Tetra-substituted pyridinylimidazoles as dual inhibitors of p38α mitogen-activated protein kinase and c-jun N-terminal kinase 3 for potential treatment of neurodegenerative diseases

Article abstract of DOI:10.1021/jm501557a

Tetra-substituted imidazoles were designed as dual inhibitors of c-Jun N-terminal kinase (JNK) 3 and p38α mitogen-activated protein (MAP) kinase. A library of 45 derivatives was prepared and evaluated in a kinase activity assay for their ability to inhibi

Full text of DOI:10.1021/jm501557a

Article
pubs.acs.org/jmc
Tetra-Substituted Pyridinylimidazoles As Dual Inhibitors of p38α
Mitogen-Activated Protein Kinase and c‑Jun N‑Terminal Kinase 3 for
Potential Treatment of Neurodegenerative Diseases
Felix Muth,† Marcel Gunther,† Silke M. Bauer,† Eva Doring,† Sabine Fischer,† Julia Maier,‡
̈
̈
Peter Druckes,§ Jurgen Koppler,§ Jorg Trappe,§ Ulrich Rothbauer,‡ Pierre Koch,† and Stefan A. Laufer*,†
̈
̈
̈
̈
†Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard Karls Universitat Tubingen,
̈
̈
Auf der Morgenstelle 8, 72076 Tubingen, Germany
̈
‡Natural and Medical Sciences Institute at the University of Tubingen, Markwiesenstrasse 55, 72770 Reutlingen, Germany
̈
§Novartis Pharma AG, CH-4002 Basel, Switzerland
S
* Supporting Information
ABSTRACT: Tetra-substituted imidazoles were designed as dual inhibitors
of c-Jun N-terminal kinase (JNK) 3 and p38α mitogen-activated protein
(MAP) kinase. A library of 45 derivatives was prepared and evaluated in a
kinase activity assay for their ability to inhibit both kinases, JNK3 and p38α
MAP kinase. Dual inhibitors with IC50 values down to the low double-digit
nanomolar range at both enzymes were identified. The best balanced dual
JNK3/p38α MAP kinase inhibitors are 6m (IC50: JNK3, 18 nM; p38α, 30
nM) and 14d (IC50: JNK3, 26 nM; p38α, 34 nM) featuring both excellent
solubility and metabolic stability. They may serve as useful tool compounds
for preclinical proof-of-principle studies in order to validate the synergistic
role of both kinases in the progression of Huntington’s disease.
deposits.8 Symptoms of HD include bradykinesia, rigidity,
seizures, and severe dementia caused by the loss of neurons in
the striatum.9,10 HD is the only neurodegenerative disease which
can be diagnosed by genetic testing at all ages. Symptoms
typically begin in midlife, and the average life expectancy after
diagnosis is approximately 20 years.
INTRODUCTION
■
Mitogen-activated protein (MAP) kinases are a family of
evolutionary highly conserved serine/threonine kinases that
link extracellular signals to the intracellular machinery,
modulating a plethora of cellular processes (e.g., cell cycle
progression, cell death and differentiation). The ten-membered
family contains four p38 MAP kinases (p38α, β, γ, δ), three c-Jun
N-terminal kinases (JNK1, 2, 3; also known as stress-activated
protein kinases), and three extracellular-regulated kinases
(ERKs).1−4 The p38α MAP kinase is expressed ubiquitously,
while the expression of JNK3 is mainly restricted to the brain and,
to a lesser extent, to the heart and testis.5 Both kinases are
activated by two different upstream kinases, MAP kinase kinase
(MKK) 4 and 7 in the case of JNK3 and MKK3 and 6 in the case
of p38α MAP kinase (Figure 1). The activation occurs by dual
phosphorylation of the side chains of Thr221 and Tyr223
(JNK3) or Thr180 and Tyr182 (p38α MAP kinase) enclosed in a
conserved Thr-Pro-Tyr motif located in the activation loop.2
JNK3 and p38α MAP kinase phosphorylate different substrates
in response to cellular stress stimuli and pro-inflammatory
cytokines such as tumor necrosis factor-α (TNFα) and
interleukin-1β (IL-1β). However, there are numerous proteins
which have been identified as substrates for both kinases, e.g., the
activating transcription factor 2 (ATF2).6
Diseases related to neurodegeneration are multifaceted.
Several agents involved in the pathogenesis of HD, e.g., oxidative
stress, class IIa histone deacetylase, and protein kinases such as
GSK3β, p38α MAP kinase, or JNK3, have been identified.11−13
Multitarget drugs inhibiting several key drivers involved in
neurodegeneration may exhibit additive or even synergistic
effects in vivo. For the treatment of complex diseases like
neurodegeneration, therapies that act at multiple targets and
providing symptomatic as well as neuroprotective effects may
thus be more effective. Recently, Taylor et al. reported enhanced
levels of MKP-1 are neuroprotective via dephosphorylation and
therefore deactivation of JNK3 and p38α MAP kinase.14 By
simultaneously blocking both pathways, improved neuro-
protective effects were observed compared to the disruption of
only a single pathway. This resulted in complete rescue of
primary striatal neurons from polyglutamine-expanded htt
Special Issue: New Frontiers in Kinases
Huntington’s disease (HD) is an autosomal dominant genetic
Received: October 8, 2014
disease characterized by mutant huntington protein (htt)
Published: November 21, 2014
© 2014 American Chemical Society
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Figure 1. JNK and p38 MAP kinase pathways are activated by inflammatory cytokines, growth factors, and environmental stresses such as UV-
irradiation, heat and osmotic shock, genotoxic agents, anisomycin, and toxins. Different upstream MAP kinase kinase (MAP2K) and MAP kinase kinase
kinase (MAP3K) family members activate the JNKs and p38 MAPKs. MAP kinase phosphatase-1 (MKP-1), also known as dual specificity
phosphatase‑1 (DUSP-1), acts as suppressor of the activation cascade (modified from ref 7).
Figure 2. Tetra-substituted pyridinylimidazoles 2, derived from the early lead compounds SKF86002, and reference compound SB203580 as inhibitors
of p38α MAP kinase.
toxicity. Therefore, the design of dual JNK3/p38α inhibitors
might be a promising strategy for treatment of HD.
However, neither a JNK3-selective nor a dual selective JNK3/
p38α inhibitor has been reported so far.
Although the ATP-binding site is conserved among all kinases,
two selective p38α MAP kinase inhibitors, namely Skepinone-L
and VX-745, were recently highlighted as chemical probes.15
During the past decade, we reported different series of 1,2,4,5-
tetra-substituted imidazoles (e.g., 2a and 2b) as inhibitors of
p38α MAP kinase, which can be regarded as open chain
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Figure 3. Schematic p38α MAP kinase binding mode of tetra-substituted imidazoles 2 (hydrogen bonds are drawn in dashed lines; HR, hydrophobic
region).
analogues of the early dihydrothiazoline-derived lead compound
SKF86002 (Figure 2).16−19
pyridine−C2-amino function (R1), (functionalized) alkyl
moieties at the imidazole−N1 position (R2), and carboxylic
acid as well as carboxylic acid derivatives were introduced at the
imidazole−C2-S position (R3) of the imidazole scaffold 2 in
order to modulate the compounds’ biological activities (Figure
5).
Compounds 2a and 2b are both adenosine triphosphate
(ATP) competitive inhibitors of p38α MAP kinase. For optimal
interaction with the enzyme, the tetra-substituted imidazoles
must comprise a 4-pyridinyl moiety, a 4-fluorophenyl group, and
an unsubstituted N-atom in the imidazole ring adjacent to the 4-
fluorophenyl group. The nitrogen atom of 4-pyridinyl moiety
interacts with the backbone amide-NH of Met109 located in the
hinge region via a hydrogen bond. The 4-fluorophenyl ring is
buried in the hydrophobic region I (selectivity pocket), which is
limited by the so-called gatekeeper amino acid Thr106 (Figure
3). The imidazole N-3 accepts a hydrogen bond from the ε-
amino group of the Lys53 side chain.20 In contrast to SKF86002,
the 1-alkyl-4-(4-fluorophenyl)-2-methylsulfanyl-5-pyridin-4-yli-
midazoles 2 possess an acylated amino function at the pyridine−
C2 position, which can act as a hydrogen bond donor to the
backbone carbonyl function of Met109, and the moieties R1 and
R2 address the hydrophobic region II.
The potent but nonselective ATP competitive p38α MAP
kinase inhibitor SB203580 (Figure 2) derived from early lead
compound SKF86002 as well as compound 2b show moderate to
good inhibition of JNK3.21,22 Because these pyridinylimidazoles
were initially optimized for targeting p38α MAP kinase, they
show some selectivity for p38α MAP kinase over JNK3. Our
general aim was to increase the JNK3 inhibition of compounds 2
and simultaneously maintain the good inhibitory potency at
p38α MAP kinase. The former series of tetra-substituted
pyridinylimidazoles reported generally had a small methylsul-
fanyl moiety at the imidazole−C2 position. To establish further
interactions of 2 with the enzyme, we introduced bulkier,
functionalized moieties at the imidazole−C2-S position. Because
Arg67 (p38α numbering) is present in both enzymes (Figure 4)
targeting this amino acid, e.g., by substituents that are
functionalized with carboxylic acids or carboxylic acid derivatives
was probed in order to obtain dual JNK3/p38α inhibitors.
Herein, we report on the synthesis of a series of novel tri- and
tetra-substituted imidazoles and their evaluation as dual
inhibitors of JNK3 and p38α MAP kinase. Alkyl and aryl
residues bearing a variety of substituents were introduced at the
RESULTS
■
Chemistry. The tetra-substituted imidazoles 6 bearing an N-
acetyl moiety at the pyridine−C2-amino position were prepared
using a versatile and diverse synthetic strategy starting from N-
(4-(2-(4-fluorophenyl)-2-(hydroxyimino)acetyl)pyridin-2-yl)-
acetamide (3a)24 (Scheme 1) that has been developed in our
group.16−19 This synthesis allows the introduction of a broad
variety of aliphatic and functionalized moieties at the imidazole−
N1 as well as the imidazole−C2-S position. Treatment of oxime
3a with different 1,3,5-trialkyl-1,3,5-triazinanes resulted in the
regioselective introduction of the imidazole−N1 substituent
(R2), giving the corresponding imidazole-N-oxides 4a−j that
were subsequently converted into imidazole-2-thiones 5a−j by
using 2,2,4,4-tetramethylcyclobutane-1,3-dithione as sulfuriza-
tion agent.
Compounds 6a−d were synthesized in good to acceptable
yields by treatment of imidazole-2-thione 5a and the
corresponding alkyl halide under known Williamson ether
synthesis conditions using potassium carbonate as the base and
dry THF at mild conditions.
To link (Z)-acrylic acid to the imidazole-2-thione, compounds
5a−j were refluxed in methanol with propiolic acid. Because of
the strongly nucleophilic nature of the thiol-tautomer of 5a−j as
well as the strong Michael acceptor properties of propiolic acid,
this 1,4-addition proceeded without the need for a catalyst or
base, yielding moderate to good amounts of imidazoles 6g−p.
1
The resulting (Z)-configuration was confirmed by H NMR
spectroscopy (3J H,H ∼ 9.8 Hz).
Similar to the Michael addition described above, compounds
6e and 6f were treated with acrylic acid and a catalytic amount of
iodine to obtain the desired saturated propionic acid derivative.
To determine the preferred length and geometry of the linker
between the imidazole core scaffold and carboxyl group, 2-
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Figure 4. (a) General structure of MAP kinases (generated from PDB structure 3TTJ). Residues contributing to the binding cleft are depicted as spheres
and the colors were chosen according to the sequence alignment below. (b) Differences in the ATP binding site of JNK3 and p38α MAP kinase.
Residues discriminating between the two kinases and their contribution to the binding pockets surface are highlighted in red and yellow. The key
differences between JNKs and p38α MAP kinase are found in the gatekeeper residue (Met vs Thr, highlighted in magenta) and in the solvent exposed
front region (Cys vs Asn, highlighted in yellow). (c) Sequence alignment of residues surrounding the ATP binding site in JNK3 and p38α MAP kinase.
Differing amino acids are highlighted using darker colors (modified from ref 23).
as a mixture of r,r,r and s,s,s enantiomers, see Supporting
Information). The reaction of imidazole-2-thiones 5b,f,i with
ethyl (E)-3-iodoacrylate (9) resulted in esters 6u−w. Ethyl (E)-
3-iodoacrylate (9) was synthesized starting from ethyl propiolate
(7) via the iodinated (Z)-isomer 8,25 which was isomerized to the
desired E-isomer 9 (3J
= 15.0 Hz) (Scheme 2) by applying
H,H
hydroiodic acid according to a protocol of Tackenuchi et al.26
Subsequent saponification of ester 6w under mild conditions
using KOHaq (10%) in THF at room temperature led to the
desired (E)-acrylic acid 6x (3J
= 15.0 Hz). Cleavage of the
H,H
acetyl group at the pyridine−C2-amino position was not
observed during this step, but a certain degree of isomerization
at the acryl double bond was unavoidable.
Figure 5. Structural modifications of imidazole scaffold 2.
For one example (compound 6y), a 6-iodopyridin-2-yl moiety
was introduced at the imidazole−C2-S position. Therefore, 2,6-
diiodopyridine was reacted with imidazole-2-thione 5e under
Ullmann-like reaction conditions by applying copper(I)oxide as
catalyst (Scheme 3).27
The synthetic route toward tetra-substituted imidazoles
bearing an alkyl or aryl moiety at the pyridine−C2-amino
position (compounds 14a−g) starting from 2-(4-fluorophenyl)-
hydroxycyclohexane-4-carboxylic acid as well as (E)-acrylic acid
were introduced as substituents at the imidazole−C2-S position.
The tetra-substituted imidazoles 6q−t bearing a 4-carboxy-2-
hydroxycyclohexyl moiety at the imidazole−C2-S position were
obtained by treatment of imidazole-2-thiones 5a,e,h,i with 3-
hydroxy-4-iodocyclohexane-1-carboxylic acid (12) using classi-
cal Williamson conditions (for synthesis of the carboxylic acid 12
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a
Scheme 1. Synthesis of Tetra-Substituted Imidazoles 6a−x (N-Acetyl Moiety at the Pyridine−C2-amino Position)
a
Reagents and conditions: (i) 1,3,5-trialkyl-1,3,5-triazinane (1.0 equiv), ethanol(abs), 80 °C, overnight; (ii) 2,2,4,4-tetramethylcyclobutane-1,3-
dithione, DCM, rt, 2 h; (iii) K2CO3 (1.25 equiv), corresponding alkyl halide (1.2 equiv), THF(abs), rt, 24 h; (iv) acrylic acid (0.67 equiv), iodine (0.14
equiv), rt, 24 h; (v) propiolic acid (1.05 equiv), methanol(abs), reflux, 2 h; (vi) imidazole-2-thione (1.0 equiv), potassium tert-butoxide (4.0 equiv),
THF(abs), 80 °C, 4 h; (vii) DABCO (2.0 equiv), ethyl (E)-3-iodoacrylate (1.0 equiv), imidazole-2-thione (1.1 equiv), DMF(abs), rt, 4 h; (viii) KOHaq
(10%), THF, rt, overnight.
a
Scheme 2. Preparation of Ethyl (E)-3-Iodoacrylate
Scheme 3. Synthesis of N-(4-(1-Cyclohexyl-4-(4-
fluorophenyl)-2-((6-iodopyridin-2-yl)thio)-1H-imidazol-5-
a
yl)pyridin-2-yl)acetamide (6y)
a
Reagents and conditions: (i) sodium iodide (1.5 equiv), ethyl
propiolate (1.0 equiv), acetic acid, 70 °C, overnight; (ii) (Z)-isomer 8
(1.0 equiv), hydroiodic acid (5.0 equiv), toluene, 80 °C, overnight.
1-(2-fluoropyridin-4-yl)-2-(hydroxyimino)ethan-1-one (3b) is
depicted in Schemes 4 and 5. This novel strategy allows the
modification of both the pyridine-2-amino function and the
imidazole−C2-S position of the 1-alkyl-4-(4-fluorophenyl)-5-(2-
fluoropyridin-4-yl)-1,3-dihydro-2H-imidazole-2-thiones 5k−m
in the last steps of the synthesis. Using this approach, aliphatic
as well as aromatic substituents were introduced at the pyridine−
C2-amino position and combined with different substituents at
the sulfur atom. At the latter position, (E)- and (Z)-acrylic acid,
acrylates, and acetonitrile were introduced to the imidazole-2-
thiones under the same conditions as described in Scheme 1.
Substitution of compound 5l with aniline was carried out
under acidic conditions (Scheme 4). Compound 5l was heated at
200 °C in concentrated hydrochloric acid and 1-methylpyrro-
lidin-2-one (NMP) to achieve complete protonation of the
pyridine−N1 atom. The lowered electron density of the resulting
a
Reagents and conditions: (i) potassium tert-butoxide (2.0 equiv),
Cu2O (0.005 equiv), 2,6-diiodopyridine (1.0 equiv), dioxane(abs), 110
°C, overnight.
protonated 2-fluoropyridin-1-ium allowed a nucleophilic attack
by aniline obtaining imidazole-2-thione 13b in a short reaction
time of 30 min. These harsh conditions are only compatible with
imidazole-2-thiones, bearing nonfunctionalized aliphatic moi-
eties at the imidazole−N1 position. However, substituted
anilines could not be introduced at the pyridine−C2 position
using these reaction conditions. Aliphatic substituents at the 2-
fluoropyridine of 5k were introduced via nucleophilic aromatic
substitution (SNAr), leading to compounds 13c−d. The liquid
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a
Scheme 4. Synthesis of Tetra-Substituted Imidazoles 14a−d,f,g,k (Aryl or Alkyl Moiety at the Pyridine−C2-amino Position)
a
Reagents and conditions: (i) 1,3,5-trialkyl-1,3,5-triazinane (1.0 equiv), ethanol(abs), 80 °C, overnight; (ii) 2,2,4,4-tetramethylcyclobutane-1,3-
dithione, DCM, rt, 2 h; (iii) corresponding thione (1.0 equiv), 2-chloroacetonitrile (1.1 equiv), potassium carbonate (3.0 equiv), THF, reflux, 3.5 h;
(iv) compound 5k (1.0 equiv), corresponding amine (11 equiv), 100 °C for 14 h; (v) in case of aniline derivatives, 5l (1.0 equiv), HCl(aq,conc) (4.0
equiv), NMP, 200 °C for 30 min, aniline (10.0 equiv), 200 °C for further 30 min; (vi) propiolic acid or ethyl propiolate (1.05 equiv), methanol(abs)
,
reflux, 2 h; (vii) DABCO (2.0 equiv), ethyl (E)-3-iodoacrylate (1.0 equiv), imidazole-2-thione (1.1 equiv), DMF(abs), rt, 4 h; (viii) KOHaq (10%) and
THF, 2 h, 95 °C.
Scheme 5. Synthesis of Tetra-Substituted Imidazole (Z)-3-((5-(2-((1-(Ethylcarbamoyl)piperidin-4-yl)amino)pyridin-4-yl)-4-(4-
a
fluorophenyl)-1-methyl-1H-imidazol-2-yl)thio)acrylic Acid (14e)
a
Reagents and conditions: (i) 1,3,5-trimethyl-1,3,5-triazinane (1.0 equiv), ethanol(abs), 80 °C, overnight; (ii) 2,2,4,4-tetramethylcyclobutane-1,3-
dithione, DCM, rt, 2 h; (iii) 5m (1.0 equiv), tert-butyl 4-aminopiperidine-1-carboxylate (11.0 equiv), PEG 400 (catalytic amount), 120 °C, 6 h,
microwave; (iv) HCl in dioxane (20.0 equiv), DCM, 20 min, rt; (v) ethyl propiolate (1.05 equiv), methanol(abs), reflux, 2 h; (vi) isocyanatoethane
(1.5 equiv), DCM(abs), 4.5 h, rt, argon atmosphere; (viii) KOHaq (10%) and THF, 2 h, 95 °C.
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Scheme 6. Synthesis of Tri-Substituted Imidazole (Z)-3-((4-(4-Fluorophenyl)-5-(2-(phenylamino)pyridin-4-yl)-1H-imidazol-2-
a
yl)thio)acrylic Acid (14h)
a
Reagents and conditions: (i) 5n (1.0 equiv), HCl(aq,conc) (4.0 equiv), NMP, 200 °C for 30 min, aniline (10.0 equiv), 200 °C for further 30 min; (ii)
ethyl propiolate (1.05 equiv), methanol(abs), reflux, 2 h; (iii) KOHaq (10%) and THF, 2 h, 95 °C.
amine was used as the solvent and the reaction mixture heated to
100 °C for 14 h.
Scheme 7. Buchwald−Hartwig Cross Coupling Reaction for
Synthesis of Tetra-Substituted Imidazoles 16a−k (Bearing
a
(Z)-Acrylic acid derivative 14e was prepared using a different
approach (Scheme 5). The moiety at the pyridine−C2-amino
position (compound 13e) was introduced via SNAr by melting 4-
(4-fluorophenyl)-5-(2-fluoropyridin-4-yl)-1-methyl-1,3-dihy-
dro-2H-imidazole-2-thione (5m) and the solid amine (tert-butyl
4-aminopiperidine-1-carboxylate) at 120 °C and 35 W in a
microwave reactor. Small amounts of PEG 400 were necessary to
ensure complete conversion in an appropriate time. Cleavage of
the Boc-protecting group under acidic conditions yielded thione
18. Compound 18 was first transferred into the ethyl (Z)-acrylate
14j, followed by conversion of the cyclic amine with isocyanato-
ethane, resulting in ethylcarbamoyl derivative 19. Finally,
saponification of compound 19 by treatment with KOHaq
resulted in imidazole 14e.
Substituted Anilines at the Pyridine−C2 Position)
a
Reagents and conditions: (i) 2.5 mol % BrettPhos precatalyst, 15 (1.0
equiv), cesium carbonate (4.0 equiv), aryl bromide (1.1 equiv),
dioxane/tert-butanol 4:1, 110 °C, 2 h.
The tri-substituted imidazole derivative (Z)-3-((4-(4-fluoro-
phenyl)-5-(2-(phenylamino)pyridin-4-yl)-1H-imidazol-2-yl)-
thio)acrylic acid 14h was synthesized starting from known 4-(4-
fluorophenyl)-5-(2-fluoropyridin-4-yl)-1,3-dihydro-2H-imida-
zole-2-thione (5n) (Scheme 6). Aniline was introduced at the
pyridine−C2 position by SNAr followed by reaction of
intermediate 13a with ethyl propiolate, resulting in imidazole
14i. Finally, saponification of 14i yielded (Z)-acrylic acid 14h.
To bypass the limiting conditions for introducing substituted
aniline derivatives at the 2-fluoropyridine using the SNAr
conditions described above (Scheme 4), a Buchwald−Hartwig
cross coupling reaction was applied (Scheme 7). First, the
conditions for the Buchwald−Hartwig arylamination were
optimized in a model system using 4-(4-(4-fluorophenyl)-1-
methyl-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-amine (15)
and m-halogenated nitrobenzene (for details, see Table S1 in
the Supporting Information). The influence of the solvent
system, ligand, base, and the halogen atom on the conversion
and/or yield of this prototype reaction was investigated.
Optimized conditions involve the use of dry dioxane/t-butanol
as the solvent, BrettPhos as ligand, cesium carbonate as base
(which forms cesium-tert-butoxide in combination with tert-
butanol in situ), and m-bromonitrobenzene as aryl halide. Using
these optimized conditions, a set of 11 imidazoles bearing
substituted aniline moieties at the pyridine−C2 position
(compounds 16a−k) were synthesized from 15 by treatment
with functionalized bromobenzene derivatives (e.g., phenylureas,
phenylamides and anisoles).
Biological Evaluation. The synthesized tri- and tetra-
substituted imidazoles were evaluated for their ability to inhibit
JNK3 and p38α MAP kinase in enzyme-linked immunosorbent
assays (ELISA).21,22 Selected compounds were further screened
in a panel against 60 protein kinases to determine their selectivity
within the kinome. Results are presented in Tables 1−3, Table S2
(Supporting Information) and Figures 6, 9, and 10.
Within the series of tetra-substituted imidazoles having a N-
acetyl function at the pyridine−C2 amino position (6a−y),
compounds bearing a (Z)- (6g−p) and a (E)-acrylic acid (6x) at
the imidazole−C2 position display good to excellent affinity
toward both enzymes, p38α MAP kinase and JNK3 with a slight
preference (1.2 to 10-fold) for JNK3 over p38α MAP kinase
(Table 1, Figure 6). The series of compounds 6a−e,g shown in
Table 1 was derived from compound 2a, maintaining the methyl
moiety in order to avoid steric hindrance with substituents
introduced at the imidazole−C2 position. To examine the size of
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Table 1. Biological Activities of Tetra-Substituted Pyridinylimidazoles 6 (n = 3)
a
Ratio IC50 (JNK3)/IC50 (p38α).
substituents tolerated by both enzymes and increasing the
inhibitory effect 6a was synthesized showing low double-digit
nanomolar inhibition of p38α MAP kinase and moderate
inhibition of JNK3. The formation of an additional H-bond
between the propan-2-one and Asp-168 in p38α MAP kinase was
assumed decisive for the improved potency. To further
investigate the contribution of the carbonyl oxygen to the
inhibitory potency, 6b−d were synthesized. Introduction of
methyl acetate in compound 6b led to a slight improvement of
JNK3 inhibition and a 8-fold decreased p38α MAP kinase activity
compared to 6a (6b IC50: JNK3, 199 nM; p38α, 297 nM).
Compound 6c displays a 10-fold selectivity for p38α MAP
kinase, whereas the selectivity profile is inversed for compound
6d, which establishes an H-bond to the conserved but steric
diverse Asn196 in JNK3 presumably. Encouraged by this
observation, compounds with a rigid and a flexible linker for
the carboxylic acid moiety were synthesized in order to avoid
repulsion of the anionic moiety with Asp168 in p38α MAP kinase
and form additional H-bonds with the cationic Arg107 in JNK3
represented by compounds 6e and 6g. Compound 6e, bearing a
propanoic acid moiety, was the most promising due to its double-
digit nanomolar inhibitory effect on JNK3 (6e IC50: JNK3, 91
nM; p38α, 246 nM) but was not compatible with large
hydrophobic substituents at imidazole-−N1 position directed
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respectively. The cyclohexylmethyl moiety at the imidazole−
N1 position points toward the hydrophobic region II. For JNK3,
the carboxyl function can form three hydrogen bond interactions
with the enzyme, two to Arg107, as expected, and one to the side
chain of Lys93. In p38α MAP kinase, the imidazole−N3 atom as
well as the carboxylic acid group may interact via hydrogen bond
interactions with the Lys53 side chain. However, the docking
algorithm suggested no interaction with the side chain of Arg67.
Compound 6h bearing a dimethoxymethyl moiety at the
imidazole−N1 position (IC50: JNK3, 33 nM; p38α, 335 nM)
displays a 10-fold selectivity for JNK3. The preference for JNK3
may be caused by the displacement of a conserved water
molecule stabilized by the Asn152 side chain (Figure 8).
Figure 6. Comparison of pIC50 values at JNK3 versus p38α MAP kinase
of the inhibitor series 6.
toward the hydrophobic region II (6f IC50: JNK3, 269 nM; p38α,
1.699 μM). Compound 6h in contrast, bearing the rigid acrylic
acid in (Z)-configuration was well tolerated by both enzymes
with a 10-fold preference for JNK3 (6h, IC50: JNK3, 33 nM;
p38α, 335 nM).
Compared to compound 6m with the (Z)-configuration,
compound 6x with the acrylic acid in (E)-configuration exhibits a
10-fold decreased potency. Cycloalkyl moieties like cyclohexyl
(6l IC50: JNK3, 133 nM; p38α, 165 nM) and cyclopropyl (6i
IC50: JNK3, 82 nM; p38α, 123 nM) at the imidazole−N1
position were well tolerated by both enzymes. Introduction of a
methylene linker between the carbocyclic ring and the imidazole
core resulted in an increase of inhibitory activity, leading to the
most potent dual inhibitors of this series, compounds 6m (IC50:
JNK3, 18 nM; p38α, 30 nM) and 6n (IC50: JNK3, 25 nM; p38α,
39 nM).
The suggested binding mode for compound 6n at both
enzymes is illustrated in Figure 7. Docking supposes a binding
mode typical for pyridinylimidazoles. The 4-fluorophenyl ring is
located at the hydrophobic pocket I. The pyridine nitrogen atom
as well as the pyridine−C2 amino function interact with the
hinge region via a bidentate hydrogen bond to the backbone of
Met149 and Met109 in JNK3 and p38α MAP kinase,
Figure 8. Suggested binding mode for compound 6h in JNK3. As a
protein model, the X-ray structure for JNK3 with the PDB code 1PMN
was used. The side chain of the gatekeeper residue (Met146) was treated
as flexible. The water molecule was added after the docking experiment.
All compounds bearing a 4-carboxy-2-hydroxycyclohexyl
moiety at the imidazole−C2-S position (6q−t) possess IC50
values for the p38α MAP kinase in the nanomolar range. For
JNK3, the inhibitory effect of compounds 6q−t depends on the
size of the imidazole−N1 moiety. Imidazoles 6s and 6t with
flexible moieties at the imidazole−N1 position show a decreased
JNK3 inhibition, resulting in a 15- and 4-fold selectivity for p38α
MAP kinase. However, compounds 6q and 6r having a small
methyl or a more rigid cyclohexyl moiety at the imidazole−N1
position exhibited good JNK3 inhibitory activity and a reversed
selectivity profile. In case of compound 6s, the combination of
the flexible dimethoxymethyl moiety at the imidazole−N1
Figure 7. A suggested binding mode for compound 6n in JNK3 (left) and p38α MAP kinase (right). As a protein model, the X-ray structures for JNK3
(PDB code: 1PMN) and p38α MAP kinase (PDB code: 1A9U) were used. In the case of JNK3, the side chain of the gatekeeper residue (Met146) was
treated as flexible.
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Table 2. Biological Activities of Tri- and Tetra-Substituted Pyridinylimidazoles 14 (n = 3)
a
Ratio IC50 (JNK3)/IC50 (p38α).
position (R2) and the bulky 4-carboxy-2-hydroxycyclohexyl
substituent at the imidazole−C2-S position (R3) may result in a
sterical clash and therefore in the loss of orienting all crucial
interactions properly.
Inhibitor 6y bearing a 6-iodopyridin-2-moiety at the
imidazole−C2-S position shows a 15-fold selectivity for p38α
MAP kinase over JNK3, presumably due to a possible π−π
interaction with the side chain of Tyr35 in p38α MAP kinase,
while JNK3 contains a glutamine at this position (Figure 4).
Inhibition data of tri- and tetra-substituted imidazoles bearing
an alkyl or aniline moiety at the pyridine−C2-amino position are
summarized in Table 2 and Figure 9. The comparison of
imidazole 14a and 14b emphasizes the importance of the
presence of a hydrogen bond donor (secondary amino function)
at the pyridine−C2 position, resulting in an additional hydrogen
bond interaction with the hinge region in both enzymes.
Compound 14c bearing a (Z)-acrylic acid at the imidazole−C2-S
position shows a better inhibition profile than 14b having an
acetonitrile moiety at this position.
Initially, we assumed that the dimethoxymethyl moiety at
imidazole−N1 position (R2) might form a hydrogen bond
mediated by the conserved water molecule. To further
investigate this hypothesis, we synthesized 6p (R2 = 2,3-
dihydroxypropyl) in order to replace the water and establish
the hydrogen bond directly between the inhibitor and Asn152.
Against our expectations, the decreased inhibitory effect of 6p
compared to 6h disproved this theory. Consequently, we assume
that 6h and also 6o in fact do not develop a hydrogen bond but
replace the conserved water, resulting in increase of JNK3
potency.
In contrast to the free carboxylic acid 6x, the ethyl ester 6w
displays a reduced inhibitory activity for both kinases and a less
balanced dual inhibition profile.
Small aliphatic amines at the pyridine−C2 position (R1) were
better tolerated by both enzymes than the aromatic aniline.
Compounds 14c and 14d having a cyclopropylamine and sec-
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Table 3. Biological Activities of Tetra-Substituted
Pyridinylimidazoles 16 (n = 3)
Figure 9. Comparison of pIC50 values at JNK3 versus p38α MAP kinase
of inhibitor series 14.
butylamine as R1, respectively, showed an increased inhibitory
potency, especially in case of p38α MAP kinase, compared to
compound 6h bearing an N-acetyl group at this position. On the
other side, (E)-acrylic acid derivative 14i bearing an aniline at the
pyridine−C2 position is a weaker inhibitor compared to its N-
acetyl counterpart (6m).
Similar to the structure−activity relationships (SARs)
observed for series 6, the derivative containing a free carboxylic
acid (14h) at the imidazole−C2-S position was a more potent
inhibitor compared to the corresponding ethyl ester (14i).
Moreover, compounds having the acrylic acid in (Z)-
configuration (14c−f and 14h) show a slight preference for
JNK3.
Compound 14h lacking the imidazole−N1 substituent
possessed an increased inhibitory activity for both tested
enzymes compared to the tetra-substituted imidazole 14f (R2 =
cyclopropylmethyl). Tri-substituted imidazole 14h (R2 = H) is a
potent dual JNK3/p38α MAP kinase inhibitor (IC50: JNK3, 17
nM; p38α, 36 nM).
a
Ratio IC50 (JNK3)/IC50 (p38α).
Within series 14, the combination of a small branched
alkylamine moiety at the pyridine−C2 amino function, a
dimethoxymethyl residue at the imidazole−N1 position, and a
(Z)-acrylic acid at the imidazole−C2-S position result in the best
balanced dual inhibitor (14d), featuring IC50 values in the low
double-digit nanomolar range IC50 (JNK3, 26 nM; p38α, 34
nM).
Biological data of tetra-substituted pyridinylimidazoles 16 are
listed in Table 3 and Figure 10. The potential of exploiting
interactions with the hydrophobic region II for dual inhibition
was explored in this series. Thus, different substituted benzenes
were introduced at the pyridine−C2-amino function (R1). To
minimize torsional effects caused by intramolecular steric
repulsion between the substituents (R2) and (R3) or to avoid a
sterical clash of R3 and the kinase due to a different binding
orientation resulted by bigger moieties at the pyridine−C2
position, a small methyl substituent was introduced at positions
R2 and R3.
Figure 10. Comparison of pIC50 values at JNK3 versus p38α MAP
kinase of the inhibitor series 16.
The SARs of this series provide restricted information. All
substituted anilines introduced at the pyridine−C2 position were
well tolerated by both enzymes (Figure 10). The compounds of
this series are good dual inhibitors of JNK3 and p38α MAP
kinase with IC50 values ranging from 50 to 299 nM. In the case of
compound 16a,b,d, the position of the methoxy group at the
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aniline ring has no influence on JNK3 inhibition. However, in the
case of p38α MAP kinase, a p-methoxybenzene substituent at the
pyridine−C2 amino position (16b) seems to be favored.
Within series 16, compound 16c bearing a p-morpholino-
aniline at the pyridine−C2 position showed best balanced dual
inhibition (IC50: JNK3, 85 nM; p38α, 50 nM).
sulfoxide (m/z = 517.5) as the main metabolite (Figure S3c,
Supporting Information). Furthermore, compounds 6m and 14d
display excellent aqueous solubility in PBS buffer of 1.20 mg/mL
(2.06 μmol/mL) and 0.93 mg/mL (3.59 μmol/mL), respec-
tively.
To estimate selectivity against other kinases, a total of five
inhibitors from series 6 were tested against a panel of 60 protein
kinases (Table S2, Supporting Information). Out of the three
tested dual JNK3/p38α MAP kinase inhibitors, compounds 6l
and 6o showed a clean profile for JNKs and p38α MAP kinase.
No other kinase was inhibited more than 50% at the tested
concentration (5.5 μM). However, dual inhibitor 6i shows some
affinity for EGFR(HER1) (51.8% inhibition) and ACVR1-
(ALK2) (60.6% inhibition) Both tested p38α MAP kinase
inhibitors 6s (IC50: p38α, 135 nM) and 6v (IC50: p38α, 356 nM)
showed a clean profile in this screen, too.
The tetra-substituted pyridinylimidazoles 6m, 14b, 14d, and
14b and as well as the tri-substituted pyridinylimidazole 14h
were further tested for potential cytotoxicity in a simple cell
count experiment. Therefore, human U2OS cells were subjected
to hyperosmotic stress followed by treatment with increasing
concentrations up to 50 μM of these compounds. The number
and morphology of treated cells were subsequently analyzed
using high-content analysis based on fluorescent microscopy.
The results show that none of the compounds induces any
changes in cell number or morphology compared to DMSO used
as control (Figures S1 and S2, Supporting Information).
To further profile the two most promising dual JNK3/p38α
MAP kinase inhibitors, metabolic stability and solubility of
compounds 6m and 14d were examined (Table 4). The
CONCLUSIONS
■
A series of 45 novel tri- and tetra-substituted imidazoles was
designed as dual inhibitors of JNK3 and p38α MAP kinase. Two
dual inhibitors of series 6 (6l and 6o) show an excellent
selectivity profile. We identified compounds with IC50 values
down to the low double-digit nanomolar range at both enzymes.
Compounds 6m (IC50: JNK3, 18 nM; p38α, 30 nM) and 14d
(IC50: JNK3, 26 nM; p38α, 34 nM) were the best balanced dual
JNK3/p38α MAP kinase inhibitors identified in this study. Both
compounds show a good metabolic profile and an outstanding
aqueous solubility in PBS puffer. They may serve as promising
tool compounds for preclinical proof-of-principle studies in
order to validate the role of both kinases in the progression of
HD. Because of the complexity of this pathology, these dual
target drugs are expected to be superior to single-target drugs for
the treatment of HD.
EXPERIMENTAL SECTION
■
All reagents and anhydrous solvents are commercially available and were
used without further purification. Purification was performed on silica
phase Davisil LC60A 20−45 μm silica from Grace for the column, using
an Interchim PuriFlash 430 automated flash chromatography system.
1H- and 13C NMR spectra were obtained with Bruker Avance 200 or
with Bruker 400 Avance. The spectra were obtained in the indicated
solvent and calibrated against the residual proton peak of the deuterated
solvent. Chemical shifts (δ) are reported in parts per million.
GC/MS analyses were carried out on a Hewlett-Packard HP 6890
series GC system equipped with a HP-5MS capillary column (0.25 μm
film thickness, 30 m × 250 μm) and a HP 5973 mass selective detector
(EI ionization). Helium was used as carrier gas.
Table 4. Metabolic Stability and Solubility Data for Tetra-
Substituted Pyridinylimidazoles 6m and 14d
The purity of all tested compounds is >95% and was determined via
reverse phase high performance liquid chromatography on Hewlett-
Packard HP 1090 series II LC equipped with a UV diode array detector
(DAD, detection at 230 and 254 nm). The chromatographic separation
was performed on a Phenomenex Luna 5μ C8 column (150 mm × 4.6
mm, 5 μm) at 35 °C oven temperature. The injection volume was 5 μL,
and the gradient of the used method was (flow: 1.5 mL/min): 0.01 M
KH2PO4, pH 2.3 (solvent A), methanol (solvent B): 40% B to 85% B in
8 min, 85% B for 5 min, 85% to 40% B in 1 min, 40% B for 2 min, stop
time 16 min.
For detailed syntheses and characterization of all shown compounds
and their precursors, see Supporting Information.
General Procedure II: Synthesis of Compounds 6g−p, 14c−f,
and 14h−i. A solution of propiolic acid or ethyl propiolate (1.05 equiv)
and corresponding thione (1.0 equiv) in absolute methanol (80 mL/
mmol thione) was refluxed for 2 h until complete conversion. The
solvent was removed under reduced pressure, and the residue was
purified by silica flash-chromatography.
(Z)-3-((5-(2-(sec-Butylamino)pyridin-4-yl)-1-(2,2-dimethoxyeth-
yl)-4-(4-fluorophenyl)-1H-imidazol-2-yl)thio)acrylic Acid (14d). 14d
was synthesized from 13d according to general procedure II. The crude
product was purified by silica flash chromatography using a gradient of
DCM/acetic acid 99/1 → DCM/ethanol/acetic acid 89/10/1 as eluent
afforded 43% of the title compound. 1H NMR (200 MHz, methanol-d4)
δ 8.01 (d, J = 5.9 Hz, 1H), 7.53−7.35 (m, 3H), 7.06−6.94 (m, 2H),
6.56−6.47 (m, 2H), 6.09 (d, J = 9.6 Hz, 1H), 4.39 (t, J = 4.9 Hz, 1H),
4.16 (d, J = 5.1 Hz, 2H), 3.25 (s, 6H), 1.59−1.44 (m, 2H), 1.16 (d, J = 6.5
Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H). NH- and carboxylic acid-protons were
not detected due to a proton/deuterium exchange with deuterated
methanol. ESI-HRMS: [M + H]+ calculated, 501.19663; found,
501.19724.
a
b
Percent remaining after 240 min incubation with HLM. Solubility in
aqueous PBS-buffer (pH 7.4).
biotransformation of both dual inhibitors was investigated in
male human liver microsomes (HLM). Although both
substances seem to undergo metabolic degradation (see Figure
S3a, Supporting Information), none reached its half-life during
the longest incubation time of 240 min. The concentration of
compounds 6m and 14d declined to about 30% and 44% relating
to the starting concentrations, respectively. As expected,
metabolic profiling of inhibitor 14d identified the corresponding
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General Procedure III for Compounds 6q−t. To a solution of the
corresponding thione derivative in dry THF (60 mL/1 mmol thione),
potassium tert-butoxide (4.0 equiv) and the corresponding alkyl halide
were added under ice cooling. The mixture was allowed to warm to
room temperature, thereafter heated to 80 °C and stirred for 4 h. After
cooling to room temperature, the mixture was quenched with saturated
ammonium chloride solution and extracted with ethyl acetate three
times. The organic layers were combined, dried over sodium sulfate, and
evaporated to dryness under reduced pressure to be purified by silica
flash chromatography.
4-((5-(2-Acetamidopyridin-4-yl)-4-(4-fluorophenyl)-1-methyl-1H-
imidazol-2-yl)thio)-3-hydroxycyclohexane-1-carboxylic Acid (6r).
According to general procedure III, 6r was synthesized from 5a and
12. Purified by silica flash chromatography using a gradient of DCM →
DCM/iPrOH 90/10 as eluent afforded 28% of the title compound. 1H
NMR (200 MHz, DMSO-d6) δ 12.13 (s, 1H), 10.67 (s, 1H), 8.40 (d, J =
5.0 Hz, 1H), 8.04 (s, 1H), 7.46−7.27 (m, 2H), 7.20−6.95 (m, 3H), 5.31
(s, 1H), 3.47 (s, 3H), 3.23−3.03 (m, 1H), 2.43−2.22 (m, 1H), 2.22−
1.94 (m, 5H), 1.83 (d, J = 13.0 Hz, 1H), 1.65−1.10 (m, 3H). ESI-
HRMS: [M + H]+ calculated, 485.16533; found, 485.16508.
General Procedure IV for Compounds 6u−y and 14k. DABCO
(2 equiv) and ethyl (E)-3-iodoacrylate (1 equiv) were dissolved in dry
DMF (8 mL/mmol ethyl (E)-3-iodoacrylate) and stirred under argon
atmosphere for 10 min. The respective thione (1.1 equiv) was dissolved
in dry DMF (3 mL/mmol ethyl (E)-3-iodoacrylate) and added dropwise
to the solution. After 3 h of stirring at room temperature, the reaction
mixture was evaporated to dryness and purified by silica flash-
chromatography.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 7071 2972459. E-mail: stefan.laufer@uni-
tuebingen.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Peter Keck for the help with the processing of the raw
data. We are grateful to Matthias Gehringer for contributing
■
Figure 3. Daniela Muller and Katharina Bauer are also
̈
acknowledged for skillful technical assistance in compound
testing.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; HD, Huntington’s disease; HR,
hydrophobic region; htt, huntingtin protein; JNK, c-Jun N-
terminal kinases; MAP, mitogen-activated protein; MKK, MAP
kinase kinases; NMP, 1-methylpyrrolidin-2-one; SAR, struc-
ture−activity relationships; SNAr, nucleophilic aromatic sub-
stitution
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ASSOCIATED CONTENT
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S
* Supporting Information
Details for the synthesis and characterization of compounds
5−19; Buchwald−Hartwig cross coupling optimization; screen-
ing data; description of the ELISA; cell toxicity data; solubility
data; metabolism in Human Male Liver Microsomes. This
material is available free of charge via the Internet at http://pubs.
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