

OX1390

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

OH

o-OH

1

12.7

<6.5

2

p-OCF₃

17

9.1

m-

OX1398

OX1453

A

4.9±0.1 263 ^a

2.1

nd

OH

NHSONMe₂

p-OCF₃

nd

28.2±8.1 8.2

4.9±2.8 11.2

<2.9

>120*

N

HO

N

OX1579

OX0999

A

o-OH

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 R_Aand R_Bpositions and

biological activities against PIM1, *MLM S9.¹ΔT_min °C; IC₅₀in nM, n = 2 unless otherwise stated. ^an

= 1, R²> 0.90, R²values listed in SI; MLM t_1/2: half-life measured in mouse liver microsomes in min;

KinSol: kinetic solubility in µM; MV4-11 IC₅₀in µM; nd: not determined.

¹MLM 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 (R_A) 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

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 (IC₅₀3.1 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 t_1/2: >120 min

HLM t_1/2:>120 min

HN

S

N

S

F₃CO

Murine Hepatocytes t_1/2: 52.3 min

Caco-2 ER: 26.9 (AB: 0.21, BA: 5.57)

No inhibition against 5 CYP P450s

No PPAR activation

N

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.²⁶We 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.²⁷FLT3 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.²⁸It 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.^29-30Therefore

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

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,³¹with multiple 4E-BP1

residues being phosphorylated in vivo.³²Both the PI3 kinase/Akt pathway and FRAP/mTOR kinase

regulate 4E-BP1 activity,^33-34and 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 Thr70.³⁵As discussed previously, PIMs are important in the upregulation of proteins

involved in cell cycle regulation and cell survival via cap-dependent translation in cancer.³⁶Pim-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).³⁷Western 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).³OX0999 was found to inhibit hERG with an IC₅₀of 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

arom

H

N

F₃CO

O

OX0998-1453

SGI-1776, 2

ID

SGI-1776

OX0999

OX0998

OX1453

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

Table 5: ADME properties of various amine-substituted analogues and biological activities against

PIM1. IC₅₀in nM, n = 2. hERG IC₅₀in 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

F₃CO

OH

N

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

N

S

R

S

OH

R

N

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

t_1/2(min)

>120

Caco-2

ER

MV4-11

ID

R_A

R_B

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

15.1^a

0.42

0.50

10.5

4.2^a

48.5*

33.5*

0.4

0.3

OH

19

OX1484

o-OH

^OH13.5± 2.9 2.6

3.8^a

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

6.9± 0.5

147±6

3.9

nd

<20 ^a

65 ^a

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

o-OH,

OH

m-OCF₃

Table 7: Physical properties of various substitutions on the R_Aand R_Bpositions and biological activities

against PIM1, *MLM S9.⁴IC₅₀in nM, n = 2 unless otherwise stated. ^an = 1, R²> 0.90, R²values listed

in SI; MLM t_1/2: half-life measured in mouse liver microsomes in min; KinSol: kinetic solubility in µM;

MV4-11 IC₅₀in 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).

⁴MLM 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

OH

O

OX1399

OX1401

IC₅₀: 15.1 nM

KinSol: 10.5 M

MLM S9 t_1/2: 48.5 min

Caco-2 ER: 0.31 (AB: 19.5, BA: 6.16)

IC₅₀: 15.1 nM

KinSol: 4.2 M

MLM S9 t_1/2: 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.

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Article Doi

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

DOI: 10.1016/j.bmc.2020.115724

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

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

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