Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family

Article abstract of DOI:10.1016/j.bmc.2017.02.056

The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug development, particularly for certain hematological malignancies. Here, we describe the discovery of a series of inhibitors of the PIM kinase family using a high throughput screening strategy. Through a combination of molecular modeling and optimization studies, the intrinsic potencies and molecular properties of this series of compounds was significantly improved. An excellent pan-PIM isoform inhibition profile was observed across the series, while optimized examples show good selectivity over other kinases. Two PIM-expressing leukemic cancer cell lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of selected inhibitors. Encouraging activities were observed for many examples, with the best example (44) giving an IC₅₀ of 0.75 μM against the K562 cell line. These data provide a promising starting point for further development of this series as a new cancer therapy through PIM kinase inhibition.

Full text of DOI:10.1016/j.bmc.2017.02.056

Accepted Manuscript
Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase
family
Carole J.R. Bataille, Méabh B. Brennan, Simon Byrne, Stephen G. Davies,
Matthew Durbin, Oleg Fedorov, Kilian V.M. Huber, Alan M. Jones, Stefan
Knapp, Gu Liu, Anna Nadali, Camilo E. Quevedo, Angela J. Russell, Roderick
G. Walker, Robert Westwood, Graham M. Wynne
PII:
S0968-0896(16)31421-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.056
BMC 13586
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 December 2016
23 February 2017
25 February 2017
Please cite this article as: 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, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.02.056
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Thiazolidine derivatives as potent and
selective inhibitors of the PIM kinase family
Carole J. R. Bataille, Méabh B. Brennan, Simon Byrne, Stephen G. Davies*, Matthew Durbin, Oleg Fedorov,
Kilian V. M. Huber, Alan M. Jones, Stefan Knapp, Gu Liu, Anna Nadali, Camilo E. Quevedo, Angela J.
Russell∗, Roderick G. Walker, Robert Westwood and Graham M. Wynne
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1
3TA, U.K.; Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building,
Roosevelt Drive, Oxford, OX3 7DQ, U.K.; cDepartment of Pharmacology, University of Oxford, Mansfield
Road, Oxford OX1 3QT, U.K.

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Thiazolidine derivatives as potent and selective inhibitors of the PIM kinase family
Carole J. R. Bataillea, Méabh B. Brennana, Simon Byrnea, Stephen G. Daviesa *, Matthew Durbina, Oleg
Fedorovb, Kilian V. M. Hubera, Alan M. Jonesa, Stefan Knappb, Gu Liua, Anna Nadalic, Camilo E.
Quevedoa, Angela J. Russella,c ∗, Roderick G. Walkerc, Robert Westwooda and Graham M. Wynnea
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.
ARTICLE INFO
ABSTRACT
Article history:
Received
The PIM family of serine/threonine kinases have become an attractive target for anti-cancer drug
development, particularly for certain haematological malignancies. Here, we describe the
discovery of a series of inhibitors of the PIM kinase family using a high throughput screening
strategy. Through a combination of molecular modelling and optimisation studies, the intrinsic
potencies and molecular properties of this series of compounds was significantly improved. An
excellent pan-PIM isoform inhibition profile was observed across the series, while optimised
examples show good selectivity over other kinases. Two PIM-expressing leukaemic cancer cell
lines, MV4-11 and K562, were employed to evaluate the in vitro anti-proliferative effects of
selected inhibitors. Encouraging activities were observed for many examples, with the best
example (44) giving an IC50 of 0.75 µM against the K562 cell line. These data provide a
promising starting point for further development of this series as a new cancer therapy through
PIM kinase inhibition
Received in revised form
Accepted
Available online
Keywords:
Thiazolidine
PIM kinase
Anti-cancer
High throughput screen
Kinase inhibitor
2009 Elsevier Ltd. All rights reserved.
1. Introduction
knock-out models in mice led to the discovery of the two other
family members, Pim 2 and Pim 3.[9],[10]
Most eukaryotic cellular processes and cell signaling pathways
are regulated by protein phosphorylation mediated by protein
kinases and phosphatases.[1] Protein kinases are the largest
enzyme superfamily involved in cell signal transduction.[2, 3] It
has been established that protein kinases and protein
phosphatases represent compelling therapeutic targets for a range
of diseases, including leukemias, tumors, cardiovascular diseases,
diabetes mellitus and immune/ inflammatory disorders.[4, 5]
Kinases are divided into those that phosphorylate serine or
threonine residues (388 kinases) and those that phosphorylate
tyrosine residues (90 kinases). Atypical kinases are proteins
reported to have biochemical kinase activity but lack sequence
similarity to the conventional eukaryotic kinases.
The three family members have six different isoforms from
alternate translation initiating sites. All PIM kinases are
constitutively active.[11, 12] Their expression is mediated by the
JAK/STAT signalling pathway, which is activated by various
cytokines and hormones.[13] PIM kinases are broadly expressed
in many cell lineages, as well as their corresponding progenitors
and also embryonic stem cells.[14, 15]
The expression of Pim genes in human cancer has been studied
extensively since their discovery as oncogenes in the 1980s;[16,
17] they were first implicated in human acute myeloid leukemia
(AML) cases, and have now been found to be over-expressed in
many different types of malignancies including hematologic and
solid tumors.[13, 18, 19] The PIM kinases have a variety of
downstream targets that are thought to contribute to tumour
growth and survival. In particular, PIM kinases target the pro-
apoptotic Bcl-2-associated death promoter (BAD) family
members and inhibit apoptosis.[20] In addition, it has been
shown in prostate tumours that PIM1 and c-MYC associate
together, resulting in transcriptional upregulation and
stabilization of c-MYC; such prostate tumours exhibited higher
Gleason scores and are poorly differentiated.[21] Furthermore,
The three PIM kinases comprise
a
small family of
serine/threonine kinases regulating several signaling pathways
that are fundamental to cancer development and progression. Pim
genes were originally identified as oncogenes in the early
1980s.[6] They form an independent branch of the kinase family
tree, and are related to the CAMK (calcium/calmodulin kinase)
super-family. The Pim 1 gene was identified initially in 1987,[7]
as a pro-viral insertion site for the Moloney Murine Leukemia
V—ir—us —(MoMuLV).[8] Follow-up experiments involving Pim 1
∗ Corresponding author. Tel.: +44-1865-275643; fax: +44-1865-285002; e-mail: angela.russell@chem.ox.ac.uk/steve.davies@chem.ox.ac.uk

selective and pan-PIM inhibitors may offer other therapeutic
opportunities. In addition to cancer, PIM1 kinase has been
described to be over-expressed in an array of diseases;[22-25] it
has also been reported to play a role in several autoimmune
diseases, mainly in inducing and increasing inflammatory
responses.[26] PIM1 kinase has also shown to exert potent
cardioprotective effects in the myocardium downstream of AKT,
and protect mitochondrial integrity in cardiomyocytes;[27-29]
PIM1 could therefore be of high interest in regenerative medicine
therapies.
This paper describes efforts towards the identification and
preliminary optimization of pan-PIM inhibitors using in silico
modeling.
2. Results and Discussion
Our in-house library of ~20000 drug-like compounds was
screened using
a
high-throughput differential scanning
fluorimetry (DSF) assay, against the recombinant PIM1
enzyme.[58] Inhibitors were screened at 10 µM and a threshold
thermal shift (Tm) value of >3 °C was defined as the minimum.
Active compounds were identified and divided into eleven
distinct structural classes. DSF assay of the most active 40
compounds against recombinant PIM2 isoenzyme was used to
establish their ‘pan-inhibitory’ properties (Tm value of >2 °C)
and multiple families of small molecules were identified. A
coupled kinetic assay was used as a secondary orthogonal assay
to determine IC50 values of the hits compounds against PIM1 and
Crystal structures for both PIM1 and PIM2 have been used to
understand their unique ATP binding pocket and for
computational and medicinal chemistry efforts to develop
inhibitors.[30, 31] The hinge region of PIM kinases is unusual as
it contains a proline residue, which is a component atypically
present in serine/threonine kinase hinges, as well as other unique
residues in the ATP binding cleft.[32] The backbone nitrogen
required to donate a hydrogen bond to the adenine ring of ATP is
therefore absent and conventional ATP mimetic kinase inhibitors
are not optimized for this site. This particular feature reduces
affinity for ATP, and could therefore lead to the discovery and
development of inhibitors selective to PIM against other
kinases.[33, 34]
PIM kinases are highly homologous at the amino acid level
(PIM1 and PIM2 are 85% identical; PIM1 and PIM3, 93%),[35,
36] yet differ partially in their tissue distribution.[37] Functional
redundancy of the three PIM kinases has been shown in vitro[38,
39] and in vivo[40]. This characteristic can be used
advantageously in the development of pan-PIM inhibitors.[41]
rank
their
activity.[59]
Amongst
those,
the
thioxothiazolidinedione (TTZD) family indicated consistent
activities against PIM1 and PIM2 isoenzymes.
Structurally related rhodanine containing compounds have been
identified as hits against a diverse range of drug discovery targets
in multiple therapeutic areas, and despite the fact that they have
attracted considerable attention and commentary relating to their
viability as starting points for medicinal chemistry over recent
years,[60-62] examples in drug discovery are well precedented
(Figure 1 - SIM-4a, 6, AZD1208, 2).[63] It was therefore
decided to use TTZD 7 as a lead compound in our SAR studies.
A co-crystal structure of the PIM1 enzyme and a known
inhibitor, containing a thioxothiazolidinedione motif (PDB code:
3QF9),[57] was used in in silico screening to guide further design
and synthesis of our initially identified PIM1 inhibitors.
An additional advantage to this particular structure is the
tractable and modular synthesis, based on a Knovenagel
condensation[64] between rhodanine and a range of aryl and
heteroaryl aldehydes, which allowed rapid access to a wide
selection of analogues.
Table 1. Synthetic scheme for the Knoevenagel condensation.
Reagents and conditions: (i) rhodanine, cat. piperidine, EtOH, 70
°C, 16 h. Physical properties of various substitution on the R
position and biological activities against PIM1. ∆Tm n = 1; IC50
n
= 3 unless otherwise stated. an = 1, R2 value > 0.90, values listed
in SI.
S
O
(i)
S
NH
Figure 1. Representative reported PIM inhibitors
R
R
Several investigations have reported novel competitive PIM
inhibitors, including SGI-1776,[42] AZD1208,[43] (AML)
BYL719,[44] CX-6258,[45] SMI-4a,[46] LGB321 (multiple
myeloma, MM), which have advanced to clinical trials.[47-56]
Those compounds have had various outcomes; SGI-1776 clinical
trials were discontinued in phase 1 due to dose limiting toxicity
of cardiac QTc prolongation; additional cardiac and
pharmacokinetic data evaluation has failed to demonstrate a safe
therapeutic window to prudently continue clinical development
of this molecule. Phase 1 trials were completed for AZD1208,
but the study was terminated whilst the drug was being tested for
safety, tolerability, pharmacokinetics and efficacy in AML
patients; the reason was not disclosed.[57] BYL719 and
LGH447’s Phase 1 trials have now finished and those
compounds are now ongoing phase 2 trials. Those examples
show that non-selective PIM inhibitors likely represent the way
forward toward finding the appropriate tool to treat PIM-
dependent cancers.
O
PIM1
∆Tm (°C) IC50 (nM)
Yield
(%)
64
84
73
87
60
80
59
86
90
72
84
66
95
79
81
73
PIM1
Cpd Substituent (R)
7
phenyl
2-Cl phenyl
thien-2-yl
2.6
5.5
4.7
2.8
-
-
8
320 69
458 40
-
624a
974a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
thiazol-2-yl
thiazol-4-yl
thiazol-5-yl
naphth-1-yl
3.9
8.3
7.6
10.6
10.4
6.8
6.4
10.4
8.0
10.4
6.3
4.7
11.0
62 30
113a
55a
naphth-2-yl
benzothien-3-yl
benzothien-7-yl
benzofuran-2-yl
benzofuran-3-yl
indol-3-yl
68 9
67a
156 18
184 69
55a
quinolin-2-yl
quinoxalin-2-yl
7-azaindol-3-yl
126 77
175a
1,8-naphthyridin-2-yl 69
4-methyl-4H-thieno
[3,2-b]pyrrol-5-yl
775 54
36a
71

Thus, a range of mono and bicyclic aryl and heteroaryl
derivatives were prepared. The (Z)-alkene geometry within the
products were confirmed by NOE 1H-13C NMR spectroscopy
analysis.
PIM1 ATP-binding pocket reaching out to the solvent front;
therefore substituents ought to be tolerated on this ring. To
validate these predictions, a range of groups were systematically
introduced onto the 2, 3, and 4 position of the C-ring following
the procedure described previously (Table 3).
While the larger, more demanding bicyclic-bearing TTZDs
generally had better activities, as exemplified by the quinolinyl
and quinoxalinyl derivatives (20, 21, Table 1), the monocyclic
derivatives still displayed good levels of activity. It was therefore
decided to base our selection of a starting point on a synthetically
versatile low molecular weight exemplar from which analogues
could be designed, prepared and developed in a straightforward
manner. Compound 11 was selected for optimization based on
these criteria, as it demonstrated the best activity compared to its
regioisomeric counterparts (10 and 12).
Next, additional groups were introduced onto the thiazole moiety
at C2 in order to improve the activity. Palladium-catalyzed
Suzuki-Miyaura coupling using the appropriate bromo thiazole
with an aryl boronic acid, followed by Knoevenagel
condensation[64] of the resulting aldehyde with rhodanine
afforded the desired tricyclic compounds (Table 2). The
inhibitory activity was dramatically improved by addition of a
phenyl group; 26 showed approximately a 90-fold increase in
activity, (IC50 = 624 nM (11) to IC50 = 6.7 3.1 nM (26)).
Heterocyclic substituents were also introduced at C2 by the same
synthetic method using the corresponding boronic acids.
Although good activities against PIM1 were observed with
derivatives such as (27) and (28), due to the relatively narrow
chemical scope for further optimization, the synthetic effort
focused on improving the aryl substitution pattern.
Figure 2. Representative docking picture of 26 in PIM1 active site.
All the synthesized compounds with additional substituents on
the phenyl ring showed that they retained similar activity to 26.
Interestingly, as predicted by the docking study, the size of the
aryl substituents seemed to play an important role in influencing
inhibitory activity against PIM1 (Table 3). Compound 31 (IC50
=
9.2 5.4 nM) bearing a strongly electron withdrawing CF3 group
had similar activity to compound 34 (IC50 = 11 3 nM) bearing a
strongly electron donating methoxy group at the same position,
indicating that electronic properties in this region do not appear
to play a significant role. The introduction of a hydrogen bonding
motif did not lead to a noticeable effect on the enzymatic activity
either, with the H-bond donor phenolic group (39, IC50 = 2.4 0.9
nM) showing comparable activity to an H-bond acceptor SO2Me
Table 2. Scheme for the TTZD synthesis via a Suzuki reaction
and a Knoevenagel condensation. Physical properties of various
substitution on the R position and biological activities against
PIM1. Reagents and conditions: (i) RB(OH)2, Pd(PPh3)4,
Na2CO3, EtOH/DME (1:1), 100 °C, 24 h. (ii) cat. piperidine,
rhodanine, EtOH, 70 °C, 16 h. ∆Tm n = 1; IC50 n = 3 unless
otherwise stated. an = 1, R2 > 0.90, R2- values listed in SI.
(40, IC50 = 3.6 nM) or halogen bonding group Cl (41, IC50
=
36 7 nM). Furthermore, in agreement with the performed
docking studies, the regiochemistry of substitution on the aryl
ring is also well tolerated. It therefore provided a useful handle
for further optimization of the physical properties of this series of
inhibitors.
Table 3. Scheme for TTZD synthesis via a Suzuki reaction and a
Knoevenagel condensation. Physical properties of various
substitution on the R position and biological activities against
PIM1. Reagents and conditions: (i) Ar-B(OH)2, Pd(PPh3)4,
Na2CO3, EtOH/DME (1:1), 100 °C, 24 h. (ii) cat. piperidine,
rhodanine, EtOH, 70 °C, 16 h. ∆Tm n = 1; IC50 n = 3 unless
Yield %
(i) (ii)
98 14
PIM1
PIM1
∆Tm (°C) IC50 (nM)
Cpd Substituent (R)
26
27
12.8
12.8
6.7 3.1
25 6
otherwise stated. an = 1, R2 > 0.90, R2- values listed in SI.
S
72 55
73 53
58 68
S
S
B
S
S
A NH
O
(i)
(ii)
R
R
O
28
29
13.4
12.1
6.7 3.8
85 1
C
O
Br
N
N
N
25
PIM1
Yield %
PIM1
IC50 (nM)
Cpd Substituent (R)
30
57 32
16.6
1.4 0.6
∆Tm (°C)
9.4
(i)
87
63
71
74
71
55
65
24
68
74
62
(ii)
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
2-CF3
3-CF3
34
51
26
53
69
36
31
34
32
32
22
9.2 5.4
49 21
16 8
12.3
11.7
9.3
An in silico study suggested that 26 shared a similar binding
mode with 11 against PIM1 (Figure 2, PIM1 crystal structure:
PDB code 2C3I),[65] with the oxygen atom on the rhodanine
head group of 26 forming an H-bonding interaction with the
water molecule which tightly associated with the Lys67
residue.[65] The molecule is predicted to align well within the
PIM binding pocket, forming a sandwich-like interaction with the
lipophilic area of the pocket; the additional aryl ring is
anticipated to serve as a space-filling feature.
Molecular modeling studies were carried out to assist in the
design and optimization of 26 analogues. The docking study
suggested that the ortho-, meta-, and para- positions of the
pendant aryl ring are located in a solvent exposed area of the
4-CF3
2-OMe
11 3
21 9
11 6
3-OMe
4-OMe
14.3
13.2
14.8
12.7
14
2-OH
3-OH
8.0 5.7
5.0 0.6
2.4 0.9
3.6a
36 7
25 4
4-OH
4-SO2Me
3-Cl
10.3
13.2
9.6
3-NHSO2Me
3-piperidine
3-NHSO2NMe2
4-OCF3
100 70
93
28
91
14
21
44
13.5
16
11.7
10.4
7
2.2 1.8
2.9 1.3
6.8 4.6
4-CH2-piperidine 100 35

With good enzymatic activities obtained from the SAR studies, a
selection of compounds was examined for their broader
physicochemical properties and in vitro metabolic stability using
kinetic solubility, and mouse liver microsome (MLM) stability
assays (Table 4).
(to reproduce assay conditions), but the compound was recovered
in quantitative amount with no trace of the degradation product
49. It was hypothesized that the presence of an N-substituent
within rhodanine 47 could increase susceptibility towards
hydrolysis, and thus does not present a significant stability issue
for analogues such as 7.
Table 4. Physicochemical properties and in vitro metabolic
stability. MLM t1/2: half-life measured in mouse liver
microsomes, KinSol: kinetic solubility.
Cl
Cl
S
N
Cl
Cl
S
SH
(i)
OH
NH
N
O
Cl
47
O
48
Cl
O
N
MLM t1/2 KinSol
Cpd Substituent
(min)
(µM)
33
41
42
4-CF3
3-Cl
49
5
<5
<5
34
S
(ii)
(i)
SH
OH
S
NH
3-NHSO2Me <5
O
49
O
7
Across the series, compound solubility is in the low µM range
and half-life in MLMs is < 1 hour. Solubility can be improved by
exploiting the tolerance of polar substituents on the phenyl ring,
as exemplified by 42 that bears a sulfonamide group and for
which the PIM1 inhibitory activity remained excellent (IC50 25 4
nM). Kinetic solubility of this compound was improved from < 5
µM (33, 41) to 34 µM (42). Unfortunately, the half-life in MLMs
dropped significantly from 49 min (33) to less than 5 min (42).
The enzymatic activities were excellent throughout this series of
inhibitors, but the physical and metabolic properties are
suboptimal, which poses a significant challenge in order to
progress beyond cellular, to in vivo studies. We therefore focused
our efforts on improving the physical and metabolic properties of
this inhibitor series. We also sought to mitigate any potential
issues with chemical instability of this series.
Whilst in our MLM stability studies we observed no degradation
in the control experiments in the absence of co-factor, it has been
reported in the literature that N-substituted rhodanines readily
undergo basic hydrolysis to afford α,β-unsaturated-α-
mercaptocarboxylic acids (Scheme 1),[66] a functional group that
could potentially cause toxicity in biological systems.
Furthermore Schofield et al. have recently described the cleavage
of an N-substituted rhodanine-containing compound 47 in Tris
buffer pH 7.5 (Scheme 1).[67]
Scheme 1. Reagents and conditions: (i) Tris buffer pH 7.5, 37 °C, 4 h; (ii)
NaOH (1M aq, 5 eq), 40 °C, 2 h.
Another possible concern cited for rhodanine derivatives in the
literature is the potential for isomerisation of the double bond.
While the (Z)-isomer of conjugated rhodanines is usually the
more thermodynamically stable diastereomer,[69, 70] it can be
isomerised under certain conditions, such as photoirradiation, to
the alternative geometric isomer. Whilst we did not observe any
such photoisomerisation, the combination of the propensity for
low photostability and poor solubility / metabolism properties led
us to investigate if the introduction of structural and/or steric
constraints would improve the overall profile of the series. In
order to explore alternative scaffolds to compound 7 we designed
a range of linked and ring fused scaffolds which could potential
interact in a bioisosteric fashion (Figure 3-A). Using Forge®, a
powerful computation suite to understand SAR and design, the
3D electronic and shape of each input scaffold were then aligned
with template molecule 7. The tricyclic scaffold 50 provided an
effective consensus overlay with a similarity coefficient of 0.79
(see Figure 3-B) A docking study of 50 against PIM1 (PDB code
2C3I) suggested that the carbonyl group in the rhodanine head
group has the potential to form similar interaction as 26 through a
water molecule-mediated interaction with Lys67 (Figure 3-B-D).
The planar conformation of the molecule, suggested to be
important to form the lipophilic sandwich within the active site
cleft was predicted to be maintained, with the newly formed
carbonyl linker pointing towards the solvent exposed surface
region of the pocket.
Both set of conditions were carried out on 7, as the corresponding
mercaptocarboxilic acid and hydrolysis are known in the
literature. Hydrolysis of 7 with 12M aq. NaOH to afford 49
proceeded with complete conversion in 2 h, and the data were in
accordance with the literature.[68] It is interesting to note that the
compound did not hydrolyze in Tris buffer. As solubility could
be an issue, the reaction was repeated with the addition of DMSO
Figure 3. Molecular modeling studies comparing TTZD series with the tricycle series. (A) Chemical structure of 50 (B) Overlay of 50 (ball) with 26 (diamond).
Blue represents electron negative field, red represents electron positive field, and yellow represents lipophilic interactions. (C) Overlay of 50 (green) with 26
(purple) in the PIM1 active site. (D) Representative docking picture of 26 in the PIM1 active site. (E) Representative docking picture of 50 in the PIM1 active site;
x = H2O.

A synthesis was designed that allowed the rapid preparation of an
array of representative molecules. The requisite ortho-formyl aryl
esters, which are commercially available or readily synthetically
accessible, were condensed with 2-thiohydantoin to afford the
desired tricycle (Table 5).1 The benzo-fused derivative, 50, was
obtained in good yield and showed moderate activity against
PIM1 (IC50 = 1341 nM, ∆Tm 3.8 °C). This was comparable to the
data obtained for 7 (∆Tm 2.6 °C) and was therefore a promising
start to this series. From the molecular modeling study suggesting
that the tricycle series shared the same binding pose with the
TTZD series, it was also predicted that the 7- position on the C-
ring of the tricycle would be the optimal position for further
functionalization. This hypothesis was probed by preparing three
regioisomers of the chloro-substituted tricycle, 51, 52 and 53.
Introducing a chlorine to the 6- and 8- positions (53 and 51
respectively) led to complete loss of inhibitory activity whilst the
7- position (52) showed a modest increase in inhibitory activity
(IC50 = 784 nM, ∆Tm 4.9 °C). Substituents at this position were
next systematically changed to assess their impact on the activity
against PIM1.
linker led to loss of activity (58). Substitution on the pendent aryl
ring had very little effect on the potency, as shown by the results
obtained for the fluoro-, tolyl, and methoxyl analogues (60, 61,
62). Interestingly, the addition of an O- linker gave an increase in
solubility, however, the in vitro metabolic stability remained
modest (57, 65 µM and 28 min). The level of activity was
dramatically improved by replacing the phenyl moiety by a 4,6-
dichloroindole (63, IC50 = 131 nM). This analogue also gave a
noticeably improved in vitro metabolic half-life (>100 min),
however, the solubility was poor (<5 µM). The potency increase
may be due to the possibility for alternative binding caused by
the presence of the two vicinal chloro substituents, as has been
reported previously in related systems.[71] With the aim of
improving the solubility by addition of rotatable bonds,
substituents were introduced to the N8 position of the indole but
to no avail (65). To eliminate the potential oxidative liability of
the C=S group of this series, the corresponding carbonyl
derivative was prepared, unfortunately, this led to significant
reduction of activity (66). Reduction of the thio-carbonyl group
to a methylene using NiCl2 and NaBH4[72] resulted in complete
loss of activity (67). Due to the increased complexity of this
series and narrow window for the improvement of physical
properties while maintaining the potency, it was decided not to
pursue the fused tricyclic scaffold further.
Table 5. Scheme for polycycle synthesis via a Knoevenagel
condensation and subsequent ring closure. Physical properties
and in vitro metabolic stability of various substitutions on the R
position and biological activities against PIM1. Reagents and
As an alternative strategy it was decided to also investigate
circumventing the intrinsic metabolic instability of the rhodanine
group; it was suspected that the C=S functionality within the
rhodanine could be the cause of metabolic instability.[62]
Following the same methodology described to prepare 50, 1,3-
pseudothiohydantoin and thiazolidine-2,4-dione were used as
rhodanine mimics to afford 69 and 70, respectively (Table 6). 70
gave decreased activity against PIM1 (IC50 = 171 46 nM), and 69
gave only a modest reduction in activity (IC50 = 70 1 nM)
compared with 33 (IC50 = 16 8 nM). Although the solubility of
69 remained low, the metabolic half-life was dramatically
improved (>100 min).
conditions: (i) NaOAc, AcOH, reflux, 4h. ∆Tm n = 1; IC50 n = 3
unless otherwise stated. an = 1, R2 > 0.90, R2- values listed in SI.
MLM t1/2: half-life measured in mouse liver microsomes, KinSol:
kinetic solubility
Z
O
O
O
X
X
6
9
Z
4
6
7
8
N
OMe
CHO
N
(i)
(i)
NH
R1
R1
NH
N
H
O
O
PIM1
PIM1 KinSol MLM
IC50 (nM) (µM ) (min)
Cpd R1
X
∆Tm (°C)
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
H
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
O
3.8
5.1
4.9
1.9
5.5
2
1341a
Inact
Table 6. Scheme for TTZD synthesis via a Suzuki reaction and a
Knoevenagel condensation. Physical properties and in vitro
metabolic stability of various substitution on the R position and
biological activities against PIM1. Reagents and conditions: (i) p-
CF3PhB(OH)2, Pd(PPh3)4, Na2CO3, EtOH/DME (1:1), 100 °C, 24
h. (ii) cat. piperidine, rhodanine or analogue, EtOH, 70 °C, 16 h.
∆Tm n = 1; IC50 n = 3 unless otherwise stated. an = 1, R2 > 0.90,
R2- values listed in SI. MLM t1/2: half-life measured in mouse
8-Cl
7-Cl
6-Cl
7-Ph
784a
Inact
554 192
Inact
7-(pyrid-4-yl)
7-(furan-2-yl)
7-O-Ph
1.9
6.2
1.9
3.6
0.3
9.0
2.3
7.7
5.5
ND
ND
107 10
267 119
Inact
127a
304 9
335 156
332a
131 13
273 4
100 7
804 37
Inact
11.5
65
14
28
7-NH-Ph
7-S-Ph
7-O-(4-F Ph)
7-S-(4-tolyl)
7-O-(4-OMe Ph)
4,6-diCl indole
4,6-indole
65
36
liver microsomes, KinSol: kinetic solubility
X
S
S
S
NH
S
(i)
(ii)
<5
<1
>100
18
O
O
N
Br
F3C
N
F3C
N
25
O
68
N-benzyl-4,6-diCl indole
7-O-Ph
H
PIM1 ∆Tm PIM1 IC50 MLM KinSol
Cpd
X
S
H2 ND
(nM)
(min)
(µM )
(°C)
11.7
33
69
70
16 8
70 1
171 46
49
>100
ND
<5
<5
Phenyl analogue 54 exhibited similar activity compared to the
chloro analogue 52 (IC50 = 554 nM vs IC50 = 784 nM).
Introduction of a pyridyl moiety (55) was not well tolerated,
whereas a furanyl group (56) gave a significant increase in
activity (Table 5). Unfortunately, the solubility and metabolic
half-life of 56 (11.5 µM and 14 min respectively) were
disappointing. Introducing additional O- and S- linkers between
C-ring and 7- position substituents to improve the structural and
flexibility also improved the potency (57, 59), whereas an NH-
NH 9.4
7.5
O
ND
The ability to inhibit all members of the PIM family is important
for anticancer treatment and representative examples were
therefore evaluated against PIM1, 2, and 3 using DSF to establish
their PIM isoform selectivity profile (Table 7). Most of the
examples showed comparable inhibitory potencies against all of
the PIM kinase family members.
———
1 see supporting information for synthesis.

Table 7. PIM isoform selectivity.
cell line)[73] and K562 (human immortalised myelogenous
leukemia cell line), both previously reported to be PIM
sensitive;[74] modest activities were observed in most cases
(Table 9). In the TTZD series, antiproliferative activities were
observed with representative exemplars across the series, with the
most active analogue 44 giving an IC50 of 3.4 µM against MV4-
11 cells and 0.75 µM against K562 cells. In all cases a significant
reduction in potency was observed between the enzymatic
inhibitory activities and whole cell activities. This observation
may be attributed to a lower free concentration of compound due
to binding to serum proteins present in the cell growth media, a
phenomenon previously reported for similar molecules. Effective
cell permeability may also be a contributory factor. In the tricycle
series, the indole motif containing analogues showed the best
cellular activities across the series, which is in an agreement with
the enzymatic activities discussed previously, with 64 giving an
IC50 of 3.9 µM. The substitution on the N8 position did not
produce a notable increase in cellular activities despite the
noticeable improvement in the enzymatic activities. The lack of
improvement in cellular activities could be due to cleavage of the
weak C-N bond formed between the substituent and indole
nitrogen in situ.
PIM1 ∆Tm PIM2 ∆Tm PIM3 ∆Tm
Cpd
(°C)
12.8
9.4
(°C)
12.0
8.2
(°C)
13.0
8.8
26
31
32
33
35
36
45
37
38
12.3
11.7
14.3
13.2
11.7
14.8
12.7
11.5
9.7
9.1
6.1
13.3
11.5
9.7
13.4
12.3
12
17.0
12.2
18.6
14.9
Representative examples from each series were also selected for
inhibitory evaluation in a 24-kinase panel to establish their
selectivity profiles (Table 8).[57] Those kinases were of
particular interest to measure off target activity as they have been
shown to bind polycyclic heteroaromatic structures, and also as
some of the kinases play a role in the cell lines utilized (e.g. Flt3
is over expressed in MV4-11 cells) and thus inhibition of such
kinases could, in principle confound the cell proliferation read
out.2 All classes showed a high level of selectivity towards the
PIM kinase isoforms (both PIM1 and PIM2 were included for
reference), the exceptions being off-target effects in 33 and 69
(GSK3β) and in 11 (CK2 and DYRK2). The selectivity profile of
63 was inconclusive and likely due to the poor solubility of the
compound. These results are encouraging and suggest that
selectivity for PIM isoforms over other kinases is achievable and
not compromised by the commonly reported promiscuity of the
rhodanine motif.
Table 9. Cellular antiproliferative activity of selected PIM
inhibitors determined by an MTT assay. ND= not determined.
Inact. = inactive.
PIM1 IC50
(nM)
Cellular activity IC50 (µM)
Cpd
MV4-11
inact
ND
8.1
K562
ND
16.8
11.9
7.1
11
31
32
33
35
36
37
38
40
42
43
44
45
46
63
65
69
70
624a
9.2 5.4
49 21
16 8
Table 8. 24-kinase selectivity evaluation (performed using 10
µM of compound). Data is provided as a percentage of residual
activity compared to vehicle alone.
3.8
21 9
11 6
ND
5.7
2.0
6.1
kinase
63
11
69
33
8.0 5.7
5.0 0.6
3.6a
2.4
2.4
4.3
ND
3.2
BTK(h)
CHK1(h)
CK2(h)
94
48
88
90
14.9
10.9
4.3
105 108 108 108
25 4
7
9.9
8.9
82
-3
117 53
108 101
113 106
103 102
2.2 1.8
2.9 1.3
6.8 4.6
131 13
100 7
70 1
3.4
8.7
0.75
6.9
cKit(h)
100 nd
8.5
3.9
5.1
cSRC(h)
DAPK1(h)
DYRK2(h)
EGFR(h)
Flt3(h)
98
90
74
92
ND
2.8
ND
inact
11.4
ND
11.1
117 15
110 nd
87
95
171 46
114 113
94
68
99
44
28
94
6
81
4
Although changing the C=S (33) to C=O (70) resulted in the
notable reduction in the enzymatic activity, it seems to have little
impact on the respective cellular activities (70 against MV4-11 =
11.4 µM, K562 = 11.1 µM).
GSK3β(h)
IR(h)
143 102 101
JAK2(h)
JNK2α2(h)
KDR(h)
PIM1(h)
PIM2(h)
PKBα(h)
Plk1(h)
100 89
116 106
103 105
99
99
49
43
76
81
95
3. Conclusion
114 88
110
1
In this paper, a high throughput screen and in silico study were
described to identify series of PIM kinase inhibitors. SAR studies
were carried out on the initial hit molecule and the thiazole core
was identified to be a suitable B-ring group with the optimal
balance of properties. Extending the pharmacophore of the initial
hit to a fused 3-ring system significantly improved the activity of
this family. Functionalization of the C-ring not only improved the
potency but also provided an additional option for optimization
of physical properties. Metabolic stability was highlighted as an
issue to address, and two strategies were proposed for
improvement. Additional constraints were introduced into the
system to minimise the potential reactivity for hydrolysis and
conjugate addition, and resulted in the identification of a fused
tricycle series. This series of inhibitors had good activity and led
17
nd
0
2
2
106 101 63
98
94
103
102
SAPK2a(h) 103 109 94
SIK(h) 106 82
119 103
Compounds were evaluated for antiproliferative activity against
two cancer cell lines MV4-11 (human acute monocytic leukemia
———
2 See supporting information for full protocol.

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to improvement in both metabolic stability and solubility. The
second strategy involved replacement of the rhodanine head
group with a pseudothiohydantoin, which resulted in a significant
8.
9.
increase
in
metabolic
stability.
Additionally,
the
pseudothiohydantoin head group provided further opportunity for
future optimisation. Selected inhibitors were evaluated to
establish the selectivity profile against the three PIM isoforms.
An excellent PIM pan-inhibition profile was observed across the
series. A kinase selectivity profile was established, and few off-
target activities were also observed, supporting our initial
hypothesis that the activity of this family of compounds was not
obtained through the suggested promiscuous nature of the
rhodanine, but instead from its specific affinity towards the PIM
family. Two leukaemia cancer cell lines, MV4-11 and K562,
were employed to evaluate the antiproliferative activities of
selected inhibitors. Moderate activities were observed for a
number of examples, with the best example showing IC50 value
of 0.75 µM against K562 cells. Together this data represents a
very promising starting point for the development of in vitro and
in vivo pharmacological probes and drug candidates. Future
efforts will focus on the optimisation of physical properties and
cellular activity of this series of compounds for potential in vivo
evaluation.
10.
11.
12.
13.
14.
Acknowledgments
This work was supported by Cancer Research UK through a
Small Molecule Cancer Drug Discovery Award (C17468/A9332,
MBB, SB, MD, KVMH, AMJ, GL, AN, CEQ and RGW). We
would also like to thank Research Councils United Kingdom for
a fellowship (AJR). SK and OF 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).
15.
16.
17.
18.
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