3,4-Diaryl-isoxazoles and -imidazoles as potent dual inhibitors of p38α mitogen activated protein kinase and casein kinase 1δ

Article abstract of DOI:10.1021/jm9005127

In this study, we report on the discovery of isoxazole 1 as a potent dual inhibitor of p38α (IC₅₀ = 0.45 μM) and CK1δ (IC ₅₀ = 0.23 μM). Because only a few effective small molecule inhibitors of CK1 have been described so far, we aim

Full text of DOI:10.1021/jm9005127

7618 J. Med. Chem. 2009, 52, 7618–7630
DOI: 10.1021/jm9005127
3,4-Diaryl-isoxazoles and -imidazoles as Potent Dual Inhibitors of p38r Mitogen Activated Protein
Kinase and Casein Kinase 1δ†
Christian Peifer,*,‡, Mohammed Abadleh,‡ Joachim Bischof,§ Dominik Hauser,‡,^ Verena Schattel,‡ Heidrun Hirner,§
Uwe Knippschild,§ and Stefan Laufer‡
‡Department of Pharmacy, Eberhard-Karls-University, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany, §Department of General, Visceral
and Transplantation Surgery, Surgery Centre, University of Ulm, Steinhoevelstrasse 9, D-89075 Ulm, Germany, MRC Protein Phosphorylation
Unit, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland/U.K., and ^c-a-i-r biosciences
GmbH, Paul-Ehrlich-Strasse 15, 72076 Tuebingen, Germany
Received April 22, 2009
In this study, we report on the discovery of isoxazole 1 as a potent dual inhibitor of p38R (IC50=0.45
μM) and CK1δ (IC50=0.23 μM). Because only a few effective small molecule inhibitors of CK1 have
been described so far, we aimed to develop this structural class toward specific agents. Molecular
modeling studies comparing p38R/CK1δ suggested an optimization strategy leading to design, synth-
esis, biological characterization, and SAR of highly potent compounds including 9 (IC50 p38R=0.006
μM; IC50 CK1δ= 1.6 μM), 13 (IC50 p38R=2.52 μM; IC50 CK1δ=0.033 μM), 17 (IC50 p38R=0.019 μM;
IC50 CK1δ = 0.004 μM; IC50 CK1ε = 0.073 μM), and 18 (CKP138) (IC50 p38R = 0.041 μM; IC50
CK1δ =0.005 μM; IC50 CK1ε =0.447 μM) possessing differentiated specificity. Selected compounds
were profiled over 76 kinases and evaluation of their cellular efficacy showed 18 (CKP138) to be a highly
potent and dual-specific inhibitor of CK1δ and p38R.
Introduction
less potently inhibited by 1 (JNK2 87% inhibition at 10 μM,
IC50=1.28μM; JNK3 72% inhibition at 10 μM, IC50=0.54μM).
These results were in line with data from recent studies, which
identified classical p38R MAPK inhibitors such as 19 and
20 sharing the same pharmacophore moiety as 1 being able
to inhibit CK1δ (e.g., 19: CK1δ 92% inhibition at 1 μM,
IC50 = 0.12 μM, Scheme 1).5-7 Hence, we became interested
Small molecule inhibitors of various protein kinases are
utilized extensively in research and drug development. The
human kinome consists of more than 500 protein kinases, and
kinase inhibitors typically bind in the highly conserved ATP
pocket of these enzymes.1 Thus the specificity of ATP compe-
titive kinase inhibitors is of significant interest and represents
a crucial factor for their use in, e.g., signal transduction
research or therapeutic applications. Recently, we reported
on the substituted isoxazoles 1 and 2 (Table 1), which have
been originally designed and characterized as ATP competi-
tive p38R mitogen activated protein kinase (MAPKa) inhibi-
tors (IC50 p38R MAPK 1=0.45μM and 2=2.2 μM).2,3 These
compoundspossessthe typical vicinalpyridin-4-yl/4-F-phenyl
pharmacophore of first generation p38R MAPK inhibitors.
The well characterized p38R MAPK is deemed to be a key
player in the signal transduction of severe diseases such as
rheumatoid arthritis, asthma, and autoimmune disorders and
is considered to be a validated drug target for anti-inflamma-
tory agents.4 However, profiling compounds 1 and 2 in a
panel of 78 protein kinases at a concentration of 10 μM also
revealed significant inhibition of casein kinase 1 δ (CK1δ)
for 1 (90% inhibition), while its isomer 2 was less potent
(CK1δ 40% inhibition, Table 1). Subsequently, 1 was deter-
mined to inhibit CK1δ with an IC50 value of 0.23 μM.
Furthermore, in this panel, JNK2 and JNK3 were slightly
a Abbreviations: AMPK, 50AMP activated protein kinase; BRSK2, BR
serine-threonine kinase-2; BTK, Bruton agammaglobulinemia protein ki-
nase; CAMK1, Ca2þ/calmodulin-dependent kinase; CAMKKb, Ca2þ/cal-
modulin-dependent kinase kinase β; CDK, cyclin dependent protein kinase;
CHK, cell cycle checkpoint kinase; CK, casein kinase; CSK, C-terminal Src
kinase; DYRK, dual-specificity tyrosine-phosphorylated and -regulated
kinase; EF2K, elongation factor 2 kinase; EPH A2, epithelial cell kinase;
ERK, mitogen activated protein kinase 1; FGF-R1, fibroblast growth factor
receptor 1; GSK3, glycogen synthase kinase 3; HIPK2, homeodomain
interacting protein kinase 2; HP, hydrophobic pocket; HR, hydrophobic
region; IGF, insulin-like growth factor; IGF-1R, insulin-like growth factor
1 receptor; IKKb, I-κ-B-kinase β; IRR, insulin receptor-related receptor;
JNK, c-Jun N-terminal kinase; kd, kinase domain; Lck, lymphocyte-specific
protein tyrosine kinase; MAPK, mitogen activated protein kinase; MAPKAP-
K2, MAPK activated protein kinase-2; MARK, microtubule affinity regulat-
ing kinase; MELK, maternal embryonic leucine zipper kinase; MKK1,
MAPK kinase-1; MNK, MAPK interacting kinase; MSK1, mitogen and
stress activated protein kinase-1; MST2, mammalian STE20-like protein
kinase; PAK, p21 protein (cdc42/Rac)-activated protein kinase; PDK1, 3-
phosphoinositide-dependent protein kinase-1; PHK, phosphorylase kinase;
PI3K, phosphoinositide 3-kinase; PIM, proto-oncogene serine-threonine
protein kinase; PKA, protein kinase A; PKB/Akt, protein kinase B; PKC,
protein kinase C; PKD1, protein kinase D1; PLK, polo-like kinase; PRAK,
p38-regulated/activated protein kinase; PRK2, PKC-related kinase-2;
ROCK, F-dependent protein kinase; RSK, p90 ribosomal S6 kinase; S6K,
p70 ribosomal S6 kinase; SGK, serum- and glucocorticoid-induced kinase-1;
SmMLCK, smooth-muscle light chain kinase; Src, proto-oncogene tyrosine
kinase; SRPK1, serine/arginine protein kinase 1; SYK, spleen tyrosine
kinase; TBK1, NF-κ-B-activating kinase; VEG-FR, vascular endothelial
growth factor receptor; YES, proto-oncogene tyrosine protein kinase.
† Contribution to celebrate the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Correspondence to:
Phone: 0049 7071 29 75278. Fax: 0049 7071 29 5037. E-mail: Christian.
Peifer@uni-tuebingen.de.
r
pubs.acs.org/jmc
Published on Web 07/10/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7619
Table 1. Activity Profile of 1 and 2 at a Concentration of 10 μM in a Panel of 78 Protein Kinases Presented as Percentage of Kinase Activity Relative to
Control Assays without Compounda
1
2
1
2
MKK1
ERK1
76 ( 6
92 ( 8
86 ( 1
67 ( 1
13 ( 1
28 ( 0
20 ( 6
95 ( 13
84 ( 0
86 ( 6
84 ( 6
79 ( 2
113 ( 4
82 ( 5
94 ( 7
101 ( 4
76 ( 2
89 ( 2
28 ( 2
76 ( 6
88 ( 2
95 ( 7
81 ( 15
60 ( 1
95 ( 12
96 ( 5
110 ( 5
105 ( 3
104 ( 9
88 ( 3
91 ( 6
99 ( 5
75 ( 8
99 ( 7
96 ( 11
91 ( 6
104 ( 0
75 ( 0
96 ( 0
88 ( 12
87 ( 7
88 ( 1
98 ( 0
59 ( 6
63 ( 2
50 ( 4
98 ( 7
94 ( 9
95 ( 3
88 ( 6
83 ( 3
120 ( 2
85 ( 9
94 ( 9
104 ( 6
91 ( 3
98 ( 0
90 ( 4
77 ( 3
98 ( 10
104 ( 10
91 ( 4
89 ( 8
113 ( 2
89 ( 4
101 ( 7
100 ( 8
113 ( 5
98 ( 11
103 ( 9
97 ( 3
76 ( 9
110 ( 1
99 ( 2
102 ( 6
109 ( 2
85 ( 8
98 ( 8
PLK1
104 ( 5
104 ( 9
101 ( 7
110 ( 6
88 ( 8
95 ( 1
87 ( 4
78 ( 3
6 ( 2
96 ( 10
100 ( 10
101 ( 4
97 ( 5
93 ( 10
105 ( 10
22 ( 2
35 ( 3
73 ( 6
92 ( 2
93 ( 7
108 ( 10
55 ( 8
95 ( 3
PLK1 (OA)
AURORA B
AURORA C
AMPK
MARK3
BRSK2
MELK
CK1δ
ERK2
JNK1
JNK2
JNK3
p38R MAPK
p38β MAPK
p38γ MAPK
p38δ MAPK
ERK8
CK2
DYRK1A
DYRK2
DYRK3
NEK2a
NEK6
NEK7
IKKb
95 ( 8
RSK1
RSK2
104 ( 3
101 ( 5
96 ( 8
108 ( 13
100 ( 1
103 ( 8
89 ( 2
96 ( 1
PDK1
PKBR
PKBβ
SGK1
S6K1
98 ( 7
112 ( 12
104 ( 7
103 ( 0
100 ( 4
105 ( 8
81 ( 4
81 ( 7
PIM1
PIM2
PKA
ROCK 2
PRK2
PIM3
78 ( 5
86 ( 8
95 ( 2
SRPK1
MST2
EF2K
PKCR
PKCζ
PKD1
95 ( 2
107 ( 1
103 ( 0
92 ( 4
106 ( 12
98 ( 6
100 ( 1
72 ( 4
42 ( 0
87 ( 13
81 ( 10
93 ( 3
79 ( 5
88 ( 9
131 ( 1
82 ( 7
81 ( 2
107 ( 4
103 ( 6
100 ( 5
107 ( 11
94 ( 7
102 ( 3
90 ( 1
64 ( 1
95 ( 1
95 ( 10
94 ( 4
81 ( 14
102 ( 1
114 ( 8
101 ( 2
71 ( 10
80 ( 6
HIPK2
HIPK3
PAK4
MSK1
MNK1
MNK2
PAK5
PAK6
MAPKAP-K2
MAPKAP-K3
PRAK
Src
Lck
CAMKKa
CAMKKb
CAMK1
SmMLCK
PHK
CSK
FGF-R1
IRR
EPH A2
MST4
SYK
CHK1
CHK2
YES1
IKKe
TBK1
GSK3b
CDK2-cyclin A
90 ( 1
a Results are means ( SD of triplicates. OA: okadaic acid, further abbreviations of protein kinases are given in the list.
in investigating molecular similarities of p38R/CK1δ and
in the question if 1 (and related compounds) could serve
as lead for the development of potent and selective CK1
inhibitors.
cleavage of the C-terminal domain, and subcellular localiza-
tion.15 Physiologically, CK1 isoforms are involved in the
regulation of many different cellular processes such as
canonical Wnt signaling, DNA damage response, and cell
cycle progression.13,16 Regarding the fact that oversti-
mulation of CK1 isoforms has been linked to the incidence
of various types of disorders such as cancer (CK1R/δ/ε),17
neurodegenerative diseases (CK1δ),18 and inflammation,19
the use of CK1 (isoform)-specific inhibitors may have ther-
apeutic potential in the cure of these diseases.20 Moreover,
potent, (isoform)-specific and cell membrane perme-
able CK1 inhibitors would be useful reagents to further
investigate the complex biological roles of this protein
kinase family. Despite the actual need, only a few CK1-
specific small molecule inhibitors have been described so far
Biological Roles of CK1 and Inhibitors. Protein kinase CK1
represents a highly conserved Ser/Thr protein kinase family,
which is ubiquitously expressed in all eukaryotic organ-
isms. The seven mammalian isoforms CK1 R, β, γ1, γ2, γ3,
δ, and ε (with various splice variants) are all highly conserved
within their kinase domains (∼290 residues) but differ
significantly within their regulatory short N-terminal and
variable C-terminal (ranging in size from 40-180 residues)
domains.13 CK1 becomes constitutively activated due to pri-
ming phosphorylation by another kinase such as GSK3β.14
CK1 can be regulated by inhibitory autophosphorylation,

7620 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Scheme 1. Synthesis of Isoxazole Derivatives 6-10a
Peifer et al.
Table 2. Selected Compounds Reported to Have Potency against CK1δ
and p38R MAPK (nd*: To the Best of Our Knowledge, No Data Has
Been Published)
a (i) (a) LDA/THF, -78 ꢀC, 1 h; (b) isobutyraldeyde, 3 h; (c) Jones
oxidation K2Cr2O7/acetone, 0 ꢀC, 2 h. (ii) (a) Hydroxylamine HCl/
ethanol, 50%NaOH, rt, 2 h; (b) NCS/DMF, rt, 2 h. (iii) Triethylamine/
ethanol, 0 ꢀC, 2 h. (iv) RNH2, autoclave, 160 ꢀC, 16 h. (v) Pd2dba3,
Xantphos, CS2CO3, CH3CONH2, dioxane, reflux.
(Table 2). These include agents such as 21,9 22,10 and the
more potent and relatively CK1δ-specific compound 23,
which has been recommended for use in cell-based assays.6
Interestingly, 23 is actually chemically related to the com-
pounds described in this study and has been reported to also
inhibit p38R MAPK (62% at a concentration of 10 μM).
Molecular Modeling. To study a rationale for 1 being a
potent dual p38R MAPK/CK1δ inhibitor, we compared
the molecular architecture of their ATP binding pockets,
respectively. In fact, the highly conserved ATP binding
sites of p38R and CK1δ (Homologue) revealed a significant
structural difference in the gatekeeper position (Figure 1A).
Consistent with the in vitro assay data, modeling studies for 1
demonstrated reasonable binding modes in the ATP pocket
of both p38R and CK1δ (Figure 1B). In an analogous
manner to ATP binding, the pyridine ring nitrogen is accept-
ing a hydrogen bond from the backbone NH/Met109 (p38R)
and NH/Leu88 (CK1δ), respectively. The 4-fluor-phenyl
moiety is situated in the hydrophobic pocket I (HPI, “selec-
tivity pocket”) lined by gatekeeper residue Thr106 (p38R)
and Met85 (CK1δ), which are embraced by the vicinal
aromatic systems of the inhibitor, respectively.
Concerning an optimization strategy for lead 1, the ra-
tional binding modes suggested a second vicinal H-bond
interaction to the carbonyl oxygen of Met109/p38R and
Leu88/CK1δ in the hinge region (binding mode exemplified
for compound 10, see Figure 1C). Accordingly to this con-
cept, we hypothesized that such an interaction could be
achieved by introducing an NH moiety suitable as H-bond
donor at the 20 position of pyridine in 1.21 To prove this
hypothesis, we synthesized a small variety of amines (6-9)
and amides (10-13) and evaluated their SAR toward p38R
and CK1δ.
proceeds as a nucleophilic aromatic substitution (SN-Ar/
addition-elimination mechanism) and yielded derivatives
6-9. (X-ray analysis for 7, 8, and 9 is available in Supporting
Information). In contrast, substitution of the 2-fluoro-pyr-
idine moiety in 5 by amides turned out to be more difficult.
Initially, a strategy involving a modified Hartwig-Buch-
wald27 reaction was employed for the preparation of 10
(Scheme 1).
This reaction involved cross-coupling of 5 and 3 equiv of
acetamide in the presence of 2 mol % of Pd2dba3, 6 mol %
Xantphos, and 1.4 equiv of Cs2CO3 in anhydrous dioxane
under reflux. However, under these reaction conditions, the
yield of 10 was not satisfying (15%) and the purification
process was rather tedious. All attempts to increase the yield
by varying reaction conditions such as using other ligands,
bases, longer reaction time (>24 h), and higher temperature
(toluene, reflux) were unsuccessful. A major reason could be
the low reactivity of 2-fluoropyridine 5 in this reaction
whereby the strong fluorine-carbon bond disfavors the
oxidative insertion of the palladium catalyst. Accordingly,
we established a more convenient approach for the synthesis
of further amides by using the intermediate 2-aminopyridyl
12 as suitable precursor (Scheme 2).
Chemistry. To investigate SAR for the NH-substituted
20-pyridine position of lead 1, a flexible and straightforward
synthesis toward 20-aminopyridin-4-yl/4-fluorophenyl-iso-
xazoles was developed (Scheme 1). Key intermediate 5 was
prepared in high yield by our established method for the
synthesis of 3,4-diarylisoxazoles by 1,3-cycloaddition.2 Sub-
sequent substitution of the 2-fluoro-pyridine moiety by
secondary amines was achieved by conventional amination
at 160 ꢀC under high pressure in an autoclave.26 This reaction
An effective procedure for the preparation of 2-aminopyr-
idine 12 proved to be the reaction of 2-fluoropyridine
derivative 5 with sodium azide28 to yield the corresponding
tetrazolo derivative 11, which was reduced to 12 by several
techniques (Scheme 2). Two reduction methods were inves-
tigated for the preparation of 12 from 11: tin(II)chloride
dihydrate29 in methanol at 50 ꢀC and catalytic hydrogena-
tion performed at low hydrogen pressure with 10% Pd/C.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7621
Figure 1. (A, left) Sequence alignment of CK1δ Homologue (PDB code 1EH49), and p38R (PDB code 1YWR22) with highlighted residues of
the hinge region; (right) Overlay of ATP binding pocket of p38R (PDB 1A9U,23 gray) and CK1δ (PDB 1CKI,24 blue). Binding modes of 1 (B)
and 10 (C) in the ATP binding pocket of p38R (left) and CK1δ (right). Key residues and H-bond interactions to the hinge region (Met109/p38R;
Leu88/CK1δ) are shown. The vicinal pyridin-4-yl/4-F-phenyl moieties clasping around the gatekeeper residue (Thr106/p38R; Met85/CK1δ)
positioning the 4-F-phenyl in HPI (“selectivity pocket”).3 In the pose of 1 and 10 in p38R the isoxazole ring nitrogen is accepting an H-bond
from conserved but flexible Lys53.25 However, a comparable interaction was not calculated for Lys41 in CK1δ although this residue is situated
in a similar position.
The coupling reagent N,N0-carbonylimidazole (CDI) was
Scheme 2. Synthesis of Building Block 12 from 5 via Tetrazole
11a
utilized to activate (E)-(2,4-dimethoxyphenyl)-cinnamic acid
for the final step for the preparation of 15. Taken together,
intermediate 12 can be used as a building block to generate
2-amido substituted pyridine derivatives.
Biological Evaluation and Discussion
Compounds 6-10 and 13-15 were initially screened for
their biological activity in a p38R (IC50)8 assay and CK1δkd
a Reagents: (i) sodium azide, DMSO, 140 ꢀC, 3 h. (ii) Reduction
method (A) tin(II)chloride dihydrate, MeOH, 50 ꢀC, 3 h; or method (B)
autoclave H2 (5 bar), 10% Pd/C, MeOH, catalytic amount of HCl, room
temperature, 24 h.
(kinase domain)30 assay at a concentration of 10 μM. Agents
showing significant inhibition of CK1δkd were further
characterized for their IC50 value against CK1δkd and
GST-CK1δ (GST-rat CK1δ fusion protein full length;31
results are presented in Table 3). While the unsubstituted
lead 1 (3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl-pyridine)
actually proved to be a submicromolar dual inhibitor of these
kinases (IC50 p38R=0.45 μM, IC50 GST-CK1δ=0.23 μM),
the 20- pyridine-butylamine derivative 6 was determined with
an IC50 value of 0.03 μM against p38R but single digit μM-
activity against CK1δkd. Similar results were obtained for 7
(N-tetrahydro-2H-pyran-4-ylpyridin-2-amine) as well as for
enantioselective synthesized N-(1R/S-1-phenylethyl)pyridin-
2-amines 8 and 9 (structures proven by X-ray analysis, see
Although the latter method proceeded in a cleaner reaction,
the yield obtained from the former technique was higher.
Having 12 in hand, we next investigated methods for the
substitution of the aminopyridine moiety. The synthesis of
13 was successfully carried out by reacting 12 with freshly
prepared 2-(4-fluorophenyl)acyl chloride and triethyla-
mine in toluene under reflux. Accordingly, 14 was prepared
by using commercially available 2-phenoxypropanoylchlo-
ride and 4-(dimethylamino)pyridine in toluene at 60 ꢀC.

7622 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Peifer et al.
Table 3. Isoxazole Derivatives 6-10 and 13-15 and Their Biological
Activity against p38R, CK1δkd (Kinase Domaina) and GST-CK1δ
(GST-ratCK1δb)c
Leu88/CK1δ, not shown for clarity). The 4-F-phenyl is
situated in the “selectivity pocket” (HPI), whereas the pyridine
20-substituted moiety is accommodated by the hydrophobic
region II (HRII) toward the solvent accessible part of the ATP
binding site. In particular, the S-1-phenylethylpyridine side
chain moiety in 9 addresses a lipophilic cleft in HRII of p38R
(surrounded by Gly110/Ala111/Asp112/Ala157). This pose
is consistent with the stereochemically defined S-orientation
of 9, and a comparable binding mode was not found for its
R-enantiomer 8. In contrast to the situation in p38R, the
less spacious and planar HRII of CK1δ is forcing a
tilted conformation of 9, resulting in poorer overall geo-
metry of binding interactions. However, these binding
modes were in line with biological data and may explain the
excellent potency and partial selectivity of 9 for p38R over
CK1δ.
On the other hand, the binding modes of 15 in p38R and
CK1δ, respectively, revealed further differences in HRII,
which may account for the potency and relative selectivity
of 13 for CK1δ over p38R. Most importantly, the elongated
planar (E)-3-(2,4-dimethoxyphenyl)-N-acrylamide moiety in
15 is faintly situated in HRII of p38R, actually reaching well
into the solvent space. By contrast, in CK1δ the 2,4-dimethox-
yphenyl moiety is accommodated by Pro142/Arg141/Glu37/
Leu87 belonging to an extended loop (termed L7/8 loop)32
that is not present in p38R (Figure 3). Again, the binding
modes described above go parallel with the in vitro assay data
and therefore could explain the partial selectivity of 15 for
CK1δ over p38R.
Given this reasonable binding modes, it is noteworthy that
in principle the acrylamide moiety in 15 could function as a
Michael acceptor addressing a chemically reactive amino acid
residue (such as cysteine), thereby forming a covalent bond to
the protein. However, a cysteine residue capable of reacting
through this mechanism cannot be found either in HRII of
p38R or in HRII of CK1δ. Moreover, related agents 17 and 18
were shown to be ATP dependent inhibitors of CK1δ
(Figure 5, for details and discussion see below), providing
further evidence against the Michael reactivity.
Scaffold Hopping: From Isoxazoles to Imidazoles. Moti-
vated by the CK1δ-selective isoxazole-based inhibitor 15, we
wondered if the concept involving the (E)-3-(2,4-dime-
thoxyphenyl)-N-acrylamide moiety could be applied to an
imidazole scaffold that is present in prototypical p38R
inhibitors.4 To address this question, we reacted 4-[5-(4-
fluoro-phenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-
2-ylamine (16)33 with activated 2,4-dimethoxycinnamic acid
(CDI method) to yield compound 17. Subsequent sulfoxida-
tion using potassium peroxomonosulfate (Oxone) as an
oxidating agent gave compound 18 (Scheme 3).
Biological Characterization of Compounds 17 and 18
(CKP138). Compounds 17 and 18 were tested for their
potency against p38R and the GST-CK1δ fusion protein
(Table 4). Fortunately, these compounds could be identified
to be extremely effective in blocking GST-CK1δ kinase
activity (17 IC50 = 0.00448 μM; 18 IC50 = 0.01077 μM)
and thus are among the most potent inhibitors for CK1δ
known so far (see Table 2). However, low nM IC50 values
against p38R were also found (17 IC50=0.019 μM; 18 IC50=
0.041 μM). To assess CK1 isoform specificity, these two
inhibitors were additionally tested for their ability to inhibit
CK1ε, which is exhibiting the highest relation to CK1δ
among the CK1 isoforms. Interestingly, IC50 values deter-
mined for CK1ε are reduced by 15-fold for 17 and even
a Isoxazole derivatives 6-10 and 13-15 and their biological activity
against p38R, CK1δkd (kinase domain). b Isoxazole derivatives 6-10
and 13-15 and their biological activity against GST-CK1δ (GST-
ratCK1δ). c Results are presented as mean ( SD from experiments
performed at least in duplicate.
Supporting Information), the latter S-enantiomer having
excellent potency against p38R (IC50=0.006 μM) with strong
selectivity over CK1δ (IC50 CK1δkd = 1.6 μM, ratio IC50
p38R/CK1δkd = 0.0038).
In contrast, the amides 10, 13, 14, and 15 showed dual
activity against p38R and CK1δ. In detail, N-acetamide 10
and racemicN-2-phenoxypropanamide14weredetermined to
have comparable potency against p38R MAPK and CK1δ.
These results were in line with the binding mode of 10 in
both kinases (Figure 1C). Because 14 was prepared and
assayed as a racemic mixture, no inhibition data for the
defined enantiomers can be revealed. However, notable
selectivity for CK1δ was observed for N-(2-(4-fluorophe-
nyl)acetamide 13 (ratio IC50 CK1δkd/p38R = 0.07). More-
over, (E)-3-(2,4-dimethoxyphenyl)-N-acrylamide derivative
15 was determined to be the most potent and selective
CK1δ inhibitor in this series (IC50 CK1δkd = 0.033 μM,
IC50 p38R=2.52 μM; ratio IC50 CK1δkd/p38R=0.013).
To investigate the partial selectivityof amine 9 for p38R and
of amide 15 for CK1δ, we compared their modeled binding
modes in these kinases, respectively (Figure 2). Consistent
with the pose of lead 1 (Figure 1B) and according to the
design idea of pyridine 20 substitution (Figure 1C), similar
plausible binding modes for 9 and 15 in the ATP binding
pockets of p38R and CK1δ were calculated (Figure 2). These
involve central bidentate H-bond interactions of the pyridine-
nitrogen/20-NH-moiety to the hinge region (Met109/p38R;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7623
Figure 2. Comparison of modeled binding modes of 9 and 15 in the ATP binding pocket of p38R MAPK (PDB code 1YWR)22 and CK1δ
Homologue (PDB code 1EH4),9 respectively. The proteins are presented with their Connolly surface (colored in gray) to illustrate more clearly
structural differences especially in the solvent accessible part of HRII.
Scheme 3. Synthesis of 2-Acylaminopyridine-4-yl-imidazole
Derivatives 17 and 18a
Figure 3. Structure alignment of p38R MAPK (illustrated in black,
PDB code 1YWR)22 and CK1δ Homologue (illustrated in red, PDB
code 1EH4)9 with modeled binding mode of 15 (highlighted in
a (i) CDI, 2,4-dimethoxycinnamic acid, dioxane, room temperature.
(ii) THF, water, potassium peroxomonosulfate (Oxone reagent).
green) in the ATP binding pocket of CK1δ. Herein, the 2,4-
dimethoxyphenyl moiety of 15 interacts with a loop (termed L7/8
loop)32 not present in p38R.
block CK1δ activity independent from ATP concentrations
in the assay. However, this experiment provided evidence
that the acrylamide moiety in 17 and 18 is not covalently
linking the compounds to the protein.
41-fold for 18 (Figure 4) compared to those determined for
CK1δ. These results impressively demonstrate that in low
concentrations near the determined CK1δ IC50 values, com-
pounds 17 and especially 18 preferentially inhibit CK1δ.
Compounds 17 and 18 are ATP Competitive Inhibitors of
CK1δ. To prove these compounds as ATP competitive
inhibitors, 17 and 18 were tested at a concentration matching
the IC50 value for their potency to inhibit CK1δ in the
presence of varying amounts of ATP (Figure 5). This experi-
ment revealed that the IC50 values increased progressively as
the concentration of ATP escalated, thus confirming the
ATP competitive properties of 17 and 18. However, even in
presence of highest ATP concentration (500 μM), the com-
pounds still show an inhibition of kinase activity by 18% (17)
and 10% (18).
Compounds 17 and 18 Show No Significant Inhibition of a
CK1δ-Gatekeeper Mutant. In line with the binding modes of
isoxazoles (Figure 2), a comparable pose was determined for
18 in CK1δ (Figure 6, left). As discussed above, in this model,
amino acid 85 (methionine) takes the central role of gate-
keeper residue, which is embraced by the aromatic systems of
this compound thereby controlling access of the 4-F-phenyl
moiety to the “selectivity pocket”. To verify the gatekeep-
ing property of methionine a GST-CK1δM82F mutant
(corresponding to position 85 in the docking model) was
generated by site directed mutagenesis. Instead of methio-
nine, the mutant (referred to as GST-CK1δM82F) carries the
more bulky amino acid phenylalanine and therefore was
expected to prevent access of the compounds to the ATP
binding pocket while still binding ATP (cofactor ATP in
Regarding a possible irreversible binding mode of 17
and 18 bearing the acrylamide moiety (Michael reactivity
as discussed above), one would expect the compounds to

7624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Peifer et al.
Table 4. Imidazole Derivatives 17 and 18 and Their Biological Activity against p38R,8 CK1δkd (Kinase Domain), GST-CK1δ (GST-ratCK1δ Fusion
Protein), and CK1ε (Full Length Protein31 a
)
no.
IC50 (μM) p38R
IC50 (μM) CK1δkdb
IC50 (μM) GST-CK1δc
IC50 (μM) CK1εd
17
18
0.019
0.041
0.004 ( 0
0.005 ( 0
0.005 ( 0
0.011 ( 0.03
0.073 ( 0.01
0.447 ( 0.08
a Results are presented as mean ( SD from experiments performed at least in duplicate. b Imidazole derivatives 17 and 18 and their biological activity
against p38R, CK1δkd8 (kinase domain). c Imidazole derivatives 17 and 18 and their biological activity against GST-CK1δ (GST-ratCK1δ fusion
protein). d Imidazole derivatives 17 and 18 and their biological activity against CK1ε (full length protein).
Figure 4. Determination of CK1 isoform specificity. Compounds
17 (A) and 18 (B) exhibit different effects on GST-CK1δ (filled
circles/squares) and CK1ε (open circles/squares). Both compounds
more potently inhibit CK1δ, showing specificity toward this iso-
form in concentrations around the IC50’s determined for GST-
CK1δ.
Figure 5. Compounds 17 and 18 inhibit CK1δ in an ATP compe-
titive manner. Inhibitors 17 (5 nM) (A) and 18 (10 nM) (B) were
assayed on CK1δkd in the presence of the indicated ATP concen-
general is not occupying the selectivity pocket in kinases). In
vitro kinase assays were performed in the absence and pre-
sence of 17 and 18 at concentrations of 5 and 10 nM,
respectively, using GST-CK1δ or GST-CK1δM82F as a
source of enzyme. As expected, GST-CK1δ kinase activity
was clearly decreased in the presence of 17 (5 nM) and 18
(10 nM). In contrast, kinase activity of GST-CK1δM82F was
not affected in reactions containing 5 nM 17 or 10 nM 18,
respectively (Figure 6, right), thus strongly supporting the
modeled binding mode. However, kinase activity of GST-
CK1δM82F was increased in control reactions by 42% com-
pared to GST-CK1δ wild-type controls, suggesting a stron-
ger ATP turnover rate for the mutant. The increase in kinase
activity detected for GST-CK1δM82F may result from con-
formational alterations occurring in the mutant, which may
lead to a more effective binding of ATP or substrate leading
to enhanced incorporation of phosphate into the substrate.
Selectivity Profiling of Compounds 17 and 18 in a Panel of
76 Protein Kinases. To investigate the specificity of these
compounds, we studied their effect at concentrations of 0.1
μM in a panel of 76 protein kinases (Table 5, further details
available in Supporting Information). The assays used ATP
concentrations for each kinase which approximate the Km
constant for ATP.6 In this panel, CK1δ and p38R were
trations. Kinase assays were perfomed using CK1δkd as enzyme
and GST-p531-64 fusion protein (FP267) as substrate. Kinase
activity in reactions containing inhibitor was calculated relative
to the control reaction for each ATP concentration (n=3 ( SD).
While ATP concentrations increase, incorporation of radioactive
labeled phosphate into GST-p531-64 fusion protein FP267 de-
creases, leading to weakened signals in the autoradiographs.
potently inhibited by 17 and 18, while most of the kinases
were not affected significantly. However, at 0.1 μM, both
compounds blocked very few kinases to a similar extent (17:
p38β by 83%, JNK2 by 69%, MKK1 by 73% and YES1 by
76%; 18: p38β by 54%, JNK2 by 88%, MKK1 by 82%, and
YES1 by 86%). Importantly, in this panel, kinases involved
in regulation of CK1 such as GSK3β, PKC, PKA, and
CHK1 were not blocked, suggesting 18 could be a suitable
pharmacological tool to further investigate the biological
roles of CK1δ in cells.
Cellular Efficacy of Compounds: Inhibition of Growth of the
Human Extravillous Trophoblast Hybrid Cells AC1-M88.
Although potent inhibition of CK1δ could be shown for
a variety of compounds against the isolated enzyme,
they might not necessarily act the same way in cellular

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7625
experiments. To identify inhibitors that are able to pass cell
membranes and to show efficacy in more physiological
conditions, compounds were tested using the human immor-
tal, invasive extravillous trophoblast hybrid cell line (AC1-
M88) using apoptosis for readout as described previously.34
Because of the heterogeneous expression of a p53 poly-
morphism, AC1-M88 cells generally show a higher apoptosis
rate upon treatment with the CK1-specific inhibitor 21
(IC261) than the wild-type p53 expressing CV-1 cells.34 On
the basis of preliminary experiments (data not shown), in this
study AC1-M88 cells were treated with compounds 15, 17,
and 18 using a 50-fold concentration of their respective IC50
values. Namely AC1-M88 cells were incubated with 15 (3.32
μM), 17 (0.38 μM), 18 (0.26 μM), or CK1 inhibitor 21
(IC261, 8 μM)9 for 48 h, fixed and stained with propidium
iodide for flow cytometrical analysis. Treatment of AC1-
M88 cells with 21 (8 μM) as positive control9 led to a high
amount of dead cells after 48 h (84.0%; Figure 7). Interest-
ingly, application of test compounds also resulted in cell
death to a similar extend. After 48 h, almost all cells either
treated with 17 (87.1%), or 18 (77.1%) were dead, whereas
less amount of dead cells (34.4%) were observed upon
treatment with 15. In contrast, cells applied with solvent
alone (negative control, final assay concentration 0.004%
DMSO) had no effect on cell cycle progression. These
results correlate with the data from CK1δ enzyme assays
and clearly indicate cell permeant inhibitors 17 and 18 to
be highly effective in AC1-M88 cells at submicromolar
concentrations.
Figure 6. (left) Modeled binding mode of 18 in the ATP binding
pocket of CK1δ Homologue (PDB code 1EH49) to illustrate the role
of gatekeeper (center) controlling access of the 4-F-phenyl moiety to
the “selectivity pocket” (background). (right) Inhibition of CK1δ
wild-type and a M82F gatekeeper mutant by 17 and 18 as judged by
autoradiographs using GST-p531-64 fusion protein (FP267) as
substrate. While GST-CK1δ wild-type activity shows clear reduc-
tion by 17 (A) and 18 (B), the gatekeeper mutant GST-CK1δM82F is
barely influenced under the same conditions. As indicated in the
autoradiographs, the activity of GST-CK1δM82F was approxi-
mately 42% higher compared to GST-CK1δ wild-type.
Table 5. Activity Profile of 17 and 18 at a Concentration of 0.1 μM in a Panel of 76 PK Presented as % Mean Residual Activity to Control Assays
without Compound (n = 3 ( SD)a
17
18
17
18
MKK1
ERK1
27 ( 10
94 ( 10
91 ( 8
90 ( 1
31 ( 2
3 ( 4
18 ( 15
101 ( 5
93 ( 8
53 ( 2
12 ( 0
5 ( 4
AMPK
MARK3
BRSK2
MELK
CK1
92 ( 0
87 ( 2
86 ( 1
81 ( 5
10 ( 1
115 ( 8
78 ( 7
82 ( 10
104 ( 3
91 ( 4
90 ( 5
84 ( 11
98 ( 4
87 ( 22
81 ( 2
91 ( 8
97 ( 2
100 ( 2
94 ( 2
81 ( 8
94 ( 3
76 ( 2
49 ( 2
50 ( 9
81 ( 4
50 ( 0
61 ( 1
97 ( 5
90 ( 7
86 ( 1
24 ( 4
87 ( 1
33 ( 0
92 ( 2
94 ( 8
94 ( 7
103 ( 7
97 ( 0
99 ( 1
89 ( 0
97 ( 3
92 ( 5
18 ( 2
122 ( 6
86 ( 5
101 ( 13
90 ( 1
92 ( 8
111 ( 17
87 ( 2
109 ( 0
101 ( 5
90 ( 8
97 ( 7
112 ( 5
102 ( 8
104 ( 4
98 ( 1
105 ( 7
84 ( 3
63 ( 5
74 ( 7
79 ( 2
50 ( 14
83 ( 1
95 ( 21
86 ( 3
86 ( 9
14 ( 1
107 ( 7
46 ( 3
105 ( 5
94 ( 9
95 ( 17
106 ( 1
90 ( 1
ERK2
JNK1
JNK2
p38a MAPK
P38b MAPK
p38g MAPK
p38d MAPK
ERK8
CK2
17 ( 3
84 ( 17
90 ( 5
83 ( 0
78 ( 6
86 ( 7
74 ( 6
105 ( 1
98 ( 0
87 ( 7
67 ( 4
84 ( 7
81 ( 6
95 ( 7
110 ( 2
79 ( 13
88 ( 13
84 ( 2
91 ( 9
85 ( 11
82 ( 7
83 ( 4
88 ( 2
92 ( 13
82 ( 7
99 ( 8
80 ( 6
79 ( 2
81 ( 4
112 ( 16
97 ( 3
93 ( 10
46 ( 2
97 ( 15
100 ( 5
88 ( 0
80 ( 8
94 ( 14
94 ( 8
105 ( 7
100 ( 28
87 ( 4
74 ( 0
92 ( 5
88 ( 6
96 ( 2
103 ( 15
83 ( 8
97 ( 3
97 ( 3
96 ( 1
88 ( 1
78 ( 6
101 ( 6
92 ( 9
98 ( 7
93 ( 6
102 ( 3
88 ( 4
85 ( 3
101 ( 12
115 ( 4
110 ( 2
93 ( 8
DYRK1A
DYRK2
DYRK3
NEK2a
NEK6
IKKb
RSK1
RSK2
PDK1
PKBa
PIM1
PIM2
PIM3
PKBb
SGK1
SRPK1
MST2
EF2K
HIPK2
PAK4
PAK5
PAK6
Src
S6K1
PKA
ROCK 2
PRK2
PKCR
PKCζ
PKD1
MSK1
MNK1
Lck
CSK
MNK2
MAPKAP-K2
PRAK
FGF-R1
IRR
EPH A2
MST4
SYK
CAMKKb
CAMK1
SmMLCK
PHK
YES1
IGF-1R
VEG-FR
BTK
CHK1
CHK2
GSK3b
CDK2-cyclin A
PLK1
IR-HIS
EPH-B3
TBK1
IKKe
AURORA B
a Abbreviations of protein kinases are given in the list, further details of assays are available in Supporting Information.

7626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Peifer et al.
the single digit nanomolar range, these inhibitors are among
the most potent inhibitors for CK1δ known so far. Assaying
inhibitor potency on CK1ε also revealed a strong isoform
specificity of 17 and 18 toward CK1δ in low nanomolar
concentrations. However, only CK1δ and the closely related
CK1ε have been tested so far. Additional experiments are
necessary to characterize their effects on the CK1R and γ
isoforms and their various splice variants. Testing compounds
17 and 18 in the gatekeeper mutant (GST-CK1δM82F) assay
revealed that kinase activity was not influenced. This result
strongly supports the modeled binding mode in the ATP
binding pocket of CK1δ. The potent in vitro CK1δ inhibitors
17 and 18 demonstrated cellular efficacy due to their ability to
induce apoptotic processes in AC1-M88 cells, a cell line which
already has been shown to respond to treatment with 21
(IC261).34 However, CK1 is involved in a variety of cellular
functions and effects of CK1 specific inhibitors are dependent
on the cellular background.34 Therefore, further experiments
will be necessary to characterize the impact of these com-
pounds on cellular CK1δ signaling more deeply. Taken to-
gether, we recommend compounds 17 and 18 (CKP138) as
pharmacological tools to further investigate the exciting roles
of CK1δ in signal transduction pathways. Although com-
pounds described in this study can find use for applications
unraveling new functional properties of protein kinases, the
dissection of cellular effects derived from inhibition of CK1δ
and p38R MAPK, respectively, will be crucial. This could be
achieved by using the highly potent and selective p38R inhi-
bitor 9 compared to the dual p38R/CK1δ inhibitor 18
(CKP138). Targeting CK1 with specific inhibitors is effective
in particular in proliferative diseases and the demand for
potent CK1-specific inhibitors has increased over the last years.
Therefore, the development of such agents and their screening
for in vivo applications will get more in focus in future.
Experimental Section
Chemistry. Infrared spectra were recorded on a “Perkin
Elmer Spectrum One” infrared spectrophotometer. 1H (200
MHz, digital resolution 0.3768 Hz) and 13C (50 MHz, digital
resolution 1.1299 Hz) NMR were recorded on a Bruker AC
200. The data are reported as follows: chemical shift in ppm
from Me4Si as external standard, multiplicity and coupling
constant (Hz). GC/MS was performed on a HP6890 series
system. EI-Mass spectra were recorded on a Varian MAT
311A (70 eV). HRMS and FD-Mass spectra were recorded on
a MAT-95 (Finnigan). For clarity, only the highest measured
signal is given for FD-mass spectra. Melting points/decomposi-
tion temperatures were determined on a Buchi apparatus ac-
cording to Dr. Tottoli and are uncorrected. X-ray structure
determination was performed on a CAD4-Enraf-Nonius using
Cu KR radiation with graphite monochromator. Where appro-
priate, column chromatography was performed for crude pre-
cursors with Merck Silica Gel 60 (0.063-0.200 mm) or Acros
organics silica gel (0.060-0.200 mm; pore diameter ca. 60 nm).
Column chromatography for test compounds was performed
using a La-Flash-System (VWR) with Merck Silica Gel 60
(0.015-0.040 mm) or RP8 columns. The progress of the reac-
tions was monitored by thin-layer chromatography (TLC)
performed with Merck Silica Gel 60 F-245 plates. Where
necessary, reactions were carried out in a nitrogen atmosphere
Figure 7. Cell cycle FACS analysis of extravillous trophoblast
hybrid (AC1-M88) cells treated with compounds 15, 17, 18, and
21 (IC261). AC1-M88 cells were incubated with compounds 21
(IC261, 8 μM),9 15 (3.32 mM), 17 (0.38 mM), or 18 (0.26 mM) for 48
h, stained with propidium iodide, and analyzed in the flow cyt-
ometer using a Becton Dickinson “FACScan” flow cytometer.
DMSO treated control cells showed a normal cell cycle distribution
of asynchronously proliferating cells. Treatment of AC1-M88 cells
with 21 (IC261) or compounds 17 and 18 resulted in cell death to a
similar extent, whereas 15 was less effective.
Conclusion
˚
using 4 A molecular sieves. All reagents and solvents were
Derived from p38R inhibitor 1 as initial lead structure,
highly potent ATP competitive dual-specific inhibitors of
CK1δ and p38R could be identified, namely 17 (GST-CK1δ
IC50=0.004 μM; p38R IC50=0.019 μM) and 18 (GST-CK1δ
IC50=0.011 μM; p38R IC50 = 0.041 μM). With IC50 values in
obtained from commercial sources and used as received (THF
was used after distillation over K/benzophenone). Reagents
were purchased from Sigma-Aldrich Chemie, Steinheim, Ger-
many; Lancaster Synthesis, Muhlheim, Germany, or Acros,
Nidderau, Germany.

Article
HPLC analysis was performed on a Hewlett-Packard HP
1090 series II using a Thermo Betasil C8 (150 μM ꢀ 4.65 μM)
column (mobile phase flow 1.5 mL/min, gradient KH2PO4
buffer pH 2.3/methanol, UV-detection 230/254 nm). All key
compounds were proven by this method to show g95% purity.
Additionally, HRMS analysis was performed for test com-
pounds. A table summarizing the purity of key target com-
pounds can be found at Supporting Information.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7627
4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]-N-(1(R)-phe-
nylethyl)pyridin-2-amine (8). This compound was obtained ac-
cording to general procedure from compound 5 (0.3 g, 1 mmol)
and 1(R)-phenylethanamine (1.1 g, 9 mmol). The residue
was purified by flash chromatography (SiO2, eluent: petrol
ether 65%/ethyl acetate 35%) to afford the title compound
as white solid (36%). Melting point: 119.0 ꢀC. 1H NMR
(CDCl3, 200 MHz, ppm): δ (ppm) 1.17 (d, J = 6.98 Hz, 6H,
2 ꢀ CH3), 1.54 (d, J=6.77 Hz, 3H, CH3-phenylethanamine),
2.81 (m, 1H, CHMe2), 4.58 (m, 1H, CHMe), 5.23 (d, 1H,
NH), 5.98 (s, 1H, 4-Pyr), 6.30 (m, 1H, 4-Pyr), 6.97 (m, 3H, 4-
F-ph and phenylethanamine), 7.28-7.38 (m, 6H, 4-F-ph and
phenylethanamine), 8.05 (d, J = 4.95 Hz, 1H, 4-Pyr). 13C NMR
(CDCl3, 200 MHz, ppm): δ (ppm) 20.8 (2 ꢀ CH3), 24.6
(CHMe2), 26.2 (CH3), 52.2 (CHMe), 107.2 (C-5), 112.2 (C-40),
The synthetic procedures for preparation of 1 and its regioi-
somer 2 were described in our previous work.2 Experimental and
spectroscopic data for nonkey compounds 3, 4, and 5 can be
found at Supporting Information.
General Procedure for the Preparation of Compounds (6-9).
In an autoclave compound, 2-fluor-4-(3-(4-fluorophenyl)-5-iso-
propyl-4-yl)pyridine (5, 1 mmol) was suspended in an excess
volume of the respective amine (approx 10 mmol). The mixture
was stirred at 160 ꢀC for 24 h. The reaction was cooled to rt. The
resulted black residue was washed with water and extracted with
CH2Cl2. The organic phase was dried over Na2SO4 and con-
centrated in vacuo. The residue was purified by flash chroma-
tography (SiO2, eluent: petrol ether-ethyl acetate, mixing ratio
given for each compound, respectively) to afford the respective
compound.
2
114.3 (C-3), 115.5 (d, JC-F =21.7 Hz, C-300/C-500), 124.6 (d,
4JC-F =3.3 Hz, C-100), 125.5 (2 ꢀ CH3, ph), 127.0 (CH, ph),
128.6 (2 ꢀ CH3, ph), 130.1 (d, 3JC-F=8.4 Hz, C-200/C-600), 140.1
(C-4), 144.0 (CH, ph), 148.4 (C-6), 158.2 (C-2), 159.7 (C-30),
163.3 (d, 1JC-F=248 Hz, C-400), 174.9 (C-50). MS (EI) m/z: 401
(Mþ, 77%), 386 (100%). HRMS (EI) for C25H24FN3O (Mþ):
calcd, 401.19032; found, 401.18846. The compound was proven
by X-ray analysis to have the desired structure. For details see
Supporting Information. CCDC number: 727847.
N-sec-Butyl-4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]py-
ridin-2-amine (6). This compound was obtained according to
general procedure from compound 5 (0.2 g, 0.67 mmol) and
butan-2-amine (0.5 g, 7 mmol). The residue was purified by flash
chromatography (SiO2, eluent: petrol ether 75%-ethyl acetate
25%) to afford the title compound as white solid. Melting point:
136 ꢀC. 1H NMR (CDCl3, 200 MHz, ppm): δ (ppm) 0.89 (t, J =
7.48 Hz, 3H, sec-butylamine), 1.13 (d, J = 6.34 Hz, 3H, sec-ba),
1.36 (d, J=6.95 Hz, 6H, 2 ꢀ CH3), 1.47 (m, 2H, sec-butylamine),
3.19 (m, 1H, sec-butylamine), 3.49 (m, 1H, sec-butylamine), 4.55
(d, J = 7.98 Hz, 1H, NH), 6.07 (s, 1H, 4-Pyr), 6.35 (d, J = 5.13
Hz, 2H, 4-Pyr), 7.03 (m, 2H, 4-F-ph), 7.46 (m, 2H, 4-F-ph), 8.05
(d, J = 4.99 Hz, 1H, 4-Pyr). 13C NMR (CDCl3, 200 MHz, ppm):
δ (ppm) 10.2 (CH3, -2-But-amine), 20.0 (2 ꢀ CH3), 20.9 (CH3,
-2-But-amine), 26.4 (CHMe2), 29.5 (CH2, -2-But-amine), 48.5
(CH, -2-But-amine), 107.1 (C-5), 112.4 (C-40), 113.4 (C-3),
4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]-N-(1(S)-phenyl-
ethyl)pyridin-2-amine (9). This compound was obtained accord-
ing to general procedure from compound 5 (0.3 g, 1 mmol)
and 1(S)-phenylethanamine (1.15 g, 10 mmol). The residue
was purified by flash chromatography (SiO2, eluent: petrol
ether 65%/ethyl acetate 35%) to afford the title compound
as white solid (37%). Melting point: 120.5 ꢀC. 1H NMR
(CDCl3, 200 MHz, ppm): δ (ppm) 1.17 (d, J = 6.98 Hz, 6H, 2
ꢀ CH3), 1.54 (d, J = 6.77 Hz, 3H, CH3-phenylethanamine),
2.81 (m, 1H, CHMe2), 4.58 (m, 1H, CHMe), 5.18 (d, J = 5.85 Hz
1H, NH), 5.97 (s, 1H, 4-Pyr), 6.30 (m, 1H, 4-Pyr), 6.97 (m, 3H, 4-
F-ph and phenylethanamine), 7.28 (m, 6H, 4-F-ph and
phenylethanamine), 8.05 (d, J = 5.17 Hz, 1H, 4-Pyr). 13C
NMR (CDCl3, 200 MHz, ppm): δ (ppm) 20.8 (2 ꢀ CH3), 24.6
(CHMe2), 26.2 (CH3), 52.2 (CHMe), 107.2 (C-5), 112.2 (C-40),
2
114.3 (C-3), 115.5 (d, JC-F = 21.7 Hz, C-300/C-500), 124.6 (d,
2
4
115.5 (d, JC-F = 21.7 Hz, C-300/C-500), 124.8 (d, JC-F =3.3
4JC-F = 3.3 Hz, C-100), 125.5 (2 ꢀ CH3, ph), 127.0 (CH, ph),
3
Hz, C-100), 130.2 (d, JC-F =8.4 Hz, C-200/C-600), 139.9 (C-4),
3
128.6 (2 ꢀ CH3, ph), 130.1 (d, JC-F = 8.4 Hz, C-200/C-600),
148.7 (C-6), 158.5 (C-2), 159.9 (C-30), 163.4 (d, 1JC-F=248 Hz,
C-400), 174.8 (C-50). MS (EI) m/z for C21H24FN3O: 353.2 (Mþ,
100%).
140.1 (C-4), 144.0 (CH, ph), 148.4 (C-6), 158.2 (C-2), 159.7 (C-
30), 163.3 (d, 1JC-F = 248 Hz, C-400), 174.9 (C-50). (EI) m/z: 401
(Mþ, 77%), 386 (100%). HRMS (EI) for C25H24FN3O (Mþ):
calcd, 401.19032; found, 401.19286. The compound was proven
by X-ray analysis to have the desired structure. For details see
Supporting Information. CCDC number: 727848.
4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]-N-(tetrahydro-
2H-pyran-4-yl)pyridin-2-amine (7). This compound was obtained
according to general procedure from compound 5 (0.3 g, 1
mmol) and tetrahydro-2H-pyran-4-amine (0.8 g, 8 mmol). The
residue was purified by flash chromatography (SiO2, eluent:
petrol ether 25%/ethyl acetate 75%) to afford the title com-
pound as white solid (37%). Melting point: 117.0 ꢀC. 1H NMR
(CDCl3, 200 MHz, ppm): δ (ppm) 1.36 (d, J = 6.84 Hz, 6H, 2 ꢀ
CH3), 1.51 (m, 2H, tetrahydro-2H-pyran), 1.89 (m, 2H, tetra-
hydro-2H-pyran), 3.19 (m, 1H, CHMe2), 3.47 (m, 2H, tetrahy-
dro-2H-pyran), 3.73 (m, 1H, tetrahydro-2H-pyran), 3.97
(m, 2H, tetrahydro-2H-pyran), 4.49 (d, J=5.53 Hz, 1H, NH),
6.09 (s, 1H, 4-Pyr), 6.41 (d, J = 4.97 Hz, 1H, 4-Pyr), 7.04 (m, 2H,
4-F-ph), 7.46 (m, 2H, 4-F-ph), 8.09 (d, J = 5.15 Hz, 1H, 4-Pyr).
13C NMR (CDCl3, 200 MHz, ppm): δ (ppm) 20.9 (2 ꢀ CH3),
26.4 (CH), 33.3 (2 ꢀ CH2, tetrahydro-2H-pyran), 47.5 (CH,
tetrahydro-2H-pyran), 66.6 (2 ꢀ CH2, tetrahydro-2H-pyran),
107.8 (C-5), 112.2 (C-40), 113.9 (C-3), 115.7 (d, 2JC-F = 21.8 Hz,
C-300/C-500), 124.8 (d, 4JC-F=3.4 Hz, C-100), 130.3 (d, 3JC-F=8.4 Hz,
C-200/C-600), 139.9 (C-4), 148.6 (C-6), 157.8 (C-2), 159.9 (C-30),
N-[4-(3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl)pyridin-2-
yl]acetamide (10). A mixture of compound 5 (0.15 g, 0.5 mmol),
acetamide (0.50 g, 17 mmol), cesium carbonate (0.23 g, 0.7
mmol), Pd2dba3 (0.005 g, 0.005 mmol), and Xantphos (0.009 g,
0.016 mmol) was refluxed under argon atmosphere in dioxane
(10 mL). The progress of the reaction was followed by TLC.
Upon completion of the reaction, the mixture was cooled to rt
and partitioned between H2O and CH2Cl2. The aqueous phase
was separated and again extracted with CH2Cl2. The combined
organic layers were dried over Na2SO4 and filtered and the
solvent removed in vacuo. The oily residue thus obtained was
purified by flash chromatography (SiO2, eluent: petrol ether
50%/ethyl acetate 50%) to afford the title compound as white
solid (14%). Melting point: 180 ꢀC. 1H NMR (CDCl3, 200 MHz,
ppm): δ (ppm) 1.39 (d, J = 6.98 Hz, 6H, 2 ꢀ CH3), 2.23 (s, 3H,
acetamide), 3.25 (m, 1H, CHMe2), 6.73 (m, 1H, 4-Pyr), 7.04 (m,
2H, 4-F-ph), 7.41 (m, 2H, 4-F-ph), 8.20 (d, J = 4.53 Hz, 2H, 4-
Pyr), 8.33 (brs, 1H, NH). 13C NMR (CDCl3, 200 MHz, ppm): δ
(ppm) 20.8 (2 ꢀ CH3), 24.6 (CHMe2), 26.5 (CH3, acetamide),
111.8 (C-40), 114.4 (C-5), 115.7 (d, 2JC-F = 21.7 Hz, C-300/C-500),
1
163.4 (d, JC-F = 248 Hz, C-400), 175.6 (C-50). MS (EI)
m/z: 381 (Mþ, 28%), 254 (100%). HRMS (EI) for C22H24FN3O2
(Mþ): calcd, 381.18523; found, 381.18187. The compound
was proven by X-ray analysis to have the desired structure.
For details see Supporting Information. CCDC number:
727846.
120.7 (C-3) 124.5 (d, 4JC-F = 3.3 Hz, C-100), 130.4 (d, 3JC-F
8.4 Hz, C-200/C-600), 141.2 (C-4), 147.7 (C-6), 151.8 (C-2), 159.9
=

7628 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Peifer et al.
(C-30), 163.5 (d, 1JC-F=248 Hz, C-400, 168.7 (CH, acetamide),
175.5 (C-50). (EI) m/z: 339 (Mþ, 55%), 296 (100%). HRMS (EI)
for C19H18FN3O2 (Mþ): calcd, 339.13828; found, 339.13723.
7-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]tetrazolo[1,5-a]-
pyridine (11). Compound 5 (0.7 g, 2.33 mmol) and sodium azide
(0.5 g, 7 mmol) were heated in DMSO (5 mL) at 140 ꢀC for 3 h.
The title compound precipitated as beige solid upon addition of
water to the cold reaction mixture. It was collected by suction
filtration, dried, and stored at 5 ꢀC. 1H NMR (CDCl3, 200 MHz,
ppm): δ (ppm) 1.42 (d, J=6.97 Hz, 6H, 2 ꢀ CH3), 3.25 (m, 1H,
CHMe2), 6.88 (d, J=7.25 Hz, 1H, 4-Pyr), 7.07 (t, J=8.69, 2H, 4-
C-3000/C-5000) 120.9 (C-3) 124.5 (d, 4JC-F = 3.4 Hz, C-100), 129.4
(d, 4JC-F = 3.4 Hz, C-1000), 130.5 (d, 3JC-F = 8.4 Hz, C-200/C-
600), 131.0 (d, 3JC-F = 8.1 Hz, C-2000/C-6000), 141.2 (C-4), 147.9
(C-6), 151.5 (C-2), 159.8 (C-30), 162.3 (d, 1JC-F = 241.2 Hz, C-
4000), 163.4 (d, 1JC-F=248.1 Hz, C-400), 169.1 (Cq), 175.4 (C-50).
(EI) m/z: 433 (Mþ, 70%), 254 (100%). HRMS (EI) for
C25H21F2N3O2 (Mþ): calcd, 433.160155; found, 433.15855.
N-(4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl)-
2-phenoxypropanamide (14). 4-(Dimethylamino)pyridine (0.7 g)
was added to a solution of 2-phenoxypropanoyl chloride (0.14 g,
0.74 mmol) and compound 12 (0.2 g, 0.67 mmol) in toluene (5
mL). The resulting mixture was stirred for 3 h at 60 ꢀC and then
cooled to rt. Aqueous sodium hydrogen carbonate solution was
added to the reaction mixture, followed by extraction with
CH2Cl2 (2 ꢀ 20 mL). The organic phase was separated and the
combined organic extracts were dried over Na2SO4, and
the solvent was removed in vacuo. The residue was purified by
flash chromatography (SiO2, eluent: petrol ether 50%/ethyl
acetate 50%) to afford the title compound as white solid
F-ph), 7.40 (m, 2H, 4-F-ph), 8.76 (d, J=7.10 Hz, 1H, 4-Pyr). 13
C
NMR (CDCl3, 200 MHz, ppm): δ (ppm) 20.9 (2 ꢀ CH3), 26.6
(CHMe2), 110.7 (C-40), 115.7 (d, 2JC-F =22.1 Hz, C-300/C-500),
116.4 (C-6), 118.5 (C-8), 123.9 (d, 4JC-F=3.5 Hz, C-100), 125.2
(C-5), 130.4 (d, 3JC-F = 8.5 Hz, C-200/C-600), 135.3 (C-7), 148.7
1
(C-9), 159.8 (C-30), 163.7 (d, JC-F = 249.6 Hz, C-400), 176.1
(C-50).
4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-amine
(12). Method A. Compound 11 (0.4 g, 1.24 mmol) and tin(II)
chloride dihydrate (1.12 g, 5 mmol) were stirred in methanol
(10 mL) at 50 ꢀC for 3 h. The mixture was concentrated,
neutralized with sodium hydrogen carbonate and the resulting
salt was soaked with water, dichloromethane, and methanol,
and the filtrate was evaporated and then extracted by dichlor-
omethane, dried over Na2SO4, and evaporated. The residue was
purified by flash chromatography (SiO2, eluent: petrol ether
50%/ethyl acetate 50%) to afford the title compound as yellow
solid (54%).
Method B. A stirred solution of compound 11 (0.4 g, 1.24
mmol) in methanol/water (9.5 mL/0.5 mL) was treated with
hydrogen gas for 24 h at rt (pressure = 5 bar) in the presence Pd/
C (10%) and few drops of conc HCl. The mixture was soaked
with methanol (200 mL) and filtered. The filtrate was concen-
trated, neutralized with sodium hydrogen carbonate, and ex-
tracted with dichloromethane. The organic phase was dried over
Na2SO4 and the solvent was evaporated. The residue was
purified by flash chromatography (SiO2, eluent: petrol ether
25%/ethyl acetate 75%) to afford the title compound as yellow
solid (42%). 1H NMR (CDCl3, 200 MHz, ppm): δ (ppm) 1.34
(d, J = 6.99 Hz, 6H, 2 ꢀ CH3), 3.18 (m, 1H, CHMe2), 4.59 (brs,
2H, NH2), 6.29 (s, 1H, 4-Pyr), 6.45 (d, J = 3.89 Hz, 1H, 4-Pyr),
7.03 (m, 2H, 4-F-ph), 7.43 (m, 2H, 4-F-ph), 8.06 (d, J = 5.13 Hz,
1H, 4-Pyr). 13C NMR (CDCl3, 200 MHz, ppm): δ (ppm) 20.9
(2 ꢀ CH3), 26.4 (CHMe2), 109.1 (C-40), 112.0 (C-5), 115.6 (d,
2JC-F = 21.7 Hz, C-300/C-500), 116.1 (C-3), 124.7 (d, 4JC-F=3.3
Hz, C-100), 130.2 (d, 3JC-F = 8.4 Hz, C-200/C-600), 140.5 (C-4),
148.5 (C-6), 158.7 (C-2), 159.7 (C-30), 163.4 (d, 1JC-F=248.2 Hz,
C-400), 174.9 (C-50).
1
(50%). Melting point: 105 ꢀC. H NMR (CDCl3, 200 MHz,
ppm): δ (ppm) 1.41 (m, 6H, 2 ꢀ CH3), 1.67 (d, J = 6.75 Hz, 3H,
CH3 (2-phenoxypropanoylamide))3.27 (m, 1H, CHMe2), 4.82
(m, 1H, CH (2-phenoxypropanoylamide)), 6.76 (m, 1H, 4-Pyr),
6.98-7.10 (m, 5H, 4-F-ph and 2-phenoxypropanoylamide),
7.30-7.44 (m, 4H, 4-F-ph and 2-phenoxypropanoylamide),
8.24 (m, 2H, 4-Pyr), 9.04 (brs, 1H, NH). 13C NMR (CDCl3,
200 MHz, ppm): δ (ppm) 18.5 (CH3), 20.9 (2 ꢀ CH3), 26.5
(CHMe2), 74.9 (CH-phenoxypropanoylamide), 111.7 (C-40),
114.5 (C-5), 115.5 (2 ꢀ CH3, phenoxy), 115.7 (d, 2JC-F = 21.5 Hz,
C-300/C-500), 121.1 (C-3), 122.4 (CH, phenoxy), 124.5 (d, 4JC-F
=
=
3
3.3 Hz, C-100), 129.6 (2 ꢀ CH, phenoxy), 130.4 (d, JC-F
8.4 Hz, C-200/C-600), 141.3 (C-4), 147.8 (C-6), 151.1 (C-2), 156.5
1
(C-1000, phenoxy), 159.9 (C-30), 163.5 (d, JC-F=248.1 Hz,
C-400), 171.0 (Cq, CONH), 175.5 (C-50). (EI) m/z: 445 (Mþ,
5%), 121 (100%). HRMS (EI) for C26H24FN3O3 (Mþ): calcd
445.18014; found 445.18224.
(E)-3-(2,4-Dimethoxyphenyl)-N-(4-[3-(4-fluorophenyl)-5-iso-
propylisoxazol-4-yl]pyridin-2-yl)acrylamide (15). To a stirred
solution of trans-2,4-dimethoxycinnamic acid (0.2 g, 0.96 mmol)
in NMP (5 mL) was added CDI (0.16 g, 1.92 mmol). The
activation of the acid was controlled by tlc. Upon completion
of the reaction, compound 12 (0.14 g, 0.47 mmol) in NMP (2 mL)
was added and the reaction mixture heated at 120 ꢀC for 72 h
until all of 12 was consumed. The black residue obtained upon
evaporation of the solvent was washed with water and extracted
with CH2Cl2 (2 ꢀ 20 mL). The combined organic extracts were
dried over Na2SO4, and the solvent was removed in vacuo. The
residue was purified by flash chromatography (SiO2, eluent:
petrol ether 25%/ethyl acetate 75%) to afford the title com-
pound, which was further purified by preparative thick layer
chromatography (Silica Gel 60 F254, 2 mm, eluent: petrol ether
65%/ethyl acetate 35%) to afford the title compound as white
solid (18%). Melting point: 221 ꢀC. 1H NMR (CDCl3, 200 MHz):
δ (ppm) 1.41 (d, J = 6.34 Hz, 6H, 2ꢀ CH3), 3.26 (m, 1H, CH Me2)
3.86 (d, J = 4.71, 6H, 2 ꢀ -OCH3), 6.48-6.70 (m, 4H, 4-Pyr
and 3-(2,4-dimethoxyphenyl)acrylamide), 7.04 (m, 2H, 4-F-ph),
7.44 (m, 3H, 4-F-ph and 3-(2,4-dimethoxyphenyl)acrylamide),
7.94 (d, J=16.14 Hz, 1H, -CH (3-(2,4-dimethoxyphenylmac_cpb;
acrylamide)), 8.22 (d, J = 4.07 Hz, 1H, 4-Pyr), 8.42 (s, 1H,
4-Pyr), 8.60 (brs, 1H, NH). 13C NMR (CDCl3, 200 MHz, ppm):
δ (ppm) 20.7 (CH), 26.5 (2 ꢀ CH3), 55.3 (CH3, -OCH3), 55.4
(CH3, -OCH3), 98.4 (CH, Ar), 105.2 (CH, Ar), 111.7 (Cq), 114.7
(CH, Ar), 115.7 (d, 2JC-F=21.7 Hz, C-300/C-500), 116.4 (Cq, Ar),
118.3 (CH, CHdCH), 120.4 (CH, Ar), 124.6 (d, 4JC-F = 3.4 Hz,
2-(4-Fluorophenyl)-N-[4-(3-(4-fluorophenyl)-5-isopropylisox-
azol-4-yl)pyridin-2-yl]acetamide (13). 2-(4-Fluorophenyl)acetyl
chloride (0.072 g, 0.42 mmol) was added under stirring at rt
to a solution of compound 12 (0.125 g, 0.42 mmol) in toluene
(10 mL). Triethylamine (1 mL) was then added to the mixture,
and stirring was continued for 2 h at 80 ꢀC. The reaction mixture
was cooled to rt, and aqueous sodium hydrogen carbonate was
added. The resulting mixture was finally extracted with ethyl
acetate (2 ꢀ 30 mL). The combined organic layers were dried
over Na2SO4 and filtered, and the solvent was removed in vacuo.
The oily residue was purified by flash chromatography (SiO2,
eluent: petrol ether 70%/ethyl acetate 30%) to afford the title
1
compound as yellow solid (27%). Melting point: 176 ꢀC. H
NMR (CDCl3, 200 MHz, ppm): δ (ppm) 1.38 (d, J = 6.97 Hz,
6H, 2 ꢀ CH3), 3.23 (m, 1H, CH Me2),3.74 (s, 2H, 2-(4-F-
ph)acetamide), 6.71 (m, 1H, 4-Pyr), 6.98-7.14 (m, 5H, 4-F-ph
and 2-(4-F-ph)acetamide), 7.27 (m, 3H, 4-F-ph and 2-(4-F-
ph)aetamide), 7.95 (brs, 1H, NH), 8.15-8.20 (m, 2H, 4-Pyr).
13C NMR (CDCl3, 200 MHz, ppm): δ (ppm) 20.8 (2 ꢀ CH3),
26.5 (CHMe2), 43.9 (CH2), 111.8 (C-40), 114.3 (C-5), 115.7
3
C-100), 130.4 (d, JC-F = 8.4 Hz, C-200/C-600), 131.2 (CH, Ar),
139.0 (CH, CHdCH), 141.5(Cq), 147.1 (CH, Ar), 152.4 (Cq),
1
159.9 (Cq), 160.0 (Cq), 162.7 (Cq), 163.5 (d, JC-F = 248.1 Hz,
C-400), 165.4 (Cq), 175.6 (Cq). (EI) m/z: 487 (Mþ, 28%), 191
(100%). HRMS (EI) for C28H26FN3O4 (Mþ): calcd, 487.190701;
found, 487.19381.
2
2
(d, JC-F = 21.5 Hz, C-300/C-500), 116.2 (d, JC-F = 11.2 Hz,

Article
(E)-3-(2,4-Dimethoxy-phenyl)-N-(4-[5-(4-fluoro-phenyl)-2-meth-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7629
The side chain of E86 is situated in the backbone and not
affecting the binding mode of compounds. However, the “gate-
keeper” residue M85 is significantly important for the binding
mode of compounds. For clarity, the unliganded ATP-site of
CK1δ structure (PDB 1CKI24) was not suitable for modeling
experiments in this study.
Biological Evaluation. Inhibitor Solutions. All inhibitor solu-
tions were prepared freshly in DMSO prior to each experiment
and used immediately. Inhibitor solutions were light sensitive
and thus should be handled under light protection.
Plasmids. For the expression of rat CK1δ as GST- CK1δ
fusion protein the plasmidpGEX-2T (449) was used.31 The plas-
mid pGEX-2T-CK1δ1-428/M82F containing a mutation at amino
acid 82 (met f phe, “gatekeeper”) of rat CK1δ was constructed
using the QuikChange site-directed mutagenesis kit according
to manufacturers0 instructions (Stratagene, La Jolla, USA). The
plasmid pGEX-2T-CK1δ1-428 (449) served as template and the
complementary primers used were 50-tacaatgtcatggtgtttgagc-
tactgggacccagcctg-30 (50 primer) and 50-atgttacagtaccacaaactc-
gatgaccctgggtcggac-30 (30 primer).
Cell Lines. Cell line AC1-M88 was used for cell culture
experiments. AC1-M88 cells were generated by fusion of
extravillous trophoblasts with AC1-1, a mutant of the chor-
iocarcinoma cell line Jeg-3.35 AC1-M88 cells were grown in
DMEM/F-12 medium (Gibco, Karlsruhe, Germany) supple-
mented with 10% fetal calf serum (FCS; Gibco, Karlsruhe,
Germany) at 37 ꢀC in a humidified 5% CO2 atmosphere. Cells
were treated with various potential CK1 kinase inhibitors as
indicated and used for flow cytometry. Control cells were
treated with DMSO.
Flow Cytometry/FACS Analysis. For discrimination of living
and dead cells, cells were washed twice with PBS and resus-
pended in PBS containing propidium iodide. After 10 min
incubation FACS analyses were performed. Unstained cells
were regarded as living cells. For cell cycle analyses the “Cycle
Test Plus Kit” (Becton Dickinson, USA) was used according to
the manufacturer’s protocol and cells were analyzed using the
Becton Dickinson “FACScan” flow cytometer and the “Cell
Quest” software.
Overexpression and Purification of Glutathione S-Transferase
Fusion Proteins. The production and purification of the GST-
fusion proteins FP267, FP449, and GST-CK1δ1-428/M82F were
carried out as described elsewhere.30 CK1δkd was purchased
from NEB (Ipswich, USA), CK1ε from Invitrogen (Karlsruhe,
Germany).
In Vitro Kinase Assays. In vitro kinase assays were carried out
in presence of various potential inhibitors of CK1δ at an ATP
concentration of 0.1 mM and DMSO control as described
previously.31 In some cases ATP was used in concentrations of
0.05, 0.25, or 0.5 mM. Compounds 21 (IC261)9 and D446711,12
have been reported to inhibit CK1δ and were used as positive
controls. The GST-p531-64 fusion protein (FP267) was used as
substrate. Recombinant CK1δ kinase domain (CK1δkd, NEB,
Ipswich, USA), GST-CK1δ (FP449) and recombinant CK1ε
(Invitrogen, Karlsruhe, Germany) were used as sources of the
enzyme. Phosphorylated proteins were separated by SDS-
PAGE and the protein bands were visualized on dried gels by
autoradiography. The phosphorylated protein bands were ex-
cised and quantified by Cherenkov counting.
ylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl)-acrylamide (17). (E)-2,4-
Dimethoxy cinnamic acid (1.25 g, 6.0 mmol) and CDI (1.04 g,
6.4 mmol) were stirred at room temperature in 10 mL of dry
DMF for 3 h and 4-[5-(4-fluoro-phenyl)-2-methylsulfanyl-3H-
imidazol-4-yl]-pyridin-2-ylamine 16 (0.608 g, 2.02 mmol) was
added. The mixture was stirred for 10 h at 110 ꢀC. The solvent
was removed, and 50 mL of water and 50 mL of ethylacetate
were added to the residue. The phases were separated and the
organic phase was washed twice with water and once with brine.
The solvent was dried over Na2SO4, evaporated, and the residue
recrystallized from ethyl acetate. Yield: 275 mg (27.7%). Melt-
1
ing point: 225.0 ꢀC. HPLC: 7.97 min, 98.0%. H NMR (200
MHz, THF-d8): δ ppm 2.66 (s, 3 H, SOCH3), 3.81 (s, 3 H, ortho-
OMe), 3.88 (s, 3 H, para-OMe), 6.54 (m, 2 H, Ar.5-H, Ar.3-H),
6.79 (d, J = 15.66 Hz, 1 H, 2-H), 6.95 (m, 1 H, Ar6-H), 7.07 (m,
2H, meta-4FPhH), 7.53 (m, 3 H, ortho-4FPh-H, Pyr-5-H), 7.88
(d, J = 15.54 Hz, 1 H, 3-H), 8.06 (d, J = 5.31 Hz, 1 H, Pyr-6-H),
8.61 (s, 1 H, Pyr-3-H), 9.55 (s, 1 H, NH). 13C NMR (50.33 MHz,
THF-d8) δ (ppm) 14.1 (S-CH3), 54.5 (meta-OCH3), 54.6 (ortho-
OCH3), 98.0 (CH, Ar), 105.1 (CH, Ar), 110.8 (CH, ArCH),
114.9 (d, 21 Hz, ortho-4-FPhH), 116.6 (ArCH), 116.9
(ArCH),119.3 (CdCH-1), 121.2 (CqAr), 129.7 (ArCH), 134.6
(ArCq), 136.6 (Cq), 147.4 (CdCH), 153.4 (Cq), 161.1 (d, 136 Hz,
4FPh-Cq), 164.3 (CdO). MS (ESI) for C26H23FN4O3S 591.1
[M þ H]þ; HRMS calcd 490.14749, found 490.14570.
(E)-3-(2,4-Dimethoxy-phenyl)-N-(4-[5-(4-fluoro-phenyl)-2-meth-
anesulfinyl-3H-imidazol-4-yl]-pyridin-2-yl)-acrylamide (18). Co-
mpound 17 (0.176 g, 0.40 mmol) was dissolved in 3.5 mL of
THF, and 1 mL of water was added. The mixture was stirred in
an ice-water bath for 10 min, and an ice-cold solution of 0.121 g
potassium peroxomonosulfate (Oxone) in 2 mL water was
added. The mixture was stirred for 2.5 h. After completion of
the reaction, 7 mL of sodium hydrogen carbonate solution, 7
mL of water, and 15 mL of EtOAc were added. The phases were
separated, and the organic phase washed with water and brine,
dried over Na2SO4, and evaporated. Yield: 177 mg (91.7%).
Melting point: 219.0 ꢀC. HPLC: 7.97 min, 99.9%. 1H NMR (200
MHz, DMSO-d6) δ ppm: 3.09 (s, 3 H, SOCH3) 3.81 (s, 3 H,
ortho-OCH3) 3.87 (s, 3 H, para-OCH3) 6.61 (m, 2 H, Ar.5-H,
Ar.3-H) 6.93 (d, J = 15.66 Hz, 1 H, 2-H) 7.03 (dd, J = 5.05 Hz,
J = 1.26 Hz 1 H, Pyr-5-H) 7.30 (m, 2 H, meta-4FPhH) 7.52 (m,
4 H, ortho-4FPhH, Pyr-6-H, ortho-ArH) 7.71 (d, J = 15.92 Hz,
1 H, 3-H) 8.22 (m, 1 H, Pyr-3-H) 8.48 (s, 1 H, NH) 10.56* (s, 1 H,
imidazoleNH, exchangeable) 13.89* (s, 1 H, imidazoleNH,
exchangeable) (*tautomers of imidazole, stabilization by
DMSO). 13C NMR (50.33 MHz, THF-D8) δ: (ppm) 40.2
(SOCH3), 54.5 (meta-OCH3), 54.7 (ortho-OCH3), 98.0 (CH,
Ar), 105.1 (CH, Ar), 115.1 (d, ortho-4FPhC), 116.9 (CH, Ar),
119.2 (CdCH-1), 129.8 (Ar, CH), 136.8 (Cq), 147.5 (CdCH-2),
153.4 (Cq), 161.1 (d, J = 136.95, 4FPh-Cq), 164.3 (CdO). MS
(ESI) for C26H23FN4O4S: 507.1 [M þ H]þ; HRMS calcd
504.126716, found 504.12372.
Molecular Modeling. All modeling was performed on a
Fedora Core 9 Linux system. For visualization and building
the structures, Maestro (version 8.5) from Schrodinger
(Schrodinger, LLC, New York, NY, 2008) was used (for PDB
codes and references, see Figures). The illustrations of modeling
were generated by PyMol. Docking studies were performed
using the Induced fit docking tool from Schrodinger
(Schrodinger Suite 2008 Induced Fit Docking protocol; Glide
version 5.0, Schrodinger, LLC, New York, NY, 2005; Prime
version 1.7, Schrodinger, LLC, New York, NY, 2005). Chain B
from CK1 structure (with CK1δ-specific inhibitor IC261 as
original ligand, PDB code 1EH4_B)9 was used for docking
studies such as Homologue, because no suitable structure of
mammalian CK1 was available. However, to correlate the
model with biological assay data of this study, key amino acids
were mutated to residues obtained from rat sequence: I85M,
D86E, and L96F. Residue F96 is not involved in active site.
Acknowledgment. We thank Barbara Radunsky and Arn-
hild Grothey, Department of General, Visceral, and Trans-
plantation Surgery, University of Ulm, for excellent technical
support. We thank Jenny Moran for screening of compounds
in the kinase profiler at the Division of Signal Transduction
Therapy, University of Dundee. We thank Dr. D. Scholl-
meyer for X-ray analysis of compounds. Financial support by
DAAD, Deutsche Forschungsgemeinschaft, and Merckle
GmbH, Blaubeuren, is gratefully acknowledged. Work in

7630 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Peifer et al.
the lab of Uwe Knippschild is supported by Deutsche
Krebshilfe, Mildred Scheel Stiftung (108489).
formation by casein kinase I. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 4159–4164.
(19) Aud, D. M.; Peng, S. L.-Y. Methods of treating inflammatory
diseases with selective inhibitors of the casein kinase 1 isoforms. [F.
Hoffmann-La Roche A.-G.]. Patent WO. 2007-EP63339[2008071605]
2007.
(20) Giamas, G.; Stebbing, J.; Vorgias, C. E.; Knippschild, U. Protein
kinases as targets for cancer treatment. Pharmacogenomics 2007, 8,
1005–1016.
(21) Green, J.; Bemis, G.; Grillot, A.-L.; Ledeboer, M.; Salituro, F.;
Harrington, E.; Gao, H.; Baker, C.; Cao, J.; Hale, M. Preparation
of as isoxazolylpyrimidines and related compounds as inhibitors of
c-JUN N-terminal kinases and other protein kinases. PCT Int.
Appl. WO 200101262196, 2001.
Supporting Information Available: Experimental and spectro-
scopic details for compounds 3, 4, and 5. IR-data and purity of
key target compounds. X-ray analysis for compounds 1, 2, 7, 8
and 9. Assay details and ATP concentrations. This material is
available free of charge via the Internet at http://pubs.acs.org.
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