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

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

IC₅₀CK1 = 25 nM), 12a (IC₅₀CK1 = 19 nM, IC₅₀CK1

IC₅₀CK1ε = 81 nM) being among the most potent CK1

δ

with compounds 11b (IC₅₀CK1

δ

= 4 nM,

= 8 nM,

ε

δ

ε

= 227 nM), and 16b (IC₅₀CK1

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

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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

δ

ε

1–3

δ

ε

2–5

δ

–

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 (IC₅₀CK1 = 1 M, IC₅₀CK1 = 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 IC₅₀values in the low nanomolar range ( IC₅₀CK1 = 5 nM, IC₅₀CK1 = 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, IC₅₀CK1 = 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 IC₅₀values 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.

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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

sp³hybridized 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 S_N2 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

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 IC₅₀Determination

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, IC₅₀values 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, IC₅₀values measured by ProQinase appear slightly lower due to the different assay setup

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such as the ATP concentration used (compare Experimental Section/Supporting Information and

ww w .proqinase.com).

Table 2. IC₅₀values 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 IC₅₀value 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. IC₅₀values of key inhibitors for CK1δ and p38α MAPK. IC₅₀values 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 IC₅₀values 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

IC₅₀for CK1 of 5 nM [28] a side chain without both carbonyl group and -bond resulted in

considerably decreased inhibition (e.g., series 1, 10a IC₅₀CK1 = 386 nM). In contrast, compounds

possessing the carbonyl group but without -bond afforded chemically stable series 2 (e.g., 11a IC₅₀

CK1δ = 20 nM IC₅₀CK1ε = 129 nM) and thereby restored potency almost to the level reported for lead

IC₅₀CK1δ = 5 nM IC₅₀CK1ε = 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 IC₅₀values 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

δ

π

δ

π

,

1

(

,

ε

π

1

δ

δ/ε

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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 IC₅₀values 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).

Molecules 2017, 22, 522

12 of 37

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

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

IC₅₀value 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 EC₅₀Determination

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

Molecules 2017, 22, 522

13 of 37

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 EC₅₀value of 9.3 M in HT-29 cells (data can be found in Supporting Information).

For the subsequently designed compound series EC₅₀data 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 EC₅₀values can be

found in Table 4).

,

15

,

16]. In general, amines 10a

–

k

µM with 10e being most

µ

–

e

–

b

–k

µ

Table 4. EC₅₀values 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.

EC₅₀Values (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.

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 EC₅₀values 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 IC₅₀values 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-d₆with 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 CDCl₃was used, DMSO-d₆was 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⁻¹or 20.5 mL min⁻¹. Progress of reactions was monitored by thin-layer chromatography

µm) (Macherey-Nagel, Düren,

µ

·

(TLC) performed with Macherey-Nagel 0.2 mm Polygram^®SIL G/UV₂₅₄pre-coated silica gel polyester

sheets and Silicagel 60 RP-18 F₂₅₄modiﬁed 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

15 of 37

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 KH₂PO₄buffer 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. Na₂SO₄and solvent was removed under reduced pressure. Recrystallization from

ethyl acetate afforded

¹H-NMR (DMSO-d₆):

(m, 2H, C^3/5H, F-Phe), 8.11–8.19 (m, 3H, C^2/6H, F-Phe, C⁶H, Pyr) ppm; ¹³C-NMR (DMSO-d₆):

3 ;

as colorless solid. Yield 23.9 g (quant.); C₁₃H₉F₂NO (M_r233.22); 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, ⁴J_CF= 2.8 Hz, CH₂), 110.8 (d, ²J_CF= 37.6 Hz, C³H, Pyr), 115.8 (d, ²J_CF= 22.0 Hz, C^3/5H, F-Phe),

123.8 (d, ⁴J_CF= 3.8 Hz, C⁵H, Pyr), 131.3 (d, ³J_CF= 9.6 Hz, C^2/6H, F-Phe), 132.9 (d, ⁴J_CF= 2.7 Hz, C¹,

F-Phe), 147.0 (d, ⁵J_CF= 15.5 Hz, C⁶H, Pyr), 150.7 (d, ³J_CF= 8.5 Hz, C¹, Pyr), 163.1 (d, ¹J_CF= 234.3 Hz,

C²F, Pyr), 165.2 (d, ¹J_CF= 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, C^2/6H, 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, ²J_CF= 38.9 Hz, C³H, Pyr), 116.7 (d, ²J_CF= 22.5 Hz, C^3/5H, F-Phe),

4

δ

as colorless solid. Yield 2.46 g (95%); C₁₃H₈F₂N₂O₂(M_r262.22);

= 7.19 (bs, 1H, C³H, Pyr), 7.37–7.46 (m, 3H, C⁵H, Pyr and C^3/5H,

δ

118.2 (d, ⁴J_CF= 4.2 Hz, C⁵H, Pyr), 130.8 (d, ⁴J_CF= 2.7 Hz, C¹, F-Phe), 132.2 (d, ³J_CF= 10.1 Hz, C^2/6H,

F-Phe), 144.3 (d, ³J_CF= 8.4 Hz, C⁴, Pyr), 148.8 (d, ⁵J_CF= 15.6 Hz, C⁶H, Pyr), 151.9 (d, ⁴J_CF= 4.0 Hz,

CNOH), 163.5 (d, ¹J_CF= 235.6 Hz, C²F, Pyr), 166.0 (d, ¹J_CF= 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 (M_r284.69); m.p. 216 C; ¹H-NMR

ether afforded

(DMSO-d₆):

δ

= 6.56 (s, 1H, CH), 7.35–7.41 (m, 2H, C^3/5H, F-Phe), 7.52–7.54 (m, 2H, C^3/5H, Pyr), 8.21

3

4

3

(dd, J = 8.4 Hz, J = 5.6 Hz, 2H, C^2/6H, 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, J_CF= 39.3 Hz, C³H, Pyr), 116.9

2

(

d, ²J_CF= 22.1 Hz, C^3/5H, F-Phe), 122.3 (d, ⁴J_CF= 4.2 Hz, C⁵H, Pyr), 130.0 (d, ⁴J_CF= 2.7 Hz, C¹, F-Phe),

Molecules 2017, 22, 522

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132.9 (d, ³J_CF= 9.8 Hz, C^2/6H, F-Phe), 147.4 (d, ³J_CF= 8.0 Hz, C⁴, Pyr), 149.4 (d, ⁵J_CF= 15.0 Hz, C⁶H,

Pyr), 163.5 (d, ¹J_CF= 236.7 Hz, C²F, Pyr), 166.3 (d, ¹J_CF= 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 (M_r303.33);

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 C^3/5H, F-Phe), 7.49–7.54 (m, 2H, C^2/6H, 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, ²J_CF= 39.5 Hz, C³H, Pyr),

2

4

115.9 (d, J_CF= 21.6 Hz, C^3/5H, F-Phe), 118.5 (d, J_CF= 2.5 Hz, C⁵H, Pyr), 126.5 (C¹, F-Phe), 130.9

(C^2/6H, F Phe), 132.9 (d, ³J_CF= 8.6 Hz, C⁴, Pyr), 143.0 (C², Imdz), 147.6 (d, ³J_CF= 16.1 Hz, C⁶H, Pyr),

162.1 (d, ¹J_CF= 246.4 Hz, CF, F-Phe), 163.7 (d, J_CF= 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, C^3/5H, Pyr), 7.17–7.27

(m, 2H, C^3/5H, F-Phe), 7.48 (bs, 2H, C^2/6H, 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 (M_r300.36); ¹H-NMR

δ

¹³C-NMR (DMSO-d₆):

δ

= 15.1 (SCH₃), 105.1 (C⁵H, Pyr), 110.0 (C³H, Pyr), 115.7 (d, ³J_CF= 21.1 Hz,

C^3/5H, F-Phe), 127.0 (s, C⁴/C⁵, Imdz), 129.2 (s, C¹, F-Phe), 130.5 (d, ⁴J_CF= 6.6 Hz, C^2/6H, 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(M_r196.25); 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, C^3/5H, (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 (C^2/4OCH₃), 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

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oil. Yield 205 mg (60%); C₁₁H₁₅BrO (M_r259.14); 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, C^2/4OCH₃), 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, C^3/5H, Pyr and C^3/5H, (OCH₃)₂-Phe and CH₂CH₂CH₂NH),

7.01 (d, ³J = 8.2 Hz, 1H, C⁶H, (OCH₃)₂-Phe), 7.22 (m, 2H, C^3/5H, F-Phe), 7.48 (m, 2H, C^2/6H,

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 (C^3/5H,

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 (C^2/6H, 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. NaHCO₃solution 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 (M_r436.51); 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, C^3/5H, F-Phe),

7.41–7.46 (m, 2H, C^2/6H, 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, ²J_CF= 21.8 Hz, C^3/5H, F-Phe), 122.5 (C, Imdz), 123.6 (C⁶H, (OCH₃)₂-Phe), 126.8

3

(

d, ⁴J_CF= 3.1 Hz, C¹, F-Phe), 130.0 (d, J_CF= 8.1 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r420.51);

◦

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, C^3/5H, F-Phe), 7.21 (bs, 1H, NH), 7.37–7.41 (m, 2H, C^2/6H, 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, J_CF= 21.7 Hz,

C^3/5H, 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, ³J_CF= 8.1 Hz, C^2/6H, F-Phe), 143.2 (C², Imdz), 146.8 (C⁶H, Pyr), 148.7 (COEt), 155.5 (C², Pyr),

162.5 (d, ¹J_CF= 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 (M_r464.56); 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, C^2/5/6H, (OCH₃)₂-Phe), 6.91–6.96 (m, 2H, C^3/5H, F-Phe), 7.32–7.36 (m, 2H, C^2/6H,

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, ²J_CF= 21.7 Hz, C^3/5H, F-Phe),

120.7 (C⁶H, (OCH₃)₂-Phe), 130.3 (d, ³J_CF= 8.2 Hz, C^2/6H, F-Phe), 131.4 (C^4/5, Imdz), 142.9 (C⁴, Pyr),

146.7 (C⁶H, Pyr, 147.7 (C⁴OCH₃), 149.0 (C³OCH₃), 158.3 (C², Pyr), 162.6 (d, ¹J_CF= 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

◦

(M_r443.54); 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, C^3/5H, 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, C^2/6H,

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, ²J_CF= 21.6 Hz, C^3/5H, F-Phe), 118.6

(C⁷H, Indole), 119.4 (C⁶H, Indole), 122.1 (C⁵H, Indole), 122.5 (C²H, Indole), 127.2 (C^8a, Indole), 130.3

(³J_CF= 8.3 Hz, C^2/6H, F-Phe), 136.4 (C^3a, 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 (M_r385.51); 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, C^3/5H, Pyr), 6.95–7.01 (m, 2H, C^3/5H, F-Phe), 7.35–7.39 (m, 2H, C^2/6H, 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, ²J_CF= 21.5 Hz, C^3/5H, F-Phe), 130.2 (d, ³J_CF= 8.1 Hz, C^2/6H, F-Phe), 142.7 (C², Imdz), 147.9 (C⁶H,

Pyr), 159.2 (C², Pyr), 162.4 (d, ¹J_CF= 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 (M_r369.46); m.p. 146 ^◦C; ¹H-NMR (DMSO-d₆):

δ

= 2.60 (bs, 3H, SCH₃), 2.73 (t, ³J = 4.5 Hz, 4H, C^2/6H₂, Piperazine), 3.31 (t, ³J = 4.6 Hz, 4H, C^3/5H₂,

Piperazine), 6.56 (d, ³J = 5.1 Hz, 1H, C⁵H, Pyr), 6.80 (bs, 1H, C³H, Pyr), 7.20–7.26 (m, 2H, C^3/5H, F-Phe),

7.47–7.51 (m, 2H, C^2/6H, F-Phe), 7.97 (d, ³J = 5.1 Hz, 1H, C⁶H, Pyr) ppm; ¹³C-NMR (DMSO-d₆): δ = 15.1

(SCH₃), 45.3 (C^2/6H₂, Piperazine), 45.8 (C^3/5H₂, Piperazine), 103.8 (C³H, Pyr), 110.6 (C⁵H, Pyr), 115.4

(d, ²J_CF= 21.6 Hz, C^3/5H, F-Phe), 130.3 (d, ³J_CF= 7.6 Hz, C^2/6H, F-Phe), 142.3 (C², Imdz), 147.6 (C⁶H,

Pyr), 159.6 (C², Pyr), 161.6 (d, ¹J_CF= 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 (M_r383.49);

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, C^3/5H₂, Piperazine), 2.57

(s, 3H, SCH₃), 3.40 (t, ³J = 4.8 Hz, 4H, C^2/6H₂, 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, C^3/5H, F-Phe), 7.32–7.37 (m, 2H, C^2/6H, F-Phe), 7.94

(d, ³J = 5.3 Hz, 1H, C⁶H, Pyr) ppm; ¹³C-NMR (CDCl₃):

δ

= 16.5 (SCH₃), 44.7 (C^2/6H₂, Piperazine), 45.6

(CH₃), 54.4 (C^3/5H₂, Piperazine), 104.9 (C³H, Pyr), 111.9 (C⁵H, Pyr), 115.7 (d, ²J_CF= 21.6 Hz, C^3/5H,

F-Phe), 128.2 (d, ⁴J_CF= 4.1 Hz, C^C1, F-Phe), 130.2 (d, ³J_CF= 8.1 Hz, C^2/6H, F-Phe), 142.8 (C², Imdz),

147.9 (C⁶H, Pyr), 159.6 (C², Pyr), 162.5 (d, ¹J_CF= 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 (M_r445.56); m.p. 101 ^◦C; ¹H- NMR (CDCl₃):

δ

= 2.64 (s, 3H, SCH₃), 3.19 (t, ³J = 5.1 Hz, 4H, C^2/6H₂, Piperazine), 3.58 (t, ³J = 5.1 Hz, 4H, C^3/5H₂,

Piperazine), 6.64 (dd, ³J = 5.4 Hz, ⁴J = 1.2 Hz, 1H, C⁵H, Pyr), 6.93–6.85 (m, 4H, C³H, Pyr and C^2/4/6H,

Phe), 6.99–7.05 (m, 2H, C^3/5H, F-Phe), 7.26 (t, ³J = 8.0 Hz, 2H, C^3/5H, Phe), 7.39–7.44 (m, 2H, C^2/6H,

F-Phe), 8.00 (d, J = 5.5 Hz, 1H, C⁶H, Pyr) ppm; ¹³C-NMR (CDCl₃):

δ

= 16.5 (SCH₃), 45.4 (C^2/6H₂,

3

Piperazine), 49.0 (C^3/5H₂, Piperazine), 105.1 (C³H, Pyr), 111.6 (C⁵H, Pyr), 115.8 (d, J_CF= 21.7 Hz,

C^3/5H, F-Phe), 116.3 (C^2/6H, Phe), 120.2 (C⁴H, Phe), 129.2 (C^3/5H, Phe), 130.3 (d, ³J_CF= 8.1 Hz, C^2/6H,

F-Phe), 143.0 (C², Imdz), 146.9 (C⁶H, Pyr), 151.1 (C¹, Phe), 159.0 (C², Pyr), 162.6 (d, ¹J_CF= 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 CO₂was 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 (SiO₂and 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

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to afford 11a as beige solid. Yield 177 mg (27%); C₂₆H₂₅FN₄O₃S (M_r492.57); m.p. 90 ^◦C; ¹H-NMR

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, C^3/5H, 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, ²J_CF= 24.1 Hz, C^3/5H, 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 C^2/6H, 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, ¹J_CF= 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 (M_r492.57); m.p. 91 ^◦C; ¹H-NMR

(DMSO-d₆):

δ

= 2.59–2.64 (m, 5H, SCH₃and 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, C^3/5H, F-Phe), 7.46–7.51 (m, 2H, C^2/6H,

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 (C^3/5H,

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 C^2/6H, F-Phe), 134.5 (C⁴, Imdz), 142.3 (C², Imdz), 147.7 (C⁴and C⁶H, Pyr), 151.2

(C², (OCH₃)₂-Phe), 152.5 (C², Pyr), 153.0 (C⁵, (OCH₃)₂-Phe), 161.9 (d, ¹J_CF= 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

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afford 11c as colorless solid. Yield 515 mg (63%); C₂₆H₂₅FN₄O₃S (M_r492.57); 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, C^3/5H, 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, ²J_CF= 21.8 Hz, C^3/5H,

F-Phe), 116.4 (C⁵H, Pyr), 121.4 (C⁶H, (OCH₃)₂-Phe), 123.7 (C⁵H, (OCH₃)₂-Phe), 126.6 (d, ⁴J_CF= 3.1 Hz,

C¹, F-Phe), 130.1 (C⁵, Imdz), 130.7 (d, J_CF= 8.4 Hz, C^2/6H, 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, ¹J_CF= 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, C^3/5H, F-Phe), 7.46–7.50 (m, 2H, C^2/6H, F-Phe), 8.10–8.34 (m, 2H, C^3/6H 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, ²J_CF= 22.5 Hz, C^3/5H, F-Phe), 116.4 (C⁵H, Pyr), 120.0 (C⁶H, (OCH₃)₂-Phe),

130.6 (C^2/6H, 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, ¹J_CF= 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 (M_r522.60); 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, C^3/5OCH₃), 6.56 (s, 2H, C^2/6H, (OCH₃)₃-Phe), 7.01 (dd, ³J = 5.3 Hz

,

⁴J = 1.6 Hz, 1H, C⁵H, Pyr), 7.26–7.32 (m, 2H, C^3/5H, F-Phe), 7.46–7.51 (m, 2H, C^2/6H, 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

(C^3/5OCH₃), 59.9 (C⁴OCH₃), 105.5 (C^2/6H, (OCH₃)₃-Phe), 110.6 (C³H, Pyr), 115.8 (d, ²J_CF= 21.7 Hz

,

C^3/5H, F-Phe), 116.4 (C⁵H, Pyr), 126.6 (d, J_CF= 3.3 Hz, C¹, F-Phe), 130.1 (C⁵, Imdz), 130.7

4

(

d, ³J_CF= 8.4 Hz, C^2/6H, 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 (C^3/5OCH₃), 162.0 (d, ¹J_CF= 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.

NaHCO₃solution, and sat. aq. NaCl solution, dried over anhyd. Na₂SO₄, and concentrated under

reduced pressure. The crude product was puriﬁed by ﬂash chromatography (SiO₂and RP-18, eluent

and mixing ratio given for each compound, respectively) to afford the particular compound.

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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 (M_r479.53); 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, C^3/5H, F-Phe and C³H, Pyr and C^2/6H, 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 (C^2/6H, F-Phe), 119.7 (C⁶H,

(OCH₃)₂-Phe), 121.9 (C¹, (OCH₃)₂-Phe), 130.7 (C^3/5H, 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, ¹J_CF= 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

(M_r479.53); 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, C^3/5H, F-Phe),

3

4

7.47–7.51 (m, 3H, C^2/6H, 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, ²J_CF= 22.0 Hz, C^3/5H, F-Phe), 126.5 (d, ⁴J_CF= 3.3 Hz, C¹, F-Phe), 129.6 (C¹, (OCH₃)₂-Phe), 130.4

(C⁵, Imdz), 130.8 (d, ³J_CF= 8.4 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r449.50); 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, C^3/5H, H₃CO-Phe), 6.92 (dd, ³J = 5.4 Hz, ⁴J = 1.5 Hz, 1H, C⁵H, Pyr), 7.30

(bs, 2H, C^3/5H, F-Phe), 7.39–7.44 (m, 2H, C^2/6H, H₃CO-Phe), 7.47–7.51 (m, 2H, C^2/6H, 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

(C^3/5H, H₃CO-Phe), 114.6 (C⁵H, Pyr), 115.8 (d, ²J_CF= 22.0 Hz, C^3/5H, F-Phe), 120.6 (C^2/6H, H₃CO-Phe),

130.6–130.8 (C^2/6H, 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, ¹J_CF= 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

(M_r433.51); 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, C^3/5H,

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, C^2/6H, F-Phe), 7.61 (bs, 1H, C³H, Pyr), 8.13 (bs, 1H, C⁶H, Pyr), 9.44 (s, 1H, NH),

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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, J_CF= 22.3 Hz, C^3/5H, F-Phe), 116.0 (C⁶H, Tol),

119.3 (C²H, Tol), 123.2 (C⁴H, Tol), 126.6 (d, ⁴J_CF= 2.6 Hz, C¹, F-Phe), 128.7 (C⁵H, Tol), 129.7 (C⁵, Imdz),

130.8 (C^2/6H, 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, ¹J_CF= 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 (M_r467.95); 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, C^5/6H, Cl-Tol), 7.28–7.34 (m, 2H, C^3/5H, F-Phe), 7.46–7.50 (m, 2H, C^2/6H,

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, J_CF= 21.7 Hz, C^3/5H, F-Phe), 117.6 (C⁶H,

2

Cl-Tol), 118.7 (C²H, Cl-Tol), 126.5 (d, J_CF= 3.1 Hz, C¹, F-Phe), 128.9 (C⁴, Cl-Tol), 130.4 (C⁵, Imdz),

4

130.8 (d, ³J_CF= 8.3 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r487.48); 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, C^3/5H, F-Phe and C⁴H, F₃C-Phe),

7.46–7.66 (m, 5H, C³H, Pyr and C^2/6H, F-Phe and C^5/6H, 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, ²J_CF= 21.8 Hz, C^3/5H, F-Phe), 118.7 (C⁴H, F₃C-Phe), 122.5 (C⁶H, F₃C-Phe), 124.2 (q, ¹J_CF= 272.1 Hz,

CF₃, F₃C-Phe), 126.5 (d, ⁴J_CF= 2.9 Hz, C¹, F-Phe), 129.6 (q, ²J_CF= 31.3 Hz, C³, F₃C-Phe), 130.0 (C⁵H,

F₃C-Phe), 130.5 (C⁵, Imdz), 130.8 (d, ³J_CF= 8.3 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r469.54); 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, C^3/5H, F-Phe),

7.42–7.59 (m, 5H, C³H, Pyr and C^2/6H, F-Phe and C^3/6H, Naph), 7.63–7.68 (m, 2H, C^4/7H, Naph), 7.95

(

d, ³J = 8.1 Hz, 1H, C⁵H, Naph), 8.16–8.22 (m, 2H, C^2/8H, 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, ²J_CF= 21.7 Hz, C^3/5H, F-Phe), 116.7 (C²H, Naph),

120.9 (C⁸H, Naph), 123.0 (C⁴H, Naph), 125.4 (C¹, Naph), 126.0 (C^3/6H, Naph), 126.3 (C⁷H, Naph), 126.5

(d, ⁴J_CF= 3.2 Hz, C¹, F-Phe), 128.6 (C⁵H, Naph), 130.6 (C⁵, Imdz), 130.8 (d, ³J_CF= 8.3 Hz, C^2/6H, F-Phe),

133.7 (C^4a, Naph), 134.0 (C^8a, 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, ¹J_CF= 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(M_r232.08); 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. Na₂CO₃solution

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(M_r289.33); 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. Na₂CO₃solution. 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(M_r289.33); 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(M_r261.28); 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

(M_r261.28); 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 (M_r543.62); 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, C^3/5H, 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, C^2/6H, 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, ²J_CF= 21.9 Hz, C^3/5H, F-Phe), 115.9 (C¹, (OCH₃)₂-Phe), 116.3 (C⁵H, Pyr), 118.5 (C⁴, Pyrrole), 124.1

(C², Pyrrole), 126.7 (d, ⁴J_CF= 3.5 Hz, C¹, F-Phe), 127.5 (C⁶H, (OCH₃)₂-Phe), 128.5 (C³H, Pyrrole), 130.8

(d, ³J_CF= 8.4 Hz, C^2/6H, 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, ¹J_CF= 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, C^3/5H, F-Phe),

7.50–7.55 (m, 2H, C^2/6H, 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, J_CF= 21.5 Hz, C^3/5H, 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 (C^2/6H, 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, ¹J_CF= 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. NaHCO₃solution, 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 (SiO₂and 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 (M_r436.51); 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

CH₂), 6.94–6.78 (m, 4H, C^4–6H, 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, C^3/5H, F-Phe and NH), 7.46–7.50 (m, 2H, C^2/6H, 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, ²J_CF= 21.7 Hz, C^3/5H, 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, ³J_CF= 8.2 Hz, C^2/6H, F-Phe), 146.7 (C², Imdz), 148.2 (C², EtO-Phe), 148.5

(C⁶H, Pyr), 156.0 (C², Pyr), 162.9 (d, ¹J_CF= 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

(M_r480.56); 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, C^2/6H, (OCH₃)₂-Phe),

3

6.71 (d, J = 8.7 Hz, 1H, C⁵H, (OCH₃)₂-Phe), 6.96–7.02 (m, 2H, C^3/5H, 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, ²J_CF= 21.7 Hz, C^3/5H, F-Phe), 120.7 (C⁶H,

(OCH₃)₂-Phe), 127.2 (C¹, F-Phe), 130.5 (d, ³J_CF= 8.2 Hz, C^2/6H, F-Phe), 131.5 (C¹, (OCH₃)₂-Phe), 135.3

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(C^4/5, 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, ¹J_CF= 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

(M_r508.57); 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, C^3/5H, 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, ²J_CF= 21.7 Hz, C^3/5H, 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, ³J_CF= 8.2 Hz, C^2/6H, 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,

¹J_CF= 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

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, C^3/5H, F-Phe), 7.51–7.55 (m, 2H, C^2/6H, 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, ²J_CF= 21.3 Hz, C^3/5H,

F-Phe), 115.9 (C⁶H, (OCH₃)₂-Phe), 126.5 (C¹, F-Phe), 130.8 (d, J_CF= 8.3 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r495.53); 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, C^3/5H, F-Phe), 7.46 (bs, 1H, C³H, Pyr), 7.51–7.56 (m, 2H, C^2/6H, 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, ²J_CF= 21.7 Hz, C^3/5H, F-Phe), 119.8 (C⁶H, (OCH₃)₂-Phe), 121.8 (C¹, (OCH₃)₂-Phe), 127.2

(C¹, F-Phe), 130.8 (d, ³J_CF= 8.4 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r465.50);

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, C^3/5H, F-Phe),

7.41 (d, ³J = 9.1 Hz, 2H, C^2/6H, H₃CO-Phe), 7.51–7.56 (m, 2H, C^2/6H, 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 (C^3/5H, H₃CO-Phe),

115.2 (C⁵H, Pyr), 115.9 (d, ²J_CF= 21.7 Hz, C^3/5H, F-Phe), 120.6 (C^2/6H, H₃CO-Phe), 127.5 (C¹, F-Phe),

130.9 (d, J_CF= 8.7 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r449.50); 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, C^3/5H,

F-Phe and C^2/6H, Tol), 7.52–7.57 (m, 2H, C^2/6H, 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, ²J_CF= 21.5 Hz,

C^3/5H, F-Phe), 116.0 (C⁶H, Tol), 119.3 (C²H, Tol), 123.2 (C⁴H, Tol), 128.7 (C⁵H, Tol), 130.9 (C^2/6H, 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, ¹J_CF= 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 (M_r483.95); 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, C^3/5H, F-Phe), 7.52–7.56 (m, 2H, C^2/6H, 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, ²J_CF= 21.5 Hz, C^3/5H, F-Phe),

117.5 (C⁶H, Cl-Tol), 118.7 (C²H, Cl-Tol), 129.0 (C⁴, Cl-Tol), 131.1 (C^2/6H, 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, ¹J_CF= 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, C^2/6H, 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, ³J_CF= 2.5 Hz, C³H, Pyr), 115.5 (C⁵H, Pyr),

115.9 (d, ³J_CF= 21.9 Hz, C^3/5H, F-Phe), 118.7 (q, ³J_CF= 2.1 Hz, C⁴H, F₃C-Phe), 122.5 (C⁶H, F₃C-Phe),

124.2 (q, ¹J_CF= 272.2 Hz, CF₃), 125.9 (d, ⁴J_CF= 3.2 Hz, C¹, F-Phe), 129.6 (q, ²J_CF= 31.4 Hz, C³, F₃C-Phe),

130.0 (C⁵H, F₃C-Phe), 130.8 (C⁵, Imdz), 131.1 (d, ⁴J_CF= 11.4 Hz, C^2/6H, 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, ¹J_CF= 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 (M_r485.54);

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, C^3/5H, F-Phe), 7.49 (t, ³J = 7.9 Hz, 1H, C³H, Naph), 7.54–7.59 (m, 4H, C³H, Pyr

and C^2/6H, F-Phe and C⁶H, Naph), 7.63–7.68 (m, 2H, C^4/7H, Naph), 7.96 (dd, ³J = 8.1 Hz, ⁴J = 0.8 Hz

,

3

1H, C⁵H, Naph), 8.16–8.20 (m, 2H, C^2/8H, 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, ²J_CF= 21.8 Hz, C^3/5H, 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 (C^2/6H, F-Phe), 133.7 (C^4a, Naph), 134.1 (C^8a, Naph), 143.5

(C⁴, Pyr), 146.7 (C⁶H, Pyr), 148.9 (C², Imdz), 152.6 (CO), 153.6 (C², Pyr), 162.3 (d, ¹J_CF= 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 (M_r559.62); 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, C^3/5H, 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, C^2/6H, 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, ²J_CF= 22.4 Hz, C^3/5H, F-Phe),

116.1 (C⁵H, Pyr), 118.5 (C⁴, Pyrrole), 124.0 (C², Pyrrole), 125.8 (d, ⁴J_CF= 3.3 Hz, C¹, F-Phe), 127.5 (C⁶H,

(OCH₃)₂-Phe), 128.6 (C³H, Pyrrole), 131.3 (d, J_CF= 8.3 Hz, C^2/6H. 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, ¹J_CF= 244.3 Hz, CF) ppm; MS (ESI, 70 eV) m/z 560 [MH]⁺.

3.3. Kinase Assays and IC₅₀Determination

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

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 IC₅₀values were carried out using

GraphPad Prism 6 statistical software (San Diego, CA, USA).

Molecules 2017, 22, 522

30 of 37

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⁻¹penicillin,

100

µg

·

mL⁻¹streptomycin and 2 mM glutamine. All cells were grown at 37 ^◦C in a humidiﬁed 5%

carbon dioxide atmosphere.

3.4.2. Cell Assays and EC₅₀Determination

In order to determine the cytotoxic effects of investigated inhibitors conventional MTT assay

were used. 1

10⁴cells well⁻¹were 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⁻¹in 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 EC₅₀values.

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⁻¹kanamycin and

20

mL⁻¹chloramphenicol 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⁻¹L-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⁻¹wet 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

Article Doi

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

DOI: 10.3390/molecules22040522

Source and publish data:

Authors:

Article abstract of DOI:10.3390/molecules22040522

Full text of DOI:10.3390/molecules22040522

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