Optimized 4,5-diarylimidazoles as potent/selective inhibitors of Protein Kinase CK1δ and their structural relation to P38α MAPK

Article abstract of DOI:10.3390/molecules22040522

The involvement of protein kinase CK1δ in the pathogenesis of severe disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, familial advanced sleep phase syndrome, and cancer has dramatically increased interest in the development of effecti

Full text of DOI:10.3390/molecules22040522

molecules
Article
Optimized 4,5-Diarylimidazoles as Potent/Selective
Inhibitors of Protein Kinase CK1δ and Their
Structural Relation to p38α MAPK
Jakob Halekotte 1,†, Lydia Witt 1, Chiara Ianes 2,†, Marc Krüger 2, Mike Bührmann 3,
Daniel Rauh 3, Christian Pichlo 4, Elena Brunstein 4, Andreas Luxenburger 5, Ulrich Baumann 4,
Uwe Knippschild 2, Joachim Bischof 2, ,‡ and Christian Peifer 1, ,‡
* *
1
Institute of Pharmacy, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany;
jhalekotte@pharmazie.uni-kiel.de (J.H.); lwitt@pharmazie.uni-kiel.de (L.W.)
Department of General and Visceral Surgery, Ulm University Hospital, Albert-Einstein-Allee 23,
D-89081 Ulm, Germany; chiara.ianes@uni-ulm.de (C.I.); marcm3012@googlemail.com (M.K.);
uwe.knippschild@uniklinik-ulm.de (U.K.)
Institute of Chemical Biology, Dortmund University of Technology, Otto-Hahn-Straße 4a,
D-44227 Dortmund, Germany; mike.buehrmann@googlemail.com (M.B.);
daniel.rauh@tu-dortmund.de (D.R.)
Department for Chemistry, University of Cologne, Otto-Fischer-Straße 12-14, D-50674 Cologne, Germany;
pichloc@uni-koeln.de (C.P.); Elena.Brunstein@uni-koeln.de (E.B.); ubaumann@uni-koeln.de (U.B.)
The Ferrier Research Institute, Victoria University of Wellington, Gracefield Research Centre,
69 Gracefield Road, Lower Hutt P.O. Box 33-436, New Zealand; Andreas.Luxenburger@vuw.ac.nz
Correspondence: joachim.bischof@uniklinik-ulm.de (J.B.); cpeifer@pharmazie.uni-kiel.de (C.P.);
Tel.: +49-731-500-53589 (J.B.); +49-431-880-1137 (C.P.)
2
3
4
5
*
†
‡
These authors contribute equally to this work.
These two authors contribute equally to this work.
Academic Editors: Pierre Koch and Stefan Laufer
Received: 31 January 2017; Accepted: 20 March 2017; Published: 24 March 2017
Abstract: The involvement of protein kinase CK1δ in the pathogenesis of severe disorders such
as Alzheimer’s disease, amyotrophic lateral sclerosis, familial advanced sleep phase syndrome,
and cancer has dramatically increased interest in the development of effective small molecule
inhibitors for both therapeutic application and basic research. Unfortunately, the design of CK1
isoform-specific compounds has proved to be highly complicated due to the existence of six
evolutionarily conserved human CK1 members that possess similar, different, or even opposite
physiological and pathophysiological implications. Consequently, only few potent and selective
CK1
in order to establish SAR that might enable complete discrimination of CK1 isoforms and related p38
MAPK. In this study we report on design and characterization of optimized 4,5-diarylimidazoles
δ inhibitors have been reported so far and structurally divergent approaches are urgently needed
α
as highly effective ATP-competitive inhibitors of CK1
IC50 CK1 = 25 nM), 12a (IC50 CK1 = 19 nM, IC50 CK1
IC50 CK1ε = 81 nM) being among the most potent CK1
δ
with compounds 11b (IC50 CK1
δ
= 4 nM,
= 8 nM,
ε
δ
ε
= 227 nM), and 16b (IC50 CK1
δ
δ
-targeting agents published to date. Inhibitor
compound 11b, displaying potential as a pharmacological tool, has further been profiled over a panel
of 321 protein kinases exhibiting high selectivity. Cellular efficacy has been evaluated in human
pancreatic cancer cell lines Colo357 (EC50 = 3.5
µM) and Panc89 (EC50 = 1.5 µM). SAR is substantiated
by X-ray crystallographic analysis of 16b in CK1δ and 11b in p38α.
Keywords: protein kinase CK1; formerly known as casein kinase 1; p38 MAPK; kinase inhibitors;
4,5-diaryl-imidazoles; Alzheimer’s disease; amyotrophic lateral sclerosis; familial advanced sleep
phase syndrome; cancer
Molecules 2017, 22, 522; doi:10.3390/molecules22040522
www.mdpi.com/journal/molecules

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1. Introduction
Protein kinase CK1
δ
is a member of the ubiquitously expressed and constitutively active
Ser/Thr-specific CK1 (formerly known as casein kinase 1) family which comprises the six human
isoforms 1, 2, 3, , and , together with their closest relatives the tau tubulin kinases 1 and
2 (TTBK1/2) and vaccinia-related kinases 1-3 (VRK1–3) [ ]. Despite CK1 being evolutionarily
highly conserved within their catalytic domains, all isoforms differ significantly in the length and
the primary structure of their regulatory N- and C-terminal regions. Among them, isoforms and
display the highest consensus, with a 98% sequence identity within their catalytic domain and
at least 40% homology within their autoregulatory C-terminal domains [ ]. Pathophysiologically,
identification of mutations within the coding region of CK1 as well as deregulation of CK1 expression
and/or activity levels as important determinants in development and progression of severe human
disorders such as Alzheimer’s disease (AD) [ ], amyotrophic lateral sclerosis (ALS) [ ], familial
advanced sleep phase syndrome (FASPS) [10], and cancer [ 11 19] has dramatically increased
α
,
γ
γ
γ
δ
ε
1–3
δ
ε
2–5
δ
δ
2
,6
–
8
9
2,5, –
interest in the development of potent and selective small molecule kinase inhibitors for both therapeutic
approaches and basic research. However, the existence of paralogous CK1 isoforms that possess similar,
different, or even opposite physiological and pathophysiological implications render the design of
suitable candidate molecules that target CK1
difficult. The most extensively used and characterized CK1 inhibitor to date, IC261, moderately inhibits
CK1 isoforms and (IC50 CK1 = 1 M, IC50 CK1 = 10 M) and proved valuable in diverse
pharmacological studies [16 20 22]. Furthermore, IC261 even revealed therapeutic potential for the
δ in an ideally isoform-dependent manner enormously
δ
ε
δ/ε
µ
α
µ
,
–
treatment of pancreatic cancer in a subcutaneous mouse xeno-transplantation model, despite the fact
that the inhibitor is active against several other targets, including tubulin polymerization and ion
channels [16
those which are commonly referred to as “linear” [25
to their three-dimensional structure. Among the latter, a promising 4,5-diarylimidazole-based inhibitor
, originally designed as an inhibitor of p38 MAPK, was revealed in 2009 by Peifer et al. to inhibit
CK1 with IC50 values in the low nanomolar range ( IC50 CK1 = 5 nM, IC50 CK1 = 73 nM) [28].
Interestingly, sulfoxidation of thioether leading to sulfoxide significantly enhanced discrimination
of highly related isoforms and to at least 40-fold while preserving good potency for CK1 IC50
CK1 = 11 nM, IC50 CK1 = 447 nM, Figure 1) [28]. Unfortunately, the reduced chemical stability
of and in solution due to E/Z-isomerization and the presence of a Michael acceptor moiety in the
,
22
–24]. In addition, few further compounds have been reported as CK1 inhibitors, mainly
,26] and “tear-drop”-like binders [27] with respect
1
α
δ/
ε
1
δ
1
ε
1
2
δ
ε
δ (2
δ
2
ε
1
2
cinnamic acid side chain are responsible for their limited usability in vitro and in vivo.
Figure 1. ATP-competitive dual specific inhibitors 1 and 2 of CK1δ/ε and p38α MAPK.
The present follow-up [28] study reports on the optimization of lead structures
1 and 2,
respectively, leading to stable novel inhibitors of CK1δ/ε with IC50 values in the low nanomolar
range. The optimization strategy followed a well-established procedure in medicinal chemistry
including in silico design, hit synthesis, and in vitro biological evaluation [29,30].

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2. Results and Discussion
2.1. Molecular Modeling
The binding modes of ATP-competitive type-I inhibitors
1
and
2
in CK1
δ
and p38
α
have been
postulated based on structure-based molecular modeling (Figure 2) [28]. Comparable to poses of
similar “tear-drop”-like binders (e.g., pdb 3UZP [31]) two hydrogen bonds are formed between the
2-amino-pyridine moiety and CK1δ hinge residue Leu85. The positive mesomeric electron donating
effect of the amino group in ortho-position of the pyridine nitrogen positively influences electron
density and thus the H-bond acceptor strength at the pyridine-N [32].
Figure 2. Modeled binding modes of
2 in CK1δ (top, pdb 3UZP [31]) and p38α (bottom, pdb
1BMK [33]) ATP-binding pockets. Key amino acid residues and ligand-active site interactions are
shown. Left: in accordance to Traxler et al. [34], the ATP-binding pocket of protein kinases ought to be
subdivided into hydrophobic pocket I (HPI), hydrophobic region II (HRII), adenine-binding region
(AB), phosphate-binding region (PB), and sugar pocket (SP). Right: 2D ligand interaction diagrams.

Molecules 2017, 22, 522
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Furthermore, core catalytic residues Lys38 and Asp149 [13
,31] coordinate a structural water
within the catalytic cleft which donates another hydrogen bond towards an imidazole nitrogen of
the respective inhibitor. Gatekeeper residue Met82 is rotated by 180◦ towards Pro66 [13
31], thereby
permitting access to the hydrophobic pocket I (selectivity pocket, HPI) [ 34] which is ideally occupied by
the 4-fluorophenyl moiety [31 35]. Molecular modeling further suggests five-membered heterocycles
to dictate an ideal angle for positioning of the vicinal aryl moieties within the ATP-binding pocket
of CK1 36]. The (E)-configured cinnamic acid side chain of and extends into the spacious
solvent-exposed hydrophobic region II (affinity pocket, HRII) [ 34]. Modeling calculations further
,
2,
,
δ
[
1
2
2,
considered different di- or trimethoxyphenyl substitution pattern optimal within this region as they
enable flexible occupation of hydrophobic surfaces and shielding deeper cavities of the ATP-binding
pocket from surrounding water, thus entailing increased enthalpic contribution of buried hydrogen
bonds. Analogous binding poses have been achieved regarding p38α with the bidentate hinge-binding
moiety addressing Met109 and the imidazole-N accepting a hydrogen bond from Lys53. Rotation of
the smaller gatekeeper residue Thr106, however, does not seem necessary in order to occupy HPI.
The binding modes described above are based on (E)-configured cinnamic moieties of
1
and
2, respectively. In contrast, molecular modeling of the respective (Z)-configurations did not
result in plausible binding modes (not shown). Accordingly, computational analysis assume these
(Z)-stereoisomers to be less bioactive. Furthermore, the acrylamide Michael acceptor moiety of the
cinnamic acid side chain was considered responsible for the observed chemical instability of
1
and
in solution. In line with this notion, within a short period of time after preparing a solution of
and in DMSO a HPLC analysis showed an increasing number of not identifiable degradation
2
1
2
products. Consequently, our primary goal towards an optimized inhibitor was to gain chemical
stability. Thus, having identified the cinnamic side chain to be responsible for the chemical instability
issue we aimed towards stable side chains attached at the validated 2-aminopyridine core moiety.
By these modifications we set out to explore the respective hydrophobic region II formerly occupied
by the cinnamic acid moiety. At the same time, both potency and selectivity for CK1δ were taken into
account. Therefore, in our systematic approach four structurally divergent series of inhibitors with
variable side chains (Scheme 1) have been designed based on the following considerations.
Scheme 1. Structural considerations based on molecular modeling leading to lead structure
derivatives 10a (series 1), 11a (series 2), 12a (series 3), and 16a (series 4).
1

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First, removal of both the planar (sp2)
π
-bond and carbonyl group in
1
and
2
led to respective
sp3 hybridized 3-(2,4-dimethoxyphenyl)propanamine 10a and derivatives (series 1). However, at
this position of the ligand, additional degrees of freedom and enhanced conformational flexibility
are typically accompanied by losses of both potency and selectivity; Second, maintaining the
amide function but formally reducing the
π-bond resulted in presumably stable and potent
3-(2,4-dimethoxyphenyl)propionic amide derivatives (e.g., 11a, series 2). Third, a carbamide moiety
in 12a and derivatives (series 3) might enable an additional hydrogen bond towards hinge Leu85
and therefore could account for enthalpic binding energy gains. The additional fixation was
further suggested to exploit different folding of related CK1
the hinge region and thus to be a key parameter for triggering inhibitor selectivity. And fourth,
fixing the (E)-configuration of cinnamic amides and within five-membered heterocycles led to
δ, CK1ε, and p38α within range of
1
2
4-(2,4-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic amide (16a, series 4). Taken together, by
our compound design concept in the side chain Michael acceptor characteristics and inactive (Z)-isomers
were eliminated while potentially conserving beneficial impacts regarding potency and selectivity.
Based on the inhibitor categories described above we generated a virtual set of compounds being
subsequently processed in a LigPrep/Glide docking campaign using CK1δ (pdb 3UZP [31]) and p38α
(pdb 1BMK [33]) protein structures. Thereby we focused on variation of side chains addressing the
HRII while maintaining the fixed 4,5-diaryl-imidazole pharmacophore. This included different sets
of substituted lipophilic and mainly sterically demanding moieties in order to exploit this region.
As methoxy-substituents were assumed most favorable in this context, efforts have been devoted to
methoxy-screenings investigating different substitution patterns.
2.2. Synthesis
In order to effectively synthesize the designed and top ranked hits from docking, a straightforward
five-step procedure towards the building blocks 2-fluoro-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-
imidazol-4-yl)pyridine (
amine ( ) was established in accordance to literature protocols [37
of key compounds and 7, variations of side chains were introduced and thus four compound series
were prepared (Scheme 2).
6
) and 4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridine-2-
7
,
38]. In the final step, by substitution
6
Slightly deviating from the procedure depicted in Scheme 2, pyridine-2-amine 10a has
been synthesized by a SN2 reaction of single Boc-protected 2-amino-4-methylpyridine and
1-(3-bromopropyl)-2,4-dimethoxybenzene (
9) followed by subsequent formation of the 4,5-diaryl-
imidazole scaffold using the procedure developed for synthesis of
6
. Alkyl halide was synthesized
9
from 3-(2,4-dimethoxyphenyl)propionic acid by reduction [39] followed by an Appel reaction using
triphenylphosphine and N-bromosuccinimide (Scheme 3) [40]. Detailed information about the
synthesis of 10a is presented in the Supporting Information.
Series 1 pyridine-2-amines 10b–f and piperazines 10g–i were prepared by a Tschitschibabin-like
nucleophilic substitution [32,38]. Therefore, 2-fluoropyridine 6 was dissolved or suspended in an
excess of the appropriate amine or piperazine and heated to 160 ◦C (Scheme 2). In general, in this
reaction primary amines accounted for better yields.
Syntheses of series 2 included the reaction of building block
di- or trimethoxy-substituted 3-phenylpropionic acids to afford amides 11a
character of
, however, required heating to 110 ◦C in order to achieve suitable reactivity
(Scheme 2) [28].
In contrast, highly reactive isocyanates readily acylated
Wöhler-like synthesis leading to series 3 carbamide derivatives 12a
7
with CDI-activated differently
–
e. The poor nucleophilic
7
7
at room temperature in terms of a
(Scheme 2) [41]. Noteworthy,
–
g
addition of Hünig’s base led to increased yields and reactions had to be performed under protective
gas atmosphere in order to prevent formation of carbamide dimers.
Series 4 precursor 4-(2,4-dimethoxy-phenyl)pyrrole-2-carboxylic acid was prepared in a three
step synthesis starting from ethyl 4-bromo-1H-pyrrole-2-caboxylate [42]. Methylation of the pyrrole

Molecules 2017, 22, 522
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nitrogen using methyl iodide was followed by Suzuki cross-coupling with (2,4-dimethoxyphenyl)
boronic acid and subsequent simple ester hydrolysis in diluted alkali. The 2,5-dimethoxyphenyl
side chain derivative 15b has been synthesized analogously. Finally, 16a
–b were obtained by
PyBOP-supported amide coupling with building block 7 under elevated temperature (Scheme 4).
Scheme 2. Synthesis of key building blocks
6 and 7 as well as inhibitors 10b–i (series 1), 11a–e (series 2),
◦
12a (series 3), and 16a (series 4). Reagents and Conditions: (i) NaHMDS, THF, 2 h 0 C, 1 h r.t.;
–
g
–b
(ii) CH3CHOOH, NaNO2, 1 h 0 ◦C, 3.5 h r.t.; (iii) H2, Pd/C, HCl-saturated 2-propanol, 12 h r.t.;
(iv) methyl thiocyanate, DMF, 45 min reflux, 45 min r.t.; (v) NH3, 20–30◦bar, 18 h 180 ◦C; (vi) HNR2,
◦
12 h 160 C; (vii) carboxylic acid, CDI or PyBOP/DIPEA, DMF, 12 h 110 C; (viii) isocyanate, DIPEA,
DMF, 12 h r.t.
Scheme 3. Synthesis of 1-(3-bromopropyl)-2,4-dimethoxybenzene (9). Reagents and Conditions:
(i) LiAlH4, THF, 1 h r.t.; (ii) NBS, PPh3, DCM, 2 h r.t.

Molecules 2017, 22, 522
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As mentioned above, the respective sulfoxides might possess interesting potential regarding
selectivity. Therefor◦e, oxidation of selected compounds was performed in accordance to literature
using Oxone® at 0 C or at ambient temperature (Scheme 5). During thioether oxidation it was
essential to strictly monitor reaction progress for quenching at the sulfoxide level [28]. Accordingly,
sulfoxidation was performed for compounds 10c
–d, 11a–b, 12a, 12c–g, and 16a leading to 10j–k
(series 1), 11f (series 2), 12h (series 3), and 16c (series 4). It has to be noted that sulfoxides are
–
g
–m
chiral compounds and we are always referring to the racemate. However, docking analysis did not
reveal differences between the enantiomers. In the literature the racemate is reported for this inhibitor
class with only one exception, where the R-configuration was observed to possess increased affinity
for p38α MAPK [43].
Scheme 4. Synthesis of 4-(2,4-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (15a). Reagents
◦
◦
and Conditions: (i) NaH, DMF, 20 min 0 C, CH3I, 15 min 0 C, 2.5 h r.t.; (ii) 2,4-dimethoxyphenyl
boronic acid, Pd(PPh3)4, aq. NaHCO3, DMF, 4 h reflux◦, 12 h r.t.; (iii) aq. NaOH, THF/methanol, 5 h
◦
50 C, 12 h r.t., (iv) DIPEA, DMF, 30 min r.t., 7, 12 h 110 C.
Scheme 5. Synthesis of sulfoxides. Reagents and Conditions: (i) Oxone® (potassium peroxomonosulfate),
◦
THF/H2O, 30 min–2 h 0 C.
All synthesized target compounds 10a–k, 11a–g, 12a–m, 16a–c are listed in Table 1. They were
stable in DMSO solution at room temperature without detectable degradation over a period of 72 h by
HPLC analysis.

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Table 1. Synthesized test compounds of series 1, 2, 3, and 4. R refers to the side chains indicated in
Scheme 5. Only a limited selection of thioether compounds has been oxidized leading to sulfoxide
compounds (12h–12m, 16c). Abbreviation: # compound number.
Thioether #
10a
Sulfoxide #
R
Thioether #
11c
Sulfoxide #
R
10b
10c
11d
12a
10j
12h
10d
10k
12b
10e
10f
10g
10h
12c
12d
12e
12f
12i
12j
12k
12l
10i
11a
11b
12g
16a
16b
12m
16c
11f
11g
2.3. Kinase Assays and IC50 Determination
Compounds 10a (series 1), 11a
been screened for their ability to inhibit the activity of CK1
at a concentration of 10 M. Inhibitors exhibiting promising effects were further subjected to IC50
value determination (Table 2). In addition, IC50 values regarding inhibition of CK1 and p38 MAPK
have commercially been obtained from ProQinase GmbH (Freiburg, Germany) for 11b 12a, and 16b
–k
–
g
(series 2), 12a
–
m
(series 3), and 16a
–c
(series 4) have initially
δ
and CK1ε in an in vitro kinase assay
µ
δ
α
,
as these were the most potent representatives of their respective series (Table 3). In comparison to
our data, IC50 values measured by ProQinase appear slightly lower due to the different assay setup

Molecules 2017, 22, 522
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such as the ATP concentration used (compare Experimental Section/Supporting Information and
www.proqinase.com).
Table 2. IC50 values of promising inhibitors for CK1
δ
and CK1
ε
. All compounds have initially
M. The most
been screened in an in vitro assay against CK1 and CK1
δ
ε
at a concentration of 10 µ
promising agents were subjected to IC50 value determination. Results are presented as mean
from experiments performed in triplicate (n = 3). Abbreviation: # compound number.
±
SD
Compound #
CK1δ IC50 (nM)
CK1ε IC50 (nM)
10a
10d
10e
11a
11b
11c
11d
11e
11f
11g
12a
12b
12d
12e
12f
12h
12j
12k
12l
12m
16a
16b
16c
386 ± 86
644 ± 274
344 ± 104
20 ± 2
6731 ± 2680
3323 ± 1297
1753 ± 738
129 ± 22
25 ± 4
4 ± 1
14 ± 2
91 ± 12
20 ± 4
233 ± 38
204 ± 32
499 ± 70
573 ± 104
227 ± 37
186 ± 28
272 ± 52
203 ± 67
498 ± 124
910 ± 18
804 ± 160
764 ± 207
623 ± 139
182 ± 39
45 ± 7
27 ± 3
93 ± 12
87 ± 2
19 ± 3
31 ± 3
47 ± 6
35 ± 10
196 ± 22
153 ± 24
160 ± 26
115 ± 18
88 ± 14
48 ± 8
9 ± 1
8 ± 1
81 ± 15
32 ± 4
181 ± 32
Table 3. IC50 values of key inhibitors for CK1δ and p38α MAPK. IC50 values of the most promising
agents of series 2 (11b), series 3 (12a), and series 4 (16b) have commercially been obtained from
ProQinase GmbH (Freiburg, Germany). Single experiments have been performed (n = 1). Abbreviation:
# compound number.
Compound #
CK1δ IC50 (nM)
p38α IC50 (nM)
11b
12a
16b
<3
10
<3
10
28
10
Fortunately, potent CK1
modes for the majority of compounds. Especially 11b
with IC50 values in the single-digit nanomolar range in standardized kinase assays and are therefore
among the most potent CK1 inhibitors reported to date. In general, loss of potency for series
1 inhibitors when compared to lead structure has been observed in vitro: while has an
IC50 for CK1 of 5 nM [28] a side chain without both carbonyl group and -bond resulted in
considerably decreased inhibition (e.g., series 1, 10a IC50 CK1 = 386 nM). In contrast, compounds
possessing the carbonyl group but without -bond afforded chemically stable series 2 (e.g., 11a IC50
CK1δ = 20 nM IC50 CK1ε = 129 nM) and thereby restored potency almost to the level reported for lead
IC50 CK1δ = 5 nM IC50 CK1ε = 73 nM). Isoform selectivity regarding CK1 , however, seemed to be
slightly decreased. Consequently, the -bond of lead can be identified to be responsible for compound
instability, while the carbonyl depicts an important determinant concerning potency. In addition,
series 3 and 4 both provided potent inhibitors of CK1 with IC50 values in the low nanomolar range.
However, the aimed impact on CK1 isoform selectivity by rigidification of the double bond in
δ
inhibition and appropriate SAR correlated with the calculated binding
,
16a, and 16b exhibited excellent efficacy
δ
1
1
δ
π
δ
π
,
1
(
,
ε
π
1
δ
δ/ε

Molecules 2017, 22, 522
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the (E)-configuration has not been fully achieved. Unfortunately, tridentate hinge binding carbamide
derivatives 12a–m (series 3) were actually limited by their poor solubility in vitro.
Oxidation of the exocyclic sulfur at the imidazole-2-position of thioether
reflects in vivo metabolization by phase I enzymes [44]. Interestingly, this sulfoxidation increased
CK1 isoform selectivity in one reported previously study [28]. In our hands, however, this effect
1 to afford sulfoxide
2
δ/ε
could not be confirmed for the newly designed and synthesized set of inhibitors as oxidation only
slightly altered potency, though without significantly affecting isoform selectivity. Nevertheless,
sulfoxidized compounds remained potent inhibitors of CK1δ with IC50 values in the low nanomolar
range. This indicates that these agents will retain activity despite metabolization.
2.4. X-ray Analysis of Binding Modes in CK1δ and p38α
In order to verify modeled binding modes and to obtain further insights regarding ligand-protein
interactions determining selectivity we set out to co-crystallize potent inhibitors of series 2, 3, and
4 (11b
be co-crystallized with a C-terminally truncated CK1
determined at 2.0 Å resolution. Unfortunately, co-crystallization of 11b and 12a failed experimentally.
Additionally, overall most active agent 11b has been co-crystallized with p38 MAPK at 1.9 Å
resolution. In fact, crystallographic data largely confirmed the calculated binding pose of 16b within
the active site of CK1 (Figure 3). As determined by X-ray analysis the 4-fluorophenyl moiety
,
12a, and 16b) in CK1
δ. In line with this notion, a ligand-protein structure of 16b could
δ
construct and the complex structure was
α
δ
deeply penetrates into HPI, enabled by rotation of gatekeeper Met82 towards Pro66. The bidentate
hinge-binding 2-aminopyridine motif forms two hydrogen bonds towards Leu85. Furthermore,
Lys38 and Asp149 coordinate a water molecule which donates another hydrogen bond accepted
by an imidazole nitrogen of 16b. HRII, however, is addressed by bulky 4-(2,5-dimethoxyphenyl)-
1-methyl-1H-pyrrole-2-carboxamide, probably displacing energetically unfavorable water molecules
and shielding deeper hydrophobic cavities of the binding pocket. Although the 2,5-dimethoxyphenyl
moiety herein mainly interacts with Pro87 by
observed within range of Phe95 which can be interpreted by different conformers of 16b, thus indicating
additional interaction. The complex of 11b in p38 MAPK predominantly confirmed the expected
4,5-diarylimidazole core fitting, similar to CK1 , with gatekeeper Thr106-lined HPI occupied by the
π-aliphatic-stacking, electron density has also been
α
δ
4-fluorophenyl moiety and the 2-aminopyridine acting as bidentate hinge binder addressing Met109.
The ATP-binding pocket in this structure does not contain structural water molecules and Lys53 directly
interacts with 11b imidazole nitrogen by formation of a hydrogen bond. Interestingly, DFG-motif
Phe169 is coordinated between 11b imidazole-N and Lys53 by
respectively, thereby interrupting the hydrophobic spine consisting of Leu75, Leu86, Phe169, and
His148 [45 47]. This pose is stabilizing the kinase in an intermediate conformation between DFG-in
and DFG-out (DFG-in-between) [48], thereby showing several characteristics similar to type-I12 -like
inhibition of p38 MAPK [49 50], although molecular alignMent studies may suggest the DFG-in state
π–π- and, most likely, π-cation-stacking,
–
α
,
most likely to be inhibited by 11b as well. However, the DFG-out conformation can be assumed to be
sterically prohibited (Figure 4, left). Interestingly, the 3-(2,5-dimethoxyphenyl)propionic amide side
chain of 11b addressing HRII has not been definitely resolvable by X-ray analysis in terms of electron
density (Figure 4, right). In fact, this is in agreement with our hypothesis postulating high flexibility of
this ligand part within HRII. In line with this notion, we originally designed bulky inhibitor side chains
to suboptimal fit into less extensive hydrophobic region II of p38
affinity for CK1 . Thus, in that way paying enthalpic and entropic penalty should result in decreased
affinity for p38 MAPK when compared to the situation in CK1 51]. However, in contrast to our
hypothesis inhibitors actually showing the bulky side chain, e.g., 11b 12a, and 16b, were determined
to be nanomolar inhibitors of p38 MAPK as well as of CK1 . This finding suggests that—despite
α MAPK, in turn allowing higher
δ
α
δ [
,
α
δ
bulky side chains—the core 4,5-diaryl-imidazole scaffold addressing both ATP sites of CK1
is able to determine high affinity for these kinases.
δ and p38α

Molecules 2017, 22, 522
11 of 37
Figure 3. Similar binding modes of 16b in CK1δ have been gained by co-crystallization (top, pdb
5MQV) and molecular modeling (bottom, pdb 3UZP [31]). The poses are presented by key residues
and hydrogen bond interactions (left) and Connolly molecular surface (right).
Figure 4. Co-crystallization of 11b in p38
residues and hydrogen bond interactions as well as
α
MAPK (pdb 5ML5). The binding mode is presented by key
-stacking (left). DFG Phe169 is stabilized
π-π
in an intermediate state between active DFG-in (purple, pdb 1BMK [33]) and inactive DFG-out
(yellow, pdb 1WBT [52]) conformation. DFG-in/out states were represented by structure alignment
using Schrödinger software. The inhibitor side chain addressing HRII has not been definitely resolvable
in terms of electron density (right, generated with PyMOL).

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2.5. Selectivity Profiling of 11b
In order to further characterize potent inhibitor 11b selectivity profiling has been performed at a
concentration of 100 nM in a panel of 321 protein kinases (ProQinase GmbH). These data revealed high
selectivity of 11b for CK1
from CK1 (residual activity = 3%) and CK1
unaffected). Among these, CK1 (residual activity = 26%) and p38
δ
hitting only six additional kinases with residual activities less than 50% apart
(residual activity = 7%, CK1 isoforms 1–3 remained
MAPK (residual activity = 22%)
δ
ε
γ
α
α
were found as prominent targets. Also the highly related kinases JNK2 (residual activity = 21%),
JNK3 (residual activity = 37%), and RIPK2 (residual activity = 45%) were identified as additional hits.
Furthermore, Tyr-specific kinase LCK (residual activity = 26%) was inhibited (Figure 5 and Supporting
Information). Consequently, 11b represents an agent highly selective for CK1δ within the range of its
IC50 value of 4 nM with an overall selectivity score S(50) of 0.027. The S-Score(50) has been calculated
in accordance to Karaman et al. [53] describing the portion of kinases with a residual activity >50% in
relation to all tested kinases included in this project.
Figure 5. Dendrogram representation of selectivity profiling of 11b screened over 321 protein kinases
at a concentration of 100 nM (ProQinase GmbH, Freiburg, Germany). Residual activity was determined
compared to DMSO control. The S-Score(50) of 11b is 0.027. The dendrogram was generated utilizing
TREEspot Software Tool, DISCOVERX CORPORATION 2010. Corresponding raw data are given in
Supplementary Table S5.
2.6. Cellular Assays and EC50 Determination
Synthesized compounds have further been evaluated regarding their efficacy in cellular systems
in MTT viability assays using colorectal HT-29 or pancreatic Colo357, Panc89, Panc-1, and MiaPaCa-2

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carcinoma cell lines. The cell lines were chosen as they are reportedly overexpressing CK1
δ
and CK1
ε
and exhibit resistance against a variety of chemotherapeutic agents [12
(series 1) were rather ineffective, even at an elevated concentration of 20
active exhibiting an EC50 value of 9.3 M in HT-29 cells (data can be found in Supporting Information).
For the subsequently designed compound series EC50 data were only obtained for compounds which
showed the most promising inhibition of CK1δ in vitro. Inhibitors from series 2 (11a ) showed
significant increases of potency, presumably referable to the amide carbonyl group which has already
been reported to beneficially affect metabolic stability [44]. As expected, 16a (series 4) were only
slightly less active. In contrast, carbamides 12a (series 3) were only moderately active, presumably
suffering from poor solubility. Among all series, 11b again proved the most effective agent with EC50
values of 3.5 M and 1.5 M in Colo357 and Panc89 cells, respectively (determined EC50 values can be
found in Table 4).
,
15
,
16]. In general, amines 10a
–
k
µM with 10e being most
µ
–
e
–
b
–k
µ
µ
Table 4. EC50 values of selected inhibitor compounds as determined for Colo357, Panc89, Panc-1,
and MiaPaCa-2 cell lines. Results are presented as mean SD from experiments performed in triplicate
±
(n = 3). Abbreviations: # compound number; n.d., not determined.
EC50 Values (µM)
Compound #
Colo357
Panc89
MiaPaCa-2
Panc-1
11a
11b
11c
11d
11e
12a
12b
12e
16a
16b
16c
4.1 ± 1.7
3.5 ± 0.3
1.9 ± 0.6
5.9 ± 2.4
4.5 ± 1.3
17.4 ± 0.04
15.5 ± 1.6
14.9 ± 2.6
n.d.
2.5 ± 1
1.5 ± 0.4
6.1 ± 3.0
7.8 ± 0.4
8.3 ± 2.2
19.6 ± 3.8
n.d.
4.9 ± 0.8
7.0 ± 0.7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
15.22 ± 0.04
11.6 ± 1.1
n.d.
2.4 ± 0.7
2.3 ± 0.2
7.7 ± 2.5
16.1 ± 5.6
15.2 ± 2.6
n.d.
5.1 ± 1.6
5.4 ± 1.5
10.2 ± 3.2
7.5 ± 3.0
n.d.
5.7 ± 2.2
7.3 ± 1.9
n.d.
16.7 ± 5.6
If the EC50 values determined in cell-based assays are compared with in vitro determined IC50
data, massive differences can be observed and compounds which appeared to be extremely potent
are apparently less efficient in the treatment of living cells. This difference is compound and cell
line dependent, can be due to different reasons, and has already been documented in previous
reports [26,54]. Firstly, limited cell permeability of small molecule inhibitors may limit their cellular
uptake resulting in lower potency; Secondly, once compounds successfully crossed the cell membrane,
ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy
between IC50 values determined by enzymatic versus cellular assays. Additionally, time of binding in
the ATP pocket and drug export systems can reduce the inhibitory effect. Therefore, cell-based assays
are essential in order to validate the inhibitory effects of newlx identified small molecule inhibitors.
3. Materials and Methods
3.1. Molecular Modeling
Molecular modeling was performed on a DELL Precision T5500 eight core workstation.
For visualization Maestro, version 10.6, 2016 (Schrödinger LLC, New York, NY, USA) was used.
Protein crystal structures were prepared prior to docking by the Protein Preparation Wizard [55
]
synchronizing the following modules: Epik, version 3.6, 2016 [56]; Impact, version 7.1, 2016; Prime,
version 4.4, 2016 [57]. In order to achieve high Enrichment-factors, the common refinement protocol
by Sastry et al. [55] has been adjusted: the process involved assignment of bond orders, addition of
hydrogen atoms, identification of disulfide bonds, and the conversion of artifical selenomethionines to

Molecules 2017, 22, 522
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methionines (default settings). Missing side chains were filled in using Prime. Missing loops have
not been detected. Water molecules beyond 5 Å from hetero atoms have been deleted automatically.
H-bond optimization was performed in a standard sampling, the Root-mean-square deviation for
atomic positions cutoff for heavy atoms in subsequent protein minimization was set to 0.3 Å
Ligands were prepared to generate energetically minimized three-dimensional coordinates with
an extended cutoff by MacroModel, version 11.2, 2016 (Schrödinger LLC). Ionization and tautomeric
states were estimated at pH 7
Hammet and Taft methodology-based Epik [56]. Additionally, Epik state penalties (kcal
±
2 by LigPrep, version 3.8, 2016 (Schrödinger LLC) [55], utilizing
mol−1) were
·
calculated for each ligand to quantify the energetic cost for state transition in solution [55]. In order
to indicate ligand flexibility, up to 50 bioactive conformers per ligand were identified and prioritized
utilizing the conformational search module in the fast mode (ConfGen, version 3.6, 2016, Schrödinger
LLC) [58]. Receptor grid generation was generated with Glide, version 7.1, 2016 (Schrödinger LLC) [59].
For ligand docking and screening the Glide XP workflow was used [59]. Energetically minimized
ligand conformations were docked into the active site of the protein; possible binding poses were
determined and subsequently ranked based on their calculated binding affinities.
3.2. Chemistry
Infrared spectra were recorded on an IRAffinity-1S FTIR-spectrometer (Shimadzu Europa GmbH,
Hannover, Germany). NMR spectra were recorded on an Avance III 300 spectrometer, tempered at
298 K: 1H (300 MHz), 13C-NMR (75 MHz) (Bruker Daltonik GmbH, Bremen, Germany). The data
is reported as follows: chemical shift in ppm from tetramethylsilane (TMS) as external standard,
multiplicity and coupling constant J (Hz). Spectra were either referenced to TMS or internal DMSO-d6
(1H-NMR
δ
2.50) and internal DMSO-d6 (13C-NMR
δ δ 7.26) and
39.52) or internal CHCl3 (1H-NMR
internal CDCl3 (13C-NMR
δ
77.00). The following NMR abbreviations have been used: b (broad),
s (singlet), d (doublet), t (triplet), m (unresolved multiplet). Several target compounds show spectra
of at least two isomers in DMSO-d6 with a maximal ratio of 1:3. These are due to atropisomers and
tautomers as already reported for similar 4,5-diaryl-imidazoles [32]. Although such effects have
not been observed whenever CDCl3 was used, DMSO-d6 was the most frequented solvent with
respect to its favorable solubility-mediating properties. For reasons of clarity, signals are only given
for the main isomer. The labelling scheme of structures to correlate NMR signals can be found in
Supporting Information. LC-MS was performed with an 1100 HPLC system (Agilent Technologies,
Santa Clara, CA, USA) over an Agilent Eclipse XDB-C8 column (150
0.1% acetic acid/acetonitrile gradient for mobile phase (flow rate = 1 mL
with nominal solution were recorded on a Bruker Esquire ~LC ion trap mass spectrometer with
×
4.6 mm, 5
·
µm) using a
min−1). Mass spectra
electron spray ionization (ESI) operating in the positive ion mode, with the following parameters:
◦
drying gas nitrogen 8 L
recorded on an AccuTOF
·
min−1, nebulizer 35 psi, drying temperature 350 C). HRMS spectra were
™
GCv 4G electron ionization (EI)/field desorption (FD) mass spectrometer
(JEOL Germany, Freising, Germany). For clarity, only the highest measured peak is given for mass
spectra. Melting points/decomposition temperatures were determined on a SMP3 Melting Point
Apparatus (Stuart Scientific, Keison Products, Chelmsford, Essex, UK) and are uncorrected. Column
chromatography was performed using a LaFlash system (VWR International GmbH, Darmstadt,
Germany). The crude product was loaded on silica gel 60 (63–200
Germany) or PuriFlash IR-50 C18 modified silica gel (50 m) (Interchim Deutschland GmbH,
Mannheim, Germany) and packed in Interchim PuriFlash-DLE/12G dry-load precolumns. Pre-packed
Interchim PuriFlash-30SIHP silica gel columns (30 m, 40 g) and Interchim PuriFlash-15C18HP
modified silica gel columns (15 m, 55 g) were used for separation with flow rates usually adjusted to
30 mL
min−1 or 20.5 mL min−1. Progress of reactions was monitored by thin-layer chromatography
µm) (Macherey-Nagel, Düren,
µ
µ
µ
·
·
(TLC) performed with Macherey-Nagel 0.2 mm Polygram® SIL G/UV254 pre-coated silica gel polyester
sheets and Silicagel 60 RP-18 F254 modified silica gel aluminum plates (Merck Millipore, Darmstadt,
Germany). Where necessary, reactions were carried out in a nitrogen atmosphere using 4 Å molecular

Molecules 2017, 22, 522
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sieves. All reagents and solvents were obtained from commercial sources (abcr GmbH, Karlsruhe,
Germany; Sigma-Aldrich Chemie GmbH, Munich, Germany; Merck Group, Munich, Germany;
Merck Millipore; Acros Organics Thermo Fisher Scientific, Geel, Belgium; VWR International GmbH,
Hannover, Germany and used as received: THF was used after distillation over Na/benzophenone.
HPLC analysis was performed on a Hewlett Packard HP 1050 Series using either a ZORBAX® Eclipse
XDB-C8 (150 mm
flow 1.5 mL
min−1, gradient KH2PO4 buffer 10 mM, pH 2.3/methanol, UV-detection 254 nm). All key
compounds submitted to biological assays were proven by this method to show 98% purity. Syntheses
under elevated pressure were performed in Berghof highpreactor BR-25 with corresponding heating
×
4.6 mm, 5
µ
m) or a Kinetex® C8 (150
×
4.6 mm, 5 µm) column (mobile phase
·
≥
™
block on a MR Hei-Standard laboratory heating plate (Heidolph, Schwabach, Germany). Microwave
syntheses were performed in a CEM Discover Microwave Synthesizer (CEM GmbH, Kamp-Lintfort,
Germany) under air cooling and high stirring with a maximal power of 100 W.
3.2.1. Syntheses of Key Building Blocks 3–7
1-(4-Fluorophenyl)-2-(2-fluoropyridin-4-yl)-ethan-1-one (3). NaHMDS (66.7 mL 2 M solution in THF,
133 mmol) was slowly added to a stirred solution of 2-fluoro-4-methylpyridine (10.6 mL, 103 mmol) and
◦
ethyl 4-fluorobenzoate (18.1 mL, 123 mmol) in 40 mL anhyd. THF at 0 C under a nitrogen atmosphere.
◦
After stirring at 0 C for 2 h the reaction was allowed to reach rt and stirring continued for 1 h.
The mixture was diluted with ethyl acetate and washed twice with 10% aq. HCl. The organic layer was
dried over anhyd. Na2SO4 and solvent was removed under reduced pressure. Recrystallization from
ethyl acetate afforded
1H-NMR (DMSO-d6):
(m, 2H, C3/5H, F-Phe), 8.11–8.19 (m, 3H, C2/6H, F-Phe, C6H, Pyr) ppm; 13C-NMR (DMSO-d6):
3 ;
as colorless solid. Yield 23.9 g (quant.); C13H9F2NO (Mr 233.22); m.p. 102 ◦C
δ
= 4.59 (s, 2H, CH2), 7.10 (s, 1H, C3H, Pyr), 7.25–7.27 (m, 1H, C5H, Pyr), 7.37–7.43
= 43.6
δ
(d, 4JCF = 2.8 Hz, CH2), 110.8 (d, 2JCF = 37.6 Hz, C3H, Pyr), 115.8 (d, 2JCF = 22.0 Hz, C3/5H, F-Phe),
123.8 (d, 4JCF = 3.8 Hz, C5H, Pyr), 131.3 (d, 3JCF = 9.6 Hz, C2/6H, F-Phe), 132.9 (d, 4JCF = 2.7 Hz, C1,
F-Phe), 147.0 (d, 5JCF = 15.5 Hz, C6H, Pyr), 150.7 (d, 3JCF = 8.5 Hz, C1, Pyr), 163.1 (d, 1JCF = 234.3 Hz,
C2F, Pyr), 165.2 (d, 1JCF = 252.0 Hz, C4F, F-Phe), 194.6 (CO) ppm; MS (ESI, 70 eV) m/z 234 [MH]+.
1-(4-Fluorophenyl)-2-(2-fluoropyridin-4-yl)-2-(hydroximino)ethan-1-one (
4). NaNO2 (2.06 g, 29.8 mmol)
in 12 mL H2O was slowly added to a stirred solution of
3 (2.30 g, 9.86 mmol) in 17 mL glacial
acetic acid at 10 ◦C. After stirring at r.t. for 1 h, 30 mL H2O were added and stirring continued for
3.5 h. The suspension was cooled to 8 ◦C, filtered, and the residue was washed with H2O and dried
under reduced pressure to afford
m.p. 185 ◦C; 1H-NMR (DMSO-d6):
F-Phe), 7.92–7.98 (m, 2H, C2/6H, F-Phe), 8.30 (d, 3J = 5.3 Hz, 1H, C6H, Pyr), 12.67 (s, 1H, OH) ppm;
13C-NMR (DMSO-d6): = 105.4 (d, 2JCF = 38.9 Hz, C3H, Pyr), 116.7 (d, 2JCF = 22.5 Hz, C3/5H, F-Phe),
4
δ
as colorless solid. Yield 2.46 g (95%); C13H8F2N2O2 (Mr 262.22);
= 7.19 (bs, 1H, C3H, Pyr), 7.37–7.46 (m, 3H, C5H, Pyr and C3/5H,
δ
118.2 (d, 4JCF = 4.2 Hz, C5H, Pyr), 130.8 (d, 4JCF = 2.7 Hz, C1, F-Phe), 132.2 (d, 3JCF = 10.1 Hz, C2/6H,
F-Phe), 144.3 (d, 3JCF = 8.4 Hz, C4, Pyr), 148.8 (d, 5JCF = 15.6 Hz, C6H, Pyr), 151.9 (d, 4JCF = 4.0 Hz,
CNOH), 163.5 (d, 1JCF = 235.6 Hz, C2F, Pyr), 166.0 (d, 1JCF = 254.8 Hz, C4F, F-Phe), 192.04 (CO) ppm;
MS (ESI, 70 eV) m/z = 263 [MH]+.
2-(4-Fluorophenyl)-1-(2-fluoropyridin-4-yl)-2-oxoethan-1-aminium chloride (
5). Pd/C 10% (279 mg) was
added in one portion under a nitrogen atmosphere to an intensely stirred solution of
4
(1.60 g,
6.10 mmol) in 15 mL 2-propanol and 20 mL HCl sat. 2-propanol and stirring continued for 12 h at
r.t. The crude product was obtained by filtration, the residue was resuspended in methanol, filtered
again, and the solvent was removed under reduced pressure. Recrystallization from methanol/diethyl
◦
5
as beige solid. Yield 1.43 g (82%); C13H11ClF2N2O (Mr 284.69); m.p. 216 C; 1H-NMR
ether afforded
(DMSO-d6):
δ
= 6.56 (s, 1H, CH), 7.35–7.41 (m, 2H, C3/5H, F-Phe), 7.52–7.54 (m, 2H, C3/5H, Pyr), 8.21
3
4
3
(dd, J = 8.4 Hz, J = 5.6 Hz, 2H, C2/6H, F-Phe), 8.31 (d, J = 5.4 Hz, 1H, C6H, Pyr), 9.36 (bs, 3H,
NH3+) ppm; 13C-NMR (DMSO-d6):
δ
= 56.8 (CNH3+), 110.5 (d, JCF = 39.3 Hz, C3H, Pyr), 116.9
2
(
d, 2JCF = 22.1 Hz, C3/5H, F-Phe), 122.3 (d, 4JCF = 4.2 Hz, C5H, Pyr), 130.0 (d, 4JCF = 2.7 Hz, C1, F-Phe),

Molecules 2017, 22, 522
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132.9 (d, 3JCF = 9.8 Hz, C2/6H, F-Phe), 147.4 (d, 3JCF = 8.0 Hz, C4, Pyr), 149.4 (d, 5JCF = 15.0 Hz, C6H,
Pyr), 163.5 (d, 1JCF = 236.7 Hz, C2F, Pyr), 166.3 (d, 1JCF = 255.1 Hz, C4F, F-Phe), 191.4 (CO) ppm; MS
(ESI, 70 eV) m/z 249 [MCl]+.
2-Fluoro-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridine (6). A mixture of 5 (4.41 g,
15.5 mmol) and methyl thiocyanate (3.18 mL, 46.5 mmol) was refluxed under a nitrogen atmosphere
for 45 min in 140 mL anhyd. DMF and stirred 45 min at r.t. before ice-cold H2O (400 mL) was
added. The suspension was cooled to 8 ◦C, filtered, the residue was washed with H2O, and dried
under reduced pressure to afford
6 as fine yellow solid. Yield 3.59 g (76%); C15H11F2N3S (Mr 303.33);
δ
m.p. 205 ◦C; 1H-NMR (DMSO-d6):
= 2.63 (s, 3H, SCH3), 7.09 (bs, 1H, C3H, Pyr), 7.26–7.34 (m, 3H,
C5H, Pyr and C3/5H, F-Phe), 7.49–7.54 (m, 2H, C2/6H, F-Phe), 8.09 (d, J = 5.3 Hz, 1H, C6H, Pyr),
3
12.84 (bs, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ
= 14.9 (SCH3), 105.0 (d, 2JCF = 39.5 Hz, C3H, Pyr),
2
4
115.9 (d, JCF = 21.6 Hz, C3/5H, F-Phe), 118.5 (d, JCF = 2.5 Hz, C5H, Pyr), 126.5 (C1, F-Phe), 130.9
(C2/6H, F Phe), 132.9 (d, 3JCF = 8.6 Hz, C4, Pyr), 143.0 (C2, Imdz), 147.6 (d, 3JCF = 16.1 Hz, C6H, Pyr),
162.1 (d, 1JCF = 246.4 Hz, CF, F-Phe), 163.7 (d, JCF = 233.3 Hz, CF, Pyr) ppm; MS (ESI, 70 eV) m/z
1
304 [MH]+.
4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-amine (
7
). A solution of ◦
6 (1.00 g,
3.30 mmol) in 15 mL 32% aq. ammonia solution was heated in a high pressure reactor at 180 C for 18 h.
The reactor was allowed to reach r.t. and H2O was added. The crude product was obtained by filtration,
washed with H2O and diisopropyl ether and purified by flash chromatography (SiO2, 50%–100% ethyl
acetate/petrol ether) to afford
(DMSO-d6):
= 2.60 (s, 1H, SCH3), 5.79–5.95 (m, 2H, NH2), 6.42–6.67 (m, 2H, C3/5H, Pyr), 7.17–7.27
(m, 2H, C3/5H, F-Phe), 7.48 (bs, 2H, C2/6H, F-Phe), 7.74–7.86 (m, 1H, C6H, Pyr), 12.58 (s, 1H, NH) ppm;
7
as beige solid. Yield 852 mg (86%); C15H13FN4S (Mr 300.36); 1H-NMR
δ
13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 105.1 (C5H, Pyr), 110.0 (C3H, Pyr), 115.7 (d, 3JCF = 21.1 Hz,
C3/5H, F-Phe), 127.0 (s, C4/C5, Imdz), 129.2 (s, C1, F-Phe), 130.5 (d, 4JCF = 6.6 Hz, C2/6H, F-Phe), 147.5
(C6H, Pyr), 160.1 (CF) ppm; MS (ESI, 70 eV) m/z 301 [MH]+.
3.2.2. Synthesis of 4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-amines and
-piperazines 8, 9, 10a–i
3-(2,4-Dimethoxyphenyl)propan-1-ol (8). Under a nitrogen atmosphere a solution of LiAlH4 (120 mg,
3.16 mmol) in 3 mL anhyd. THF was slowly added to 3-(2,4-dimethoxyphenyl)propionic acid
(508 mg, 2.42 mmol) in 6 mL anhyd. THF at r.t. with intense stirring that continued for 1 h.
After completion, the reaction was cooled down in an ice-bath and quenched by successive
addition of H2O (120 µL), 15% aq. NaOH solution (120 µL), and H2O (360 µL). The precipitate
was filtered off, washed with THF, and the filtrate was concentrated under reduced pressure.
The crude product was purified by flash chromatography (SiO2, 20%–30% ethyl acetate/petrol
1
ether) to afford
8
as colorless oil. Yield 457 mg (96%); C11H16O3 (Mr 196.25); H-NMR (CDCl3):
δ = 1.78–1.83 (m, 2H, CH2CH2CH2OH), 2.65 (t, 3J = 7.3 Hz, 2H, CH2CH2CH2OH), 3.59 (t, 3J = 6.3 Hz
1H, CH2CH2CH2OH), 3.78 (s, 1H, OH), 3.79 (s, 3H, C4OCH3), 3.81 (s, 3H, C2OCH3), 6.42–6.46
(m, 2H, C3/5H, (OCH3)2-Phe), 7.03 (dd, 3J = 7.7 Hz 5J = 0.6 Hz, 1H, C6H, (OCH3)2-Phe) ppm;
13C-NMR (CDCl3):
,
,
δ
= 25.4 (CH2CH2CH2OH), 33.2 (CH2CH2CH2OH), 55.5 (C2/4OCH3), 62.1
(CH2CH2CH2OH), 98.6 (C3H, (OCH3)2-Phe), 104.3 (C5H, (OCH3)2-Phe), 122.4 (C1, (OCH3)2-Phe),
130.4 (C6H, (OCH3)2-Phe), 158.3 (C2OCH3), 159.3 (C4OCH3) ppm.
1-(3-Bromopropyl)-2,4-dimethoxybenzene (9). To an ice-cold solution of 8 (260 mg, 1.33 mmol) in 4 mL
anhyd. DCM, triphenylphosphine (412 mg, 1.57 mmol) and N-bromosuccinimide (263 mg, 1.48 mmol)
were added under a nitrogen atmosphere and the reaction was stirred for 2 h. The mixture was washed
with H2O and sat. aq. NaCl solution, dried over anhyd. Na2SO4, and the solvent was removed under
reduced pressure. Diethyl ether was added, the precipitate filtered off, and the filtrate was concentrated
and purified by flash chromatography (SiO2, 2%–10% ethyl acetate/petrol ether) to afford 9 as colorless

Molecules 2017, 22, 522
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oil. Yield 205 mg (60%); C11H15BrO (Mr 259.14); 1H-NMR (CDCl3):
δ
= 2.11 (quint, 3J = 5.6 Hz, 2H,
CH2CH2CH2Br), 2.70 (t, 3J = 7.2 Hz, 2H, CH2CH2CH2Br), 3.39 (t, 3J = 6.8 Hz, 2H, CH2CH2CH2Br),
3.80 (s, 6H, C2/4OCH3), 6.42 (dd, 3J = 8.0 Hz, 4J = 2.5 Hz, 1H, C5H, (OCH3)2-Phe), 6.45 (d, 4J = 2.3 Hz,
1H, C3H, (OCH3)2-Phe), 7.05 (dd, 3J = 8.0 Hz, 5J = 0.5 Hz, 1H, C6H, (OCH3)2-Phe) ppm; 13C-NMR
(CDCl3):
δ
= 28.4 (CH2CH2CH2Br), 33.0 (CH2CH2CH2Br), 33.9 (CH2CH2CH2Br), 55.3 (C4OCH3),
55.5 (C2OCH3), 98.7 (C3H, (OCH3)2-Phe), 103.9 (C5H, (OCH3)2-Phe), 121.4 (C1, (OCH3)2-Phe), 130.5
(C6H, (OCH3)2-Phe), 158.5 (C2OCH3), 159.6 (C4OCH3) ppm.
N-(3-(2,4-Dimethoxyphenyl)propyl)-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-amine
(
10a). The synthetic protocol starting from Boc-protected 2-amino-4-methylpyridine and
predominantly matches the procedure described for and can be found in the Supporting
Information. 1H-NMR (DMSO-d6):
= 1.67–1.72 (m, 2H, CH2CH2CH2NH), 2.48–2.52 (m, 2H,
9
7
δ
CH2CH2CH2NH), 2.60 (s, 3H, SCH3), 3.13–3.17 (m, 2H, CH2CH2CH2NH), 3.72 (s, 3H, C4OCH3),
3.74 (s, 3H, C2OCH3), 6.41–6.50 (m, 5H, C3/5H, Pyr and C3/5H, (OCH3)2-Phe and CH2CH2CH2NH),
7.01 (d, 3J = 8.2 Hz, 1H, C6H, (OCH3)2-Phe), 7.22 (m, 2H, C3/5H, F-Phe), 7.48 (m, 2H, C2/6H,
F-Phe), 7.85 (bs, C6H, Pyr), 12.58 (bs, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3), 26.6
(CH2CH2CH2NH), 29.3 (CH2CH2CH2NH), 40.7 (CH2CH2CH2NH), 55.1 (C4OCH3), 55.2 (C2OCH3),
98.3 (C3H, (OCH3)2-Phe), 104.3 (C5H, (OCH3)2-Phe), 104.9 (C3H, Pyr), 109.7 (C5H, Pyr), 115.4 (C3/5H,
F-Phe), 121.8 (C1, (OCH3)2-Phe), 126.5 (C4, Imdz), 127.0 (C5, Imdz), 129.7 (C1, F-Phe), 129.7 (C6H,
(OCH3)2-Phe), 130.1 (C2/6H, F-Phe), 142.1 (C2, Imdz), 147.5 (C6H, Pyr), 157.9 (C2OCH3), 158.8
(C4OCH3), 159.3 (C2, Pyr) ppm; MS (ESI, 70 eV) m/z 479 [MH]+; HRMS (EI, 70 eV) m/z [M]+ calcd. for
C26H27FN4O2S, 478.1839; found, 478.1839.
General Procedure for the Preparation Compounds 10b–i
Compound 6 (1.0 equiv) was suspended in the appropriate amine/piperazine (4.0 to 6.0 equiv)
and the intensely stirred mixture was heated to 160 ◦C. Progress of the reaction was monitored by
HPLC control. After complete conversion the mixture was diluted with ethyl acetate, washed with sat.
aq. NaHCO3 solution and H2O, dried over anhyd. Na2SO4, and concentrated under reduced pressure.
The crude product was purified by flash chromatography (stationary phase, eluent, and mixing ratio
given for each compound, respectively) to afford the particular compound.
N-(2,4-Dimethoxyphenyl)-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-amine
(10b).
Synthesis was performed according to the general procedure from (400 mg, 1.32 mmol) and
6
2,4-dimethoxyaniline (808 mg, 5.27 mmol). Purification was achieved by flash chromatography
(SiO2, 20%–90% ethyl acetate/petrol ether) to afford 10b as greyish crystals. Yield 312 mg (54%);
1
C23H21FN4O2S (Mr 436.51); m.p. 206 ◦C; H-NMR (DMSO-d6):
δ = 2.60 (s, 3H, SCH3), 3.75
(s, 6H, 2 OCH3), 6.39–6.44 (m, 1H, C5H, (OCH3)2-Phe), 6.56–6.58 (m, 1H, C3H, (OCH3)2-Phe), 6.63
dd, 3J = 5.3 Hz, 4J = 1.2 Hz, 1H, C5H, Pyr), 6.82 (s, 1H, C3H, Pyr), 7.18–7.28 (m, 2H, C3/5H, F-Phe),
7.41–7.46 (m, 2H, C2/6H, F-Phe), 7.52 (d, 3J = 8.7 Hz, 1H, C6H, (OCH3)2-Phe), 7.71 (s, 1H, NH), 7.95
(
(
d, 3J = 5.3 Hz, 1H, C6H, Pyr), 12.60 (s, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3),
55.2 (OCH3), 55.2 (OCH3), 99.0 (C3H, (OCH3)2-Phe), 104.0 (C5H, (OCH3)2-Phe), 105.4 (C3H, Pyr), 111.5
(C5H, Pyr), 115.4 (dd, 2JCF = 21.8 Hz, C3/5H, F-Phe), 122.5 (C, Imdz), 123.6 (C6H, (OCH3)2-Phe), 126.8
3
(
d, 4JCF = 3.1 Hz, C1, F-Phe), 130.0 (d, JCF = 8.1 Hz, C2/6H, F-Phe), 134.9 (C1, (OCH3)2-Phe), 138.8
(C, Imdz), 141.7 (C2OCH3), 142.7 (C2, Imdz), 147.6 (C6H, Pyr), 152.2 (C4OCH3), 155.9 (C4, Pyr), 157.4
(C2, Pyr), 161.8 (d, 1JCF = 245.0 Hz, CF) ppm; MS (ESI, 70 eV) m/z 437 [MH]+.
N-(2-Ethoxyphenyl)-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-amine (10c). Synthesis
was performed according to the general procedure from
6 (1.50 g, 4.95 mmol) and 2-ethoxyaniline
(3.88 mL, 29.7 mmol). Purification was achieved by flash chromatography (SiO2, 20%–90% ethyl
acetate/petrol ether) to afford 10c as dark red crystals. Yield 1.10 g (52%); C23H21FN4OS (Mr 420.51);
◦
1
3
m.p. 179 C; H-NMR (CDCl3):
(
δ = 1.39 (t, J = 7.0 Hz, 3H, CH2CH3), 2.63 (s, 3H, SCH3), 4.03
q, 3J = 7.0 Hz, 2H, CH2CH3), 6.76 (t, 3J = 7.7 Hz, 1H, C5H, EtO-Phe), 6.81–6.84 (m, 2H, C5H, Pyr and

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C3H, EtO-Phe), 6.91 (t, 3J = 7.7 Hz, 1H, C4H, EtO-Phe), 6.97 (bs, 1H, C3H, Pyr), 7.00–7.05 (t, 3J = 8.7 Hz,
2H, C3/5H, F-Phe), 7.21 (bs, 1H, NH), 7.37–7.41 (m, 2H, C2/6H, F-Phe), 7.49 (d, 3J = 7.7 Hz, 1H, C6H,
EtO-Phe), 8.00 (d, 3J = 5.5 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ
= 15.0 (CH3CH2), 16.6 (SCH3),
64.3 (CH2CH3), 106.5 (C3H, Pyr), 111.7 (C3H, EtO-Phe), 112.9 (C5H, Pyr), 116.0 (d, JCF = 21.7 Hz,
C3/5H, F-Phe), 120.0 (C6H, EtO-Phe), 120.7 (C5H, EtO-Phe), 122.6 (C4H, EtO-Phe), 129.4 (C1, EtO-Phe),
130.5 (d, 3JCF = 8.1 Hz, C2/6H, F-Phe), 143.2 (C2, Imdz), 146.8 (C6H, Pyr), 148.7 (COEt), 155.5 (C2, Pyr),
162.5 (d, 1JCF = 249.0 Hz, CF) ppm; MS (ESI, 70 eV) m/z = 421 [MH]+.
2
N-(3,4-Dimethoxyphenethyl)-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-amine (10d).
Synthesis was performed according to the general procedure from
and 3,4-dimethoxyphenethylamine (900 L, 5.31 mmol). Purification was achieved by flash
chromatography (SiO2, 20%–90% ethyl acetate/petrol ether) to afford 10d as yellow crystals. Yield
550 mg (90%); C25H25FN4O2S (Mr 464.56); m.p. 87 ◦C; 1H-NMR (CDCl3):
= 2.55 (s, 3H, SCH3), 2.69
(t, 3J = 6.9 Hz, 2H, CH2CH2NH), 3.29 (m, 2H, CH2CH2NH), 3.74 (s, 3H, C3OCH3), 3.75 (s, 3H, C4OCH3),
6 (400 mg, 1.32 mmol)
µ
δ
3
4
4.86 (bs, 1H, CH2CH2NH), 6.51 (d, J = 5.2 Hz, 1H, C5H, Pyr), 6.57 (d, J = 2.0 Hz, 1H, C3H, Pyr),
6.57–6.70 (m, 3H, C2/5/6H, (OCH3)2-Phe), 6.91–6.96 (m, 2H, C3/5H, F-Phe), 7.32–7.36 (m, 2H, C2/6H,
F-Phe), 7.74 (d, 3J = 5.8 Hz, 1H, C6H, Pyr), 10.77 (bs, 1H, NH) ppm; 13C-NMR (CDCl3): δ = 16.4 (SCH3),
35.1 (CH2CH2NH), 43.5 (CH2CH2NH), 55.9 (C3OCH3), 55.9 (C4OCH3), 104.1 (C3H, Pyr), 111.0 (C5H,
Pyr), 111.4 (C5H, (OCH3)2-Phe), 112.1 (C2H, (OCH3)2-Phe), 115.7 (d, 2JCF = 21.7 Hz, C3/5H, F-Phe),
120.7 (C6H, (OCH3)2-Phe), 130.3 (d, 3JCF = 8.2 Hz, C2/6H, F-Phe), 131.4 (C4/5, Imdz), 142.9 (C4, Pyr),
146.7 (C6H, Pyr, 147.7 (C4OCH3), 149.0 (C3OCH3), 158.3 (C2, Pyr), 162.6 (d, 1JCF = 248.5 Hz, CF) ppm;
MS (ESI, 70 eV) m/z 465 [MH]+.
N-(2-(1H-Indol-3-yl)-ethyl)-4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-amine (10e).
Synthesis was performed according to the general procedure from
6 (400 mg, 1.32 mmol) and
tryptamine (845 mg, 5.27 mmol). Purification was achieved by flash chromatography (SiO2, 20%–90%
ethyl acetate/petrol ether) to afford 10e as yellowish-brown crystals. Yield 427 mg (73%); C25H22FN5S
◦
(Mr 443.54); m.p. 122 C; 1H-NMR (CDCl3):
δ
= 2.61 (s, 3H, SCH3), 2.95 (t, 3J = 6.6 Hz, 2H, CH2CH2NH),
3.39 (t, 3J = 6.6 Hz, 2H, CH2CH2NH), 6.52 (d, 3J = 5.7 Hz, 1H, C5H, Pyr), 6.52 (bs, 1H, C2H, Indole), 6.87
(d, 4J = 2.1 Hz, 1H, C3H, Pyr), 6.95–7.01 (m, 2H, C3/5H, F-Phe), 7.08 (t, 3J = 7.8 Hz, 1H, C6H, Indole),
7.16 (t, 3J = 7.9 Hz, 1H, C5H, Indole), 7.30 (d, 3J = 7.9 Hz, 1H, C4H, Indole), 7.35–7.39 (m, 2H, C2/6H,
F-Phe), 7.52 (d, 3J = 7.7 Hz, 1H, C7H, Indole), 7.71 (d, 3J = 5.7 Hz, 1H, C6H, Pyr), 8.38 (s, 1H, NH, Indole)
ppm; 13C-NMR (CDCl3):
δ
= 16.4 (SCH3), 25.1 (CH2CH2NH), 42.2 (CH2CH2NH), 104.0 (C3H, Pyr),
110.6 (C5H, Pyr), 111.3 (C4H, Indole), 112.6 (C3, Indole), 115.8 (d, 2JCF = 21.6 Hz, C3/5H, F-Phe), 118.6
(C7H, Indole), 119.4 (C6H, Indole), 122.1 (C5H, Indole), 122.5 (C2H, Indole), 127.2 (C8a, Indole), 130.3
(3JCF = 8.3 Hz, C2/6H, F-Phe), 136.4 (C3a, Indole), 143.3 (C2, Imdz), 145.1 (C6H, Pyr), 158.0 (C2, Pyr)
ppm; MS (ESI, 70 eV) m/z 444 [MH]+.
N-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-N0,N0-dimethyl-propan-1,3-diamine
(
10f). Synthesis was performed according to the general procedure from 6 (400 mg, 1.32 mmol)
and N1,N1-dimethylpropane-1,3-diamine (1.00 mL, 7.93 mmol). Purification was achieved by
flash chromatography (SiO2, 20%–90% ethyl acetate/petrol ether, then methanol) and subsequent
◦
filtration, to afford 10f as yellow solid. Yield 175 mg (35%); C20H24FN5S (Mr 385.51); m.p. 129 C;
1H-NMR (CDCl3):
δ
= 1.61 (t, 3J = 6.8 Hz, 2H, CH2CH2CH2), 2.13 (s, 6H, N(CH3)2), 2.29 (t, 3J = 6.9 Hz
2H, CH2N(CH3)2), 2.58 (s, 3H, SCH3), 3.11 (t, J = 6.7 Hz, 2H, CH2NH), 5.22 (bs, 1H, CH2NH),
6.42–6.46 (m, 2H, C3/5H, Pyr), 6.95–7.01 (m, 2H, C3/5H, F-Phe), 7.35–7.39 (m, 2H, C2/6H, F-Phe),
,
3
5
7.78 (dd, 3J = 5.4 Hz, J = 0.6 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ = 16.5 (SCH3), 26.7
(CH2CH2CH2), 45.2 (N(CH3)2), 57.6 (CH2N(CH3)2), 104.0 (C3H, Pyr), 111.1 (C5H, Pyr), 115.5
d, 2JCF = 21.5 Hz, C3/5H, F-Phe), 130.2 (d, 3JCF = 8.1 Hz, C2/6H, F-Phe), 142.7 (C2, Imdz), 147.9 (C6H,
Pyr), 159.2 (C2, Pyr), 162.4 (d, 1JCF = 247.7 Hz, CF) ppm; MS (ESI, 70 eV) m/z 386 [MH]+.
(

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1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-piperazine (10g). Synthesis was
performed according to the general procedure from (300 mg, 989 mol) and piperazine (341 mg,
6
µ
3.96 mmol). Purification was achieved by flash chromatography (bas. Al2O3, 100% methanol) to afford
10g as yellow solid. Yield 63.0 mg (17%); C19H20FN5S (Mr 369.46); m.p. 146 ◦C; 1H-NMR (DMSO-d6):
δ
= 2.60 (bs, 3H, SCH3), 2.73 (t, 3J = 4.5 Hz, 4H, C2/6H2, Piperazine), 3.31 (t, 3J = 4.6 Hz, 4H, C3/5H2,
Piperazine), 6.56 (d, 3J = 5.1 Hz, 1H, C5H, Pyr), 6.80 (bs, 1H, C3H, Pyr), 7.20–7.26 (m, 2H, C3/5H, F-Phe),
7.47–7.51 (m, 2H, C2/6H, F-Phe), 7.97 (d, 3J = 5.1 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (DMSO-d6): δ = 15.1
(SCH3), 45.3 (C2/6H2, Piperazine), 45.8 (C3/5H2, Piperazine), 103.8 (C3H, Pyr), 110.6 (C5H, Pyr), 115.4
(d, 2JCF = 21.6 Hz, C3/5H, F-Phe), 130.3 (d, 3JCF = 7.6 Hz, C2/6H, F-Phe), 142.3 (C2, Imdz), 147.6 (C6H,
Pyr), 159.6 (C2, Pyr), 161.6 (d, 1JCF = 245.1 Hz, CF) ppm; MS (ESI, 70 eV) m/z 370 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-4-methyl-piperazine (10h). Synthesis
was performed according to the general procedure from
(600 L, 5.39 mmol). Purification was achieved by flash chromatography (RP-18, 20%–90%
methanol/H2O) to afford 10h as pale yellow solid. Yield 115 mg (23%); C20H22FN5S (Mr 383.49);
6 (400 mg, 1.32 mmol) and N-methylpiperazine
µ
◦
m.p. 111 C; 1H-NMR (CDCl3):
δ
= 2.28 (s, 3H, CH3), 2.45 (t, 3J = 4.8 Hz, 4H, C3/5H2, Piperazine), 2.57
(s, 3H, SCH3), 3.40 (t, 3J = 4.8 Hz, 4H, C2/6H2, Piperazine), 6.54 (dd, 3J = 5.3 Hz, 4J = 1.1 Hz, 1H, C5H,
Pyr), 6.73 (bs, 1H, C3H, Pyr), 6.91–6.97 (m, 2H, C3/5H, F-Phe), 7.32–7.37 (m, 2H, C2/6H, F-Phe), 7.94
(d, 3J = 5.3 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ
= 16.5 (SCH3), 44.7 (C2/6H2, Piperazine), 45.6
(CH3), 54.4 (C3/5H2, Piperazine), 104.9 (C3H, Pyr), 111.9 (C5H, Pyr), 115.7 (d, 2JCF = 21.6 Hz, C3/5H,
F-Phe), 128.2 (d, 4JCF = 4.1 Hz, CC1, F-Phe), 130.2 (d, 3JCF = 8.1 Hz, C2/6H, F-Phe), 142.8 (C2, Imdz),
147.9 (C6H, Pyr), 159.6 (C2, Pyr), 162.5 (d, 1JCF = 248.3 Hz, CF) ppm; MS (ESI, 70 eV) m/z 384 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-4-phenyl-piperazine (10i). Synthesis
was performed according to the general procedure from
6 (400 mg, 1.32 mmol) and N-phenylpiperazine
(800 L, 5.23 mmol). The combined organic phases were extracted with 2 M aq. HCl solution, the aq.
µ
layer was neutralized with 2 M aq. KOH solution, and the pale yellow precipitate was collected by
filtration, and purified by flash chromatography (SiO2, 20%–90% ethyl acetate/petrol ether) to afford
10i as yellow crystals. Yield 291 mg (50%); C20H22FN5S (Mr 445.56); m.p. 101 ◦C; 1H- NMR (CDCl3):
δ
= 2.64 (s, 3H, SCH3), 3.19 (t, 3J = 5.1 Hz, 4H, C2/6H2, Piperazine), 3.58 (t, 3J = 5.1 Hz, 4H, C3/5H2,
Piperazine), 6.64 (dd, 3J = 5.4 Hz, 4J = 1.2 Hz, 1H, C5H, Pyr), 6.93–6.85 (m, 4H, C3H, Pyr and C2/4/6H,
Phe), 6.99–7.05 (m, 2H, C3/5H, F-Phe), 7.26 (t, 3J = 8.0 Hz, 2H, C3/5H, Phe), 7.39–7.44 (m, 2H, C2/6H,
F-Phe), 8.00 (d, J = 5.5 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ
= 16.5 (SCH3), 45.4 (C2/6H2,
3
Piperazine), 49.0 (C3/5H2, Piperazine), 105.1 (C3H, Pyr), 111.6 (C5H, Pyr), 115.8 (d, JCF = 21.7 Hz,
C3/5H, F-Phe), 116.3 (C2/6H, Phe), 120.2 (C4H, Phe), 129.2 (C3/5H, Phe), 130.3 (d, 3JCF = 8.1 Hz, C2/6H,
F-Phe), 143.0 (C2, Imdz), 146.9 (C6H, Pyr), 151.1 (C1, Phe), 159.0 (C2, Pyr), 162.6 (d, 1JCF = 248.5 Hz, CF)
ppm; MS (ESI, 70 eV) m/z 446 [MH]+.
2
3.2.3. Synthesis of N-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-
phenyl-propanamides 11a–e
The appropriate methoxy-substituted 3-phenylpropionic acid and CDI (1.1 equiv) were stirred
in 3–4 mL anhyd. DMF until formation of CO2 was undetectable.
7 was added to the reaction and
◦
the mixture was heated at 110 C under a nitrogen atmosphere for 12 h. The reaction was cooled
to rt and ethyl acetate was added. The resulting mixture was washed with H2O and sat. aq. NaCl
solution, dried over anhyd. Na2SO4, and the solvent was removed under reduced pressure. The crude
product was purified by flash chromatography (SiO2 and RP-18, eluent and mixing ratio given for
each compound, respectively) to afford the particular compound.
3-(2,4-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)propanamide
(
11a). Synthesis was performed according to the general procedure from 3-(2,4-dimethoxyphenyl)
propionic acid (560 mg, 2.66 mmol) and (400 mg, 1.33 mmol). Purification was achieved by flash
chromatography (SiO2, 30%–50% ethyl acetate/petrol ether and RP-18, 30%–80% methanol/H2O)
7

Molecules 2017, 22, 522
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to afford 11a as beige solid. Yield 177 mg (27%); C26H25FN4O3S (Mr 492.57); m.p. 90 ◦C; 1H-NMR
3
3
(DMSO-d6):
δ = 2.57 (t, J = 7.4 Hz, 2H, COCH2CH2), 2.62 (s, 3H, SCH3), 2.75 (t, J = 7.5 Hz, 2H,
COCH2CH2), 3.72 (s, 3H, C2OH3), 3.77 (s, 3H, C4OCH3), 6.42 (dd, J = 8.3 Hz, J = 2.4 Hz, 1H,
3
4
C5H, (OCH3)2-Phe), 6.51 (d, J = 2.4 Hz, 1H, C3H, (OCH3)2-Phe), 6.99 (dd, J = 5.3 Hz, 1H, C5H,
4
3
Pyr), 7.03 (d, J = 8.3 Hz, 1H, C6H, (OCH3)2-Phe), 7.28 (bs, 2H, C3/5H, F-Phe), 7.46–7.51 (m, 2H,
3
C
2/6H, F-Phe), 8.12 (bs, 1H, C6H, Pyr), 8.32 (bs, 1H, C3H, Pyr), 10.29 (bs, 1H, CONH), 12.72 (bs, 1H,
NH) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3), 24.7 (COCH2CH2), 36.3 (COCH2CH2), 55.1
(C2OCH3), 55.3 (C4OCH3), 98.3 (C3H, (OCH3)2-Phe), 104.3 (C5H, (OCH3)2-Phe), 110.6 (C3H, Pyr),
115.8 (d, 2JCF = 24.1 Hz, C3/5H, F-Phe), 116.4 (C5H, Pyr), 120.9 (C1, (OCH3)2-Phe), 126.7 (C1, F-Phe),
129.8 (C6H, (OCH3)2-Phe), 130.7 (C5, Imdz and C2/6H, F-Phe), 134.5 (C4, Imdz), 143.7 (C2, Imdz), 147.6
(C6H, Pyr), 148.0 (C4, Pyr), 152.5 (C2, Pyr), 157.9 (C4, (OCH3)2-Phe), 159.0 (C2, (OCH3)2-Phe), 161.9
(
d, 1JCF = 246.7 Hz, CF), 171.4 (CO) ppm; IR (ATR):
ν =2935, 1670, 1609, 1547, 1505, 1414, 1289, 1262,
1221, 1207, 1153, 1121, 1036, 835 cm−1; MS (ESI, 70 eV) m/z 493 [MH]+; HRMS (EI, 70 eV) m/z [M]+
calcd. for C26H25FN4O3S, 492.1631; found, 492.1631.
3-(2,5-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-propanamide
(11b). Synthesis was performed according to the general procedure from 3-(2,5-dimethoxyphenyl)
propionic acid (560 mg, 2.66 mmol) and (400 mg, 1.33 mmol). Purification was achieved by flash
7
chromatography (SiO2, 20%–100% ethyl acetate/petrol ether and RP-18, 20%–100% methanol/H2O) to
afford 11b as colorless solid. Yield 255 mg (39%); C26H25FN4O3S (Mr 492.57); m.p. 91 ◦C; 1H-NMR
(DMSO-d6):
δ
= 2.59–2.64 (m, 5H, SCH3 and COCH2CH2), 2.80 (t, 3J = 7.5 Hz, 2H, COCH2CH2), 3.66
3
4
(s, 3H, C5OCH3), 3.73 (s, 3H, C2OCH3), 6.72 (dd, J = 8.8 Hz, J = 3.1 Hz, 1H, C4H, (OCH3)2-Phe),
4
3
6.78 (d, J = 3.0 Hz, 1H, C6H, (OCH3)2-Phe), 6.86 (d, J = 8.8 Hz, 1H, C3H, (OCH3)2-Phe), 6.99
4
(
dd, 3J = 5.2 Hz, J = 1.6 Hz, 1H, C5H, Pyr), 7.26 (bs, 2H, C3/5H, F-Phe), 7.46–7.51 (m, 2H, C2/6H,
F-Phe), 8.13 (bs, 1H, C6H, Pyr), 8.32 (bs, 1H, C3H, Pyr), 10.34 (bs, 1H, CONH), 12.72 (bs, 1H, NH)
ppm; 13C-NMR (DMSO-d6): = 15.1 (SCH3), 25.3 (COCH2CH2), 36.0 (COCH2CH2), 55.2 (C5OCH3),
δ
55.8 (C2OCH3), 110.6 (C3H, Pyr), 111.2 (C4H, (OCH3)2-Phe), 111.5 (C3H, (OCH3)2-Phe), 115.6 (C3/5H,
F-Phe), 115.9 (C6H, (OCH3)2-Phe), 116.5 (C5H, Pyr), 126.7 (C1, F-Phe), 130.0 (C1, (OCH3)2-Phe), 130.6
(C5, Imdz and C2/6H, F-Phe), 134.5 (C4, Imdz), 142.3 (C2, Imdz), 147.7 (C4 and C6H, Pyr), 151.2
(C2, (OCH3)2-Phe), 152.5 (C2, Pyr), 153.0 (C5, (OCH3)2-Phe), 161.9 (d, 1JCF = 247.3, CF), 171.3 (CO) ppm;
MS (ESI, 70 eV) m/z 494 [MH]+; HRMS (EI, 70 eV) m/z [M]+ calcd. for C26H25FN4O3S, 492.1631;
found, 492.1631. The compound was demonstrated to have the desired structure by small molecule
X-ray structure determination (Figure 6). For further details see CCDC and Supporting Information.
Figure 6. Small molecule X-ray crystal structure determination of compound 11b.
3-(2,3-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-propanamide
(
11c). Synthesis was performed according to the general procedure from 3-(2,3-dimethoxyphenyl)
propionic acid (700 mg, 3.33 mmol) and (500 mg, 1.67 mmol). Purification was achieved by flash
chromatography (SiO2, 30%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O) to
7

Molecules 2017, 22, 522
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afford 11c as colorless solid. Yield 515 mg (63%); C26H25FN4O3S (Mr 492.57); 1H-NMR (DMSO-d6):
3
δ = 2.62 (s, 3H, SCH3), 2.59–2.66 (m, 2H, COCH2CH2), 2.84 (t, J = 7.7 Hz, 2H, COCH2CH2), 3.73
3
4
(s, 3H, C2OCH3), 3.78 (s, 3H, C3OCH3), 6.79 (dd, J = 7.5 Hz, J = 1.6 Hz, 1H, C6H, (OCH3)2-Phe),
6.88 (dd, 3J = 8.1 Hz, 4J = 1.2 Hz, 1H, C4H, (OCH3)2-Phe), 6.97 (t, 3J = 7.8 Hz, 1H, C5H, (OCH3)2-Phe),
7.01 (dd, 3J = 5.3 Hz, 4J = 1.5 Hz, 1H, C5H, Pyr), 7.26–7.32 (m, 2H, C3/5H, F-Phe), 7.45–7.51 (m, 2H,
C
2/6H, F-Phe), 8.11 (d, 3J = 5.3 Hz, 1H, C6H, Pyr), 8.34 (bs, 1H, C3H, Pyr), 10.35 (s, 1H, CONH), 12.70
(s, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3), 24.8 (COCH2CH2), 36.8 (COCH2CH2), 55.3
(C3OCH3), 60.0 (C2OCH3), 110.6 (C3H, Pyr), 110.9 (C4H, (OCH3)2-Phe), 115.8 (d, 2JCF = 21.8 Hz, C3/5H,
F-Phe), 116.4 (C5H, Pyr), 121.4 (C6H, (OCH3)2-Phe), 123.7 (C5H, (OCH3)2-Phe), 126.6 (d, 4JCF = 3.1 Hz,
C1, F-Phe), 130.1 (C5, Imdz), 130.7 (d, JCF = 8.4 Hz, C2/6H, F-Phe), 134.4 (C1, (OCH3)2-Phe), 134.5
3
(
C4, Imdz), 142.2 (C2, Imdz), 143.8 (C4, Pyr), 146.6 (C2OCH3), 147.6 (C6H, Pyr), 152.4 (C3OCH3), 152.5
(C2, Pyr), 162.0 (d, 1JCF = 244.7 Hz, CF), 171.1 (CO) ppm; MS (ESI, 70 eV) m/z 493 [MH]+.
3-(3,4-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-propanamide
(11d). Synthesis was performed according to the general procedure from 3-(3,4-dimethoxyphenyl)
propionic acid (700 mg, 3.33 mmol) and (500 mg, 1.67 mmol). Purification was achieved by flash
7
chromatography (SiO2, 30%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O)
1
to afford 11d as colorless solid. Yield 468 mg (57%); H-NMR (DMSO-d6):
δ = 2.62 (s, 3H, SCH3),
2.62–2.67 (m, 2H, COCH2CH2), 2.81 (t, 3J = 7.4 Hz, 2H, COCH2CH2), 3.70 (s, 3H, C4OCH3), 3.72 (s, 3H,
C3OCH3), 6.73 (dd, J = 8.2 Hz, J = 1.9 Hz, 1H, C6H, (OCH3)2-Phe), 6.84 (d, J = 8.3 Hz, 1H, C5H,
3
4
3
(OCH3)2-Phe), 6.85 (bs, 1H, C2H, (OCH3)2-Phe), 7.00 (dd, J = 5.2 Hz, J = 1.5 Hz, 1H, C5H, Pyr),
3
4
7.26–7.31 (m, 2H, C3/5H, F-Phe), 7.46–7.50 (m, 2H, C2/6H, F-Phe), 8.10–8.34 (m, 2H, C3/6H Pyr), 10.33
(s, 1H, CONH), 12.71 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3), 30.4 (COCH2CH2), 38.0
(COCH2CH2), 55.4 (C4OCH3), 55.5 (C3OCH3), 110.6 (C3H, Pyr), 111.9 (C5H, (OCH3)2-Phe), 112.3 (C2H,
(OCH3)2-Phe), 115.8 (d, 2JCF = 22.5 Hz, C3/5H, F-Phe), 116.4 (C5H, Pyr), 120.0 (C6H, (OCH3)2-Phe),
130.6 (C2/6H, F-Phe), 133.5 (C1, (OCH3)2-Phe), 147.1 (C4OCH3), 147.6 (C6H, Pyr), 148.6 (C3OCH3),
152.5 (C2, Pyr), 165.8 (d, 1JCF = 242.6 Hz, CF), 171.2 (CO) ppm; MS (ESI, 70 eV) m/z 493 [MH]+.
3-(3,4,5-Trimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-propanamide
(11e). Synthesis was performed according to the general procedure from 3-(3,4,5-dimethoxyphenyl)
propionic acid (800 mg, 3.33 mmol) and (500 mg, 1.67 mmol). Purification was achieved by flash
7
chromatography (SiO2, 20%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O)
1
to afford 11e as pale yellowish solid. Yield 422 mg (49%); C27H27FN4O4S (Mr 522.60); H-NMR
(DMSO-d6):
δ = 2.62 (s, 3H, SCH3), 2.62–2.69 (m, 2H, COCH2CH2), 2.80–2.85 (m, 2H, COCH2CH2),
3.61 (s, 3H, C4OCH3), 3.74 (bs, 6H, C3/5OCH3), 6.56 (s, 2H, C2/6H, (OCH3)3-Phe), 7.01 (dd, 3J = 5.3 Hz
,
4J = 1.6 Hz, 1H, C5H, Pyr), 7.26–7.32 (m, 2H, C3/5H, F-Phe), 7.46–7.51 (m, 2H, C2/6H, F-Phe), 8.11
5
(
dd, 3J = 5.3 Hz, J = 0.5 Hz, 1H, C6H, Pyr), 8.36 (bs, 1H, C3H, Pyr), 10.36 (s, 1H, CONH), 12.70
(s, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3), 31.3 (COCH2CH2), 37.9 (COCH2CH2), 55.7
(C3/5OCH3), 59.9 (C4OCH3), 105.5 (C2/6H, (OCH3)3-Phe), 110.6 (C3H, Pyr), 115.8 (d, 2JCF = 21.7 Hz
,
C3/5H, F-Phe), 116.4 (C5H, Pyr), 126.6 (d, JCF = 3.3 Hz, C1, F-Phe), 130.1 (C5, Imdz), 130.7
4
(
d, 3JCF = 8.4 Hz, C2/6H, F-Phe), 134.5 (C4, Imdz), 135.7 (C4OCH3), 136.8 (C1, (OCH3)3-Phe), 142.2
(C2, Imdz), 143.8 (C4, Pyr), 147.6 (C6H, Pyr), 152.5 (C2, Pyr), 152.7 (C3/5OCH3), 162.0 (d, 1JCF = 245.4 Hz
CF), 171.2 (CO) ppm; MS (ESI, 70 eV) m/z 523 [MH]+.
,
3.2.4. Synthesis of 4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-carbamides 12a
–g
A solution of (1.0 equiv), the appropriate isocyanate (1.1 equiv) and DIPEA (1.2 equiv) in 5 mL
7
anhyd. DMF was stirred under a nitrogen atmosphere at rt for 12 h. The solvent was removed under
reduced pressure, the residue resuspended in ethyl acetate, and washed with 0.1 M aq. HCl, sat. aq.
NaHCO3 solution, and sat. aq. NaCl solution, dried over anhyd. Na2SO4, and concentrated under
reduced pressure. The crude product was purified by flash chromatography (SiO2 and RP-18, eluent
and mixing ratio given for each compound, respectively) to afford the particular compound.

Molecules 2017, 22, 522
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1-(2,4-Dimethoxyphenyl)-3-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)carbamide
12a). Synthesis was performed according to the general procedure from 2,4-dimethoxyphenyl
(
isocyanate (262 mg, 1.47 mmol). Purification was achieved by flash chromatography (SiO2, 0%–10%
methanol/DCM and RP-18, 50%–100% methanol/H2O) to afford 12a as colorless solid. Yield 135 mg
◦
(21%); C24H22FN5O3S (Mr 479.53); m.p. 214 C; 1H-NMR (DMSO-d6):
δ = 2.63 (s, 3H, SCH3), 3.74
(s, 3H, C4OCH3), 3.88 (s, 3H, C2OCH3), 6.48 (dd, 3J = 8.9 Hz, 4J = 2.7 Hz, 1H, C5H, (OCH3)2-Phe), 6.62
(d, 4J = 2.7 Hz, 1H, C3H, (OCH3)2-Phe), 6.92 (dd, J = 5.4 Hz, J = 1.5 Hz, 1H, C5H, Pyr), 7.30–7.51
(m, 5H, C3/5H, F-Phe and C3H, Pyr and C2/6H, F-Phe), 8.03 (d, 3J = 8.9 Hz, 1H, C6H, (OCH3)2-Phe),
8.12 (bs, 1H, C6H, Pyr), 9.64 (bs, 1H, NH), 10.89–11.26 (m, 1H, NH), 12.74 (bs, 1H, NH, Imdz) ppm;
3
4
13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 55.3 (C4OCH3), 56.0 (C2OCH3), 98.8 (C3H, (OCH3)2-Phe),
104.1 (C5H, (OCH3)2-Phe), 108.6 (C3H, Pyr), 114.6 (C5H, Pyr), 115.7 (C2/6H, F-Phe), 119.7 (C6H,
(OCH3)2-Phe), 121.9 (C1, (OCH3)2-Phe), 130.7 (C3/5H, F-Phe), 134.2 (C4, Pyr), 142.3 (C2, Imdz), 146.1
(C6H, Pyr), 149.4 (C2, (OCH3)2-Phe), 152.2 (CO), 153.6 (C2, Pyr), 155.2 (C4, (OCH3)2-Phe), 161.8
(d, 1JCF = 241.6 Hz, CF) ppm; MS (ESI, 70 eV) m/z 480 [MH]+.
1-(2,5-Dimethoxyphenyl)-3-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)carbamide
(12b). Synthesis was performed according to the general procedure from 2,5-dimethoxyphenyl
isocyanate (300 mg, 1.67 mmol). Purification was achieved by flash chromatography (SiO2, 20%–100%
ethyl acetate/petrol ether) to afford 12b as pale yellowish solid. Yield 79.9 mg (15%); C24H22FN5O3S
◦
1
(Mr 479.53); m.p. 184 C; H-NMR (DMSO-d6):
δ
= 2.63 (s, 3H, SCH3), 3.70 (s, 3H, C5OCH3), 3.84
(s, 3H, C2OCH3), 6.52 (dd, 3J = 8.8 Hz, 4J = 2.9 Hz, 1H, C4H, (OCH3)2-Phe), 6.91–6.96 (m, 1H, C3H,
(OCH3)2-Phe), 6.95 (dd, J = 5.5 Hz, J = 1.4 Hz, 1H, C5H, Pyr), 7.28–7.38 (m, 2H, C3/5H, F-Phe),
3
4
7.47–7.51 (m, 3H, C2/6H, F-Phe and C3H, Pyr), 7.92 (d, J = 2.9 Hz, 1H, C6H, (OCH3)2-Phe), 8.13
4
(d, 3J = 5.4 Hz, 1H, C6H, Pyr), 9.76 (bs, 1H, NH), 11.52 (vbs, 1H, NH), 12.74 (bs, 1H, NH, Imdz) ppm;
13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 55.3 (C5OCH3), 55.6 (C2OCH3), 105.6 (C6H, (OCH3)2-Phe),
105.8 (C4H, (OCH3)2-Phe), 108.5 (C3H, Pyr), 111.7 (C3H, (OCH3)2-Phe), 114.7 (C5H, Pyr), 115.9
(
d, 2JCF = 22.0 Hz, C3/5H, F-Phe), 126.5 (d, 4JCF = 3.3 Hz, C1, F-Phe), 129.6 (C1, (OCH3)2-Phe), 130.4
(C5, Imdz), 130.8 (d, 3JCF = 8.4 Hz, C2/6H, F-Phe), 134.1 (C4, Imdz), 142.3 (C2, Imdz), 142.4 (C2OCH3),
144.2 (C4, Pyr), 146.1 (C6H, Pyr), 152.2 (CO), 153.4 (C5OCH3), 153.4 (C2, Pyr), 162.1 (d, 1JCF = 245.9 Hz,
CF) ppm; MS (ESI, 70 eV) m/z 480 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-(4-methoxy-phenyl)carbamide (12c).
Synthesis was performed according to the general procedure from 4-methoxyphenyl isocyanate (165 µL,
1.27 mmol). Purification was achieved by flash chromatography (SiO2, 50%–100% ethyl acetate/petrol
ether and RP-18, 60%–100% methanol/H2O) to afford 12c as pale yellow solid. Yield 137 mg (26%);
◦
C23H20FN5O2S (Mr 449.50); m.p. 201 C; 1H-NMR (DMSO-d6):
δ = 2.62 (s, 3H, SCH3), 3.72 (s, 3H,
OCH3), 6.86–6.90 (m, 2H, C3/5H, H3CO-Phe), 6.92 (dd, 3J = 5.4 Hz, 4J = 1.5 Hz, 1H, C5H, Pyr), 7.30
(bs, 2H, C3/5H, F-Phe), 7.39–7.44 (m, 2H, C2/6H, H3CO-Phe), 7.47–7.51 (m, 2H, C2/6H, F-Phe), 7.58
(bs, 1H, C3H, Pyr), 8.11 (bs, 1H, C6H, Pyr), 9.40 (bs, 1H, NH), 10.35–10.71 (m, 1H, NH), 12.74 (s, 1H, NH,
Imdz) ppm; 13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 55.2 (OCH3), 105.3 (C4, Pyr), 108.7 (C3H, Pyr), 114.0
(C3/5H, H3CO-Phe), 114.6 (C5H, Pyr), 115.8 (d, 2JCF = 22.0 Hz, C3/5H, F-Phe), 120.6 (C2/6H, H3CO-Phe),
130.6–130.8 (C2/6H, F-Phe), 132.0 (C1, H3CO-Phe), 142.3 (C2, Imdz), 146.4 (C6H, Pyr), 152.3 (CO), 153.5
(C2, Pyr), 154.9 (C4, H3CO-Phe), 162.1 (d, 1JCF = 252.6 Hz, CF) ppm; MS (ESI, 70 eV) m/z 450 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-(m-tolyl)-carbamide (12d). Synthesis
was performed according to the general procedure from 3-methylphenyl isocyanate (284 µL, 2.20 mmol).
Purification was achieved by flash chromatography (SiO2, 30%–100% ethyl acetate/petrol ether and
RP-18, 60%–80% methanol/H2O) to afford 12d as colorless solid. Yield 241 mg (28%); C23H20FN5OS
◦
1
(Mr 433.51); m.p. 211 C; H-NMR (DMSO-d6):
(
δ = 2.29 (s, 3H, CH3), 2.63 (s, 3H, SCH3), 6.83
d, 3J = 7.4 Hz, 1H, C4H, Tol), 6.95 (dd, 3J = 5.4 Hz, 4J = 1.5 Hz, 1H, C5H, Pyr), 7.15–7.35 (m, 2H, C3/5H,
F-Phe), 7.17 (t, 3J = 7.6 Hz, 1H, C5H, Tol), 7.31 (d, 3J = 8.0 Hz, 1H, C6H, Tol), 7.35 (s, 1H, C2H, Tol),
7.47–7.52 (m, 2H, C2/6H, F-Phe), 7.61 (bs, 1H, C3H, Pyr), 8.13 (bs, 1H, C6H, Pyr), 9.44 (s, 1H, NH),

Molecules 2017, 22, 522
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10.42–10.78 (m, 1H, NH), 12.74 (bs, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 21.2
(CH3), 108.7 (C3H, Pyr), 114.8 (C5H, Pyr), 115.8 (d, JCF = 22.3 Hz, C3/5H, F-Phe), 116.0 (C6H, Tol),
119.3 (C2H, Tol), 123.2 (C4H, Tol), 126.6 (d, 4JCF = 2.6 Hz, C1, F-Phe), 128.7 (C5H, Tol), 129.7 (C5, Imdz),
130.8 (C2/6H, F-Phe), 134.2 (C4, Imdz), 138.1 (C3, Tol), 139.0 (C1, Tol), 142.4 (C2, Imdz), 144.3 (C4, Pyr),
146.4 (C6H, Pyr), 152.1 (CO), 153.4 (C2, Pyr), 162.0 (d, 1JCF = 243.0 Hz, CF) ppm; MS (ESI, 70 eV) m/z
434 [MH]+.
2
1-(3-Chloro-4-methylphenyl)-3-(4-(5-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-carbamide
(12e). Synthesis was performed according to the general procedure from 3-chloro-4-methylyphenyl
isocyanate (205 L, 1.47 mmol). Purification was achieved by flash chromatography (SiO2, 0%–10%
µ
methanol/DCM and RP-18, 70%–100% methanol/H2O and SiO2, 20%–100% ethyl acetate/petrol
ether) to afford 12e as colorless solid. Yield 210 mg (34%); C23H19ClFN5OS (Mr 467.95); m.p. 210 ◦C;
1H-NMR (DMSO-d6):
δ
= 2.27 (s, 3H, CH3), 2.63 (s, 3H, SCH3), 6.96 (dd, 3J = 5.5 Hz, 4J = 1.4 Hz, 1H,
C5H, Pyr), 7.26 (m, 2H, C5/6H, Cl-Tol), 7.28–7.34 (m, 2H, C3/5H, F-Phe), 7.46–7.50 (m, 2H, C2/6H,
F-Phe), 7.59 (bs, 1H, C3H, Pyr), 7.77 (s, 1H, C2H, Cl-Tol), 8.12 (d, J = 5.4 Hz, 1H, C6H, Pyr), 9.51
3
(s, 1H, NH), 10.99 (s, 1H, NH), 12.73 (s, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ = 15.1 (SCH3),
18.8 (CH3), 108.7 (C3H, Pyr), 114.8 (C5H, Pyr), 115.9 (d, JCF = 21.7 Hz, C3/5H, F-Phe), 117.6 (C6H,
2
Cl-Tol), 118.7 (C2H, Cl-Tol), 126.5 (d, JCF = 3.1 Hz, C1, F-Phe), 128.9 (C4, Cl-Tol), 130.4 (C5, Imdz),
4
130.8 (d, 3JCF = 8.3 Hz, C2/6H, F-Phe), 131.2 (C5H, Cl-Tol), 133.2 (C3, Cl-Tol), 134.1 (C4, Imdz), 138.2
(
C1, Cl-Tol), 142.3 (C2, Imdz), 144.3 (C4, Pyr), 146.4 (C6H, Pyr), 152.2 (CO), 153.2 (C2, Pyr), 162.1
(d, 1JCF = 245.5 Hz, CF) ppm; MS (ESI, 70 eV) m/z 468 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-(3-(trifluoromethyl)phenyl)-carbamide
(12f). Synthesis was performed according to the general procedure from 3-(trifluoromethyl)phenyl
isocyanate (300 L, 2.20 mmol). Purification was achieved by flash chromatography (SiO2, 40%
µ
methanol/DCM) and subsequent filtration to afford 12f as colorless solid. Yield 255 mg (26%);
C23H17FN5OS (Mr 487.48); m.p. 234 ◦C; 1H-NMR (DMSO-d6):
δ = 2.63 (s, 3H, SCH3), 6.99
4
(dd, 3J = 5.6 Hz, J = 1.2 Hz, 1H, C5H, Pyr), 7.28–7.37 (m, 3H, C3/5H, F-Phe and C4H, F3C-Phe),
7.46–7.66 (m, 5H, C3H, Pyr and C2/6H, F-Phe and C5/6H, F3C-Phe), 8.08 (bs, 1H, C2H, F3C-Phe), 8.14
3
(d, J = 5.5 Hz, 1H, C6H, Pyr), 9.56 (s, 1H, NH), 11.12 (s, 1H, NH), 12.74 (s, 1H, NH, Imdz) ppm;
13C-NMR (DMSO-d6):
δ
= 15.1 (SCH3), 108.7 (C3H, Pyr), 114.7 (C2H, F3C-Phe), 115.0 (C5H, Pyr), 115.9
(d, 2JCF = 21.8 Hz, C3/5H, F-Phe), 118.7 (C4H, F3C-Phe), 122.5 (C6H, F3C-Phe), 124.2 (q, 1JCF = 272.1 Hz,
CF3, F3C-Phe), 126.5 (d, 4JCF = 2.9 Hz, C1, F-Phe), 129.6 (q, 2JCF = 31.3 Hz, C3, F3C-Phe), 130.0 (C5H,
F3C-Phe), 130.5 (C5, Imdz), 130.8 (d, 3JCF = 8.3 Hz, C2/6H, F-Phe), 134.0 (C4, Imdz), 139.9 (C1, F3C-Phe),
142.3 (C2, Imdz), 144.3 (C4, Pyr), 146.6 (C6H, Pyr), 152.2 (CO), 153.0 (C2, Pyr), 162.0 (d, 1JCF = 245.2
Hz, CF) ppm; MS (ESI, 70 eV) m/z 488 [MH]+; HRMS (EI, 70 eV) m/z [M]+ calcd. for C23H17FN5OS,
487.1090; found 487.1090.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-(naphthalen-1-yl)carbamide (12g).
Synthesis was performed according to the general procedure from naphthyl isocyanate (211 µL,
1.47 mmol). Purification was achieved by flash chromatography (SiO2, 0%–10% methanol/DCM and
SiO2, 30%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O) to afford 12g as
pale yellow solid. Yield 257 mg (41%); C26H20FN5OS (Mr 469.54); m.p. 249 ◦C; 1H-NMR (DMSO-d6):
δ = 2.64 (s, 3H, SCH3), 7.01 (dd, 3J = 5.5 Hz, 4J = 1.4 Hz, 1H, C5H, Pyr), 7.30–7.36 (m, 2H, C3/5H, F-Phe),
7.42–7.59 (m, 5H, C3H, Pyr and C2/6H, F-Phe and C3/6H, Naph), 7.63–7.68 (m, 2H, C4/7H, Naph), 7.95
(
d, 3J = 8.1 Hz, 1H, C5H, Naph), 8.16–8.22 (m, 2H, C2/8H, Naph), 8.27 (d, 3J = 5.5 Hz, 1H, C6H, Pyr),
9.87 (s, 1H, NH), 11.86 (bs, 1H, NH), 12.77 (s, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
= 15.1
δ
(SCH3), 108.7 (C3H, Pyr), 114.7 (C5H, Pyr), 115.9 (d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 116.7 (C2H, Naph),
120.9 (C8H, Naph), 123.0 (C4H, Naph), 125.4 (C1, Naph), 126.0 (C3/6H, Naph), 126.3 (C7H, Naph), 126.5
(d, 4JCF = 3.2 Hz, C1, F-Phe), 128.6 (C5H, Naph), 130.6 (C5, Imdz), 130.8 (d, 3JCF = 8.3 Hz, C2/6H, F-Phe),
133.7 (C4a, Naph), 134.0 (C8a, Naph), 134.2 (C4, Imdz), 142.4 (C2, Imdz), 144.5 (C4, Pyr), 146.1 (C6H,
Pyr), 152.6 (CO), 153.5 (C2, Pyr), 162.1 (d, 1JCF = 246.2 Hz, CF) ppm; MS (ESI, 70 eV) m/z 471 [MH]+.

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3.2.5. Synthesis of N-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-1-methyl-
4-phenyl-1H-pyrrole-2-carboxamides 13, 14a–b, 15a–b, 16a–b
Ethyl 4-bromo-1-methyl-1H-pyrrole-2-carboxylate (13). NaH (240 mg 60% dispersion in mineral oil,
6.05 mmol) was added in one portion to a solution of 4-bromo-1H-pyrrole-2-carboxylate (1.17 g,
5.34 mmol) in 15 mL anhyd. DMF at 0 ◦C under a nitrogen atmosphere. The suspension was stirred
for 20 min at the same temp. before methyl iodide (400 µL, 6.43 mmol) was carefully added and
stirring continued at 0 ◦C for 15 min and another 2.5 h at r.t. The reaction was quenched with H2O
and extracted with ethyl acetate. The combined organic phases were washed with H2O and sat. aq.
NaCl solution, dried over anhyd. Na2SO4, and the solvent was removed under reduced pressure.
Purification was achieved by flash chromatography (SiO2, 2%–10% ethyl acetate/petrol ether) to
afford 13 as clear colorless oil that crystallized on standing as colorless needles. Yield 1.22 g (98%);
C8H10BrNO2 (Mr 232.08); m.p. 43 ◦C; 1H-NMR (DMSO-d6):
δ
= 1.26 (t, 3J = 7.1 Hz, 3H, COOCH2CH3),
3.83 (d, J = 0.2 Hz, 3H, CH3), 4.21 (q, 3J = 7.1 Hz, 2H, COOCH2CH3), 6.84 (d, 4J = 2.0 Hz, 1H, C3H,
Pyrrole), 7.28 (d, 4J = 1.7 Hz, 1H, C5H, Pyrrole) ppm; 13C-NMR (DMSO-d6):
δ = 14.2 (COOCH2CH3),
36.6 (CH3), 59.8 (COOCH2CH3), 93.8 (CBr), 118.1 (C3H, Pyrrole), 122.7 (C2, Pyrrole), 129.5 (C5H,
Pyrrole), 159.5 (COOCH2CH3) ppm.
Ethyl 4-(2,4-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (14a). 27 mL 2 M aq. Na2CO3 solution
were added to a stirred solution of 13 (1.22 g, 5.26 mmol), 2,4-dimethoxyphenyl boronic acid (2.89 g,
15.9 mmol), and Pd(PPh3)4 (306 mg, 265 µmol) in 80 mL DMF. Stirring continued for 4 h under reflux
and 12 h at r.t. H2O was added and the mixture was extracted with ethyl acetate. The combined organic
phases were washed with H2O and sat. aq. NaCl solution, dried over anhyd. Na2SO4, and the solvent
was removed under reduced pressure. The crude product was purified by flash chromatography (SiO2,
5%–10% ethyl acetate/petrol ether and RP-18, 50%–70% methanol/H2O) to afford 14a as colorless
solid. Yield 1.02 g (67%); C16H19NO4 (Mr 289.33); 1H-NMR (DMSO-d6): δ = 1.29 (t, 3J = 7.1 Hz, 3H,
COOCH2CH3), 3.77 (s, 3H, C4OCH3), 3.84 (s, 3H, C2OCH3), 3.87 (s, 3H, CH3), 4.23 (q, 3J = 7.1 Hz, 2H,
COOCH2CH3), 6.53 (dd, 3J = 8.5 Hz, 4J = 2.5 Hz, 1H, C5H, (OCH3)2-Phe), 6.60 (d, 4J = 2.5 Hz, 1H, C3H,
(OCH3)2-Phe), 7.17 (d, 4J = 2.0 Hz, 1H, C3H, Pyrrole), 7.44 (d, 3J = 8.5 Hz, 1H, C6H, (OCH3)2-Phe), 7.46
(dd, 4J = 2.0 Hz, 5J = 0.4 Hz, 1H, C5H, Pyrrole) ppm; 13C-NMR (DMSO-d6): δ = 14.4 (COOCH2CH3), 36.4
(CH3), 55.2 (C4OCH3), 55.4 (C2OCH3), 59.4 (CH2), 98.8 (C3H, (OCH3)2-Phe), 105.2 (C5H, (OCH3)2-Phe),
115.4 (C3H, Pyrrole), 115.5 (C1, (OCH3)2-Phe), 119.0 (C4, Pyrrole), 121.3 (C2, Pyrrole), 127.7 (C6H,
(OCH3)2-Phe), 129.0 (C5H, Pyrrole), 156.7 (C4OCH3), 158.74 (C2OCH3), 160.5 (COOCH2CH3) ppm; MS
(ESI, 70 eV) m/z 290 [MH]+.
Ethyl 4-(2,5-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (14b). Synthesis was performed
according to the procedure described for 14a starting from 13 (1.11 g, 4.78 mmol), 2,5-dimethoxyphenyl
boronic acid (2.65 g, 14.6 mmol), Pd(PPh3)4 (289 mg, 250 µmol) in 80 mL DMF, and 25 mL 2 M
aq. Na2CO3 solution. Purification was achieved by flash chromatography (SiO2, 2%–10% ethyl
acetate/petrol ether and RP-18, 65% methanol/H2O) to afford 14b as colorless solid. Yield 984 mg
(71%); C16H19NO4 (Mr 289.33); 1H-NMR (DMSO-d6):
δ
= 1.30 (t, 3J = 7.1 Hz, 3H, COOCH2CH3), 3.74
(s, 3H, C5OCH3), 3.79 (s, 3H, C2OCH3), 3.89 (s, 3H, CH3), 4.24 (q, 3J = 7.1 Hz, 2H, COOCH2CH3), 6.74
dd, 3J = 8.9 Hz, 4J = 3.1 Hz, 1H, C4H, (OCH3)2-Phe), 6.96 (d, 3J = 8.9 Hz, 1H, C3H, (OCH3)2-Phe), 7.09
(d, 4J = 3.1 Hz, 1H, C6H, (OCH3)2-Phe), 7.28 (d, 4J = 2.0 Hz, 1H, C3H, Pyrrole), 7.60 (dd, 4J = 2.0 Hz
5J = 0.4 Hz, 1H, C5H, Pyrrole) ppm; 13C-NMR (DMSO-d6):
= 14.3 (COOCH2CH3), 36.5 (CH3), 55.4
(
,
δ
(C5OCH3), 55.9 (C2OCH3), 59.4 (COOCH2CH3), 111.7 (C4H, (OCH3)2-Phe), 112.5 (C6H, (OCH3)2-Phe),
112.9 (C3H, (OCH3)2-Phe), 116.1 (C3H, Pyrrole), 118.9 (C4, Pyrrole), 121.6 (C1, Pyrrole), 123.4
(C1, (OCH3)2-Phe), 130.0 (C5H, Pyrrole), 150.0 (C2OCH3), 153.4 (C5OCH3), 160.5 (COOCH2CH3)
ppm; MS (ESI, 70 eV) m/z 290 [MH]+.
4-(2,4-Dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (15a). 7 mL 4 M aq. NaOH were added to
a solution of 14a (990 mg, 3.42 mmol) in 18 mL THF and 9 mL methanol and the mixture was stirred

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at 50 ◦C for 5 h and then 12 h at r.t. H2O was added to the reaction, the pH was adjusted to 3 using
1 M aq. HCl, and the mixture was extracted with ethyl acetate. The combined organic phases were
washed with H2O and sat. aq. NaCl solution, the organic phase was dried over anhyd. Na2SO4,
and the solvent was removed under reduced pressure to afford 15a as brown solid. Yield 894 mg
(quant.); C14H15NO4 (Mr 261.28); m.p. 164 ◦C; 1H-NMR (DMSO-d6):
δ
= 3.76 (s, 3H, C4OCH3), 3.83
(s, 3H, C2OCH3), 3.86 (s, 3H, CH3), 6.53 (dd, 3J = 8.5 Hz, 4J = 2.4 Hz, 1H, C5H, (OCH3)2-Phe), 6.59
(d, 4J = 2.4 Hz, 1H, C3H, (OCH3)2-Phe), 7.14 (d, 4J = 2.0 Hz, 1H, C3H, Pyrrole), 7.41 (d, 4J = 2.0 Hz,
1H, C5H, Pyrrole), 7.42 (d, 3J = 8.4 Hz, 1H, C6H, (OCH3)2-Phe), 12.17 (bs, 1H, COOH) ppm; 13C-NMR
(DMSO-d6):
δ
= 36.3 (CH3), 55.2 (C4OCH3), 55.4 (C2OCH3), 98.8 (C3H, (OCH3)2-Phe), 105.2 (C5H,
(OCH3)2-Phe), 115.7 (C3H, Pyrrole), 115.7 (C1, (OCH3)2-Phe), 118.8 (C4, Pyrrole), 122.0 (C2, Pyrrole),
127.6 (C6H, (OCH3)2-Phe), 128.6 (C5H, Pyrrole), 156.7 (C2OCH3), 158.7 (C4OCH3), 162.1 (COOH) ppm;
MS (ESI, 70 eV) m/z 262 [MH]+.
4-(2,5-Dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (15b). Synthesis was performed according
to the procedure described for 15a starting from 14b (980 mg, 3.39 mmol) in 19 mL THF and 10 mL
methanol, and 7 mL 4 M aq. NaOH to afford 15b as brown solid. Yield 885 mg (quant.); C14H15NO4
◦
1
(Mr 261.28); m.p. 149 C; H-NMR (DMSO-d6):
δ
= 3.74 (s, 3H, C2OCH3), 3.79 (s, 3H, C5OCH3),
3.88 (CH3), 6.72 (dd, 3J = 8.9 Hz, 4J = 3.1 Hz, 1H, C4H, (OCH3)2-Phe), 6.95 (d, 3J = 8.9 Hz, 1H, C3H,
(OCH3)2-Phe), 7.08 (d, 4J = 3.1 Hz, 1H, C6H, (OCH3)2-Phe), 7.25 (d, 4J = 2.0 Hz, 1H, C3H, Pyrrole), 7.55
(d, 4J = 2.0 Hz, 1H, C5H, Pyrrole), 12.15 (bs, 1H, COOH) ppm; 13C-NMR (DMSO-d6):
δ = 36.4 (CH3),
55.4 (C2OCH3), 55.9 (C5OCH3), 111.5 (C4H, (OCH3)2-Phe), 112.5 (C6H, (OCH3)2-Phe), 112.9 (C3H,
(OCH3)2-Phe), 116.2 (C3H, Pyrrole), 118.7 (C4, Pyrrole), 122.4 (C2, Pyrrole), 123.6 (C1, (OCH3)2-Phe),
129.6 (C5H, Pyrrole), 150.1 (C5OCH3), 153.4 (C2OCH3), 162.1 (COOH) ppm; MS (ESI, 70 eV) m/z
262 [MH]+.
4-(2,4-Dimethoxyphenyl)-N-(4-(5-(4-fluorphenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-1-methyl-
1H-pyrrole-2-carboxamide (16a). A solution of 15a (1.01 g, 4.07 mmol), PyBOP (2.54 g, 4.88 mmol), and
DIPEA (2.15 mL, 12.3 mmol) was stirred in 14 mL anhyd. DMF under a nitrogen atmosphere for
30 min at r.t.
7 (1.60 g, 5.31 mmol) was added in one portion and the mixture was stirred for 12 h at
110 ◦C. The reaction was quenched with H2O and extracted with ethyl acetate. The combined organic
phases were washed with H2O and sat. aq. NaCl solution, dried over anhyd. Na2SO4, and the solvent
was removed under reduced pressure. The crude product was purified by flash chromatography (SiO2,
30%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O) to afford 16a as beige
solid. Yield 303 mg (24%); C29H26FN5O3S (Mr 543.62); m.p. 236 ◦C; 1H-NMR (DMSO-d6): δ = 2.64
3
(
s, 3H, SCH3), 3.78 (s, 3H, C2OCH3), 3.86 (s, 3H, C4OCH3), 3.90 (s, 3H, CH3), 6.57 (dd, J = 8.5 Hz,
4J = 2.3 Hz, 1H, C5H, (OCH3)2-Phe), 6.61 (d, 4J = 2.3 Hz, 1H, C3H, (OCH3)2-Phe), 7.06 (dd, 3J = 5.3 Hz
,
4J = 1.2 Hz, 1H, C5H, Pyr), 7.28–7.34 (m, 2H, C3/5H, F-Phe), 7.41 (d, 4J = 1.3 Hz, 1H, C3H, Pyrrole),
7.46 (d, 3J = 8.4 Hz, 1H, C6H, (OCH3)2-Phe), 7.49–7.54 (m, 2H, C2/6H, F-Phe), 7.66 (d, 4J = 1.4 Hz, 1H,
C5H, Pyrrole), 8.18 (d, 3J = 5.2 Hz, 1H, C6H, Pyr), 8.38 (bs, 1H, C3H, Pyr), 10.10 (bs, 1H, CONH), 12.73
(bs, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ
= 15.3 (SCH3), 36.5 (CH3), 55.2 (C2OCH3), 55.5 (C4OCH3),
98.8 (C3H, (OCH3)2-Phe), 105.2 (C5H, (OCH3)2-Phe), 111.3 (C3H, Pyr), 113.2 (C5H, Pyrrole), 115.9
(d, 2JCF = 21.9 Hz, C3/5H, F-Phe), 115.9 (C1, (OCH3)2-Phe), 116.3 (C5H, Pyr), 118.5 (C4, Pyrrole), 124.1
(C2, Pyrrole), 126.7 (d, 4JCF = 3.5 Hz, C1, F-Phe), 127.5 (C6H, (OCH3)2-Phe), 128.5 (C3H, Pyrrole), 130.8
(d, 3JCF = 8.4 Hz, C2/6H, F-Phe), 134.5 (C5, Imdz), 142.1 (C2, Imdz), 143.6 (C4, Imdz), 147.6 (C6H, Pyr),
152.7 (C2, Pyr), 156.7 (C4OCH3), 158.6 (C2OCH3), 159.8 (CONH), 162.0 (d, 1JCF = 245.6 Hz, CF) ppm;
MS (ESI, 70 eV) m/z 544 [MH]+; HRMS (EI, 70 eV) m/z [M]+ calcd. for C29H26FN5O3S, 543.1740;
found, 543.1740.
4-(2,5-Dimethoxyphenyl)-N-(4-(5-(4-fluorphenyl)-2-(methylthio)-1H-imidazol-4-yl)-pyridin-2-yl)-1-methyl-
1H-pyrrole-2-carboxamide (16b). Synthesis was performed according to the procedure described for 16a
starting from 15b (604 mg, 2.31 mmol), PyBOP (1.45 g, 2.79 mmol), DIPEA (1.20 mL, 6.87 mmol), and
7
(904 mg, 3.01 mmol) in 12 mL anhyd. DMF. The crude product was purified by flash chromatography

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(SiO2, 30%–100% ethyl acetate/petrol ether and RP-18, 50%–100% methanol/H2O) to afford 16b as
pale yellowish solid. Yield 255 mg (20%); m.p. 127 ◦C; 1H-NMR (DMSO-d6):
δ
= 2.64 (s, 3H, SCH3),
3.76 (s, 3H, C5OCH3), 3.81 (s, 3H, C2OCH3), 3.92 (s, 3H, CH3), 6.73 (dd, J = 8.9 Hz, J = 3.1 Hz,
3
4
1H, C4H, (OCH3)2-Phe), 6.96 (d, 3J = 9.0 Hz, 1H, C3H, (OCH3)2-Phe), 7.06 (dd, 3J = 5.3 Hz, 4J = 1.6
4
Hz, 1H, C5H, Pyr), 7.17 (d, J = 3.1 Hz, 1H, C6H, (OCH3)2-Phe), 7.24–7.30 (m, 2H, C3/5H, F-Phe),
7.50–7.55 (m, 2H, C2/6H, F-Phe), 7.56 (d, 4J = 1.7 Hz, 1H, C5H, Pyrrole), 7.80 (d, 4J = 1.8 Hz, 1H, C3H,
3
Pyrrole), 8.22 (d, J = 5.2 Hz, 1H, C6H, Pyr), 8.35 (bs, 1H, C3H, Pyr), 10.15 (bs, 1H, CONH), 12.77
(vbs, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ
= 15.2 (SCH3), 36.7 (CH3), 55.4 (C5OCH3), 55.9 (C2OCH3),
111.3 (C4H, (OCH3)2-Phe and C3H, Pyr), 112.5 (C6H, (OCH3)2-Phe), 112.8 (C3H, (OCH3)2-Phe), 113.6
2
(C3H, Pyrrole), 115.7 (d, JCF = 21.5 Hz, C3/5H, F-Phe), 116.5 (C5H, Pyr), 118.3 (C4, Pyrrole), 123.8
(C1, (OCH3)2-Phe), 124.3 (C2, Pyrrole), 129.7 (C5H, Pyrrole), 130.42 (C2/6H, F-Phe), 142.5 (C2, Imdz
and C4, Pyr), 147.7 (C6H, Pyr), 150.1 (C2OCH3), 152.7 (C1, Pyr), 153.4 (C5OCH3), 159.8 (CONH), 161.9
(d, 1JCF = 247.7 Hz, CF) ppm; MS (ESI, 70 eV) m/z 544 [MH]+; HRMS (EI, 70 eV) m/z [M]+ calcd. for
C29H26FN5O3S, 543.1740; found, 543.1740.
3.2.6. Synthesis of Sulfoxides 10j–k, 11f–g, 12h–m, 16c
The sulfide (1.0 equiv) was dissolved in THF and H2O was added (approx. 3:1). The mixture was
stirred at 0 ◦C for 10 min before an ice-cold aq. solution of potassium peroxomonosulfate (Oxone®,
0.6 equiv) was added and stirring continued for 0.5–2 h at the same temp. After completion of the
reaction sat. aq. NaHCO3 solution, H2O, and ethyl acetate were added and the phases were separated.
The organic layer was washed with H2O and sat. aq. NaCl solution, dried over anhyd. Na2SO4, and
the solvent was removed under reduced pressure. Purification of the crude products was achieved by
crystallization from ethyl acetate or flash chromatography (SiO2 and RP-18, eluent and mixing ratio
given for each compound) to afford the appropriate compound.
N-(2-Ethoxyphenyl)-4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)pyridin-2-amine (10j). Synthesis
was performed according to the general procedure for sulfoxidation starting from 10c (300 mg,
713
µmol) in 5 mL THF and 2 mL H2O. Flash chromatography (RP-18, 20%–90% methanol/H2O)
afforded 10j as voluminous yellow solid. Yield 255 mg (82%); C23H21FN4O2S (Mr 436.51); m.p. 152 ◦C
;
1H-NMR (CDCl3):
δ = 1.42 (t, J = 7.0 Hz, 3H, CH3), 3.03 (s, 3H, SCH3), 4.07 (q, J = 7.0 Hz, 2H,
3
3
CH2), 6.94–6.78 (m, 4H, C4–6H, EtO-Phe and C5H, Pyr), 7.03 (dd, 4J = 1.4 Hz, 5J = 0.7 Hz, 1H, C3H,
Pyr), 7.07–7.13 (m, 3H, C3/5H, F-Phe and NH), 7.46–7.50 (m, 2H, C2/6H, F-Phe), 7.62 (dd, 3J = 7.8 Hz,
4J = 1.5 Hz, 1H, C3H, EtO-Phe), 8.14 (dd, 3J = 5.3 Hz, 5J = 0.7 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ
= 14.9 (CH3), 40.8 (SCH3), 64.1 (CH2), 106.9 (C3H, Pyr), 111.5 (C3H, EtO-Phe), 113.3 (C5H, Pyr), 116.0
(d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 118.2 (C6H, EtO-Phe), 120.6 (C5H, EtO-Phe), 121.9 (C4H, EtO-Phe),
129.9 (C1, EtO-Phe), 130.6 (d, 3JCF = 8.2 Hz, C2/6H, F-Phe), 146.7 (C2, Imdz), 148.2 (C2, EtO-Phe), 148.5
(C6H, Pyr), 156.0 (C2, Pyr), 162.9 (d, 1JCF = 248.8 Hz, CF) ppm; MS (ESI, 70 eV) m/z 437 [MH]+.
N-(3,4-Dimethoxyphenethyl)-4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)-pyridin-2-amine (10k).
Synthesis was performed according to the general procedure for sulfoxidation starting from 10d
(500 mg, 1.08 mmol) in 5 mL H2O in 9 mL THF and 3 mL H2O. Flash chromatography (RP-18,
20%–90% methanol/H2O) afforded 10d as pale yellow solid. Yield 208 mg (39%); C25H25FN4O3S
◦
3
(Mr 480.56); m.p. 183 C; 1H-NMR (CDCl3):
δ = 2.74 (t, J = 6.8 Hz, 1H, CH2CH2NH), 2.97 (s, 3H,
SCH3), 3.36 (m, 2H, CH2CH2NH), 3.76 (s, 6H, 2 OCH3), 5.00 (bs, 1H, CH2CH2NH), 6.49 (bs, 1H,
C3H, Pyr), 6.52 (dd, 3J = 5.4 Hz, 4J = 1.3 Hz, 1H, C5H, Pyr), 6.60–6.63 (m, 2H, C2/6H, (OCH3)2-Phe),
3
6.71 (d, J = 8.7 Hz, 1H, C5H, (OCH3)2-Phe), 6.96–7.02 (m, 2H, C3/5H, F-Phe), 7.36–7.41 (m, 2H,
C
2/6H, F-Phe), 7.80 (d, 3J = 5.5 Hz, 1H, C6H, Pyr) ppm; 13C-NMR (CDCl3):
δ = 35.1 (CH2CH2NH), 40.7
(SCH3), 43.3 (CH2CH2NH), 55.9 (C3OCH3), 55.9 (C4OCH3), 104.6 (C3H, Pyr), 111.2 (C5H, Pyr), 111.4
(C5H, (OCH3)2-Phe), 112.1 (C2H, (OCH3)2-Phe), 115.9 (d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 120.7 (C6H,
(OCH3)2-Phe), 127.2 (C1, F-Phe), 130.5 (d, 3JCF = 8.2 Hz, C2/6H, F-Phe), 131.5 (C1, (OCH3)2-Phe), 135.3

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(C4/5, Imdz), 141.6 (C4, Pyr), 146.8 (C2, Imdz), 147.4 (C6H, Pyr), 147.7 (C4OCH3), 149.0 (C3OCH3), 158.6
(C2, Pyr), 162.9 (d, 1JCF = 249.2 Hz, CF) ppm; MS (ESI, 70 eV) m/z 481 [MH]+.
3-(2,4-Dimethoxyphenyl)-N-(4-(5-(4-fluorphenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)-pyridin-2-yl)-propanamide
(
11f). Synthesis was performed according to the general procedure for sulfoxidation starting from
11a (100 mg, 203 mol) in 2 mL THF and 0.6 mL H2O. Flash chromatography (SiO2, 40%–100%
ethyl acetate/petrol ether) afforded 11f as colorless solid. Yield 96.1 mg (93%); C26H25FN4O4S
µ
◦
1
3
(Mr 508.57); m.p. 209 C; H-NMR (DMSO-d6):
δ = 2.58 (t, J = 7.5 Hz, 2H, CH2CH2CO), 2.75
(
(
t, 3J = 7.4 Hz, 2H, CH2CH2CO), 3.08 (s, 3H, SCH3), 3.72 (s, 3H, C2OCH3), 3.77 (s, 3H, C4OCH3), 6.42
dd, 3J = 8.3 Hz
,
4J = 2.4 Hz, 1H, C5H, (OCH3)2-Phe), 6.51 (d, J = 2.4 Hz, 1H, C3H, (OCH3)2-Phe),
4
7.01–7.05 (m, 2H, C5H, Pyr and C6H, (OCH3)2-Phe), 7.26–7.32 (m, 2H, C3/5H, F-Phe), 7.51–7.55 (m, 2H,
C
2/6H, F-Phe), 8.19 (d, 3J = 5.2 Hz, 1H, C6H, Pyr), 8.34 (bs, 1H, C3H, Pyr), 10.40 (s, 1H, CONH), 13.89
(bs, NH) ppm; 13C-NMR (DMSO-d6):
= 24.7 (CH2CH2CO), 36.3 (CH2CH2CO), 39.1 (SCH3), 55.1
(C2OCH3), 55.3 (C4OCH3), 98.3 (C3H, (OCH3)2-Phe), 104.3 (C5H, (OCH3)2-Phe), 111.1 (C3H, Pyr), 115.8
d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 117.0 (C5H, Pyr), 120.9 (C1, (OCH3)2-Phe), 127.3 (C1, F-Phe), 129.8
δ
(
(C6H, (OCH3)2-Phe), 130.8 (d, 3JCF = 8.2 Hz, C2/6H, F-Phe), 133.4 (C4, Imdz), 133.9 (C5, Imdz), 142.2
(C4, Pyr), 147.9 (C6H, Pyr), 148.9 (C2, Imdz), 152.6 (C2, Pyr), 157.9 (C4OCH3), 159.0 (C2OCH3), 162.1 (d,
1JCF = 245.5 Hz, CF), 171.54 (CO) ppm; MS (ESI, 70 eV) m/z 509 [MH]+; HRMS (EI, 70 eV) m/z [M]+
calcd. for C26H25FN4O4S, 508.1581; found, 508.1581.
3-(2,5-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)-pyridin-2-yl)propanamide
(11g). Synthesis was performed according to the general procedure for sulfoxidation starting from
11b (100 mg, 203 mol) in 2 mL THF and 0.6 mL H2O. Crystallization from ethyl acetate afforded
µ
11g as colorless solid. Yield 26.6 mg (26%); m.p. 204 ◦C; 1H-NMR (DMSO-d6):
δ
= 2.62 (t, 3J = 7.7 Hz,
2H, CH2CH2CO), 2.80 (t, 3J = 7.5 Hz, 2H, CH2CH2CO), 3.09 (s, 3H, SCH3), 3.66 (s, 3H, C5OCH3), 3.72
(s, 3H, C2OCH3), 6.72 (dd, 3J = 8.8 Hz, 4J = 3.1 Hz, 1H, C4H, (OCH3)2-Phe), 6.77 (d, 4J = 3.0 Hz, 1H,
3
3
C6H, (OCH3)2-Phe), 6.86 (d, J = 8.8 Hz, 1H, C3H, (OCH3)2-Phe), 7.02 (dd, J = 5.2 Hz, 4J = 1.5 Hz
,
,
1H, C5H, Pyr), 7.25–7.31 (m, 2H, C3/5H, F-Phe), 7.51–7.55 (m, 2H, C2/6H, F-Phe), 8.19 (d, 3J = 5.0 Hz
1H, C6H, Pyr), 8.33 (s, 1H, C3H, Pyr), 10.45 (s, 1H, CONH), 14.00 (bs, 1H, NH) ppm; 13C-NMR
(DMSO-d6):
δ
= 25.3 (CH2CH2CO), 36.0 (CH2CH2CO), 39.0 (SCH3), 55.2 (C5OCH3), 55.8 (C2OCH3),
111.2 (C4H, (OCH3)2-Phe), 111.3 (C3H, Pyr), 111.5 (C3H, (OCH3)2-Phe), 115.8 (d, 2JCF = 21.3 Hz, C3/5H,
F-Phe), 115.9 (C6H, (OCH3)2-Phe), 126.5 (C1, F-Phe), 130.8 (d, JCF = 8.3 Hz, C2/6H, F-Phe), 142.1
3
(C4, Pyr), 148.0 (C6H, Pyr), 148.8 (C2, Imdz), 151.2 (C2OCH3), 152.58 (C2, Pyr), 153.0 (C5OCH3), 162.1
(d, 1JCF = 245.6 Hz, CF), 171.42 (CO) ppm; MS (ESI, 70 eV) m/z 509 [MH]+.
1-(2,4-Dimethoxyphenyl)-3-(4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)-pyridin-2-yl)-carbamide
(
12h). Synthesis was performed according to the general procedure for sulfoxidation starting from
12a (85.0 mg, 177 mol) in 3.4 mL THF and 1 mL H2O. Flash chromatography (SiO2, 35%–100%
ethyl acetate/petrol ether and RP-18, 55%–100% methanol/H2O) afforded 12h as colorless solid.
µ
Yield 18.9 mg (22%); C24H22FN5O4S (Mr 495.53); m.p. 227 ◦C; 1H-NMR (DMSO-d6):
δ = 3.08 (s, 3H,
3
4
SCH3), 3.74 (s, 3H, C4OCH3), 3.88 (s, 3H, C2OCH3), 6.48 (dd, J = 8.9 Hz, J = 2.7 Hz, 1H, C5H,
(OCH3)2-Phe), 6.62 (d, 4J = 2.7 Hz, 1H, C3H, (OCH3)2-Phe), 6.95 (dd, 3J = 5.4 Hz, 4J = 1.5 Hz, 1H, C5H,
Pyr), 7.27–7.33 (m, 2H, C3/5H, F-Phe), 7.46 (bs, 1H, C3H, Pyr), 7.51–7.56 (m, 2H, C2/6H, F-Phe), 8.02
3
(
d, 3J = 8.9 Hz, 1H, C6H, (OCH3)2-Phe), 8.19 (d, J = 5.3 Hz, 1H, C6H, Pyr), 9.70 (s, 1H, NH), 11.07
(bs, 1H, NH), 13.87 (bs, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ
= 39.1 (SCH3), 55.3 (C2OCH3),
56.0 (C4OCH3), 98.8 (C3H, (OCH3)2-Phe), 104.1 (C5H, (OCH3)2-Phe), 109.2 (C3H, Pyr), 115.1 (C5H,
Pyr), 115.8 (d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 119.8 (C6H, (OCH3)2-Phe), 121.8 (C1, (OCH3)2-Phe), 127.2
(C1, F-Phe), 130.8 (d, 3JCF = 8.4 Hz, C2/6H, F-Phe), 132.7 (C4, Imdz), 134.1 (C5, Imdz), 142.8 (C4, Pyr),
146.5 (C6H, Pyr), 149.1 (C2, Imdz), 149.4 (C2OCH3), 152.2 (CO), 153.6 (C2, Pyr), 155.2 (C4OCH3), 162.14
(d, 1JCF = 244.6 Hz, CF) ppm; MS (ESI, 70 eV) m/z 496 [MH]+.

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1-(4-(5-(4-Fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)pyridin-2-yl)-3-(4-meth-oxyphenyl)carbamide
12i). Synthesis was performed according to the general procedure for sulfoxidation starting from
12c (100 mg, 225 mol) in 2 mL THF and 0.6 mL H2O. Flash chromatography (RP-18, 50%–70%
(
µ
methano◦l/H2O) afforded 12i as colorless solid. Yield 62.6 mg (61%); C23H20FN5O3S (Mr 465.50);
m.p. 233 C; 1H-NMR (DMSO-d6):
δ
= 3.09 (s, 3H, SCH3), 3.73 (s, 3H, C4OCH3), 6.89 (d, 3J = 9.1 Hz, 2H,
C
3/5H, H3CO-Phe), 6.95 (dd, 3J = 5.4 Hz, 4J = 1.5 Hz, 1H, C5H, Pyr), 7.28–7.34 (m, 2H, C3/5H, F-Phe),
7.41 (d, 3J = 9.1 Hz, 2H, C2/6H, H3CO-Phe), 7.51–7.56 (m, 2H, C2/6H, F-Phe), 7.65 (bs, 1H, C3H, Pyr),
8.18 (d, 3J = 5.4 Hz, 1H, C6H, Pyr), 9.43 (s, 1H, NH), 10.47 (bs, 1H, NH), 13.92 (bs, 1H, NH, Imdz) ppm;
13C-NMR (DMSO-d6):
δ
= 39.1 (SCH3), 55.2 (C4OCH3), 109.4 (C3H, Pyr), 114.0 (C3/5H, H3CO-Phe),
115.2 (C5H, Pyr), 115.9 (d, 2JCF = 21.7 Hz, C3/5H, F-Phe), 120.6 (C2/6H, H3CO-Phe), 127.5 (C1, F-Phe),
130.9 (d, JCF = 8.7 Hz, C2/6H, F-Phe), 132.0 (C1, H3CO-Phe), 146.9 (C6H, Pyr), 148.8 (C2, Imdz),
3
152.2 (CO), 153.5 (C2, Pyr), 154.9 (C4OCH3), 162.2 (d, 1JCF = 246.7 Hz, CF) ppm; MS (ESI, 70 eV) m/z
466 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)pyridin-2-yl)-3-(m-tolyl)-carbamide (12j). Synthesis
was performed according to the general procedure for sulfoxidation starting from 12d (100 mg,
231
µmol) in 2 mL THF and 0.6 mL H2O. Flash chromatography (SiO2, 20%–100% ethyl acetate/petrol
ether) afforded 12j as colorless solid. Yield 74.8 mg (72%); C23H20FN5O2S (Mr 449.50); m.p. 235 ◦C;
1H-NMR (DMSO-d6):
δ
= 2.29 (s, 3H, CH3), 3.09 (s, 3H, SCH3), 6.83 (d, 3J = 7.4 Hz, 1H, C4H, Tol), 6.97
(dd, 3J = 5.4 Hz, 4J = 1.4 Hz, 1H, C5H, Pyr), 7.18 (t, 3J = 7.7 Hz, 1H, C5H, Tol), 7.29–7.35 (m, 4H, C3/5H,
F-Phe and C2/6H, Tol), 7.52–7.57 (m, 2H, C2/6H, F-Phe), 7.69 (s, 1H, C3H, Pyr), 8.19 (d, 3J = 5.1 Hz, 1H,
C6H, Pyr), 9.48 (s, 1H, NH), 10.54 (bs, 1H, NH), 13.92 (bs, 1H, NH, Imdz) ppm; 13C-NMR (75 MHz,
DMSO-d6):
δ
= 21.2 (CH3), 39.1 (SCH3), 109.3 (C3H, Pyr), 115.3 (C5H, Pyr), 115.9 (d, 2JCF = 21.5 Hz,
C3/5H, F-Phe), 116.0 (C6H, Tol), 119.3 (C2H, Tol), 123.2 (C4H, Tol), 128.7 (C5H, Tol), 130.9 (C2/6H, F-Phe),
138.1 (C3, Tol), 138.9 (C1, Tol), 143.1 (C4, Pyr), 147.0 (C6H, Pyr), 148.7 (C2, Imdz), 152.1 (CO), 153.4
(C2, Pyr), 162.2 (d, 1JCF = 244.0 Hz, CF) ppm; MS (ESI, 70 eV) m/z 450 [MH]+.
1-(3-Chloro-4-methylphenyl)-3-(4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)pyridin-2-yl)-carbamide
(
12k). Synthesis was performed according to the general procedure for sulfoxidation starting from
12e (100 mg, 214 mol) in 4 mL THF and 1 mL H2O. Flash chromatography (SiO2, 20%–100% ethyl
acetate/petrol ether and RP-18, 50%–100% methanol/H2O) afforded 12k as beige solid. Yield 78.0 mg
µ
(75%); C23H19ClFN5O2S (Mr 483.95); 1H-NMR (DMSO-d6):
δ = 2.27 (s, 3H, CH3), 3.09 (s, 3H, SCH3),
6.99 (dd, 3J = 5.4 Hz, 4J = 1.3 Hz, 1H, C5H, Pyr), 7.26 (s, 1H, C5H, Cl-Tol), 7.26 (s, 1H, C6H, Cl-Tol),
7.29–7.35 (m, 2H, C3/5H, F-Phe), 7.52–7.56 (m, 2H, C2/6H, F-Phe), 7.65 (bs, 1H, C3H, Pyr), 7.76 (bs, 1H,
C2H, Cl-Tol), 9.55 (s, 1H, NH), 10.77 (bs, 1H, NH), 13.93 (bs, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ
= 18.8 (CH3), 39.1 (SCH3), 109.2 (C3H, Pyr), 115.4 (C5H, Pyr), 115.9 (d, 2JCF = 21.5 Hz, C3/5H, F-Phe),
117.5 (C6H, Cl-Tol), 118.7 (C2H, Cl-Tol), 129.0 (C4, Cl-Tol), 131.1 (C2/6H, F-Phe), 131.2 (C5, Imdz and
C5H, Cl-Tol), 133.2 (C3, Cl-Tol), 134.6 (C4, Imdz), 138.2 (C1, Cl-Tol), 147.0 (C6H, Pyr), 148.7 (C2, Imdz),
152.1 (CO), 153.2 (C2, Pyr), 162.3 (d, 1JCF = 249.9 Hz, CF) ppm; MS (ESI, 70 eV) m/z 484 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)pyridin-2-yl)-3-(3-(trifluormethyl)phenyl)carbamide
(12l). Synthesis was performed according to the general procedure for sulfoxidation starting from 12f
(20.0 mg, 41.0 mol) in 0.4 mL THF and 0.1 mL water. Flash chromatography (SiO2, 40%–100% ethyl
µ
◦
acetate/petrol ether) afforded 12l as a colorless solid. Yield 15.1 mg (73%); m.p. 235 C; 1H-NMR
(DMSO-d6):
δ
= 3.10 (s, 3H, SCH3), 7.02 (dd, 3J = 5.4 Hz, 4J = 1.4 Hz, 1H, C5H, Pyr), 7.30–7.37 (m, 2H,
3
C
3/5H, F-Phe), 7.36 (d, J = 7.7 Hz, 1H, C4H, F3C-Phe), 7.51–7.57 (m, 3H, C2/6H, F-Phe and C5H,
F3C-Phe), 7.65 (d, 3J = 8.3 Hz, 1H, C6H, F3C-Phe), 7.71 (s, 1H, C2H, F3C-Phe), 8.08 (bs, 1H, C3H, Pyr),
8.22 (bs, 1H, C6H, Pyr), 9.59 (s, 1H, NH), 10.90 (bs, 1H, NH), 13.93 (bs, 1H, NH, Imdz) ppm; 13C-NMR
(DMSO-d6):
δ
= 39.1 (SCH3), 109.3 (C2H, F3C-Phe), 114.7 (q, 3JCF = 2.5 Hz, C3H, Pyr), 115.5 (C5H, Pyr),
115.9 (d, 3JCF = 21.9 Hz, C3/5H, F-Phe), 118.7 (q, 3JCF = 2.1 Hz, C4H, F3C-Phe), 122.5 (C6H, F3C-Phe),
124.2 (q, 1JCF = 272.2 Hz, CF3), 125.9 (d, 4JCF = 3.2 Hz, C1, F-Phe), 129.6 (q, 2JCF = 31.4 Hz, C3, F3C-Phe),
130.0 (C5H, F3C-Phe), 130.8 (C5, Imdz), 131.1 (d, 4JCF = 11.4 Hz, C2/6H, F-Phe), 134.5 (C4, Imdz), 139.9

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(C1, F3C-Phe), 143.4 (C4, Pyr), 147.1 (C6H, Pyr), 148.7 (C2, Imdz), 152.2 (CO), 162.4 (d, 1JCF = 242.12 Hz,
CF) ppm; MS (ESI, 70 eV) m/z 504 [MH]+.
1-(4-(5-(4-Fluorophenyl)-2-(methylthio)-1H-imidazol-4-yl)pyridin-2-yl)-3-(naphthalen-1-yl)carbamide (12m).
Synthesis was performed according to the general procedure for sulfoxidation starting from 12g
(51.0 mg, 109
acetate/petrol ether) afforded 12m as colorless solid. Yield 37.0 mg (70%); C26H20FN5O2S (Mr 485.54);
m.p. 244 ◦C; 1H-NMR (DMSO-d6): = 3.10 (s, 3H, SCH3), 7.04 (dd, 3J = 5.4 Hz, 4J = 1.4 Hz, 1H, C5H,
µmol) in 5 mL THF and 1.5 mL H2O. Flash chromatography (SiO2, 20%–100% ethyl
δ
Pyr), 7.31–7.36 (m, 2H, C3/5H, F-Phe), 7.49 (t, 3J = 7.9 Hz, 1H, C3H, Naph), 7.54–7.59 (m, 4H, C3H, Pyr
and C2/6H, F-Phe and C6H, Naph), 7.63–7.68 (m, 2H, C4/7H, Naph), 7.96 (dd, 3J = 8.1 Hz, 4J = 0.8 Hz
,
3
1H, C5H, Naph), 8.16–8.20 (m, 2H, C2/8H, Naph), 8.34 (d, J = 5.4 Hz, 1H, C6H, Pyr), 8.93 (s, 1H,
NH), 11.58 (bs, 1H, NH), 13.95 (bs, 1H, NH, Imdz) ppm; 13C-NMR (DMSO-d6):
δ = 39.1 (SCH3), 109.4
(C3H, Pyr), 115.3 (C5H, Pyr), 115.9 (d, 2JCF = 21.8 Hz, C3/5H, F-Phe), 116.9 (C2H, Naph), 120.9 (C8H,
Naph), 123.1 (C4H, Naph), 125.5 (C1, Naph), 126.0 (C6H, Naph), 126.0 (C3H, Naph), 126.3 (C7H, Naph),
128.6 (C5H, Naph), 130.7 (C5, Imdz), 130.9 (C2/6H, F-Phe), 133.7 (C4a, Naph), 134.1 (C8a, Naph), 143.5
(C4, Pyr), 146.7 (C6H, Pyr), 148.9 (C2, Imdz), 152.6 (CO), 153.6 (C2, Pyr), 162.3 (d, 1JCF = 246.9 Hz, CF)
ppm; MS (ESI, 70 eV) m/z 486 [MH]+.
4-(2,4-Dimethoxyphenyl)-N-(4-(5-(4-fluorophenyl)-2-(methylsulfinyl)-1H-imidazol-4-yl)-pyridin-2-yl)-1-methyl
-1H-pyrrol-2-carboxamide (16c). Synthesis was performed according to the general procedure for
sulfoxidation starting from 16a (196 mg, 368
µmol) in 4 mL THF and 1 mL H2O. Flash chromatography
(SiO2, 40%–100% ethyl acetate/petrol ether) afforded 16c as yellow solid. Yield 182 mg (88%);
◦
C29H26FN5O4S (Mr 559.62); m.p. 136 C; 1H-NMR (DMSO-d6):
δ
= 3.11 (s, 3H, SCH3), 3.78 (s, 3H,
C2OCH3), 3.86 (s, 3H, C4OCH3), 3.90 (s, 3H, CH3), 6.57 (dd, J = 8.5 Hz, J = 2.4 Hz, 1H, C5H,
3
4
(OCH3)2-Phe), 6.61 (d, 4J = 2.3 Hz, 1H, C3H, (OCH3)2-Phe), 7.07 (d, 3J = 4.8 Hz, 1H, C5H, Pyr), 7.23–7.38
(m, 2H, C3/5H, F-Phe), 7.42 (bs, 1H, C3H, Pyrrole), 7.46 (d, J = 8.4 Hz, 1H, C6H, (OCH3)2-Phe),
3
7.54–7.59 (m, 2H, C2/6H, F-Phe), 7.68 (bs, 1H, C5H, Pyrrole), 8.22 (d, 3J = 4.5 Hz, 1H, C6H, Pyr), 8.42
(s, 1H, C3H, Pyr), 10.17 (s, 1H, CONH), 13.89 (s, 1H, NH) ppm; 13C-NMR (DMSO-d6):
δ = 36.6 (CH3),
39.2 (SCH3), 55.2 (C2OCH3), 55.4 (C4OCH3), 98.8 (C3H, (OCH3)2-Phe), 105.2 (C5H, (OCH3)2-Phe),
111.8 (C3H, Pyr), 113.3 (C5H, Pyrrole), 115.9 (C1, (OCH3)2-Phe), 116.0 (d, 2JCF = 22.4 Hz, C3/5H, F-Phe),
116.1 (C5H, Pyr), 118.5 (C4, Pyrrole), 124.0 (C2, Pyrrole), 125.8 (d, 4JCF = 3.3 Hz, C1, F-Phe), 127.5 (C6H,
(OCH3)2-Phe), 128.6 (C3H, Pyrrole), 131.3 (d, JCF = 8.3 Hz, C2/6H. F-Phe), 131.9 (C4, Imdz), 135.1
3
(C5, Imdz), 143.0 (C4, Pyr), 147.8 (C6H, Pyr), 148.5 (C2, Imdz), 152.9 (C2, Pyr), 156.7 (C4OCH3), 158.6
(C2OCH3), 159.9 (CO), 162.4 (d, 1JCF = 244.3 Hz, CF) ppm; MS (ESI, 70 eV) m/z 560 [MH]+.
3.3. Kinase Assays and IC50 Determination
In vitro kinase assays using 2
buffer containing 25 mM Tris-HCl [pH 7.5], 10 mM MgCl2, 100
inhibitor compounds were used in a dilution series ranging from 10
concentration which was prepared by serial dilution in DMSO. Recombinant human CK1
variant 1 (expressed and purified as GST fusion protein as described earlier [60]) and human GST-CK1
(Invitrogen) were used as sources of enzyme while -casein (C6780; Sigma-Aldrich) was used as
µ
Ci 32P-
γ
-ATP per reaction as co-factor were carried out in kinase
M EDTA, and 10 M ATP. Potential
M to 5 nM final reaction
transcription
µ
µ
µ
δ
ε
α
◦
substrate. Kinase reactions were incubated for 30 min at 30 C. Subsequently, reactions were separated
by SDS-PAGE and phosphorylated protein bands were visualized on dried gels by autoradiography.
The phosphorylated substrate protein bands were excised and phosphorylation was quantified by
Cherenkov counting. Dose-response analyses and calculation of IC50 values were carried out using
GraphPad Prism 6 statistical software (San Diego, CA, USA).

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3.4. Cell Culture
3.4.1. Cell Lines
The human pancreatic cancer cell lines Colo357 [61], Panc-1 [62], and MiaPaCa-2 [63] were grown
in Dulbecco’s modified Eagle’s medium (DMEM). Panc89 [64] was grown in DMEM:RPMI-1640
medium in ratio 1:1. The human colon adenocarcinoma cell line HT-29 [65] was grown in McCoy’s 5A
medium. All media were supplemented with 10% fetal calf serum (FCS), 100 units
·
mL−1 penicillin,
100
µg
·
mL−1 streptomycin and 2 mM glutamine. All cells were grown at 37 ◦C in a humidified 5%
carbon dioxide atmosphere.
3.4.2. Cell Assays and EC50 Determination
In order to determine the cytotoxic effects of investigated inhibitors conventional MTT assay
were used. 1
104 cells well−1 were seeded in 96-well cell culture plates and cultivated for 24 h. Cell
lines were exposed to increasing concentrations of the indicated inhibitor compounds, with untreated
and DMSO-treated cells serving as control. After an incubation period of 48 h 10 L of MTT solution
(5 mg
mL−1 in PBS) were added and cells were incubated for 4 h. MTT-containing medium was
carefully removed and 100 L acidic isopropanol (0.04 N HCl in isopropanol) per well were added.
×
·
µ
·
µ
For dissolution of formazan crystals plates were shaken for 30 min on an orbital shaker in the dark.
Finally, dissolved crystals were measured spectrophotometrically at 570 nM. All experiments were
performed in triplicate with four technical replicates per assay. Results were normalized considering
the mean optical density value of control wells as 100%. GraphPad Prism 6 (La Jolla, CA, USA)
software was used to calculate EC50 values.
3.5. X-ray Crystallography
3.5.1. Protein Expression, Purification, and Crystallisation of CK1δ
BL21 (DE3) TaKaRa 2 cells (Clontech) were transformed with the plasmid pET28a-tCK1
contains a codon-optimized construct of CK1 spanning residues 1–294 in a pET28a vector (NdeI and
XhoI restriction sites) and streaked out on LB agar plates containing 50
mL−1 kanamycin and
20
mL−1 chloramphenicol as selective antibiotics. A pre-culture was prepared by inoculating
cells from a single colony in LB medium supplemented with selective antibiotics and 4 mg
mL−1
δ which
δ
µg
·
µg
·
·
L-arabinose and overnight cultivation at 37 ◦C. Expression cultures were prepared by diluting a fresh
pre-culture with LB medium (plus selective antibiotics and 4 mg
·
mL−1 L-arabinose) to an optical
◦
density of 0.1 at 600 nm (OD600). Expression cultures were c◦ultivated at 37 C until an OD600 of
0.6 was reached, then cultivation temperature was reduced to 20 C and tCK1
δ
expression was induced
by adding 0.5 mM IPTG (isopropyl-
β
-D-1-thiogalactopyranoside). 16 h after IPTG addition, cells were
harvested by centrifugation (4000
×
g, 20 min 4 ◦C), washed with TBS (20 mM Tris pH 7.5, 300 mM
NaCl), and stored at −80 ◦C until purification.
Cells were thawed and resuspended (4 mL
g−1 wet cell pellet) in TBS plus 0.5 mM TCEP and
·
subsequently lysed on ice by sonication (Vibra-Cell VCX500, Sonics, Newtown, CT, USA). The resultant
lysate was clarified by ultracentrifugation (165,000
g at 4 ◦C for 30 min) and supplemented with
5 mM imidazole pH 7.5 before loading it on 2 mL TALON® (Clontech Takara Bio Europe SAS,
Saint-Germain-en-Laye, France) resin. After a wash step with TBS containing 10 mM imidazole, tCK1
×
δ
was eluted with TBS and 120 mM imidazole, concentrated, and applied to a Superdex S200 16/600
size exclusion chromatography column using TBS as chromatography buffer. Fractions containing
monomeric tCK1δ were pooled and concentrated to 10 mg/mL and stored at −80 ◦C.
For co-crystallization of tCK1
δ
with 16b, protein stock solution (10 mg
mL−1) was mixed 30:1
·
with 10 mM 16b (solubilized in DMSO) and incubated for 30 min at room temperature. Sitting drop
crystallization trials were set up at 4 ◦C with drop ratios of 3
L protein/inhibitor solution to 2
precipitant solution. Crystals appeared after three to four days in drops containing 0.1 M Hepes pH
µ
µL