Pyridinylimidazoles as dual glycogen synthase kinase 3β/p38α mitogen-activated protein kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.04.035

Compounds simultaneously inhibiting two targets that are involved in the progression of the same complex disease may exhibit additive or even synergistic therapeutic effects. Here we unveil 2,4,5-trisubstituted imidazoles as dual inhibitors of p38α mitoge

Full text of DOI:10.1016/j.ejmech.2019.04.035

Accepted Manuscript
Pyridinylimidazoles as dual glycogen synthase kinase 3β/p38α mitogen-activated
protein kinase inhibitors
Fabian Heider, Francesco Ansideri, Roberta Tesch, Tatu Pantsar, Urs Haun, Eva
Döring, Mark Kudolo, Antti Poso, Wolfgang Albrecht, Stefan A. Laufer, Pierre Koch
PII:
S0223-5234(19)30350-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.035
EJMECH 11270
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 January 2019
Revised Date: 29 March 2019
Accepted Date: 13 April 2019
Please cite this article as: F. Heider, F. Ansideri, R. Tesch, T. Pantsar, U. Haun, E. Döring, M. Kudolo, A.
Poso, W. Albrecht, S.A. Laufer, P. Koch, Pyridinylimidazoles as dual glycogen synthase kinase 3β/p38α
mitogen-activated protein kinase inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.04.035.
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Graphical Abstract

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Pyridinylimidazoles as dual Glycogen Synthase Kinase
3β/p38α Mitogen-activated Protein Kinase Inhibitors
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Fabian Heider, Francesco Ansideri, Roberta Tesch, Tatu Pantsar, Urs Haun, Eva
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Döring, Mark Kudolo, Antti Poso, Wolfgang Albrecht, Stefan A. Laufer and Pierre
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,5
Koch *
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Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical
Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen,
Germany
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School of Pharmacy, University of Eastern Finland, P.O.BOX 1627, 70211 Kuopio,
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Finland
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Department of Internal Medicine VIII, University Hospital Tübingen, Otfried-Müller-Strasse
14, 72076 Tübingen, Germany
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Teva-ratiopharm, Graf-Arco-Str. 3, 89079 Ulm, Germany
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Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy, University of
Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany
*Corresponding author. Email address: pierre.koch@uni-tuebingen.de (P. Koch)
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Abstract
Compounds simultaneously inhibiting two targets that are involved in the progression of
the same complex disease may exhibit additive or even synergistic therapeutic effects.
Here we unveil 2,4,5-trisubstituted imidazoles as dual inhibitors of p38α mitogen-activated
protein kinase and glycogen synthase kinase 3β (GSK3β). Both enzymes are potential
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therapeutic targets for neurodegenerative disorders, like Alzheimer’s disease. A set of 39
compounds was synthesized and evaluated in kinase activity assays for their ability to
inhibit both target kinases. Among the synthesized compounds, potent dual-target-directed
inhibitors showing IC50 values down to the low double-digit nanomolar range, were
identified. One of the best balanced dual inhibitors presented in here is N-(4-(2-ethyl-4-(4-
fluorophenyl)-1H-imidazol-5-yl)pyridin-2-yl)cyclopropanecarboxamide (20c) (IC50 p38α: 16
nM; IC50 GSK3β: 35 nM) featuring an excellent metabolic stability and an appreciable
isoform selectivity over the closely related GSK3α. Our findings were rationalized by
computational docking studies based on previously published X-ray structures.
Keywords
Kinase inhibitors, pyridinylimidazoles, glycogen synthase kinase 3β, p38α MAP kinase,
dual inhibitors, Alzheimer’s disease.
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1. Introduction
Neurodegenerative disorders, like Alzheimer’s disease (AD), present one of the major
st
medical challenges of the 21 century. It was estimated that in 2015 more than 46 million
people worldwide were affected by AD.[1] With globally increasing life expectancy, the
prevalence of AD is going to rise constantly. Currently, there are four active
pharmaceutical ingredients approved by the FDA for the treatment of AD. However, none
of them is able to stop or cure the disease. Therefore, there is a high unmet medical need
for the development of new treatment options and strategies.
In the last decade, much effort has been put into the development of new therapeutic
concepts – with little success. One of the reasons for the low clinical success rate of new
therapies is undoubtedly the still poor understanding of the pathophysiology of AD. As AD
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is a complex disease with a multicausal pathogenesis, a multi-target approach might be a
promising and required strategy for the treatment of this devastating condition.
Several kinases have been implicated in the pathology of AD. Glycogen synthase kinase-
3β (GSK3β) is a serine/threonine kinase for which significant evidence has been collected
for its involvement in AD pathology over the years.[2-5] GSK3β is responsible for the
hyperphosphorylation of the microtubule-associated protein tau, and is often referred to as
,
a tau-kinase.[6, 7] Overactivity of GSK3β has also been connected to an increased
production of β-amyloids (Aβ),[8] memory deficits[9], oxidative stress and
neuroinflammation.[10] Thus, the kinase plays a crucial role in almost every pathway
leading to the hallmarks of AD.[11] The p38α mitogen-activated protein (MAP) kinase[12]
is a serine/threonine kinase, which has been identified as a drug target for chronic
inflammatory diseases since the mid-1990s.[13] This enzyme plays a central role in the
biosynthesis of proinflammatory cytokines, like the tumor necrosis factor (TNF)-α, both at
translational and transcriptional levels. The connection between the kinase and AD was
drawn by Hensley et al., who found increased activity of p38α MAP kinase in human
postmortem brain tissue from AD patients.[14] Since then, an increasing amount of
evidence has been piling up to establish a link between cytokine overproduction in the
central nervous system (CNS) modulated by p38α MAP kinase and
neuroinflammation.[15-18] Furthermore, the kinase has also been suspected to be
involved in the hyperphosphorylation of tau proteins. A study with a brain-penetrant,
selective p38α inhibitor significantly diminished tau phosphorylation and signs of insoluble
tau aggregates on top of the expected reduction of proinflammatory cytokine released in a
tauopathy mouse model.[19] Recently, two phase II studies on the selective p38α MAP
kinase inhibitor Neflamapimod for the treatment of AD were completed successfully.[20,
21] Two follow-up phase II studies were launched in early 2018 (NCT03435861 &
NCT03402659).
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GSK3β and p38α MAP kinase both belong to the CMGC group and share an overall
sequence similarity of 42 %. A closer analysis of their ATP binding pockets reveals several
distinctions between the two enzymes (Figure 1). Most importantly the gatekeeper residue
in p38α MAP kinase is Thr106, whereas a slightly bulkier Leu132 occupies this position in
GSK3β. Moreover, the hinge region is composed of fairly different amino acids.
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9
0
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Figure 1. (A) Structural overlay of GSK3β and p38α MAP kinase. The most crucial
differences in the amino acids of the ATP binding site are highlighted in green (GSK3β)
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and purple (p38α MAP kinase). (B) Sequence alignment of GSK3β (UniProt code P49841)
and p38α MAP kinase (UniProt code Q16539). The amino acids belonging to each region
are highlighted as depicted in (A).
Pyridinylimidazoles are versatile scaffolds in kinase drug discovery and have been used to
target several kinases such as p38α MAP kinase,[22-26] CK1δ,[27] TEK,[28] (triple
20
mutant) EGFR and the JNK family.[29, 30] Recently, synthetic strategies towards 2-
alkylsulfanyl-4-aryl-5-(pyridin-4-yl)imidazoles as well as their biological activities were
reviewed by our group.[31]
A screening of pyridinylimidazole-type p38α MAP kinase inhibitors from our in-house
library (TüKIC, Tübingen Kinase Inhibitor Collection) versus GSK3β led to the identification
of compound 1a, serving as a lead compound for this study to design potent dual
GSK3β/p38α MAP kinase inhibitors. Independently from us, compound 1a has recently
been disclosed by Halekotte et al. in an effort to synthesize CK1δ inhibitors.[27]
F
N
CH3
S
N
H
OCH3
IC50 (p38 ) =
IC50 (GSK3 ) = 363 nM
88 nM
N
OCH3
HN
OCH3
O
1
a
9
7
8
9
Figure 2. Structure and biological data of in-house library screening hit 1a.
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Based on the initial screening data, first structure-activity relationships (SARs) were
generated. An amide at the pyridine-C2 position seemed to be crucial for GSK3β activity. It
was also assumed that 2,4,5-trisubstituted imidazoles, in general, show a higher affinity
towards GSK3β than 1,2,4,5-tetrasubstituted imidazole derivatives. Previous SAR studies
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on the p38α MAP kinase revealed that a tri- and tetrasubstitution pattern on the central
imidazole core as well as a wide variety of substituents at the pyridine-C2 position
(including amides) are well tolerated by the kinase.[31]
The aim of the present study was to design brain penetrant and metabolically stable dual
inhibitors of GSK3β and p38α MAP kinase, which might serve as potential drugs for the
treatment of neurodegenerative diseases. A series of 39 pyridinylimidazoles was
synthesized and variations at the pyridine-C2 position, the imidazole-C2 and C4 positions
as well as on both imidazole-N atoms were performed to further improve the GSK3β
inhibitory activity while maintaining the excellent potency on p38α MAP kinase.
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2. Results and Discussion
2.1. Chemistry
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The 2-methylsulfanylimidazoles 1a-u and 2-benzylsulfanylimidazoles 2a-e were
synthesized by a linear approach. Diversity at the pyridine-C2 position was introduced in
the last step of the synthetic sequences (Schemes 1 and 2). The key intermediates 5-(2-
aminopyridin-4-yl)imidazoles (10a, 10b) were synthesized by a novel synthetic approach
derived from a strategy published by our group in 2008 (Scheme 1).[24] Starting from N-
Boc protected 2-amino-4-methylpyridine 3, a p-methoxybenzyl (pMB) moiety was
introduced as second protecting group of the amino function. The fully protected 4-picoline
4 was reacted with sodium hexamethyldisilazide (NaHMDS) in THF and ethyl 4-
fluorobenzoate to obtain ethanone 5. An excess of sodium nitrite dissolved in water was
added dropwise to an acidic solution of 5 to give α-oximino ketone 6. Subsequent
treatment with Pd/C using hydrogen at atmospheric pressure in methanolic HCl resulted in
both, reduction of the oximino group into a primary amino group as well as cleavage of the
Boc group. Conveniently, the acid labile pMB was stable under these conditions.
Cyclization of aminoketone 7 with potassium thiocyanate followed by the reaction with
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methyl iodide or benzyl bromide resulted in S-substituted intermediates 9a and 9b,
respectively. Finally, the pMB group was removed in neat trifluoroacetic acid (TFA) with
gentle heating to yield the key intermediates 10a and 10b.
a
Scheme 1. Synthesis of key intermediates 10a and 10b.
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a
1
Reagents and conditions: (a) NaH, 4-methoxybenzyl chloride, DMF, 0 °C then rt, 18 h; (b)
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NaHMDS, ethyl 4-fluorobenzoate, THF, 0 °C then rt, 2 h, 67% (over 2 steps); (c) NaNO2,
acetic acid, rt, 1 h, 100%; (d) H , Pd/C 10 %, methanolic HCl, 45 °C, 6 h; (e) KSCN, DMF,
2
160 °C, 2 h, 62% (over 2 steps); (f) CH I, NaOtBu, MeOH, 55 °C, 2 h, 84% (in case of
3
preparation of 11a) or benzyl bromide, Cs CO , DMF, rt, 36 h, 35% (in case of preparation
2
3
of 11b); (g) TFA, 45 °C, 96-97%.
The acylamino function at the pyridine-C2 position of imidazoles 1b, 1c, 1s, 2b, and 2c
was introduced via Buchwald-Hartwig arylamidation by reacting known 2-chloropyridine
derivatives 11a-b[26, 32] with the corresponding amides in the presence of a Pd catalyst
(Scheme 2). In case of imidazoles 1a,d-r,t-u and 2a,d-e, the amide moiety was installed
the amide moiety was installed depending on the commercial availability of the starting
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material either by direct acylation of 2-aminopridine derivates 10a and 10b with acid
chlorides or by reacting 10a and 10b with the corresponding carboxylic acids in the
presence of a coupling reagents. The sulfoxides 12a and 12b were obtained by oxidation
of 2-methylsulfanylimidazoles 1c and 1m with hydrogen peroxide respectively. (Scheme
2).
a
Scheme 2. Synthesis of compounds 1a-u, 2a-e and sulfoxides 12a-b.
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a
1
Reagents and conditions: (a) carboxylic acid, PyBOP, DIPEA, DCM, rt, 40-93%; (b)
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carboxylic acid, HATU, DIPEA, DCM, rt, 21-85%; (c) acyl chloride, pyridine, 0 °C then rt;
(d) amide, Pd (dba) , XantPhos, Cs CO , DMF, 100 °C, 16 h, 19-78%; (e) H O , MeCN, rt,
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3
2
3
2
2
72-96 h, 42-51%. For R , see Tables 1-2.
2
The synthesis of tetrasubstituted imidazoles 14 and 16 started from known 4-(4-(4-
fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-amine 13[25] and 2-
chloro-4-(5-(4-fluorophenyl)-1-methyl-2-(methylthio)-1H-imidazol-4-yl)pyridine
15,[22]
respectively (Scheme 3). The cyclopropylamide moiety at the pyridine-C2 position was
introduced either by reaction of pyridine-2-amine compound 13 and cyclopropanecarbonyl
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chloride and the or by Buchwald-Hartwig arylamidation of 2-chloropyridine derivative 15
and cyclopropanecarboxamide.
a
Scheme 3. Synthesis of tetrasubstituted 2-acylaminopyridinylimidazole
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a
1
Reagents and conditions: (a) cyclopropanecarbonyl chloride, pyridine, 0 °C then rt, 2 h,
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31%; (b) cyclopropanecarboxamide, Pd (dba) , XantPhos, Cs CO , DMF, 100 °C, 16 h,
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3
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3
63%.
4,5-Disubstituted imidazole 20a and 2,4,5-trisubstituted imidazoles 20b-g were prepared in
analogy to our recently reported protocol[33] based on the Radziszewski imidazole
synthesis in four steps starting from ethanone 5 (Scheme 4). Treatment of 5 under Riley-
Oxidation conditions yielded the corresponding α-diketone 17. Under these acidic
conditions, the Boc protecting group was already cleaved and in the next step, the pMB
protecting group was removed using neat TFA. Then, the primary aromatic amine 18 was
acylated with cyclopropanecarbonyl chloride. Interestingly, even using only one equivalent
of acyl chloride, beside the expected monoacylated main product 19a a substantial
amount of double acylation (compound 19b) was observed. Since the monoacylated
product 19a, however, could not be isolated, we used cyclopropanecarbonyl chloride in
excess and continued our synthetic sequence with the double-acylated intermediate 19b.
Ring closing reaction to the imidazoles 20a-g was achieved by reacting the α-diketone 19b
with ammonium acetate and different (hetero)aromatic, aliphatic aldehydes as well as
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formaldehyde. Conveniently, the second acyl was cleaved during this step. The yields in
this reaction fluctuated between 5% and 65%. The reaction of non-enolizable aldehydes
gave generally significantly higher yields than aldehydes with an enolizable system. In
case of latter ones, an acid catalyzed aldol-reaction took place before the ring closing
reaction. For these compounds, we observed a second relatively distinct spot on the TLC
and the mass of this spot corresponded to a molecule formed by ring closing reaction with
the aldol product.
Scheme 4. Synthesis of 4-aryl-5-heteroaryl-substituted imidazole 20a and 2-alkyl/aryl-4-
a
aryl-5-heteroaryl-substituted Imidazole 20b-g.
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a
1
Reagents and conditions: (a) SeO , acetic acid, 130 °C, 1.5 h, 44%; (b) TFA, rt, 97%; (c)
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cyclopropanecarbonyl chloride, DIPEA, DCM, rt, 18 h, 21%; (d) R -CHO, NH OAc, acetic
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4
acid, 130 °C, 3-4 h, 5-65%. For R , see Table 4.
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The disubstituted imidazole 25 was prepared starting from commercially available 2-
methylimidazole (21), which was protected in the first step using 2-
(trimethylsilyl)ethoxymethyl (SEM) chloride (Scheme 5). A procedure previously described
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by Markey and Kelly was used,[34] employing a directed ortho-metalation using n-
butyllithium followed by the addition of tributyltin chloride, to obtain imidazole-5-stannane
23. Intermediate 23 was reacted via a Stille coupling with N-(4-bromopyridin-2-
yl)cyclopropanecarboxamide to yield imidazole 24. Final product 25 was obtained after
removal of the SEM protecting group under acidic conditions.
a
Scheme 5. Synthesis of 2-methyl-5-heteroaryl-substituted imidazole 25.
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a
2
Reagents and conditions: (a) NaH, 2-(trimethylsilyl)ethoxymethyl chloride, THF, 0 °C then
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rt, 18 h, 96%; (b) n-butyllithium, tributyltin chloride, Et O, 0 °C then rt, 2 h, 25%; (c) N-(4-
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bromopyridin-2-yl)cyclopropanecarboxamide, Pd(PPh ) , 1,4-dioxane, 105 °C, 18 h, 77%;
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(d) TFA, DCM, rt, 6 h, 89%.
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2.2. Biological Evaluation
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2.2.1. Kinase activity assays and molecular modelling
The synthesized compounds were tested for their ability to inhibit p38α MAP kinase and
GSK3β using an enzyme-linked immunosorbent assay (ELISA)[35] and the Promega ADP-
Glo™ platform,[36] respectively. As reference compounds, we selected SB203580 (IC =
50
41 nM) for the p38α MAP kinase activity assay and SB216763 (IC50 = 89 nM) for the
GSK3β assay. To estimate the GSK3 isoform selectivity, selected compounds were further
evaluated for their inhibitory potency toward GSK3α using again the ADP-Glo™ activity
assay. Specific inhibitors were further investigated for their metabolic stability in human
liver microsomes (HLM) as well as in an ex vivo experiment for their ability to inhibit the
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lipopolysaccharide (LPS)-stimulated TNF-α release from human whole blood (HWB).[37]
In latter assay, SB203580 was used as reference compound (IC = 3,202 nM). Moreover,
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the two most promising inhibitors (1c and 20c) were evaluated for their brain penetration,
their cytochrome P450 (CYP450) inhibition as well as for their affinity to the human Ether-
a-go-go-related gene (hERG) channel.
A possible binding mode for lead compound 1a (Table 1) within the ATP binding site of
p38α MAP kinase is depicted in Figure 3. The pyridine nitrogen atom as well as the NH at
the pyridine-C2 position act as a classical donor-acceptor hinge-binding motif engaging in
two H-bond interactions to the backbone of Met109 of the hinge region. A charge-assisted
H-bond interaction occurs between the side chain of the Lys53 and the imidazole-N3
nitrogen. Moreover, the imidazole ring lays on top of the Phe169 forming an aromatic π–π
stacking interaction. The 4-fluorophenyl ring occupies the hydrophobic region I (HR I).
Access to this region is controlled by the gatekeeper residue Thr106. Targeting of HR I
might be an important factor for selectivity over other protein kinases since bulkier
gatekeepers often do not tolerate large substituents.[38]
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Figure 3. Two-dimensional schematic binding mode of pyridinylimidazoles in p38α MAP
kinase. The binding mode is derived from published crystallographic data (PDB ID: 5ML5).
The 4-fluorophenyl ring occupies HR I, while the HR II is rather large and solvent exposed.
H-bonds and the π–π stacking interaction between the imidazole ring and Phe169 are
indicated with a black dashed line.
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Since different regions within the ATP binding site are highly conserved among all protein
kinases, a similar binding mode of the lead compound 1a was expected for GSK3β.
Because of the gatekeeper on GSK3β being a bulkier amino acid (Leu132, in contrast to
Thr106 in p38α MAP kinase), a simple rigid docking could lead to a misguided evaluation.
Thus, we utilized an Induced Fit docking (IFD)[39-41] approach to study the binding to
GSK3β. The IFD revealed a comparative binding mode of compound 1a with GSK3β as in
p38α MAP kinase. As depicted in Figure 4, the hinge-binding motif (pyridine-2-amine) is,
as expected, able to act as a donor-acceptor to both, the carbonyl and NH group of
Val135. Similar to the pose for p38α MAP kinase, the 4-fluorophenyl ring is located in the
HR I of GSK3β. The proposed binding mode suggested a possible cation-π interaction
between the 2,3,4-trimethoxyphenyl ring and Arg141. However, as the Lys85 in GSK3β is
in a fixed conformation between two negatively charged residues, Asp200 and Glu97, and
it is located farther away (compared to the corresponding Lys53 in p38α MAP kinase), we
did not observe the charge-assisted H-bond between the imidazole ring and the Lys85.
Instead, one of the imidazole tautomers is capable to form a H-bond with the Asp200 of
the activation loop (part of the DFG motif). Since an equilibrium of the two possible
tautomeric forms is expected, we suggest that the other imidazole tautomer (H-bond
acceptor) may interact via a water-mediated H-bond with the Lys85. This interaction is
observed in several X-ray structures of other protein kinases (e.g. JNK3) complexed with
similar pyridinylimidazole-type inhibitors.[42]
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Figure 4. Proposed binding mode of 1a on GSK3β obtained by IFD (PDB ID: 4PTC). H-
bonds between the inhibitor and the kinase as well as the cation–π interaction between
the trimethoxyphenyl ring and Arg141 are shown with black dashed lines.
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Based on our initial HTS data analysis, we concluded that GSK3β inhibition was strongly
enhanced when N-(pyridin-2-yl)amides instead of N-(pyridin-2-yl)amines were present at
the pyridine-C2 position. From previous studies, we also knew that p38α MAP kinase
tolerates both amines and amides in this position equally well.[25] Therefore, a set of
compounds bearing different amides at the pyridine-C2 position was planned to collect first
SAR, especially in regards to GSK3β. Our first synthetic efforts led to the series of
compounds presented in Table 1 (1b-u).
Compared to the lead structure 1a, the close analogs 1f and 1g bearing only one methoxy
group at the phenyl ring are less active against GSK3β. This indicates that the electronic
properties of the phenyl ring play an important role in the binding, probably, via
contributing to the cation–π interaction with the Arg141 residue. The decreased electron
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density in the phenyl ring could lead to a weakened cation–π interaction as electron-
withdrawing substituents are known to attenuate these types of interactions.
Consequently,
inhibitor
1i
(GSK3β,
IC :
995
nM)
bearing
an
3-(4-
5
0
(trifluoromethyl)phenyl)propanamide moiety showed a 3-fold reduced GSK3β inhibition
than the lead compound. These findings support our proposed orientation of our inhibitor
molecule within the ATP bindings site. Meanwhile, these inhibitors (1f, 1g and 1i) retained
their high inhibitory activity against p38α MAP kinase while displaying a substantial loss of
activity versus GSK3β. Shortening of the C-linker of 1a and 1i by one methylene group
resulted in compounds 1e and 1h, respectively. These compounds exhibited decreased
GSK3β inhibition, while p38α MAP kinase inhibition of 1e was increased almost by 7-fold
compared to 1a. To rationalize the data at hand, we decided to use already published
crystal structures of p38α MAP kinase (PDB ID: 5ML5) and GSK3β (PDB ID: 4PTC) for the
molecular modeling experiments. According to the computational approach, on GSK3β our
compounds bind to the hinge region with two H-bonds to Val135, which is followed by
Pro136, Glu137 and Thr138. Interestingly, Glu137 participates in a salt bridge interaction
with Arg141 (Figure 5A). In the case of p38α MAP kinase, the corresponding amino acids
are Met109, Gly110, Ala111 and Asp112, while the corresponding residue to the Arg141
on p38α MAP kinase is Asn115, which is unable to interact with the hinge region. Another
important structural feature on p38α MAP kinase is the “glycine flip”. The peptide bond of
Met109 and Gly110 can flip 180°, which has already been reported in the literature to
explain the selectivity of another class of compounds.[43] Instead of the flexible glycine
residue, GSK3β has a rigid Pro136 in this position. Finally, GSK3β has a bulky Tyr134
residue, which is stabilized on top of the hinge-region and forms a H-bond to the fixed
Pro136 (Figure 5B). This Tyr134 occupies more space in the hinge region compared to the
Leu108 in this position on p38α MAP kinase. Based on these three aspects, the flexibility
of the residues in the hinge region, the stabilizing salt-bridge Glu137–Arg141 and the bulky
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316
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320
Tyr134, we hypothesize that the hinge region of GSK3β is clearly more constrained and
crowded (in the vicinity of the hinge) than in p38α MAP kinase. This would explain the
hindered binding of the shortened C-linker chain compounds 1e and 1h to GSK3β. The
importance of these three aspects was further exemplified by the dramatical loss of activity
towards GSK3β with the bulky and/or planar structural properties containing compounds
1j, 1t and 1u.
3
21
22
3
Figure 5. (A) Differences in the hinge region of p38α MAP kinase (purple) and GSK3β
(green). The activation loops of both structures are omitted for clarity. The blue sphere
represents the NH group of Met109 and Val135 in each kinase that forms a H-bond with
the pyridine. The stabilizing salt bridge between Glu137 and Arg141 in GSK3β is
highlighted by a red circle. (B) The rigid environment of the GSK3β hinge region prevents
the accommodation of compounds with bulkier moieties in close proximity to the hinge
binding motif. Compound 1j is unable to fit into the GSK3β while maintaining H-bonding to
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328
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330
331
332
the hinge. The (trifluoromethyl)cyclopropyl moiety clash with the protein is illustrated here
by crossing the cavity surface (PDB ID: 4PTC).
Table 1. Structures and kinase activity of 2-methylsulfanylimidazoles 1a-u, 14, 16 and 37.
IC50 [nM] ± SEM
a
b
Cpd
R2
p38α MAP kinase
GSK3β
1a
88 ± 8
363 ± 16
1
b
45 ± 1
24 ± 0
152 ± 9
40 ± 5
1
c
1d
40 ± 0.4
136 ± 45
1e
13 ± 1
510 ± 43
1
f
55 ± 6
36 ± 2
542 ± 34
471 ± 15
1g
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1
h
29 ± 1
3,242 ± 592
956 ± 56
> 10,000
808 ± 31
215 ± 49
132 ± 9
1
i
27 ± 2
184 ± 53
15 ± 0.1
15 ± 0
27 ± 0
20 ± 1
38 ± 0
18 ± 0
21 ± 1
28 ± 2
23 ± 0.4
22 ± 1
42 ± 8
1
j
1
k
1
l
1m
1n
1o
1p
1q
183 ± 7
395 ± 69
170 ± 7
73 ± 7
1
r
527 ± 44
126 ± 23
3,654 ± 115
> 10,000
1
s
1
t
1u
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26
14
16
--
--
--
14 ± 0
> 10,000
638 ± 19
222 ± 4
1,603 ± 733
1,780 ± 16
a
b
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
n = 3; n = 2
With a molecular weight of >500 Da, the inhibitors discussed so far are fairly heavy when
considering the desired properties needed for CNS penetration.[44] Additionally, our
insights from the IFD urged us to abandon any bulky residues that may clash with the rigid
hinge region of GSK3β. Therefore, analogs of lead structure 1a bearing smaller moieties at
the pyridine-C2 position were synthesized. Compound 1b with an N-acetyl moiety at the
pyridine-C2 amino function displays improved GSK3β inhibition (GSK3β, IC : 152 nM)
50
and a promising p38α MAP kinase/GSK3β inhibition ratio of 3.3. Following this
observation, a series of inhibitors (1b-d, 1j, 1l-u) were synthesized bearing small
cycloalkanecarboxamide moieties at the pyridine-C2 position. Introduction of a
cyclopropanecarboxamide resulted in the potent and balanced dual inhibitor 1c, showing
IC50 values down to the low double-digit nanomolar range for both target kinases (p38α,
IC : 24 nM; GSK3β, IC : 40 nM).
50
50
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3
48
49
3
Figure 6. Proposed binding mode of 1c on GSK3β obtained by IFD (PDB ID: 4PTC). The
cyclopropyl moiety occupies ideally the small and rigid front pocket (HR II) area. H-bonds
between the inhibitor and the kinase are shown as black dashed line.
350
351
352
353
354
355
356
357
358
359
360
361
362
363
Modifications on the cyclopropyl ring resulted in a loss of GSK3β activity. The introduction
of a phenyl ring in position 2 of the cyclopropyl ring (1k) resulted in a slight improvement of
p38α MAP kinase inhibition but also in a substantial drop of GSK3β activity. The change of
the spacer length between the amide function and the cyclopropyl ring (1l) as well as
replacement of the small cyclopropyl ring by bigger cyclobutyl (1m) or cyclopentyl (1d)
rings decreased the GSK3β inhibitory potency slightly. The introduction of a trifluoromethyl
group in position 1 of the cyclopropyl ring (1j) led to a complete loss of GSK3β activity,
which is in good agreement with our hinge flexibility hypothesis (see Figure 5B).
Interestingly, compound 1j had also one order of magnitude reduced inhibitory potency on
p38α MAP kinase, which indicates that the trifluoromethyl group attached to the
cyclopropyl is already too close to the hinge region and may distort the H-bond interactions
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364
365
366
with Met109 (see Figure 3). Nevertheless, the flexibility of the hinge region on p38α MAP
kinase still enables the binding of this compound much better than GSK3β does.
3
67
68
3
Figure 7. The output conformation of the compound 1c after 200 ns MD simulation (PDB
ID: 4PTC). The carbonyl group of the amide and the imidazole-N3 participate in the
solvent interactions. The protein surface is illustrated with light purple color and the solvent
access to the binding site is highlighted in blue. The water molecules are displayed within
4Å from the ligand.
369
370
371
372
373
374
375
376
377
378
379
380
381
382
Attempts to address the solvent interface (Figure 7) with more polar groups attached to
small cycloalkyl groups in order to gain binding affinity through enthalpically favorable,
stabilized water networks, resulted in compounds 1n and 1q. Compared to the
cyclobutane analog 1m, imidazole 1n bearing a 3-oxocyclobutyl substituent at the amide
function displayed no improvement in GSK3β inhibition. In comparison to the parent
cyclopentyl compound 1d, pyridinylimidazole 1q having a 3-oxocyclopentyl substituent
showed a 2-fold increased GSK3β inhibitory activity. The two cis/trans 3-chlorocyclobutyl
isomers (1o and 1p) showed a roughly 2-fold difference in inhibition for both kinases with
the cis-isomer being the more potent one.
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384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
Compound 1s bearing a 2-oxopyrrolidin-1-acetamide at the pyridine-C2 position displayed
a similar p38α MAP kinase/GSK3β inhibition profile as 1d bearing a cyclopentylamide at
the same position. Imidazole 1r having 2-tetrahydrofuranyl at the amide function showed a
decreased GSK3β activity (factor 4) compared to 1d. The introduction of aromatic
heterocyclic rings directly at the amide function (1t and 1u) resulted in a tremendous loss
of GSK3β inhibitory activity as a consequence of the aforementioned rigidity of the GSK3β
hinge region (Figure 5B). In contrast, none of the modifications at the pyridine-C2 position
had any meaningful impact on p38α MAP kinase inhibition. All pyridinylimidazoles reported
in Table 1 except 1j, 14 and 16 are potent p38α MAP kinase inhibitors displaying IC50
values in the double-digit nanomolar range. Replacement of the carbonyl part of the amide
group by a methylene group resulted in a complete loss of GSK3β affinity (compare 37 vs
1c), whereas this modification slightly increased p38α MAP kinase inhibitory activity.
Those findings exemplify the importance of the amide group at the pyridine-C2 for the
binding to GSK3β. This arises most likely from solvent interactions that have been
observed to play a crucial role for the compound affinity, as demonstrated in other
studies.[45] Indeed, we noticed important solvent interactions with the amide in our
molecular dynamics (MD) simulation (Figure 7). Moreover, there seems to be a possibility
for solvent mediated, bridged interactions between the amide carbonyl and the imidazole-
N3. This may help to stabilize the active conformation of the molecule, ultimately
enhancing the overall binding affinity. Interestingly, this effect was only dramatic with
GSK3β. The bulky and charged Arg141 residue in GSK3β clearly shields the solvent
access to the binding site and influences to the water network organization. In case of the
more flexible and open p38α MAP kinase, the water network reorganization within the
pocket is tolerated thus enabling the binding of 37.
The introduction of substituents on the imidazole-N1 or -N3 atom of potent dual p38α MAP
kinase/GSK3β inhibitor 1c resulted in a drop of both GSK3β and p38α MAP kinase
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409
410
411
412
413
414
415
416
417
418
419
420
421
inhibitory activity (compounds 14 and 16). This effect was more pronounced in the case of
16. The methylation of the nitrogen atom adjacent to the 4-fluorophenyl ring resulted in two
orders of magnitude reduced p38α MAP kinase inhibitory activity, compromising the
charge-assisted H-bond to the Lys53 (see Figure 3). In addition, in the case of GSK3β, 16
would also disrupt either the H-bond to Asp200 or the potential water-mediated interaction
to Lys85 depending on the NH tautomerism (see Figure 4).
In previous studies, our group showed that bigger moieties at the imidazole-C2 position
were well tolerated by p38α MAP kinase.[24] Therefore, the inhibitory potency of a small
series of 5 compounds, wherein the S-methyl group at this position was replaced by a S-
benzyl group, was evaluated (Table 2). In case of GSK3β, imidazoles 2a-d displayed a
decreased affinity (3- to 6-fold) in comparison to their S-methyl counterparts 1a-d
Table 2. Structures and kinase activity of 2-benzylsulfanylimidazole 2a-e.
IC50 [nM] ± SEM
a
b
Cpd
R2
p38α MAP kinase
GSK3β
2a
61 ± 4
1,149 ± 167
2
b
51 ± 5
41 ± 3
560 ± 23
229 ± 3
2
c
2
3

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2d
20 ± 2
663 ± 45
2e
3 ± 0
1,499 ± 51
a
b
422
423
424
n = 3; n = 2
Computational studies of 2c with GSK3β revealed that the S-benzyl substituent is oriented
towards the solvent accessible region and displays contacts with the lipophilic glycine rich
loop residues, mainly with Phe67, Val70, and Ile62. In agreement with previous
observations, imidazoles 2a-e bearing S-benzyl moiety at the imidazole-C2 position
displayed similar p38α MAP kinase inhibitory activity as their S-methyl counterparts.
Compound 2e with a 4-fluorophenylacetamide at the pyridine-C2 position represents the
most potent p38α MAP kinase inhibitor in this series having an IC value in the low single-
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
50
digit nanomolar range and displaying a greater than 500-fold selectivity over GSK3β.
Previous studies on in vitro metabolic stability of similar p38α MAP kinase inhibitors
revealed that the alkylsulfanyl moiety at the imidazole-C2 position is susceptible to
oxidation, resulting in the corresponding 2-alkylsulfinylimidazoles as main metabolites.[33,
46] To estimate the impact of the metabolism on the inhibition profile, the corresponding
sulfoxides of 1c and 1m were additionally evaluated for their ability to inhibit both target
kinases (Table 3). Sulfoxides 12a and 12b showed a 3- and 6-fold reduced GSK3β
inhibition compared to 1c and 1m, respectively, while the two compounds displayed a
similar p38α MAP kinase inhibition profile like their S-methyl counterparts.
Table 3. Structures and kinase activity of 2-methylsulfinylimidazoles 12a and 12b.
2
4

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IC50 [nM] ± SEM
a
b
Cpd
R
1
R
2
p38α MAP kinase
GSK3β
1
2a
44 ± 0
236 ± 45
428 ± 19
12b
29 ± 6
a
b
4
4
4
4
4
4
4
4
4
4
4
42
43
44
45
46
47
48
49
50
51
52
n = 3; n = 2
In order to remove the metabolic hotspot of the 2-methylsulfanylimidazoles, another series
of pyridinylimidazoles were synthesized wherein the imidazole-C2 position was modified.
To this end, alkyl (20b,c), aryl (20d), arylalkyl (20e,f) and heteroaryl (20g) substituents
were introduced to this position (Table 4). In case of p38α MAP kinase, substitution of the
imidazole-C2 position had no influence on the inhibitory activity and compounds 20b-g
displayed IC50 values in the same range as the simple disubstituted imidazole 20a.
Looking at GSK3β, introduction of a phenyl residue (20d) led to a distinctly reduced
inhibitory potency. Compared to 20a, imidazole 20c with a small ethyl moiety at the
imidazole C2-position is the only example in this series displaying a significantly improved
inhibition as well as a p38α MAP kinase/GSK3β inhibition profile similar to the most potent
compound out of the S-methyl series (1c).
453
454
455
456
457
Table 4. Structures and kinase activity of 4,5-disubstitued imidazole 20a and 2,4,5-
trisubstitued imidazoles 20b-g.
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5

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IC50 [nM] ± SEM
a
b
Cpd
R1
p38α MAP kinase
GSK3β
20a
19 ± 1
64 ± 4
2
0b
19 ± 1
16 ± 1
27 ± 1
18 ± 0
20 ± 0.5
18 ± 1
53 ± 12
35 ± 6
2
0c
20d
570 ± 98
59 ± 11
2
0e
2
0f
133 ± 7
20g
156 ± 14
1,684 ± 120
c
2
5
>10,000 (1 %)
a
b
c
4
4
4
58
59
60
n = 3; n = 2; percent inhibition at indicated concentration, tested in a LANCE assay at Eurofins
Cerep SA, France (reference compound in this assay: SB203580, IC = 65 nM).
50
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461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
As observed in previous studies,[29, 42] the removal of the 4-fluorophenyl ring (25) led to
a complete loss of p38α MAP kinase inhibitory activity (Table 5). Moreover, this structural
modification resulted in a 32-fold reduced inhibitory activity of GSK3β (25 vs. 20b),
demonstrating the importance of this residue also for GSK3β inhibition.
2.2.2. GSK3β ATP-competitiveness
Compounds 1c and 20c were tested at three different ATP concentrations (25 µM, 100 µM
and 500 µM) to confirm their ATP-competitiveness. As we expected from our postulated
binding mode, higher concentrations of ATP led to significantly increased IC values from
50
18 nM and 46 nM at 25 µM of ATP to 423 nM and 492 nM at 500 µM of ATP in case of 1c
and 20c, respectively (Figures S3 and S4, supplementary data). This ATP-dependent
behavior of both compounds is a strong indicator for ATP-competitive binding of this
inhibitor class.
2.2.3. GSK3 isoform selectivity
The sequences of GSK3α and GSK3β are highly conserved, showing 98 % identity in the
kinase domain.[47] The few existing variations in their amino acid composition are mostly
minor changes and occur in areas that display no direct interactions with the ATP binding
site. In the hinge region, the only difference is that the Asp133 of GSK3β is replaced by the
Glu196 in GSK3α. The side-chain of Asp133/Glu196 is pointing outwards from the ATP
pocket towards the solvent and is putatively interacting with the positively charged
residues (e.g. Arg113 in GSK3β) in the solvent interface. To evaluate the structural
influence of this difference among the isoforms is tedious as there is no publicly available
crystal structures of GSK3α. During the preparation of this manuscript, however, Wagner
and co-workers demonstrated that the switch between these two amino acids may have an
influence on the topology of the ATP binding site and the HR I.[48] These changes in the
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488
489
490
binding site dynamics might explain the slight selectivity of the selected 2-methylsulfanyl
(1a,c), 2-benzylsulfanyl (2c) and 2-alkylimidazole (20c,e) derivatives for GSK3β versus
GSK3α (Table 5). The closely related analogues 1c and 20c proved to be the most
selective compounds with a 7.5- and 8.6-fold selectivity towards GSK3β, respectively.
4
91
92
4
Table 5. GSK3α/β isoform selectivity.
a
IC50 [nM] ± SEM
Cpd
GSK3β
GSK3α
1
2
1
c
c
a
40 ± 5
300 ± 31
229 ± 3
363 ± 16
236 ± 45
35 ± 6
853 ± 268
2,046 ± 220
1,337 ± 25
301 ± 22
12a
20c
20e
59 ± 11
428 ± 96
a
493
494
495
496
497
498
n = 2
2.2.4. Metabolism
The two potent dual GSK3β/p38α MAP kinase inhibitors 1c and 20c, as well as the highly
active and p38α MAP kinase selective compound 2e, were further evaluated for their
metabolic stability in mixed male&female HLM (Tables S1-S3, supplementary data). The
2
8