From 2-alkylsulfanylimidazoles to 2-alkylimidazoles: An approach towards metabolically more stable p38α MAP kinase inhibitors

Article abstract of DOI:10.3390/molecules22101729

In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine) undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38α mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4- (2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group resulted in 2-alkylimidazole derivatives 1 and 2, respectively, having a remarkably improved metabolic stability. The 2-alkylimidazole analogs 1 and 2 showed 20% and 10% biotransformation after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold increased binding affinity towards the target kinase as well as similar in vitro potency and ex vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950. For example, 2-alkylimidazole 2, the analog of LN950, inhibits both the p38α MAP kinase as well as the LPS-stimulated tumor necrosis factor-α release from human whole blood in the low double-digit nanomolar range.

Full text of DOI:10.3390/molecules22101729

molecules
Article
From 2-Alkylsulfanylimidazoles to
2-Alkylimidazoles: An Approach towards
Metabolically More Stable p38α MAP
Kinase Inhibitors
Fabian Heider 1 ID , Urs Haun 1, Eva Döring 1, Mark Kudolo 1, Catharina Sessler 1,
Wolfgang Albrecht 2, Stefan Laufer 1 and Pierre Koch 1,
*
ID
1
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences,
Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany;
fabian.heider@uni-tuebingen.de (F.H.); urs.haun@student.uni-tuebingen.de (U.H.);
eva.doering@uni-tuebingen.de (E.D.); mark.kudolo@uni-tuebingen.de (M.K.);
catharina.sessler@uni-tuebingen.de (C.S.); stefan.laufer@uni-tuebingen.de (S.L.)
Teva-ratiopharm, Graf-Arco-Str. 3, 89079 Ulm, Germany; wolfgang.albrecht@ratiopharm.de
Correspondence: pierre.koch@uni-tuebingen.de; Tel.: +49-7071-29-74579
2
*
Received: 13 September 2017; Accepted: 10 October 2017; Published: 14 October 2017
Abstract: In vitro and in vivo metabolism studies revealed that 2-alkylsulfanylimidazole
ML3403 (4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-N-(1-phenylethyl)pyridin-2-amine)
undergoes rapid oxidation to the sulfoxide. Replacing the sulfur atom present in the two potent p38
α
mitogen-activated protein (MAP) kinase inhibitors ML3403 and LN950 (2-((5-(4-fluorophenyl)-4-
(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)thio)ethan-1-ol) by a methylene group
resulted in 2-alkylimidazole derivatives
1
and
2, respectively, having a remarkably improved
metabolic stability. The 2-alkylimidazole analogs
1
and showed 20% and 10% biotransformation
2
after 4 h of incubation with human liver microsomes, respectively. They display a 4-fold
increased binding affinity towards the target kinase as well as similar in vitro potency and ex
vivo efficacy relative to their 2-alkylsulfanylimidazole counterparts ML3403 and LN950
example, 2-alkylimidazole , the analog of LN950, inhibits both the p38 MAP kinase as well as the
release from human whole blood in the low double-digit
. For
2
α
LPS-stimulated tumor necrosis factor-
nanomolar range.
α
Keywords: kinase inhibitors; p38α MAP kinase; trisubstituted imidazoles; metabolic stability;
Alzheimer’s disease; neurodegenerative diseases; cancer
1. Introduction
The p38α mitogen-activated protein (MAP) kinase is a ubiquitously expressed serine/threonine
kinase, which is implicated in various cellular processes such as cell survival, proliferation and
differentiation. Because it promotes the expression of pro-inflammatory cytokines such as tumor
necrosis factor-
α
(TNF-
α
) and interleukin-1
β
(IL-1
β
), the p38
α
MAP kinase has received a lot
MAP
of attention as a target for drug discovery programs since the mid-1990s. Several p38
α
kinase inhibitors were tested in clinical trials against cancer and chronic inflammatory diseases like
rheumatoid arthritis or chronic obstructive pulmonary disease. Despite these major efforts, up to
date there is still no p38α MAP kinase inhibitor on the market, as most of the trials were terminated
due to adverse events or lack of efficacy. However, recent studies suggest an important role of the
kinase in the pathogenesis of neurodegenerative diseases like multiple sclerosis and Alzheimer’s
Molecules 2017, 22, 1729; doi:10.3390/molecules22101729
www.mdpi.com/journal/molecules

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disease [
1–
3
]. Recently, two phase II studies of selective p38
α
MAP kinase inhibitor VX-745 (Figure S1,
supplementary materials) to treat Alzheimer’s disease have been completed [4,5].
Developed by SmithKline Beecham Pharmaceuticals, SB203580 is a trisubstituted imidazole
representing one of the first prototypical p38α MAP kinase inhibitors (Figure 1).
Figure 1. Pyridinylimidazoles as p38α MAP kinase inhibitors.
In collaboration with the University of Tübingen, Merckle GmbH disclosed a structural analog
ML3403, which has been widely investigated in a variety of studies [
study resulted in 2-(2-hydroxyethylsulfanyl)-4-(4-fluorophenyl)-5-(2-aminopyridin-4-yl)imidazole
LN950) [11]. Compared to ML3403 LN950 displays an improved p38 MAP kinase inhibitory
6–10]. A further optimization
(
,
α
activity as well as a two orders of magnitude higher inhibition of lipopolysaccharide (LPS)-stimulated
TNF-α release from human whole blood (HWB) (Figure 1).
Both inhibitors, ML3403 and LN950, possess an alkylsulfanyl moiety in the imidazole-C2 position,
which is prone to oxidation by metabolic enzymes such as cytochrome p450 (CYP). The metabolic
stability of ML3403 was extensively investigated in in vitro (animal and human liver microsomes) as
well as in vivo (Wistar rats) studies [12,13]. Sulfoxide ML3603 (Figure 1) was identified as the main
metabolite in all studies and acts as an active metabolite. The conversion of ML3403 to ML3603 is
mainly driven by the four CYP isoenzymes CYP1A2, CYP2C19, CYP2D6, and CYP3A4. The sulfoxide
is then to a certain extent further metabolized to the corresponding sulfone; in addition N-dealkylation
of the phenylethyl moiety as well as N-oxidation of the pyridine have also been observed. In phase II
metabolism, N-conjugation of the dealkylated product by N-methyltransferase was predominantly
registered. Kammerer et al. [13] showed the in vitro half-life of ML3403 in male and female human
liver microsomes (HLM) to be 32.7 min. In vitro experiments using mouse and rat liver microsomes
also showed short half-lives of 5.9 and 11.4 min, respectively.
While the metabolite ML3603 itself is active on p38α MAP kinase, it would be favorable to have
a metabolically stable compound as a pharmacological tool compound since active metabolites can
affect e.g., dosing calculations, dose-response relations and off-target effects.
In order to develop metabolically more stable p38α MAP kinase inhibitors, we removed the
metabolic hot spot in ML3403 and LN950 and synthesized analogs of both inhibitors (compounds
and 2), wherein the sulfur atom was replaced by a methylene group (Figure 2).
1
We evaluated the binding affinity as well as the inhibitory activity of the novel compounds toward
the target kinase and their ability to inhibit the LPS-stimulated TNF- release in human whole blood.
The obtained biological data of and were compared to those of the parent compounds in order
α
1
2
to estimate the influence of the sulfur atom present in ML3403 and LN950. Moreover, the metabolic
stability in human liver microsomes (HLM) of all four inhibitors was investigated. To rule out any
interference and possible side-effects due to CYP inhibition, we subjected the compounds to a CYP
inhibition assay.

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Figure 2. Modifications of ML3403 and LN950.
2. Results and Discussion
2.1. Chemistry
The synthesis of the ML3403 analog
depicted in Scheme 1. Ethanone
using selenium dioxide. The imidazole ring formation was achieved in a Radziszewski imidazole
synthesis by reacting dione in the presence of 7 M ammonia in methanol and propanal. In the
1
was performed in a concise three-step synthetic route, as
3
was oxidized to the corresponding α-diketone 4 in a Riley oxidation
4
last step, the amino moiety at the pyridine-C2 position was introduced via a nucleophilic aromatic
substitution reaction.
Scheme 1. Synthesis of 2-ethylimidazole
ammonia in methanol, reflux temperature, 4 h, 43%; (iii) 1-phenylethylamine (excess), 160 C, 72 h, 68%.
1
. (i) SeO2, acetic acid, 50 ◦C, 3 h, 62%; (ii) p◦ropanal, 7 M
For the synthesis of the LN950 analog
ethanone followed by Radziszewski imidazole synthesis was pursued (Scheme 2). Ethanone
already bears the 3-methylbut-2-ylamine moiety at the pyridine-C2 position as well as a Boc-protecting
group. Riley oxidation of ethanone under acidic conditions resulted in the -diketone , wherein the
2, a similar strategy consisting of Riley oxidation of an
6
[11]
6
α
7
Boc-protecting group was cleaved, too.
Triisopropylsilyl (TIPS)-protected aldehyde
9
was synthesized by the oxidation of primary alcohol
8
using a copper(I)/(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) catalyst system according to
Hoover and Stahl [14]. The ring closing reaction of dione and aldehyde in the presence of
7
9
ammonium acetate in methanol afforded imidazole 10. Finally, the TIPS-protecting group was removed
under acidic conditions to yield the trisubstituted imidazole 2.

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Scheme 2. Synthesis of 2-(3-hydroxypropyl)imidazole 2. (i) SeO2, acetic acid, reflux temperature,
0
1.5 h, 39%; (ii) N-methylimidazole, 2,2 -bipyridinyl, Cu(MeCN)4CF3SO3, (2,2,6,6-tetramethylpiperidin-
1-yl)oxyl, MeCN, room temperature (rt), 1.5 h, 20%; (iii) NH4OAc, methanol, reflux temperature, 4 h,
20%; (iv) HCl, methanol, rt, 1 h, 73%.
2.2. Biological Evaluation
2-Alkylsulfanyl imidazoles
(ELISA) [15] as well as in a fluorescence polarization (FP)-based assay [16] for their ability to inhibit
and bind to the p38 MAP kinase. Moreover, the ability of the novel compounds to inhibit the
LPS-stimulated TNF- release in HWB was tested [17]. In this ex vivo assay, the efficacy of the
inhibitors is determined more specifically with regard to pharmacokinetic characteristics such as
cellular permeability and plasma protein binding. The obtained data of and were compared to
those of their parent compounds ML3403 and LN950, respectively. The results are listed in Table 1.
2-Alkylimidazoles and are both potent inhibitors and potent binders of the target kinase,
displaying IC50 and Ki values down to the low single-digit nanomolar range.
1 and 2 were evaluated in an enzyme-linked immunosorbent assay
α
α
1
2
1
2
Table 1. Evaluation of trisubstituted imidazoles for their inhibition of p38
their binding affinity to p38 MAP kinase (FP assay) as well as for their inhibition of TNF-
LPS-stimulated human whole blood (HWB assay).
α
MAP kinase (ELISA assay),
α
α
release in
HWB Assay TNF-α IC50 [nM] b
Cpd
ELISA Assay p38α IC50 [nM] a
FP Assay p38α Ki [nM] a
ML3403
40 ± 5 c
38 ± 1 e
11 ± 3
4 ± 1 e
1 ± 0.2
2979 ± 874
2539 ± 20
37 ± 4 d
32 ± 1
1
25 ± 2
11 ± 0.9 d
11 ± 5
LN950
2
a
n = 3; b n = 2; c value taken from Laufer and coworkers [
7
]; d value taken from Koch and coworkers [11]; e value
taken from Ansideri et al. [16].
Comparison of the biological data of the 2-alkylsulfanylimidazole derivatives ML3403 and LN950
with their 2-alkylimidazole counterparts and reveals the replacement of the sulfur atom by a
methylene group not to have any influence on the biological activity of these inhibitors. However,
the 2-alkylimidazole derivatives display a 4-fold stronger binding affinity toward p38 MAP kinase
1
2
α
compared to their corresponding 2-alkylsulfanylimidazoles. The most potent 2-alkylimidazole derived
inhibitor 2 is 3.6-fold more active than the p38α MAP kinase reference compound ML3403.

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2.3. Microsomal Stability Studies
Inhibitors ML3403
,
LN950
,
1
and
2
were further tested for their metabolic stability in pooled adult
male & female HLM.
ML3403 is rapidly metabolized in our study displaying an in vitro half-life of 0.86 h (Figure 3).
After an incubation time of 4 h, less than 20% of ML3403 were present (Figure 3 and Table S1,
supplementary materials). Analysis of the metabolites by LC-MS (Tables S2-S5, supplementary
materials) confirmed the aforementioned sulfoxide ML3603 (m/z 421.5) to represent the main
metabolite (almost 75% of all metabolites after 4 h). Up to 6% of the corresponding sulfone (m/z
437.4) was observed. Other low abundance metabolites were detected having an m/z ratio of 301.4
(N-dealkylation) and 317.5 (N-dealkylation + sulfoxidation). These findings are in agreement with the
previously reported study by Kammerer et al. [13].
100
75
ML3403
50
ML3603
sulfone of ML3403
25
0
0
1
2
3
4
Time (h)
Figure 3. Metabolic stability of ML3403 and the formation of main metabolites (sulfoxide ML3603
and sulfone).
In contrast, 2-alkylimidazole
1 displays excellent metabolic stability remaining unmetabolized at
up to 80% after the incubation time of 4 h (Figure 4 and Table S6, supplementary materials). Among the
identified metabolites (Tables S7–S10, supplementary materials), m/z 283.6 was present, representing
the N-dealkylated metabolite. Additionally, the LC-MS analysis shows two peaks in close proximity,
both having an m/z ratio of 403.3. It is conceivable that both peaks correspond to the diastereomers
resulting from the hydroxylation of the methylene group present in the ethyl moiety. The most
prominent metabolite after the final sampling is seen at m/z 299.5 and is a combination of both
N-dealkylation and possible hydroxylation, accounting for almost 50% of all arising metabolites.
100
75
1
50
25
0
N-dealkylation + oxidation
N-dealkylation
0
1
2
3
4
Time (h)
Figure 4. Metabolic stability of
1 and the formation of main metabolites (N-dealkylation as well as
oxidation and N-dealkylation of 1).

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LN950 undergoes a similar but slightly slower biotransformation like ML3403. Over a time
span of 4 h, LN950 exhibits >70% degradation, most likely leading to the oxidation of the sulfur
atom at the imidazole-C2 position (Figure 5 and Table S11, supplementary materials). Examination
of the metabolites showed an almost identical pattern in comparison with ML3403, giving the
corresponding sulfoxide as the main metabolite, as well as the sulfone and some N-dealkylated
product (Tables S12–S14, supplementary materials).
100
75
LN950
50
sulfoxide of LN950
sulfone of LN950
25
0
0
1
2
3
4
Time (h)
Figure 5. Metabolic stability of LN950 and the formation of main metabolites (sulfoxide and sulfone
of LN950).
The LN950 analog, imidazole 2, wherein the sulfur atom was replaced by a methylene group,
shows under same conditions an exquisite metabolic profile. After 4 h, a degradation of only 10% could
be observed (Figure 6 and Table S15, supplementary materials). Several metabolites were detected, all
in low quantities (Tables S16 and S17, supplementary materials). Overall, a similar metabolite pattern
as that for compound
and to a small extent N-dealkylation occured. Comparison of the substituents at the imidazole-C2
position of 2-alkylimidazoles and leads to the assumption that the more polar 2-hydroxyethyl
moiety of is less susceptible for hydroxylation by CYP than the ethyl moiety present in when
1 was observed. In detail, CYP-mediated oxidation (hydroxylation or N-oxide)
1
2
2
1
incubated with HLM.
100
75
50
25
2
N-dealkylation + oxidation
N-dealkylation
0
0
1
2
3
4
Time (h)
Figure 6. Metabolic stability of
2 and the formation of main metabolites (N-dealkylation as well as
oxidation and N-dealkylation of 2).
2.4. CYP Inhibition
Pyridinylimidazoles, like SB203580, are known to inhibit CYP isoenzymes due to the capability of
the heterocyclic rings’ nitrogen atoms to coordinate with the iron present in these proteins [18 High
].
CYP inhibition is associated with side-effects such as hepatotoxicity and is also responsible for many
clinically relevant drug-drug interactions [19].

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In an initial screening, ML3403 and LN950 as well as imidazoles
1
and
2
were studied for the
purpose of assessing their inhibitory activity on the CYP isoforms 1A2, 2C9, 2C19, 2D6 and 3A4
(Table 2). At a test concentration of 10 M, ML3403 displays more than 50% inhibition of four out of
the five major drug metabolizing CYP isoforms. Compared to ML3403 LN950 shows a reduced CYP
inhibition profile. LN950 is a low inhibitor of CYP isoenzymes 1A2 and 2D6 and exhibits moderate to
high affinity for the other three tested CYP isoforms. The 2-alkylimidazoles and display a higher
affinity toward the tested CYP isoforms compared to their corresponding 2-alkylsulfanylimidazole
counterparts. 2-Alkylimidazoles and inhibit all five CYP isoforms by more than 70% and 60%,
respectively. The most potent inhibitor, 2-alkylimidazole , shows a similar inhibition of CYP isoforms
1A2 and 2C19 as well as a reduced 2C9 CYP inhibition in comparison to the p38 MAP kinase
reference compound ML3403. However, the CYP isoforms 2D6 and 3A4 are inhibited more strongly
by 2-alkylimidazole than by ML3403. Since the tested concentration of 10 M in the CYP inhibition
assay represents almost 1000-fold the IC50 value of imidazole in the kinase activity assay, a certain
µ
,
1
2
1
2
2
α
2
µ
2
margin of safety might be given concerning CYP inhibition-associated side effects.
Table 2. (%) inhibition of the most important CYP isoenzymes at 10 µM.
CYP
1A2
2C9
2C19
2D6
3A4
ML3403 a
1
LN950
2
66.7
85.6
28.0
69.4
79.0
85.1
91.5
61.9
83.5
71.0
63.0
83.6
31.1
90.2
41.5
78.8
79.2
97.6
61.0
92.1
a
values taken from Laufer and coworkers [6].
3. Materials and Methods
3.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without further
purification. Thin layer chromatography (TLC) reaction controls were performed for all reactions using
fluorescent silica gel 60 F254 plates (Merck, Darmstadt, Germany) and visualized under natural light
and UV illumination at 254 and 366 nm. The purity of tested compounds
1 and 2 were determined by
reverse phase high performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara,
CA, USA). HPLC was carried out on an Agilent 1100 Series HPLC system, equipped with an UV
DAD (detection at 218 nm, 254 nm and 280 nm). The chromatographic separation was perform◦ed
on an XBridgeTM C18 column (Waters, Milford, MA, USA) (150 mm
oven temperature. The injection volume was 10 µL and the flow rate was 1.5 mL/min using the
×
4.6 mm, 5 µm) at 30 C
following gradient: 0.01 M KH2PO4, pH 2.3 (solvent A), methanol (solvent B), 45% B to 85% B in
9 min; 85% B for 6 min; stop time 16 min. Flash column chromatography was performed using an
Interchim PuriFlash 430 automated flash chromatography system (Interchim, Montluçon, France)
with Davisil LC60A 20–45 µm silica from Grace Davison or PuriFlash SIHP 30 µm columns. Nuclear
magnetic resonance (NMR) spectra were measured on a Bruker Avance III HD NMR spectrometer
(Bruker Daltonik GmbH, Bremen, Germany) at 300 MHz in the Organic Chemistry Institute, Eberhard
Karls Universität Tübingen. The chemical shifts δ are reported in parts per million (ppm) relative to
tetramethylsilane. All spectra were calibrated against the (residual proton) peak of the deuterated
solvent used. Mass spectra were performed on an Advion Expression S electrospray ionization mass
spectrometer (ESI-MS) with an Advion Plate Express (TLC interface) (Advion, Ithaka, NY, USA).
3.1.1. Synthesis of 2-Alkylimidzole 1
1-(4-Fluorophenyl)-2-(2-fluoropyridin-4-yl)ethane-1,2-dione
fluoropyridin-4-yl)ethan-1-one ( ) (1500 mg, 6.43 mmol) was dissolved in glacial acetic acid
(10 mL) and selenium dioxide (928 mg, 8.36 mmol) was added. The reaction mixture was heated to
(
4)
[20].
1-(4-Fluorophenyl)-2-(2-
3

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◦
50 C for 3 h. After cooling to rt, the reaction was filtered and the solvent was removed under reduced
pressure. The crude product was taken up in ethyl acetate and washed with saturated NaHCO3
solution. The aqueous layer was adjusted to pH 10 with 1 M aq. NaOH solution and extracted twice
with ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, the solvent
was removed under reduced pressure and the residue was purified by flash chromatography (SiO2,
1
n-hexane/EtOAc 9:1 to 7:3) to give an orange solid (987 mg, 62%). H-NMR (300 MHz, DMSO-d6)
δ
7.43–7.53 (m, 2H), 7.69 (d, J = 0.8 Hz, 1H), 7.83 (dt, J = 5.0, 1.7 Hz, 1H), 8.09–8.18 (m, 2H), 8.55 (d,
J = 5.1 Hz, 1H); MS-ESI (m/z) 301.9 [M + Na + MeOH]+; HPLC: tR = 5.73 min.
4-(2-Ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)-2-fluoropyridine ( ). Compound (250 mg, 1.01 mmol) was
5
4
dissolved in methanol (5 mL). Subsequently, 7 M ammonia in methanol solution (2.89 mL, 20.23 mmol)
and propionaldehyde (88.11 mg, 1.52 mmol) were added successively. The reaction mixture was
heated to 80 ◦C for 4 h. After removing the solvent under reduced pressure, the crude product was
directly purified by flash chromatography (SiO2, DCM/EtOH 97:3) to give a white solid (125 mg, 43%).
1H-NMR (300 MHz, DMSO-d6)
δ 1.28 (t, J = 7.6 Hz, 3H), 2.64–2.77 (m, 2H), 7.09 (s, 1H), 7.17–7.40 (m,
3H), 7.47–7.56 (m, 2H), 8.06 (d, J = 5.4 Hz, 1H), 12.41 (br. s, 1H); MS-ESI (m/z) 286.0 [M + H]+, 284.0
[M − H]−; HPLC: tR = 3.68 min.
4-(2-Ethyl-4-(4-fluorophenyl)-1H-imidazol-5-yl)-N-(1-phenylethyl)pyridin-2-amine (
0.21 mmol) was dissolved in
1
). Compound
5
(60 mg,
◦
α
-methylbenzylamine (1 mL) and heated for 72 h to 160 C. After cooling
to rt, the crude mixture was purified by flash chromatography (SiO2, DCM/EtOH 97:3) to give a
1
white solid (57 mg, 68%). H-NMR (300 MHz, CDCl3)
δ
1.18–1.25 (m, 3H), 1.38–1.44 (m, 3H), 2.63 (q,
J = 7.6 Hz, 2H), 4.44 (quin, J = 6.5 Hz, 1H), 5.03 (d, J = 5.7 Hz, 1H), 6.3 (br. s, 1H), 6.65 (d, J = 4.7 Hz, 1H),
6.91–6.99 (m, 2H), 7.10–7.25 (m, 5H), 7.33 (dd, J = 8.0, 5.6 Hz, 2H), 7.90 (d, J = 5.4 Hz, 1H); 13C-NMR
(75 MHz, CDCl3)
δ 12.6, 21.7, 24.3, 51.8, 104.0, 111.4, 115.5 (d, J = 21.6 Hz), 125.6, 126.9, 128.5, 130.0
(d, J = 8.3 Hz), 144.3, 147.8, 150.2, 158.1, 162.3 (d, J = 247.7 Hz); MS-ESI (m/z) 387.1 [M + H]+; 385.1
[M − H]−; HPLC: tR = 7.23 min (100% purity).
3.1.2. Synthesis of 2-Alkylimidzole 2
4-(Triisopropylsilyl)oxy)butanal (9). 4-(Triisopropylsilyl)oxy)butan-1-ol (8) [21] (4,600 mg, 18.68 mmol)
was dissolved in MeCN (50 mL) before Cu(MeCN)4CF3SO3 (352 mg, 0.93 mmol), 2,20-bipyridyl
(149 mg, 0.93 mmol), TEMPO (146 mg, 0.93 mmol) and N-methylimidazole (154 mg, 1.87 mmol) were
added successively. The mixture was stirred at rt for 16 h. n-Hexane and water were added and the
organic layer was separated. The aqueous layer was extracted three times with diethyl ether. The
combined organic layers were washed with brine and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure and the residue was purified by flash chromatography (SiO2,
1
n-hexane/EtOAc 90:10) to yield a yellowish oil (2580 mg, 56%). H-NMR (300 MHz, CDCl3)
(m, 21H), 1.67–1.79 (m, 2H), 2.55 (m, 2H), 3.72 (dt, J = 16.6, 6.5 Hz, 2H), 9.39 (s, 1H).
δ 0.99–1.13
1-(4-Fluorophenyl)-2-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)ethane-1,2-dione (
7). tert-Butyl-(4-(2-(4-
fluorophenyl)-2-oxoethyl)pyridin-2-yl)(3-methylbutan-2-yl)carbamate ( ) (395 mg, 0.99 mmol) was
6
dissolved in glacial acetic acid (8 mL). Selenium dioxide (142 mg, 1.28 mmol) was added and the
reaction was heated to reflux temperature for 1.5 h. After cooling to rt, the reaction was filtered and
the solvent was removed under reduced pressure. The crude product was taken up in ethyl acetate
and washed with saturated NaHCO3 solution. The aqueous layer was adjusted to pH 10 with 1 M aq.
NaOH and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous
Na2SO4, the solvent was removed under reduced pressure and the residue was purified by flash
1
chromatography (SiO2, n-hexane/EtOAc 80:20 to 60:40) to give a white solid (121 mg, 39%). H-NMR
(300 MHz, CDCl3) δ 0.95 (dd, J = 10.0, 6.8 Hz, 6H), 1.15 (d, J = 6.5 Hz, 3H), 1.82 (dq, J = 12.3, 6.8 Hz,
1H), 3.69–3.78 (m, 1H), 6.82 (s, 1H), 6.91 (dd, J = 5.2, 1.4 Hz, 1H), 7.18–7.25 (m, 2H), 7.94–8.09 (m, 2H),
8.25 (dd, J = 5.2, 0.5 Hz, 1H); MS-ESI (m/z) 315.1 [M + H]+; 313.1 [M − H]−; HPLC: tR = 5.07 min.

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4-(4-(4-Fluorophenyl)-2-(3-((triisopropylsilyl)oxy)propyl)-1H-imidazol-5-yl)-N-(3-methylbutan-2-yl)pyridin-2-
amine (10). Compound (125 mg, 0.40 mmol) was dissolved in methanol (10 mL). Ammonium acetate
(613 mg, 7.95 mmol) and aldehyde (147 mg, 0.60 mmol) were added. The reaction was heated to
7
9
80 ◦C for 4 h. After cooling to rt, saturated NaHCO3 solution was added. The aqueous phase was
adjusted to pH 10 with 1 M aq. NaOH and extracted thrice with ethyl acetate. The combined organic
layers were dried over anhydrous Na2SO4, the solvent was removed under reduced pressure and
the residue was purified by flash chromatography (SiO2, n-hexane/EtOAc 50:50 to 0:100) to give a
1
colorless oil (43 mg, 20%). H-NMR (300 MHz, CDCl3)
δ
0.87 (dd, J = 6.8, 1.6 Hz, 6H), 1.03–1.05 (m,
24H), 1.71 (td, J = 6.7, 5.4 Hz, 1H), 1.93–2.06 (m, 2H), 2.96 (d, J = 6.1 Hz, 2H), 3.34–3.51 (m, 1H), 3.84
(d, J = 4.5 Hz, 2H), 4.39 (d, J = 8.2 Hz, 1H), 6.19–6.54 (m, 1H), 6.57–6.70 (m, 1H), 6.94–7.15 (m, 2H),
7.31–7.44 (m, 1H), 7.57 (br. s, 1H), 7.89 (d, J = 4.9 Hz, 1H), 9.96–10.24 (m, 1H); MS-ESI (m/z) 539.2
[M + H]+, 537.1 [M − H]−; HPLC: tR = 5.12 min.
3-(4-(4-Fluorophenyl)-5-(2-((3-methylbutan-2-yl)amino)pyridin-4-yl)-1H-imidazol-2-yl)propan-1-ol
(2).
Compound 10 (125 mg, 0.40 mmol) was dissolved in methanol (10 mL) and 2 M aq. HCl solution
was added. The reaction was stirred at rt for 1 h. The organic solvent was removed under reduced
pressure and the aqueous residue was treated with saturated NaHCO3 solution. A white precipitate
was formed, collected by filtration and taken up in ethyl acetate. The organic solution was dried over
anhydrous Na2SO4 and the compound was purified by flash chromatography (SiO2, DCM/EtOH
1
92:08 to 85:15) to give an off-white solid (22 mg, 73%). H-NMR (300 MHz, CDCl3)
δ 0.85 (d, J = 6.5 Hz,
6H), 1.02 (d, J = 6.6 Hz, 3H), 1.63–1.75 (m, 1H), 1.98 (dt, J = 11.5, 5.8 Hz, 2H), 2.86–2.96 (m, 2H),
3.32–3.43 (m, 1H), 3.79 (t, J = 5.3 Hz, 2H), 4.53 (d, J = 8.5 Hz, 1H), 6.45 (s, 1H), 6.57 (d, J = 4.5 Hz,
1H), 7.03 (t, J = 8.6 Hz, 2H), 7.46 (dd, J = 8.6, 5.4 Hz, 2H), 7.90 (d, J = 5.4 Hz, 1H); 13C-NMR (75 MHz,
DMSO-d6)
δ 16.9, 17.7, 18.8, 26.5, 29.8, 32.5, 52.2, 62.3, 103.8, 110.5, 115.6 (d, J = 21.6 Hz), 130.1 (d,
J = 7.7 Hz), 147.8, 148.9, 158.5, 162.4 (d, J = 247.7 Hz); MS-ESI (m/z) 383.1 [M + H]+, 381.1 [M
−
H]−;
HPLC: tR = 6.53 min (100% purity).
3.2. HLM Stability Test
Pooled human liver microsomes (adult male & female) were purchased from Merck (Schnelldorf,
Germany). These microsomes were characterized in protein and CYP content. All incubations (final
total volume 1050
5 mM Glucose-6-phosphate, 5 U/mL Glucose-6-phosphate dehydrogenase and 1 mM NADP+. The
substrate (100 M), the NADPH regenerating system and 4 mM MgCl2 6 H2O in 0.1 M Tris buffer
(pH 7.4 at 37 ◦C) were preincubated for 5 min in an incubator at 37 ◦C and 750 rpm. The incubation
mix was split into 50 L aliquots and the reaction was started by addition of the HLM. Thereby the
µL) were made in the presence of an NADPH-regenerating system, consisting of
µ
×
µ
microsomal protein content was standardized to 1 mg/mL. To follow the course of metabolism, the
reaction tubes were quenched at selected time points (0, 10, 20, 30, 60, 120, 180 and 240 min) by adding
100
µ
L internal standard at a concentration of 22.5 ◦
µM in MeCN. The samples were vortexed for 30 s
and centrifuged (19,800 relative centrifugal force/4 C/10 min). The supernatant was directly used for
LC-MS analysis (for detailed LC-MS conditions, see supplementary materials). All incubations were
conducted in triplicates and incubations with heat-inactivated HLM were used to prove that analyte
reduction results from metabolic degradation only. In all incubations, a limit of 1% organic solvent
was not exceeded.
3.3. CYP Inhibition Test
CYP inhibition (fluorimetric detection) assay was performed by Eurofins Panlabs Inc. (St. Charles,
MO, USA) with human recombinant CYP enzyme and the appropriate CYP substrate.

Molecules 2017, 22, 1729
10 of 11
4. Conclusions
Replacement of the sulfur atom present in both known p38
α
MAP kinase inhibitors ML3403 and
LN950 by a methylene group results in 2-alkylimidazole derivatives
improved microsomal stability. Both compounds undergo minimal hydroxylation and N-dealkylation
upon incubation with HLM. Trisubstituted imidazoles and display a 4-fold increase in binding
affinity toward the kinase and possess a similar inhibition profile of p38 MAP kinase and the
LPS-stimulated TNF- release from HWB like their 2-alkylsulfanylimidazole counterparts ML3403
and LN950. However, the exchange of the sulfur atom present in ML3403 and LN950 by a methylene
group is accompanied with a slightly higher CYP inhibition profile of and . The most potent inhibitor,
2-alkylimidazole , inhibits both the p38 MAP kinase as well as the LPS-stimulated TNF- release
from human whole blood in the low double-digit nanomolar range. Moreover, the excellent metabolic
profile of gives advantage over the mixed pharmacokinetics of the p38 MAP kinase reference
is a good alternative to
1 and 2, showing remarkably
1
2
α
α
1
2
2
α
α
2
α
inhibitor ML3403 and its active metabolite. Therefore, 2-alkylimidazole
2
evaluate the role of this kinase in in vitro and in vivo studies.
Supplementary Materials: The following are available online, Figure S1 (Structure of VX-745) and Tables S1–S17
(metabolic stability of the title compounds in HLM and metabolite formation).
Acknowledgments: We thank Katharina Bauer and Jens Strobach for their assistance in the p38
α MAP
kinase ELISA assay. Luiza Oprezka is gratefully acknowledged for her assistance in the metabolism
study. This study was supported by the Federal Ministry of Education and Research (BMBF) within the
BioPharma—Neuroallianz consortium.
Author Contributions: F.H., U.H., E.D., M.K., C.S., W.A., S.L. and P.K. conceived and designed the experiments;
F.H. and U.H. performed synthesis; E.D. performed the metabolism experiments; C.S. and M.K. performed the
biological evaluation in vitro; F.H., W.A., S.L. and P.K. analyzed the data; F.H. and P.K. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1 and 2 are available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).