Discovery of Highly Selective Inhibitors of Calmodulin-Dependent Kinases That Restore Insulin Sensitivity in the Diet-Induced Obesity in Vivo Mouse Model

Article abstract of DOI:10.1021/acs.jmedchem.9b01803

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

Full text of DOI:10.1021/acs.jmedchem.9b01803

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Article
Discovery of highly selective inhibitors of calmodulin-dependent kinases that
restore insulin sensitivity in the diet-induced obese in vivo mouse model.
Christophe Fromont, Alessio Atzori, Divneet Kaur, Lubna Hashmi, Graziella Greco, Alejandro
Cabanillas, Huy Van Nguyen, D. Heulyn Jones, Miguel Garzón, Ana Varela, Brett Stevenson,
Greg Iacobini, Marc Lenoir, Sundaresan Rajesh, Clare Box, Jitendra Kumar, Paige Grant, Vera
Novitskaia, Juliet Morgan, Fiona Sorrell, Clara Redondo, Andreas Kramer, C. John Harris, Brendan
Leighton, Steven Vickers, Sharon Cheetham, Colin Kenyon, Anna M. Grabowska, Michael Overduin,
Fedor Berditchevski, Chris J Weston, Stefan Knapp, Peter M. Fischer, and Sam Butterworth
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01803 • Publication Date (Web): 20 May 2020
Downloaded from pubs.acs.org on May 21, 2020
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Cheetham, Sharon; RenaSci Ltd
Kenyon, Colin; Stellenbosch University, DST/NRF Centre of Excellence
for Biomedical Tuberculosis Research, SAMRC Centre for Molecular and
Cellular Biology
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Grabowska, Anna; University of Nottingham, Division of Pre-Clinical
Oncology
Overduin, Michael; University of Alberta, Department of Biochemistry
Berditchevski, Fedor; University of Birmingham, Institute of Cancer and
Genomic Sciences, College of Medical and Dental Sciences
Weston, Chris; University of Birmingham, Institute of Immunology and
Immunotherapy, College of Medical and Dental Sciences
Knapp, Stefan; Goethe-Universitat Frankfurt am Main, Instutute of
Pharmaceutical Chemistry
Fischer, Peter; University of Nottingham Centre for Biomolecular
Sciences, School of Pharmacy
Butterworth, Sam; University of Manchester, Pharmacy and Optometry
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Discovery of highly selective inhibitors of calmodulin-dependent kinases that
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restore insulin sensitivity in the diet-induced obese in vivo mouse model.
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Authors
Christophe Fromont,§ Alessio Atzori, § Divneet Kaur,§ Lubna Hashmi,§ Graziella
Greco,¶ Alejandro Cabanillas,¶ Huy Van Nguyen,¶ D. Heulyn Jones,# Miguel Garzón,#
Ana Varela,# Brett Stevenson,+ Greg P. Iacobini,+ Marc Lenoir,Φ Sundaresan Rajesh,Φ
Clare Box,Φ Jitendra Kumar,ϒ Paige Grant,ϒ Vera Novitskaya,Φ Juliet Morgan,+ Fiona
J. Sorrell,ε Clara Redondo,ε Andreas Kramer,Ψ C. John Harris,ρ Brendan Leighton,Ω
Steven P. Vickers,ϑ Sharon C. Cheetham,ϑ Colin Kenyon,ς Anna M. Grabowska,ϖ
Michael Overduin,ϒ Fedor Berditchevski,Φ Chris J. Weston,λ Stefan Knapp,Ψ Peter M.
Fischer§ and Sam Butterworth#*
Affiliations
#Division of Pharmacy and Optometry, School of Health Sciences, Manchester
Academic Health Sciences Centre, University of Manchester, Manchester, M13 9PL,
UK.
§Centre for Biomolecular Sciences and School of Pharmacy, University of
Nottingham, Nottingham NG7 2RD, UK.
¶School of Pharmacy, College of Medical and Dental Sciences, University of
Birmingham, Edgbaston, B15 2TT, UK.
ΦInstitute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston,
Birmingham, B15 2TT, UK.
+Sygnature Discovery, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, UK.
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ϒDepartment of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7,
Canada.
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εStructural Genomics Consortium and Target Discovery Institute, Nuffield
Department of Clinical Medicine, University of Oxford, Old Road Campus Research
Building, Oxford OX3 7DQ, UK.
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ΨStructural Genomics Consortium and Buchmann Institute for Molecular Life
Sciences, Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-
University, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
ϑRenaSci Limited, BioCity, Pennyfoot Street, Nottingham, NG1 1GF, UK.
ρCJH Consultants, Ford Cottage, South Weirs, Burley Road, Brockenhurst, Hants,
SO42 7UQ, UK.
ΩThe Research Network, IPC 600 Discovery Park, Ramsgate Road, Sandwich CT13
9NJ.
ςDST/NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC
Centre for Molecular and Cellular Biology, Division of Molecular Biology and
Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University,
Cape Town 8000, South Africa
ϖEx Vivo Cancer Pharmacology Centre of Excellence, Cancer Biology, Division of
Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham
NG7 2RD, UK.
λCentre for Liver Research, Institute of Immunology and Immunotherapy, University
of Birmingham, Birmingham, B15 2TT, UK, and NIHR Birmingham Biomedical
Research Centre, University Hospitals Birmingham NHS Foundation Trust and
University of Birmingham, B15 2TT, UK
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ABSTRACT
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Polymorphisms in the region of the calmodulin-dependent kinase isoform D
(CaMK1D) gene are associated with increased incidence of diabetes, with the most
common polymorphism resulting in increased recognition by transcription factors and
increased protein expression. While reducing CaMK1D expression has a potentially
beneficial effect on glucose processing in human hepatocytes, there are no known
selective inhibitors of CaMK1 kinases that can be used to validate or translate these
findings. Here we describe the development of a series of potent, selective and drug-
like CaMK1 inhibitors that are able to provide significant free target cover in mouse
models and are therefore useful as in vivo tool compounds. Our results show that a
lead compound from this series improves insulin sensitivity and glucose control in the
diet-induced obesity mouse model after both acute and chronic administration,
providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.
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INTRODUCTION
The CaMK1 family of calmodulin-dependent kinases are widely expressed including
in hepatocytes, endothelia, immune cells, and the CNS.1,2 There are four CaMK1
isoforms with high similarity in the kinase domain, especially the ATP binding site,
but which differ in their overall structure and tissue distribution.
Single-nucleotide polymorphisms in the CaMK1D locus are associated with increased
incidence of diabetes in a large number of genome-wide association studies
(GWAS).3–6 While these variations are non-coding it has been demonstrated that the
diabetes-associated polymorphism rs11257655 increases FOXA1 transcription factor
binding and thereby increases CaMK1D protein expression in multiple cell models.7
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A direct role for CaMK1D in glucose processing has been observed following knock-
down of commonly observed GWAS-identified proteins in primary human
hepatocytes.8 In this model treatment with CaMK1D siRNA results in loss of nuclear
translocation of the established diabetes target CRTC2/TORC29–11 and is associated
with decreased gluconeogenesis and increased glycogen deposition.
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Increased CaMK1D expression is also implicated in triple-negative breast cancer
(TNBC). Large-scale genomic/transcriptomic analyses of breast tumours indicate that
gains at the 10p13 locus, which spans the CaMK1D gene, are observed in 80% of
TNBC tumours12 with high occurrence in estrogen receptor-negative and TNBC
tumours of younger patients.13 In a separate study, biopsies from 172 breast cancer
patients showed significant gains at the 10p13 locus among basal-like tumours,
leading to CaMK1D over-expression at transcriptional and protein level.14 When
expressed in non-tumourigenic mammary epithelial cells (MCF10A), CaMK1D was
found to lead to transformation, increasing proliferation and inducing a mesenchymal-
like phenotype.14 Mouse models also corroborate the effect of overexpressing
CaMK1D on altered cell proliferation and apoptosis.15
Despite the emergence of CaMK1D as a potentially important therapeutic target, there
are no known selective CaMK1 inhibitors. We therefore sought to develop potent and
selective inhibitors of this class of kinases for use in target validation experiments,
ahead of further translational studies.
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RESULTS AND DISCUSSION
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Compound 1 and derivatives thereof have received significant attention as inhibitors
of spleen tyrosine kinase (SYK) and may have utility in the treatment of autoimmune
disease or lymphomas.16–19 Previously published selectivity data shows compound 1
to have inhibitory activity against CaMK1D20 and as such we selected this as the basis
for a structure-based drug discovery campaign, with the aim of improving the potency
and selectivity of compounds from this series towards CaMK1D. We initially
examined the compound-induced shift in the thermal denaturation midpoint (Tm) of a
small panel of kinases, in order to allow rapid quantification of the binding to both
CaMK1D and off-target kinases (Table 1). Initial chemical efforts focussed on
alterations in the primary amide and amine regions, resulting in compounds 2-6,
which demonstrate comparatively steep structure activity relationships when either
the primary amide or primary amine are substituted. Substitution of the secondary
amine to give compound 3 is well tolerated and results in an apparent improvement in
kinase selectivity, with further derivatisation leading to compound 6, which
demonstrated a higher Tm shift against CaMK1D and improved selectivity relative to
earlier compounds. Separation of the enantiomers of this compound led to compounds
7 and 8, the latter of which has previously been reported as a SYK inhibitor17 but has
higher activity against CaMK1D than its enantiomer and demonstrates a more
favourable activity profile relative to compounds 1 and 2 in both Tm and enzymatic
assays.
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Table 1. Structure-Activity Relationships of Pyrimidine Amides.
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Tm shift (°C)
IC50 (µM)
R1
R2
0.456a
0.432
5.44
(0.58)
7.51
(0.09)
4.05
3.4
1.34
(0.28)
0.11
(0.25)
0.007a
1
2
3
4
5
6
7
8
H
Me
H
(0.29)
(0.44)
(0.084)
0.12
(0.3)
1.36
-0.27
-0.18
(0.18)
-0.53
(0.15)
-
-
-
(0.16)
(0.39)
7.35
(0.4)
5.13
(0.17)
4.48
0.84
2.13
(0.21)
0.10
(0.07)
0.851
(0.160)
-
(0.13)
(0.13)
2.43
(0.08)
2.70
0.70
3.36
(0.08)
0.11
(0.14)
-
H
-
-
3.49a
(0.27)
(0.16)
4.68
(0.11)
3.45
0.76
2.21
(0.1)
0.08
(0.23)
H
-
-
(0.31)
(0.19)
9.84
(0.17)
8.59
(0.34)
6.69
2.31
4.94
(0.08)
1.08
(0.04)
0.485
(0.017)
-
-
H
(0.11)
(0.13)
6.84
(0.07)
6.82
(0.39)
4.63
1.70
3.36
(0.14)
0.95
(0.05)
1.35
(0.018)
H
(0.03)
(0.12)
0.179a
0.186
10.5
(0.04)
9.05
(0.36)
7.01
2.50
5.09
(0.15)
1.1
(0.08)
H
0.087a
(0.01)
(0.16)
(0.027)
All data represents mean of at least n=3 independent experiments with standard
deviation in parentheses, except; a Compounds tested at Reaction Biology n=1.
Based on these results we evaluated the pan-kinome selectivity of 8 in a competitive
binding assay at 1 µM, which demonstrated a somewhat targeted profile. CaMK1D
and SYK are amongst the 11 wild-type kinases inhibited by >90% in this format,
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supported by subsequent enzymatic selectivity data against selected targets (Fig. 1
and SI).
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8
Enzyme
% residual probe
IC50
Kinase
binding at 1 µMa
(µM)b
RIPK4
CaMK1D
PIM1
0.9
2.0
2.7
5.7
6.5
7.3
8.1
8.6
8.9
9.5
9.6
0.092
0.179
0.053
0.356
9.131
0.240
-
TAK1
CK2a
MEK5
PIP5K1C
CSF1R
MLK3
BLK
0.168
0.052
0.057
0.087
SYK
Figure 1. Selectivity data against selected wild-type kinases for compound 8
aCompounds tested at Eurofins DiscoverX, n=1. bCompounds tested at Reaction
Biology, n=1.
As expected the CaMK1D-bound crystal structure of compound 8 shows the
compound binds at the ATP binding site in a type-1 fashion. A comparison of the
binding mode of related compounds in SYK (e.g. PDB: 4RX9) demonstrated a slight
shift in binding mode resulting from differences in the conformation of the loop at
residues 163-165 (corresponding to 510-512 in SYK) as well as a flip in the
orientation of the aniline meta-substituent in CaMK1D to occupy a pocket adjacent to
L100 at the edge of the hinge region. The L100 pocket appears significantly larger in
CaMK1D than the majority of the observed off-targets where structural data was
available. In addition, the second meta region that is not utilised by 8 is close to the
potentially flexible side chain of E105. We hypothesised that flipping of the
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orientation of the aniline allows the ligand to avoid the L100 pocket when binding to
some off-target kinases. This led to the design of compound 9 that removes this
ambiguous binding mode by occupying both the L100 and E105 regions.
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V37
L100
G30
L29
Hinge
M98
L29
S102
D165
N149
DFG-motif
V101
I163
S164
F166
E105
L151
CAMK1D
SYK
Figure 2. Compound 8 bound to CaMK1D (6T6F, white) and overlay with related
SYK structure (4RX9, yellow). Important residues and inhibitors are shown in stick
representation. For better visibility the P-Loop has been made transparent.
This bis-meta substitution pattern was well tolerated by CaMK1D when assessed by
Tm shift and resulted in a significant decrease in the binding to off-targets including
SYK (Table 2). Further SAR studies identified that a wide range of functional groups
can be tolerated at the L100 pocket, with introduction of groups with a wide range of
lipophilicity and bonding potential able to maintain or even enhance binding and
selectivity (Table 2).
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Table 2. Effects of varying aniline substitution on potency/selectivity.
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Enzyme
IC50 (µM)
Tm shift (°C)
Compound
R3
10.5
(0.04)
9.05
(0.36)
5.92
3.77
5.02
(0.36)
8.32
(0.28)
0.186
(0.027)
8
3-CF3
3,5-diCF3
3-tBu
(0.45)
(0.12)
13.3
(0.09)
4.13
(0.57)
4.58a
(0.33)
6.07
(0.06)
5.89
(0.1)
3.88
(0.24)
3.98
(0.05)
0.455
(0.451)
0.277b
(0.053)
9
9.61
(0.16)
3.02
3.02
2.27
(0.22)
2.09
(0.44)
10
11
12
13
(0.09)
(0.56)
7.85
(0.09)
5.23
(0.18)
4.21
5.11
5.33
(0.09)
1.78
(0.65)
0.101
(0.07)
3-SO2Me
3-Ph
(0.09)
(0.02)
11.9
(0.26)
9.32
(0.29)
4.81
3.79
4.5
(0.26)
4.96
(0.06)
0.047
(0.026)
(0.11)
(0.01)
3-(2-
cyanoiPr)
7.76
(0.24)
5.00
(0.28)
3.32
9.51
2.85
(0.28)
1.3
(0.2)
0.096
(0.05)
(0.39)
(0.19)
All data represents mean of at least n=3 independent experiments with standard
deviation in parentheses. aMeasured on racemic compound. bn=2.
The binding affinity and selectivity for CaMK1D were further improved by
combining these structural features in symmetrical and unsymmetrical bis-meta
substituted anilines, especially those containing substituents that place electron
density above and below the aniline ring plane. The clearest example of this is
compound 14, which is too lipophilic to be a useful lead compound but nonetheless
exhibits a very high Tm shift with CaMK1D and negligible off target binding.
Evaluation of 14 in pan-kinome selectivity assay reveals highly specific binding to
CaMK1D and the closely related CaMK1A and CaMK1B, again supported by
enzymatic evaluation (Figure 3 and SI).
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% residual
probe binding
at 1 µMa
Enzyme
IC50
Tm shift
(°C)
Kinase
(µM)b
14
12.4
(0.07)
CaMK1D
CaMK1B
CaMK1A
DAPK1
CK2a
1.4
0.0
8.8
81
0.399
-
-
-
-
1.13
(0.03)
-
-0.03
(0.08)
100
100
95
-
-
-0.43
(0.93)
PIM1
MEK5
-
-
>10
>10
-
MLK3
77
0.37
(0.27)
SYK
82
Figure 3. Selectivity data against selected wild-type kinases for compound 14. Tm
shift data represents mean of at least n=3 independent experiments with standard
deviation in parentheses, see supporting information for experimental details.
aCompounds tested at Eurofins DiscoverX, n=1. bCompounds tested at Reaction
Biology, n=1.
Our hypothesis is that the disconnect between the apparent high binding of 14
observed by Tm shift and the competitive binding assay at DiscoveRx, compared to
the lower activity in competitive inhibition assays, relate to it’s poor physiochemical
properties, which may lead to compound losses during serial dilution.
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Despite the issues with properties in this specific example, we believed 14
demonstrated the potential to achieve high selectivity in this series and therefore
sought to combine this selectivity with improved potency and physical properties by
exploiting the wide SAR scope at the aniline meta positions. Subsequent evaluation of
a wide range of aniline substituents generally validated our hypothesis that ‘bulky’
substituents are favoured for both potency and selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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This work led to compounds 15-18, which show good binding and selectivity in both
Tm and enzymatic assays. Selected compounds were assessed for their ability to
inhibit auto-phosphorylation of CaMK1D at activation loop residues serine 179 and
threonine 180 in over-expressing MDA-MB-231 cells. This data demonstrated that
the inhibitors have limited cell drop-off for compounds with no additional H-bond
donors or basic centres (Table 3). Pan-kinome screening data on 18 shows the high
selectivity of 14 can be maintained in compounds with more favourable
physicochemical properties, with enzymatic data on identified off-targets
demonstrating >150-fold greater activity vs CaMK1D than all non-CaMK1 kinases.
The in vitro pharmacokinetic profile of 18 reveals generally favourable properties
with high solubility, low metabolism and moderately high plasma protein binding
(PPB), but low permeability with some evidence of efflux in the CaCo2 model (Table
5). This low permeability does not appear to impact on either cellular activity or oral
pharmacokinetics, where 18 shows good bioavailability in mice and rats despite
moderately high clearance.
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Table 3. Effects of varying substitution of bis-meta substituted anilines on potency
5
6
and selectivity.
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8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Tm shift (°C)
CaMK1D IC50 (µM)
R3
R3’
11.9
8.20
4.65
4.05
5.95
0.62
(0.32)
0.115
0.285a
15
16
17
18
iPr
iPr
SO2Me
Ph
(0.15) (0.37) (0.19) (0.07) (0.17)
(0.051)
(0.013)
11.8 0.87 3.83 1.85 1.09
(0.25) (0.38) (0.19) (0.16) (0.06)
0.94
(0.46)
0.027
0.028a
tBu
(0.005)
(0.011)
2-cyano-
15.9
(0.13)
4.8
-0.21
1.95
1.11
(0.14)
0.022
0.019a
-
-
iPr
(0.2) (0.37) (0.02)
(0.009)
(0.001)
2-cyano- 2-cyano-
iPr iPr
11.5
(0.17)
3.46 0.56 0.27
(0.23) (0.16) (0.16)
0.81
(0.27)
0.031
0.008
(0.003)
(0.002)
All data represents mean of at least n=3 independent experiments with standard
deviation in parentheses, except; an=2.
18
% residual
probe binding at
1 µMa
IC50
Kinase
(µM)b
CaMK1D
CaMK1B
CaMK1A
PIP5K1C
MEK5
0.9
15
0.026c
0.011
(<0.001)
0.002
(0.001)
16
3.0
9.8
95
7.12c
7.68
(0.547)
7.86
(4.20)
MLK3
Figure 4. Selectivity data against selected wild-type kinases for compound 18.
aCompounds tested at Eurofins DiscoverX, n=1. bCompounds tested at Reaction
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Biology, n=2 independent experiments with standard deviation in parentheses except
cn=1.
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The oral pharmacokinetic profile of 18 in mice scaled well at doses up to 100 mg/kg,
and the PPB-adjusted cover over cellular IC50 observed suggested that this compound
may be suitable for in vivo target validation studies (Figure 5).
Mouse
Rat
Route
i.v.
p.o.
p.o.
p.o.
p.o.
p.o.
i.v.
p.o.
Dose (mg/kg)
2
10
25
37
50
100
200
1
7
%F
CL (mL/min/kg)
AUC (0->inf, ng.h/mL)
Cmax (ng/mL)
t1/2 (h)
-
41
-
88
-
94
-
45
-
-
73
-
88
-
1775
453
1.7
-
125
134
133
6.2
387
747
2.3
4.9
790
196
1.2
8497 18305 17522
684
136
3.3
-
1294
2.7
-
2221
2.8
-
3057
4.7
-
Vss (L/kg)
40.3
Figure 5. In vivo pharmacokinetic profile of 18 in male CD-1 mice (25-40 g) and
male Crl:CD Sprague Dawley rats (250-400 g) dosed in 10% DMSO/90%
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hydroxypropyl-beta-cyclodextrin (20% w/v), n=3 per group. Free cell IC50 was
calculated by dividing measured cellular IC50 by free fraction in mouse plasma.
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In order to improve the potential utility of these compounds, we sought to identify a
lead compound with improved potency and reduced clearance in order to allow for
greater cover in in vivo experiments at reduced doses. In common with 18, a large
number of compounds from this series demonstrated high solubility, low A-B/high B-
A CaCo2 permeability and moderate to low metabolism in mouse microsome and
hepatocyte assays. However, the in vivo clearance of the compounds was often greater
than estimated liver blood flow, suggesting that hepatic metabolism is not the key
driver of clearance in mouse.
A more detailed evaluation of the in vivo pharmacokinetics of 18 revealed a similar
picture. Despite its high in vivo clearance, metabolite identification studies on rat ex
vivo plasma samples reveals only low levels of metabolites, resulting from oxidation,
acetylation or amide hydrolysis (potentially subsequent to acetylation), while analysis
of urine collected from 18 dosed rats reveals renal excretion of unchanged drug at
least partially contributes to clearance.
Despite the poor predictivity of the in vitro pharmacokinetic assays, optimisation of
the aniline region of the compounds was continued, relying on in vivo studies to
distinguish compounds that showed suitable in vitro activity profiles. This work led to
the finding that compounds containing 4-pyridyl substituents such as seen in 19
exhibited retained or enhanced activity in CaMK1D enzyme and cell assays with
similar selectivity, but reduced in vivo clearance. Examination of the structure activity
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relationships around this structural change revealed similar SAR to that observed with
the anilines. Our hypothesis is that the pyridyl nitrogen is protonated in the bound
state, supported by experimental data revealing that 19 is di-basic, with pKa values of
8.7 and 7.5 for the primary amine and pyridine respectively.
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13
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18
19
20
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Table 4. Effects of introducing 2,6-di-iso-propylpyridine on potency and selectivity
of compound 19.
O
N
NH2
N
H
N
N
N
NH2
CaMK1D IC50
Tm shift (°C)
(µM)
16.0
(0.24)
5.25
(0.02)
1.54
(0.2)
2.34
(0.06)
0.55
(0.24)
0.008
(0.001)
0.011
(0.001)
19
All data represents mean of at least n=3 independent experiments with standard
deviation in parentheses.
The increased potency and higher in vivo blood concentrations seen with 19 result in
significantly improved free cover over the cellular IC50 in mouse (Figure 7), and
consistent with the low CaCo2 A-B/high B-A seen with this compound the free
plasma to brain ratio is ≤0.01 at all time-points to 24 hours. The compound retains
high pan-kinome selectivity, however the move to secondary (i.e. iso-propyl)
substituents does result in a reduction in selectivity over MEK5 in both affinity and
biochemical assays. These findings are reflected in the CaMK1D bound crystal
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structures of compounds 18 and 19, which show the iso-propyl groups in 19
occupying a low-energy conformation with the methyl groups out of the plane of the
aryl ring, potentially avoiding unfavourable interactions with the protonated pyridine.
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13
14
15
16
17
18
19
20
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Compound 19
% residual probe
IC50
Kinase
CaMK1D
CaMK1B
CaMK1A
CaMK1G
PIP5K1C
MEK5
binding at 1 µMa
(µM)b
19
0.9
0
0.008c
0.003
(<0.001)
0.001
(<0.001)
0.001
(<0.001)
11.2
(8.50)
0.025
(0.012)
5.69
(0.310)
2.75
0
0
0
0.95
4.5
99
RIPK4
MLK3
(2.05)
Figure 6. Selectivity data against selected wild-type kinases for compound 19
aCompounds tested at Eurofins DiscoverX, n=1. bCompounds tested at Reaction
Biology data, n=2 independent experiments with standard deviation in parentheses
except cn=1.
B
A
19
18
L100
L100
V101
Q99
Q99
M98
M98
S102
V101
S102
DFG-motif
DFG-motif
Hinge
D165
S164
S164
Hinge
F166
D165
F166
G103
G104
G103
N149
G104
E105
N149
E105
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Figure 7. Comparison of the CaMK1D bound structures of compounds 18 (6T28)
and 19 (6T29). CaMK1D is shown as grey cartoon representation, the hinge region is
highlighted in wheat and the DFG motif in green colour. Important residues and
inhibitors are shown in stick representation. For better visibility the P-Loop has been
made transparent. Hydrogen bonds are indicated as black dashed lines. The inserts on
the upper left of each figure show the electron density of the compounds as 2Fo-
Fc maps contoured at 1.5 σ.
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13
14
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18
19
20
21
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46
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49
50
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Table 5. In vitro pharmacokinetic and toxicology profile of 18 and 19.
18
19
LogD
2.3
2.6
52
>200
6 / 3 / 7
Solubility (µM)
Mouse/rat/human Mics
15 / <1 / 13
13 / 11 / -
98.4 / 94 / -
1.1 / 13.4
6 / 1
(µL/min/mg)
Mouse/rat/human Heps
14 / - / 19
94 / 92 / 89
0.4 / 7.9
6 / 10
(µL/min/106 cells)
Mouse/rat/human PPB
(% bound)
CaCo2 Papp (A-B x 10-6
cm/s)/Efflux ratio
CYP450 2C9/2C19
IC50 (µM)a
Ionworks hERG IC50
(µM) / ratio to cell IC50
7.1 / 284
3.7 / 336
aCYP450 1A2, 2D6 and 3A45 IC50 > 20 µM for all compounds.
As expected from the high active site homology in the CaMK1 family, both 18 and 19
show limited selectivity between CaMK1A, CaMK1B, CaMK1D and CaMK1G.
More detailed secondary pharmacology screening with 18 and 19 reveals some
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evidence of hERG and CYP450 inhibition (Table 5) with the compounds
demonstrating ~300-fold selectivity over hERG based on cellular IC50.
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18
19
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Mouse
i.v.
2
Rat
i.v.
Route
Dose (mg/kg)
F%
p.o.
10
44
-
p.o.
20
p.o.
40
p.o.
10
2
-
-
118
-
100
-
67.5
-
CL (mL/min/kg)
AUC (0-inf, ng.hr/mL)
Cmax (ng/mL)
t1/2 (h)
13.7
2454
503
8.3
7.46
-
36.5
913
391
7.7
18.5
-
5408
387
5.9
-
28923
2015
5.9
-
49220
3792
6.2
-
3058
199
7.0
-
Vss
Kp, uu (@tmax
)
-
-
-
0.001
Figure 8. In vivo pharmacokinetic profile of 19 in male CD-1 mice (25-40 g) and
male Crl:CD Sprague Dawley rats (250-400 g) dosed in 10% DMSO/90%
hydroxypropyl-beta-cyclodextrin (20% w/v), n=3 per group. Free cell IC50 calculated
by dividing measured cellular IC50 by free fraction in mouse plasma.
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Initial evaluation of the in vivo efficacy of these inhibitors was conducted in mice
with diet-induced obesity (DIO mice), which demonstrate impaired glucose control
mediated by reduced sensitivity to insulin. Compound 18 was selected for these
studies due to its high kinome selectivity, with sampling revealing slightly higher
exposure in DIO mice relative to earlier pharmacokinetic studies (Figure 5) and no
significant changes in exposure on repeat dosing for 14 days at 25 mg/kg. DIO mice
treated with a single dose of 25 or 50 mg/kg 18 (in 1:9 DMSO:20% aqueous 2-
hydroxypropyl-β-cyclodextrin) 4 hours prior to an oral glucose tolerance test (OGTT)
show improved glucose control and increased insulin sensitivity relative to vehicle
controls (figure 9A), with no significant difference in effect between the 25mg/kg and
50 mg/kg dose groups. Repeat administration of 18 twice daily for 14 days (in 1:9
DMSO:20% aqueous 2-hydroxypropyl-β-cyclodextrin on days 1-6, and 1% methyl
cellulose on days 7-14) resulted in reduced baseline glucose and insulin levels, as well
as reduced peak glucose levels following OGTT. While there was no effect on
glucose AUCB2 in this experiment, the improvement in apparent insulin sensitivity
was maintained and the overall profile was similar to the positive control liraglutide,
which demonstrated a similar profile to that expected based on previous data.
Unfortunately repeat dosing of 50 mg/kg 18 was not tolerated due to bloating of the
gastrointestinal tract, (resulting in change of vehicle in the 25 mg/kg group at day 7)
however this was not observed in subsequent tolerability studies with other
compounds at significantly higher exposure/free cover. For example 19 has been
dosed at 40 mg/kg uid (in 50:45:5 PEG400:water:ethanol) for 21 days in NGS mice
with no observable adverse effects, suggesting the GI effects seen with 18 are
unlikely to be target related.
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19
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53
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60
Figure 9. In vivo activity of compound 18 following oral glucose tolerance test
(OGTT) in diet induced obesity mouse model (male C57Bl/6J mice) following single
dose 4 hours prior to OGTT (A, n=6 per group) and 14 day repeat dosing (B, n=8 per
group). Significant differences (from vehicle), determined by multiple t-test
comparisons, are denoted by *p<0.05, **p<0.01 and ***p<0.001.
CHEMISTRY
Compounds 1 and 2 are known compounds and were prepared using the previously
reported synthesis.16 Analogues 3-8 were prepared from ethyl 2,4-dichloropyrimidine-
5-carboxylate 20 via the 2-HOBt pyrimidine intermediate 21, which was prepared as
previously described,16 by displacement of the 2-HOBt by the appropriate Boc-
protected amine and Boc removal under acidic conditions (scheme 1).
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Scheme 1. Synthetic route to compounds 3-8
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
29
30
31
32
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34
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60
Reagents and conditions: (i) N-Boc-diamine, THF/DMF, RT, 30 mins; then DCM, 4
N HCl in dioxane, RT, 1 h, 81%.
To explore the aniline component, the tail was fixed to the (S)-3-aminopiperidine and
a new synthetic strategy was sought to provide more rapid access to analogues with
varied aniline substituents. The commercially available 2,4-dichloropyrimidine-5-
carboxamide 22 allows formation of the final products in a typically 3-step process,
by sequential displacement using the aniline followed by protected (S)-3-
aminopiperidine and final acidic deprotection (scheme 2). The synthesis can be
abbreviated by utilising excess unprotected (S)-3-aminopiperidine directly in the
second stage, with the increased nucleophilicity of the cyclic secondary amine
resulting in <1% formation of the product resulting from reaction at the primary
amine. In general this approach complicates the purification of the final compounds
and as such has not been extensively used, however may be useful when introducing
acid-sensitive anilines.
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Scheme 2. General synthetic route to compounds 9-19
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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41
42
43
44
45
46
47
48
49
50
51
52
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58
59
60
Reagents and conditions: (i) aniline, iPr2NEt, MeCN or dioxane, reflux, 16 h; (ii) (S)-
tert-butyl piperidin-3-ylcarbamate, DIPEA, solvent, RT, 2-16 h; (iii) TFA/DCM or 4
N HCl in dioxane, RT, 1 h, 10-73% over 3 steps.
The route outlined in scheme 2 provided ready access to compounds 9-14 by utilising
commercially available anilines in stage 1. Subsequent synthetic effort focussed on
preparation of anilines designed to develop the emerging SAR in this series. The
symmetrical anilines 27 and 28 were initially synthesised by Suzuki reaction on
dibromo precursors 23 and 24, however it was later found that use of the nitro
derivatives 25 and 26 results in a slightly cleaner reaction and easier purification,
without adding extra steps as the nitro group and carbon-carbon double bonds are
reduced in a single stage by heterogeneous hydrogenation.
Scheme 3. Synthesis of di-alkyl (hetero)anilines 27 and 28, used in compounds 15
and 19.
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Reagents and conditions: (i) (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoprene,
NaHCO3, PdCl2dppf or Pd(PPh3)4, 1,4-dioxane, water, 90 °C, 4-16 h, 54-83%; (ii) H2,
Pd/C, MeOH, RT, 2-16 h, 75-98%.
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19
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21
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t
In order to prepare the mixed Bu/methylsulfone aniline 34 the commercially
available nitro compound 29 was treated with chlorosulfonic acid in chloroform at
reflux to afford the sulfonyl chloride 30, which was reduced to the thiol 31 with
triphenyl phosphine in refluxing toluene. Alkylation of 31 with methyl iodide
provided the methyl sulfide 32, which was oxidized with m-CPBA to the
corresponding sulfone 33, before nitro group reduction by heterogeneous
hydrogenation to provide aniline 34.
Scheme 4. Synthesis of aniline 34, used in compound 16
Reagents and conditions: (i) chlorosulfonic acid, CHCl3, reflux, 48 h, 85%; (ii)
P(Ph)3, toluene, RT, 10 min, 72%; (iii) NaOH, MeI, EtOH, RT, 16 h, 98%; (iv) m-
CPBA, DCM, 0 °C to RT, 2 h, 73%; (v) H2 (5 atm.), Pd/C, MeOH, 16 h, 76%.
The cyanoisopropyl anilines used in compounds 17 and 18 were prepared from 3,5-
dibromoaniline 27 by bis-benzylation to give 35 followed by treatment with
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potassium 2-cyano-2-mehtylpropanoate and palladium catalyst21 to afford a separable
mixture of the symmetrical aniline 36 and partially reacted bromo derivative 37.
Compound 37 was submitted to a second Suzuki coupling to afford intermediate 38.
De-benzylation of 36 and 39 by heterogeneous hydrogenation provided the anilines
38 and 40.
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17
18
19
20
21
22
23
24
25
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27
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40
41
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Scheme 6. Synthesis of 2-cyanoisopropyl substituted anilines used in compounds
17 and 18
Reagents and conditions: (i) Benzyl bromide, K2CO3, MeCN, reflux, 24 h, 73%; (ii)
potassium 2-cyano-2-methylpropanoate, mesitylene, Xantphos, Pd2allyl2Cl2, 140 °C,
24 h, 36 = 53%, 37 = 20%; (iii) 4,4,5,5-tetramethyl-2-phenyl-1,3,2-dioxaborolane,
Pd(PPh3)2Cl2, K2CO3, dioxane:water, 100 °C, 1 h, 74%; (iv) H2, Pd/C, MeOH:DCM,
18 h, 88-98%.
EXPERIMENTAL
Tm-Shift assays. Thermal melting experiments were carried out using a Stratagene
Mx3005p Real Time PCR machine (Agilent Technologies). Proteins were buffered in
10 mM HEPES, pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final
concentration of 2 µM in a 20-µl volume. Compounds were added at a final
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concentration of 10 µM (final DMSO concentration was 0.025%). SYPRO Orange
(Molecular Probes) was added as a fluorescence probe at a dilution of 1:1,000 (v/v).
Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and
590 nm, respectively. The temperature was raised with a step of 3 °C per minute from
25 °C to 96 °C, and fluorescence readings were taken at each interval. Experiments
were performed in triplicate and the observed temperature shifts, ΔTmobs, were
recorded as the difference between the transition midpoints of sample and reference
wells containing protein without ligand in the same plate and determined by non-
linear least squares fit, reported in °C as the mean of the values obtained from 3
independent repeats.
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CaMK1D ADP GLO Assay. Test compounds were prepared in 100% DMSO and 12
nL was dispensed to individual wells of a multiwell plate (Perkin Elmer, catalog no.
6007290). A reaction mixture containing full length His tagged CaMK1D (Fisher
Scientific, PR6770A), calmodulin (Merck, 208694) and autocamtide-2 (SignalChem,
A15-58) was prepared in assay buffer composed of 50 mM Tris-HCl pH 7.5, 10 mM
MgCl2, 0.1 CaCl2 and 2 mM DTT. 7.88 µL of reaction mixture was added to each
well to give a final assay concentration: 3 nM CaMK1D, 1 µM Calmodulin, 125 µM
Autocamtide-2. Plates were centrifuged at 300 rpm for 30 s and incubated for 15
mins at 25°C. The enzyme reaction was initiated by the 4 µL addition of 30 µM ATP
solution to give a final assay concentration 10 µM. Plates were centrifuged at 300
rpm for 30 s and then incubated at 25°C for 2 h. ADP-Glo (Promega, catalog no.
V9102) was prepared according to manufacturer’s instructions and equilibrated to
room temperature, shielded from light. A 12 µL addition of ADP-Glo reagent was
made to terminate the kinase reaction and deplete residual ATP. Plates were
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centrifuged at 300 rpm for 30 s and then incubated at 25°C for 1 h. Following ATP
depletion, 24 µL of ADP-Glo substrate was added to convert ADP to ATP and initiate
a luciferase/luciferin chemiluminescent reaction. Plates were centrifuged at 300 rpm
for 30 s and then incubated at 25°C for 30 min, shielded from light. After 30 minutes
plates read with the EnVision® Multilabel Plate Reader, using Luminescence 700.
Compound IC50 was determined using a 4-parameter equation, and reported as the
geometric mean of the IC50 values obtained from 3 independent repeats.
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pCaMK1D cell assay. MDA-MB-231 cells were purchased from the ATCC and
routinely cultured in DMEM containing 10% FCS, 5U/mL penicillin/streptomycin
(Gibco). MDA-MB-231-HA-CaMK1D cells were established by infecting MDA-MB-
231 cells with modified pLVx-HA-CaMK1D lentivirus. Transduced cells were
selected in media containing puromycin (2 µg/mL) and expression of CamK1D was
verified by Western blotting using Rabbit anti-CamK1D mAb ([EPR3536(2)]
(ab172618), used at 0.1 mg/mL). Custom made anti-pCamK1D (Ser179,Thr180)
polyclonal antibodies were prepared by LifeTein (Hillsborough, NJ, USA),
immunising MEGKGDVM(pS)(pT)ACGTPGYVA peptide and verified in a series of
western blotting experiments. MDA-MB-231-HA-CaMK1D cells were cultured in
DMEM Glutmax (31966-021, ThermoFisher) containing 10% fetal bovine serum
(FBS) and 1 % penicillin-streptomycin. For the assay cells were seeded at 1.25 x105
cells/mL in 6-well plates and maintained at 37 °C in a humidified incubator with 5%
CO2 and 95% air for 48 hours. Then, cells were treated with compound for 4 h with a
final DMSO concentration of 0.1 %, for each compound an 11-point serial dilution
was used and DMSO included as a control. Media was aspirated and the cells washed
with PBS. Cells were lysed, supernatants were recovered by centrifugation at 13 000
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rpm, protein concentrations were measured, and equal amounts of total protein were
separated by SDS-PAGE. Proteins were transferred to PVDF membranes (Bio-Rad),
which was followed by blocking for 1 h in 5% milk in TBS-T. Membranes were
incubated overnight at 4 °C with primary antibody: anti-pCamK1D (Ser179,Thr180)
(1:1000) or total CaMK1D (1:10,000, ab172618, Abcam). Membranes were incubated
with the corresponding HRP-conjugated secondary antibody (7074S, CST) for 1 h.
Specific bands were detected using the enhanced-chemiluminescence reagent (Clarity
Western substrate, Bio-Rad) and the ChemiDoc MP Gel Imaging System (Bio-Rad),
and % change in pCAMK1D from control calculated using a ratio of pCaMK1D to
total CaMK1D bands. Compound IC50 was determined using a 4-parameter equation,
and reported as the geometric mean of the IC50 values obtained from 3 independent
repeats.
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Oral glucose tolerance test after acute and chronic/sub-acute (14 day) dosing.
Male C57Bl/6J mice obtained from Charles River UK (Margate, Kent UK) at 7-8
weeks of age were group housed for 16 weeks (n = 3 in each cage) on a normal
light/dark cycle (lights on: 07:00 – 19:00 h) with ad libitum access to a high fat diet
(D12451 diet, 45% kcal as fat, 35% as carbohydrate; Research Diets, New Jersey,
USA) and filtered water. Acute dosing study. Animals were allocated to dosing
groups (6 mice per group) such that groups were balanced as closely as possible for
mean body weight. The day prior to the OGTT, all animals were deprived of food
(but not water) beginning approximately 16.45. The following morning the mice were
dosed with vehicle or either 10 mg/kg, 25 mg/kg or 50 mg/kg 18 formulated in a
vehicle of DMSO (10% final volume) and 20% (2-Hydroxypropyl)-β-cyclodextrin (90%
final volume) by the oral route (beginning at 08.45). Four hours after dosing, a blood
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sample was taken (B1) and 3 minutes later glucose administered (2 g/kg orally).
Further blood samples were taken 10, 30, 60 and 90 minutes post glucose
administration. Between blood sampling, animals were returned to the home cage
with free access to water (but not food). Blood samples (approx. 30 µl) were collected
into lithium heparinised tubes (Sarstedt Microvette CB300LH) and plasma separated
by centrifugation to produce a single aliquot of plasma which was frozen (approx. -
80°C) and subsequently assayed for glucose (in duplicate; Thermoelectron Infinity
glucose reagent TR15498) and insulin (single replicate; Alpco mouse ultrasensitive
insulin kit 80 -INSMSU-E10). Chronic dosing study. Upon completion of the
OGTT, all animals were singly housed with food provided as above for two weeks
prior to the onset of the baseline phase of the chronic study. Upon single housing after
the OGTT, mice were placed on a reverse-phase light dark cycle (lights off: 09:30 –
17:30). Following this period the animals underwent a 5-day baseline phase where
they were be dosed twice daily with vehicle at approximately 08:45 and 16:45 each
day. Towards the end of the baseline phase mice were reallocated to dosing groups (8
animals per group) such that groups within the study were balanced as closely as
possible for body weight and food and water intake and previous treatment. From Day
1 onwards, mice were dosed orally twice daily with 25 mg/kg 18 formulated in a
vehicle of DMSO (10% final volume) and 20% (2-Hydroxypropyl)-β-cyclodextrin (90%
final volume) on days 1-6 and 1% methyl cellulose at 5 mL/kg on subsequent days, or
twice daily orally with vehicle alone, or with subcutaneous dosing of 0.1 mg/kg
liraglutide (Bachem) formulated in pH 7.4 phosphate buffer solution. Oral dosing
began at approximately 08:45 and 16.45, with subcutaneous dosing at 08:45 only.
Dosing continued until the morning of Day 14, when food was removed beginning at
approximately 16:45. Approximately 16h post-fast the animals were be moved to a
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