Synthesis and structure-activity relationships of 4-fluorophenyl-imidazole p38α MAPK, CK1δ and JAK2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.080

The synthesis and structure-activity relationships of novel 4-(4′-fluorophenyl)imidazoles as selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described. Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5- disubstituted imidazoles. Carboxylate and phosphonate groups were introduced via 'click' reactions. The kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited p38α MAPK with IC₅₀ = 250 nM and 96 nM, respectively. Pyridine 3 gave CK1δ inhibition with IC₅₀ = 89 nM and pyridin-2-one 31 gave JAK2 inhibition with IC₅₀ = 62 nM.

Full text of DOI:10.1016/j.bmcl.2014.05.080

Accepted Manuscript
Synthesis and structure-activity relationships of 4-fluorophenyl-imidazole
p38α MAPK, CK1δ and JAK2 kinase inhibitors
Jean-Paul G. Seerden, Gabriela Leusink-Ionescu, Titia Woudenberg-Vrenken,
Bas Dros, Grietje Molema, Jan A.A.M. Kamps, Richard M. Kellogg
PII:
S0960-894X(14)00589-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.080
BMCL 21694
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 April 2014
20 May 2014
22 May 2014
Please cite this article as: Seerden, J.G., Leusink-Ionescu, G., Woudenberg-Vrenken, T., Dros, B., Molema, G.,
Kamps, J.A.A., Kellogg, R.M., Synthesis and structure-activity relationships of 4-fluorophenyl-imidazole p38α
MAPK, CK1δ and JAK2 kinase inhibitors, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/
10.1016/j.bmcl.2014.05.080
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Synthesis and structure-activity relationships of
4-fluorophenyl-imidazole p38α MAPK,
CK1δ and JAK2 kinase inhibitors
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∗
Jean-Paul G. Seerden , Gabriela Leusink-Ionescu, Titia Woudenberg-Vrenken, Bas Dros, Grietje Molema, Jan
A.A.M. Kamps, Richard M. Kellogg

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and structure-activity relationships of 4-fluorophenyl-imidazole p38α
MAPK, CK1δ and JAK2 kinase inhibitors
Jean-Paul G. Seerden a, ∗, Gabriela Leusink-Ionescu a , Titia Woudenberg-Vrenken b, Bas Dros a, Grietje
Molema b, Jan A.A.M. Kamps b and Richard M. Kellogg a
aSyncom B.V., Kadijk 3, Groningen, 9747 AT The Netherlands
bLaboratory for Endothelial Biomedicine & Vascular Drug Targeting Research, University Medical Center Groningen, University of Groningen, Hanzeplein 1,
Groningen 9713 GZ, The Netherlands
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The synthesis and structure-activity relationships of novel 4-(4’-fluorophenyl)imidazoles as
selective p38α MAPK, CK1δ and JAK2 inhibitors with improved water solubility are described.
Microwave-assisted multicomponent reactions afforded 4-fluorophenyl-2,5-disubstituted
imidazoles. Carboxylate and phosphonate groups were introduced via ‘click’ reactions. The
kinase selectivity was influenced by the heteroaryl group at imidazole C-5 and the position of a
carboxylic acid or tetrazole at imidazole C-2. For example, pyrimidines 15 and 34 inhibited
p38α MAPK with IC50 = 250 nM and 96 nM, respectively. Pyridine 3 gave CK1δ inhibition with
IC50 = 89 nM and pyridin-2-one 31 gave JAK2 inhibition with IC50 = 62 nM.
Keywords:
p38α MAPK, CK1δ, JAK2 inhibitors
4-fluorophenyl-imidazole
multicomponent reactions
alkyne-azide click reaction
aqueous solubility
2009 Elsevier Ltd. All rights reserved.
The discovery and development of small-molecule kinase
inhibitors to treat inflammatory and immune-related diseases and
cancer with minimal undesirable side effects are subjects of
major attention in the pharmaceutical industry. Resistance, gene
point mutations, multi-kinase inhibition and the need for
selectivity are major issues in the design of therapeutically
relevant kinase inhibitors1. Despite recent FDA approval and
successful marketing of small-molecule tyrosine kinase inhibitors
that target vascular endothelial growth factor (VEGF), epidermal
growth factor receptor (EGFR), Bcl-Abl, and Janus kinase
(JAK1, JAK2 and JAK3) for the treatment of chronic myeloid
leukemia (CML), non-small cell lung cancer, kidney and breast
cancer, myelofibrosis (MF), and rheumatoid arthritis, many
specific kinase inhibitors have not been further developed owing
to toxicity or poor ADME2. The poor developability for oral
administration is correlated to the increased lipophilicity of
kinase inhibitors and resembles a classical medicinal chemistry
problem. The highly lipophilic character of many potent kinase
inhibitors is a consequence of the highly lipophilic binding
pocket next to the small polar hinge recognition site. Improving
the water solubility of these highly lipophilic kinase inhibitors
without lowering the potency and/or proper formulation into
targeted liposomes might solve the problem and make potent
benefit of patients3. Previously we have described regioselective
direct C-H arylation and cross coupling alkynylation of 4-
fluorophenyl-imidazoles, followed by regioselective azide ‘click’
reactions, to provide access to novel triazolyl p38α MAPK
inhibitors with improved water solubility for further biological
evaluation and drug delivery studies4. The 4-fluorophenyl-
imidazole chemical scaffold is not only present in p38α MAPK
inhibitors, such as SB203580, SB202190 or the highly potent JNJ
75839795, but also in caseine kinase I delta (CK1δ) inhibitors PF-
6704626, CKP1387 and others8, as well as in some JAK
inhibitors, such as conformationally rigid pan-JAK inhibitor
Pyridone 69 and related pyridones10 (Figure 1).
Figure 1. 4-(4-Fluorophenyl)-5-aryl imidazole kinase inhibitors
k—in—ase—inhibitors become orally or intravenously available for the
∗ Corresponding author. Tel.: +31-050-5757386; fax: +31-050-5757399; e-mail: j.p.g.seerden@syncom.nl

calculated solubility (moles/L) using EPIWEB 4.1 WSKOWWIN v1.42; the
actual aqueous solubility has not been determined; d water solubility
calculated for corresponding phosphonic acid.
In addition, several p38α MAPK inhibitors such as SB203580
and SB202190 have been shown to be moderate CKδ1
inhibitors11 and cross reactivity between p38α and CK1δ in Wnt
β-catenine signaling12 has been reported. No p38α MAPK or
CK1δ kinase inhibitors have been approved yet. The discovery of
activating mutations in JAK2 and myeloproliferative leukemia
virus oncogene (MPL) in patients with myeloproliferative
neoplasms (MPN) has led to the rapid clinical development of
several JAK kinase inhibitors and recent approval of JAK1/2
inhibitor ruxolitinib for the treatment of MF. Many JAK2
inhibitors are being examined for oncological applications.
None of the 4-(4’-fluorophenyl)-pyridinylimidazoles 1-8
significantly inhibited any of the JAK1, JAK2 or JAK3 enzymes
(IC50 >100 µM) and therefore these results have been omitted in
Table 1. SB202190 was used as reference p38α MAPK inhibitor
(IC50 = 0.009 µM). It also showed CK1δ inhibition (IC50 = 0.188
µM) in agreement with the literature9. All triazolyl p38α MAPK
inhibitors 1-8 were able to inhibit CK1δ kinase with various IC50
values. The unsubstituted triazole 1 behaved as a dual p38α
MAPK/CK1δ inhibitor (IC50 ~ 0.2 µM). The water-soluble
racemic trans-3-fluoropiperidine HCl salt 6 showed the best p38α
MAPK inhibition (IC50 = 0.096 µM) and also some CK1δ
inhibition (IC50 = 0.437 µM). Interestingly, from a medicinal
chemical point of view, the difference in selectivity and potency
for p38α MAPK or CK1δ inhibition was most pronounced
between the para- and meta-benzoic acid derivatives 2 and 3,
respectively. Whereas 2 and 3 gave almost equal p38α inhibition
(IC50 = 0.166 µM and 0.185 µM respectively), the para-benzoic
acid 2 was 10-fold more selective for p38α than CK1δ, its meta-
benzoic acid analog 3 gave 18-fold stronger CK1δ inhibition
(IC50 = 0.089 µM) than 2 (IC50 = 1.6 µM). Despite its poor water-
solubility and low retention efficiency upon formulation into
SAINT-O-Somes4 the meta-benzoic acid 3 could serve as further
starting point for the design of more selective CK1δ inhibitors by
varying substituent R2. Caseine kinase 1 (CK1), a family of
conserved serine/threonine kinases (e.g. CK1δ), modulates Wnt
signaling pathways and has been implicated in the regulation of
vesicular trafficking, DNA damage repair, cell cycle progression,
cytokinesis and circadian rhythms14.
The previously developed ‘click’ strategy for the synthesis of
other triazolyl trisubstituted azoles with improved water
solubility was further explored, using the 4-fluorophenyl-
imidazoles in Figure 1 as model compounds4. The various
chemotypes of 4,5-diarylimidazoles13 were prepared from α-
bromo ketones and 2-aminopyrimidines or from 1,2-dicarbonyl
compounds in a highly efficient 3-component reaction with
ammonia and aldehydes. The replacement of the 4-pyridyl group,
essential for p38α selectivity, with a pyrimidine or 2-oxo-1,2-
dihydropyridin-4-yl group and scaffold hopping from triazolyl-
aryl to aryl triazolyl moieties were investigated in order to have
impact on the selectivity and potency for p38α MAPK, CK1δ,
JAK1, JAK2 and JAK3 kinase inhibition. A selection of our
previously prepared small library of triazolyl 4-fluorophenyl-
imidazole p38α MAPK inhibitors4 was assayed for p38α, CK1δ,
JAK1, JAK2 and JAK3 inhibition. The results are depicted in
Table 1.
Table 1. Triazolyl 4-fluorophenyl-imidazoles as dual p38α
MAPK/CK1δ inhibitors (IC50 in µM)
The highly potent p38α MAPK inhibitor JNJ 758397915 was
prepared from α-bromo ketone 9 by condensation with 2,5-
diaminopyrimidine 10 to provide imidazo[1,2-a]pyrimidine 11
(42% yield.), followed by S-oxidation and substitution with (S)-
1-phenylethylamine in low yield (Scheme 1). For applying the
‘click’ approach to enhance aqueous solubility the required
alkyne group was introduced via a condensation reaction of α-
bromo ketone 9 with 4-((trimethylsilyl)ethynyl)pyrimidin-2-
amine 1216 in refluxing EtOH for 16 hours. Subsequent
deprotection with 1M TBAF in THF afforded the key 7-ethynyl-
imidazo[1,2-a]pyrimidine 13 in 13% yield over two steps17. The
subsequent Cu-catalyzed ‘click’ reaction of 13 with azido ethyl
acetate gave the 7-(1,2,3-triazol-4-yl)imidazo[1,2-a]pyrimidine
14 in 50% yield. Saponification of 14 gave the corresponding
sodium carboxylate 15. In a similar way the Cu-catalyzed ‘click’
reaction of 13 with freshly prepared diethyl (2-
azidoethyl)phosphonate18 afforded the diethylphosphonate 16.
Deprotection of the phosphonate with TMSBr, purification by
preparative LC and subsequent treatment with stoichiometric
NaOH and lyophilization afforded water soluble disodium
phosphonate 17. The lipophilic esters 14 and 16 were synthetic
intermediates for the preparation of the water-soluble compounds
15 and 17 and were not screened for their potency on kinase
inhibition.
p38α MAPK
IC50 (µM)a
0.009
CK1δ
Compound
R2
Log Sc
IC50 (µM)a
0.188
SB202190
-
-4.35
-4.02
1
H
0.21 (0.14)b
0.202
2
0.166 (0.14)b
1.60
-5.81
3
4
0.185
0.089
0.24
-5.81
-3.48
0.783 (0.50)b
5
6
1.02
0.439
0.437
-3.95
-3.85
Scheme 1. Synthesis of 7-(1,2,3-triazol-4-yl)imidazo[1,2-
a]pyrimidines 14-17
0.096
7
8
2.48 (>100)b
3.10
-5.24
-4.0d
0.345
0.225
a IC50 determined by radiometric assay, ProQinase GmbH, Germany; JAK1,
JAK2, JAK3 IC50 >100 µM; b reported IC50 values from ref 4 in brackets; c

The ‘click’ strategy for enhancing solubility was also applied to
the 4-fluorophenylimidazoles with a pyridine group at C-5 by
introducing the solubilizing group at the imidazole C-2 position.
The 1,2,3-triazole-substituted “click” products 1-8 were less
effective p38 MAPK inhibitors than SB2035804 or SB202190
(Table 1), an effect that might originate from less efficient π-
stacking with the Tyr35 residue in the ATP-binding pocket of
p38 MAP kinase. The influence of the 1,2,3-triazole group was
now studied by scaffold hopping, interchanging the triazole and
benzoic acid group in triazolylbenzoic acid p38α MAPK
inhibitors 2 and 3. Improved p38 MAPK inhibition by successful
replacement of the essential 4-pyridine, which gives a strong
hydrogen bond between the p38 amide NH of Met109 and the
pyridine nitrogen, by a pyrimidine has been demonstrated in
many 4-fluorophenylimidazoles also to minimize cytochrome
P450 inhibition. The pyridone ring of ‘Pyridone 6’ (Figure 1)
binds in the ATP-binding site of JAK and the amide segment
forms key dual H-bonds to the hinge region of the protein1. A
Reagents and conditions: a) EtOH, reflux, 13 h, 42% c.y.; b) oxone (3 eq.),
H2O, MeOH, 20 h, room temp., 78% c.y.; c) (S)-phenylethylamine, 24% c.y.;
d) EtOH, reflux, 16 h; 1M TBAF in THF, 13% c.y.; e) CuSO4, Na-ascorbate,
H2O, room temp. 14: azido ethyl acetate (50% c.y.) or 16: diethyl (2-
azidoethyl)phosphonate (48% c.y.); f) 15: aq. NaOH, MeOH reflux, 88% c.y.;
17: TMSBr, DMF; aq. NaOH; lyophilization, 27% c.y.
double hydrogen bond of the 4-pyridone group with Met109
-
Gly110 in the p38α MAP kinase ATP-pocket might give stronger
interactions and lower p38α IC50 values. An efficient microwave-
assisted multicomponent Debus-Radziszewski22 reaction of
diketone 1823 and 4-ethynyl-benzaldehyde 19 using 10 eq.
ammonium acetate in acetic acid for 10 minutes at 180 °C in a
closed vessel afforded the new building block 4-(2-(4-
The inhibition of p38α MAPK by imidazo[1,2-a]pyrimidines
11, 13, 15 and 17, the calculated Ligand Lipophilicity Efficiency
(LLE19) and aqueous solubility (Log S) are presented in Table 2.
The reference compound JNJ7583979 was a potent and selective
p38α MAPK inhibitor with IC50 = 8.5 nM.5 No significant CK1δ,
JAK1, JAK2, or JAK3 inhibition was found (IC50 > 100 µM).
The presence of the lipophilic (S)-1-phenylethylamino group in
JNJ7583979 is crucial20 for its high potency as compared to 14-
fold less potent thiomethyl imidazo[1,2-a]pyrimidine analog 11
(IC50 = 120 nM; R4 = NH2). The replacement of the amino group
in 11 by the triazolyl acetate group in 15 (IC50 = 250 nM)
resulted in an 11-fold increase in aqueous solubility with
concomitant 2-fold decrease in p38α inhibition. This
demonstrates that introduction of a triazolyl acetate group is an
effective way of improving the physicochemical property without
a dramatic decrease in potency. The integrated physicochemical
properties of 15 (LLE = 7.9, Log S = -2.95) have met our initially
hypothesized requirements for further evaluation in formulation
studies21. Note, however, that on further increase of the aqueous
solubility by introduction of the 1,2,3-triazolyl-1-ethyl disodium
phosphonate group in 17 no p38α MAPK inhibition was
achieved.
ethynylphenyl)-1H-imidazol-5-yl)pyridin-2(1H)-one
20
on
multigram scale (Scheme 2). Subsequent regioselective Cu-
catalyzed ‘click’ reactions with azido ethanol, azido ethyl acetate
and
diethyl
(2-azidoethyl)phosphonate
afforded
the
corresponding
1H-1,2,3-triazol-4-yl)phenyl)-1H-imidazol-5-
yl)pyridin-2(1H)-ones 21-23, respectively. Saponification of the
diethylphosphonate ester 23 with TMSBr in DMF gave the
water-soluble disodium phosphonate 24. The analogous 4-(4-(4-
fluorophenyl)-1H-imidazol-5-yl)pyridin-2(1H)-ones 25-29 were
efficiently prepared in a similar way from diketone 18 via a
microwave-assisted Debus-Radziszewski
ammonium acetate and 4-hydroxybenzaldehyde,
reaction with
4-
carboxybenzaldehyde, 3-hydroxy-4-carboxybenzaldehyde, 4-
cyanobenzaldehyde and Boc-4-N-piperidine carboxaldehyde,
respectively. The corresponding water-soluble sodium salts
26.Na and 27.Na of the poorly water-soluble carboxylic acids 26
and 27 were obtained after lyophilization. Deprotection of 29
under acidic conditions afforded piperidine 30 as a water-soluble
HCl salt in 29% yield over 2 steps. The tetrazole 31 was prepared
in 16% yield from poorly soluble nitrile 28 via a 1,3-dipolar
cycloaddition with aqueous sodium azide in the presence of
ZnBr224 using 2-propanol as cosolvent, followed by purification
by preparative LC. The corresponding tetrazolyl potassium salt
of 31 was highly water-soluble. The tetrazole group is considered
to be a bioisostere of the carboxylic acid group in 26. The
palmitoyl ester prodrug 32 was prepared from phenol 25 and
palmitoyl chloride (Et3N, DCM, DMF; isolated after preparative
LC). The inhibition of p38α MAPK, CK1δ, JAK1, JAK2 and
JAK3 kinase was determined by a radiometric IC50 profiling
assay. The results in Table 3 only summarize the p38α and JAK2
IC50 data because no significant inhibition of the other kinases
was observed with IC50 > 10 µM.
Table 2. Imidazo[1,2-a]pyrimidine p38α MAPK inhibition, LLE
and Log S
compound
JNJ 7583979
11
R4
p38α IC50 (µM)a
0.0085
LLEb
3.3
Log Sc
-5.55
-4.02
NH2
0.12
4.3
13
15
2.51
0.25
1.8
7.9
-5.10
-2.95
Scheme 2. Synthesis of 4-fluorophenylimidazole-pyridin-
2(1H)-ones 20-31
17
>100
n.d.
-2.19
a IC50 determined by radiometric assay, ProQinase GmbH, Germany; no
significant inhibition of CK1δ, JAK1, JAK2, JAK3 was found (IC50 >100
µM); b LLE = pIC50 – cLogP; c calculated water solubility (moles/L) using
EPIWEB 4.1 WSKOWWIN v1.42.

JAK1 (IC50 >10 µM; except 31: IC50 = 1.68 µM), or JAK3 (IC50 >10 µM
b
except 31: IC50 = 0.783 µM) was found; Log S: calculated water solubility
(moles/L) using EPIWEB 4.1 WSKOWWIN v1.42.
No significant inhibition of p38α MAPK, CK1δ, JAK1 or JAK3
was found (IC50 >1 µM) for any of the pyridones 20-31 including
the highly water-soluble disodium phosphonate 24. However, the
JAK2-selective benzoate 26 (JAK2: IC50 = 0.274 µM; JAK1: IC50
= 14.1 µM; JAK3: IC50 = 3.90 µM) displayed a 10- to 100-fold
stronger inhibition of JAK2 against p38α (IC50 = 2.94 µM) and
CK1δ (IC50 = 24.8 µM), respectively. The bioisosteric tetrazole
potassium salt 31 was the most potent JAK2 inhibitor (IC50
=
0.061 µM) with comparable JAK2 selectivity against p38α (IC50
= 2.53 µM), CK1δ (IC50 = 12.1 µM), JAK1 (IC50 = 1.68 µM) and
JAK3 (IC50 = 0.783 µM) as benzoate 26. In addition to their
JAK2 kinase inhibition the pyridones 26.Na and 31.K show
improved water solubility with desired physicochemical
properties (LLE and Log S).
The synthetic approach shown in Scheme 3, as an obvious
modification of Scheme 2, allows investigation of the influence
of the 5-heteroaryl substituent in the 4-fluorophenylimidazoles
on the kinase selectivity and potency (IC50). This is further
exemplified by pyridinyl-imidazole SB202190, pyridonyl-
imidazole 26 and pyrimidinyl-imidazole 34. The p38α MAPK
inhibitors 34 and 35 were both prepared from a common diketone
intermediate 33 via a clean multicomponent Debus-Radziszewski
reaction using microwave heating similar to the synthesis of 20
and 25-29 respectively (Scheme 3). The pyrimidine 34 is a p38α
MAPK inhibitor with IC50 = 0.096 µM and is a 10-fold less
potent analogue of SB202190. Replacement of the 4-OH
substituent with a 4-ethynyl group in 35 caused a 8-fold drop in
potency (IC50 = 0.82 µM). No significant inhibition of CK1δd,
JAK1, JAK2, JAK3 was found (IC50 >100 µM). Note that the
alkyne 35 could serve as building block for the library synthesis
of triazole analogues with improved physicochemical properties.
Reagents and conditions: a) NH4OAc (10 eq.), AcOH, 10 min. 180 oC
microwave heating; 20: 86% c.y.; 25-29: 78-96% c.y.; b) CuSO4, Na-
ascorbate, H2O, 21: azido ethanol, 68% c.y.; 22: azido ethyl acetate, 79% c.y.;
23: diethyl (2-azidoethyl)phosphonate, 34% c.y.; c) 24: TMSBr, DMF, prep.
LC, d) 29, aq. HCl: 30 (29% c.y. over 2 steps from 18); e) 28, ZnBr2, NaN3:
31 (16%); f) 25, palmitoyl chloride, Et3N, DCM, DMF: 32 (54% c.y.).
Table 3. Selective JAK2 inhibitiona (IC50 µM) by pyridones
20-31, LLE and Log S
p38α
IC50
(µM)
JAK2
IC50
(µM)
Log
R6
LLE
S
Scheme 3. Synthesis of 4-fluorophenylimidazolyl-pyrimidines
34 and 35
9.39
18.6
32.9
14.3
0.50
0.352
1.27 -4.84
4.24 -4.18
3.38 -5.35
20
21
22
o
a) 4-hydroxybenzaldehyde (1 eq.), NH4OAc (10 eq.), AcOH, 10 min. 180 C
microwave heating, 93% ; b) aldehyde 19 (1 eq.), NH4OAc (10 eq.), AcOH,
>100
>100
4.20
45.5
2.29 -5.94
5.72 -1.93
23
24
10 min. 180 oC microwave heating, 87%.
Preliminary results show that in TNFα activated HUVEC cells
the (4-fluorophenyl)imidazolyl pyridones 25, 26.Na, 27.Na and
30 (at 10 µM concentration) did not downregulate IL-6, or COX-
2 or VCAM-1 gene expression (1 h TNFα stimulation; incubation
for 4 h, then harvesting cells for mRNA and analysis by real-time
RT-PCR) in contrast to SB203580 or the potent p38α MAPK
inhibitor 64. Poor membrane permeability and low potency might
be major reasons. Liposomal formulation of some of the prepared
kinase inhibitors might improve the cellular activity. The highly
potent p38α inhibitor JNJ583979 was too lipophilic for efficient
encapsulation into cationic liposomes (SAINT-O-Somes25) by
remote loading. Formulation of the disodium phosphonates 8, 17
and 24 into SAINT-O-Somes was not successful despite their
good water solubility. HPLC-MS analyses of eight different
formulations of the piperidines 6 and 30 showed that 92% of 6
remained in 10% SAINT-O-Somes, (DSPC : SAINT : cholesterol
: DSE-PEG = 45 : 10 : 40 : 5), but 30 was not retained at all,
although both have similar aqueous solubility. The difference in
pKa between pyridine 6 and pyridone 30 might be responsible for
4.96
2.94
0.911
0.274
3.22 -3.56
7.52 -2.59
25
26.Na
27.Na
30
6.81
1.14
2.53
3.71
6.62
0.061
5.62 -2.79
2.81 -3.69
6.72 -2.81
31.K
a
IC50 determined by radiometric assay (ProQinase GmbH, Germany); no
significant inhibition of CK1δ (IC50 >10 µM; except 30: IC50 = 1.20 µM),

this effect. In addition, the β-fluoro substituent in 6 not only
decreases the pKa of the piperidine nitrogen but also is expected
to improve the bioavailability26. As an alternative approach the
palmitoyl ester prodrug 32 was formulated (ca. 1%) into the lipid
bilayer of 18% SAINT-O-Somes (DSPC : SAINT : cholesterol :
DSE-PEG = 37 : 18 : 40 : 5) by remote loading. After E-Selectine
antibody conjugation all 32 was converted to the phenol 25
which was detected by HPLC-MS. Further studies of this prodrug
approach will reveal its applicability for liposomal formulation of
poorly soluble drugs.
inhibitor. We believe that the chosen synthetic strategy can find
wider application for the library synthesis of other triazolyl
trisubstituted azoles with improved physicochemical properties
and for the development of “clickable” photoaffinity probes in
molecular biology for finding new biological targets.27
Acknowledgments
This work was sponsored by the Top Institute Pharma (TI
Pharma project D5-301, The Netherlands). The authors gratefully
acknowledge the discussions with Eduard Talman and Marcel
Ruiters (Synvolux Therapeutics, Groningen, The Netherlands).
In summary, we have demonstrated that the introduction of a
(1H-1,2,3-triazol-1-yl)acetate or phosphonate via
a ‘click’
reaction lowered the lipophilicity and improved water solubility
of kinase inhibitors that share a common 4-fluorophenyl-
imidazole scaffold. The multicomponent Debus-Radziszewski
reaction using microwave heating is a generally applicable
synthetic method for ready access to various 2,4,5-
triarylimidazoles. For p38α binding and selectivity a basic
pyridine or pyrimidinyl group is essential but a pyridone
precludes p38α or CK1δ inhibition. Dual p38α/CK1δ inhibition
References and notes
Supplementary Material
Supplementary data associated with this article can be found
in the online version. These data include typical synthetic and in
vitro procedures, NMR and IC50 data, MOL files and InChiKeys
of the most important compounds described in this article.
was
observed
for
some
triazole-derived
4-
fluorophenylimidazoles. The pyridone series displayed good
JAK2 selectivity and culminated in tetrazole 31 as potent JAK2
7. Peifer, C.; Abadleh, M.; Bischof, J.; Hauser, D.; Schattel, V.;
Hirner, H.; Knippschild, U.; Laufer, S. J. Med. Chem. 2009, 52,
7618.
8. (a) Almario Garcia, A.; Barrague, M.; Burnier, P.; Enguehard, C.;
Gao, Z.; George, P.; Gueiffier, A.; Li, A.-T.; Puech, F.
WO2009/016286; (b) Oudot, R.; Pacaud, C.; Puech, F. WO2010/
130934.
9. Thompson, J. E.; Cubbon, R. M.; Cummings, R.T.; Wicker, L.S.;
Frankshun, R.; Cunningham, B.R.; Cameron, P. M.; Meinke, P.T.;
Liverton, N.; Weng, Y.; DeMartino, J.A. Bioorg. Med. Chem. Lett.
2002, 12, 1219.
1. (a) Protein Kinases as Drug Targets, Klebl, B.; Müller, G.;
Hamacher, M.; Eds.; Methods and Principles in Medicinal
Chemistry, Vol. 49, Wiley-VCH, 2011; (b) Davis, M.I.; Hunt,
J.P.; Herrgard, S.; Ciceri, P.; Wodicka, L.M.; Pallares, G.; Hocker,
M.; Treiber, D.K.; Zarrinkar, P.P. Nature Biotech. 2011, 29, 1046;
(c) Anastassiadis, T.; Deacon, S.W.; Devarajan, K.; Ma, H.;
Peterson, J.R. Nature Biotech. 2011, 29, 1039; (d) Müller, S.;
Knapp, S. Expert Opin. Drug Discovery 2010, 5, 867; (e) Morphy,
R. J. Med. Chem. 2010, 53, 1413.
2. (a) Zhang, J.; Yang, P.L.; Gray, N.S. Nature Rev. Cancer 2009,
9, 28; (b) Kesarwani, M.; Huber, E.; Azam, M. Blood Cancer
Journal 2013, 3, e138; (c) Wilhelm, S.M.; Dumas, J.; Adnane, L.;
Lynch, M.; Carter, C.A.; Schütz, G.; Thierauch, K.-H.; Zopf, D.
Int.J. Cancer 2011, 129, 245.
10. (a) Rodgers, J.D.; Robinson, D.J.; Arvanitis, A.G.; Maduskuie,
T.P.; Shepard, S.; Storace, L.; Wang, H.; Rafalski, M.; Jalluri,
R.K.; Combs, A.P.; Crawley, M.L. WO2005/105814; (b) Laufer,
S.A.; Wagner, G.K.; Kotschenreuther, D.A.; Albrecht, W. J. Med.
Chem. 2003, 46, 3230; (c) Laufer, S.; Striegel, H.-G.; Tollmann,
K.; Albrecht, W. WO2004/18458; (d) Ulloora, S.; Adhikari, A.V.;
Shabaraya, R.; Aamir, S. Bioorg. Med. Chem. Lett. 2013, 23,
1502; (e) Su, Q.; Ioannidis, S.; Chuaqui, C.; Almeida, L.;
Alimzhanov, M.; Bebernitz, G.; Bell, K.; Block, M.; Howard, T.;
Huang, S.; Huszar, D.; Read, J.A.; Costa, C.R.; Shi, J.; Su, M.;
Ye, M; Zinda, M. J. Med. Chem. 2014, 57, 144 and references
cited therein; (f) Wrobelski, S.T.; Pitts, W.J. Annu. Rep. Med.
Chem. 2009, 44, 247.
11. (a) Hirota, T.; Kay, S.A. Chem. Biol. 2009, 16, 291; (b) Peifer, C.;
Abadleh, M.; Bischof, J.; Hauser, D.; Schattel, V.; Hirner, H.;
Knippschild, U.; Laufer, S., J. Med. Chem. 2009, 52, 7618.
SB202190 was reported to inhibit CK1δ with IC50 = 600 nM.
12. Verkaar, F.; van der Doelen, A.A.; Smits, J.F.M.; Blankesteijn,
W.M.; Zaman, G.J.R. Chem. Biol. 2011, 18, 485.
3. (a) Stella, V.J. , Nti-Addae, K.W. Advanced Drug Delivery
Reviews 2007, 59, 677–694. For recent examples of liposomal
formulation of small molecule kinase inhibitors, see: (b) Gupta,
V.; Gupta, N.; Shaik, I.H.; Mehvar, R.; McMurtry, I.F.; Oka, M.;
Nozik-Grayck, E.; Komatsu, M.; Ahsan, F. J. Control. Release
2013, 167, 189; (c) Mukthavaram, R.; Jiang, P.; Saklecha, R.;
Simberg, D.; Bharati, I.S.; Nomura, N.; Chao, Y.; Pastorino, S.;
Pingle, S.C.; Fogal, V.; Wrasidlo, W.; Makale, M.; Kesari, S. Int.
J. Nanomedicine 2013, 8, 3991; (d) Uckun, F.M.; Qazi, S.; Cely,
I.; Sahin, K.; Shahidzadeh, A.; Ozercan, I.; Yin, Q.; Gaynon, P.;
Termuhlen, A.; Cheng, J.; Yiv, S. Blood 2013, 121, 4348.
4. Seerden, J.-P.G.; Leusink-Ionescu, G.; Leguijt, R.; Saccavini, C.;
Gelens, E.; Dros, B.; Woudenberg-Vrenken, T.; Molema, G.;
Kamps, J.A.A.M.; Kellogg, R.M. Bioorg. Med. Chem. Lett. 2014,
24, 1352.
5. Although the synthesis of JNJ7583979 is not described in the
(patent) literature, the compound has been claimed as a very
potent p38α MAPK inhibitor (IC50 = 1 nM) for treatment of
degenerative disc disease in an intervertebral disc and orthopedic
joint inflammation: Brown, L.J.; Geesin, J.C.; Fang, C.H.;
Siekierka, J.J. US2009/0162351 and US2009/0162376; For
analogues see: Rupert, K.C.; Henry, J.R.; Dodd, J.H.; Wadsworth,
S.A.; Cavender, D.E.; Olini, G.C.; Fahmy, B.; Siekierka, J.J.
Bioorg. Med. Chem. Lett. 2003, 13, 347.
13. (a) Bellina, F.; Cauteruccio, S.; Rossi, R. Tetrahedron 2007, 63,
4571; (b) Kong, T.-T.; Zhang, C.-M; Liu, Z.-P. Curr. Med. Chem.
2013, 20, 1997; (c) Karcher, S.C.; Laufer, S.A. Curr. Top. Med.
Chem. 2009, 9, 655.
14. (a) Galatsis, P.; Wager, T.T.; Offord, J.; DeMarco, G.J.; Ohren,
J.F.; Efremov, I.; Mente, S. Annu. Rep. Med. Chem. 2011, 46, 33;
(b) Bibian, M.; Rahaim, R.J.; Choi, J.Y.; Noguchi, Y.; Schürer, S.;
Chen, W.; Nakanishi, S.; Licht, K.; Rosenberg, L.H.; Li, L.; Feng,
Y.; Cameron, M.D.; Duckett, D.R.; Cleveland, J.L.; Roush, W.R.
Bioorg. Med. Chem. Lett. 2013, 23, 4374 and references cited
therein; (c) Oumata, N.; Bettayeb, K.; Ferandin, Y.; Demange, L.;
Lopez-Giral, A.; Goddard, M.L.; Myrianthopoulos, V.; Mikros,
E.; Flajolet, M.; Greengard, P.; Meijer, L.; Galons, H. J. Med.
Chem. 2008, 17, 5229.
15. The α-bromo ketone 9 was prepared in two steps from
commercially available 2-methylthio-4-methylpyrimidine
hydrochloride by lithiation of the 4-Me group and α-aroylation
with methyl 4-fluorobenzoate followed by α-bromination of the
6. (a) Walton, K.M.; Fisher, K.; Rubitski, D.; Marconi, M.; Meng,
Q.-J.; Sládek, M.; Adams, J.; Bass, M.; Chandrasekaran,
R.;Butler, T.; Griffor, M.; Rajamohan, F.; Serpa, M.; Chen, Y.;
Claffey, M.;Hastings, M.; Loudon, A.; Maywood, E.; Ohren, J.;
Doran, A.; Wager, T.T., J. Pharm. Exp. Therap. 2009, 330, 430;
(b) Boggon, T.Y.; Li. Y.; Manley, P.W.; Eck, M.J. Blood 2005,
106, 996.

ketone according to a modified patent procedure: Chae, M.Y.;
Choi, I.Y.; Han, S.B.; Kim, H.; Kim, H.M.; Lee, H.; Lee, K.;
Lee, K.; Park, S.-Y. WO2006137658 A1. Amine 11 has been
described by Dodd, J. H.; Henry, J.R.; Rupert, K.C.,
WO2001034605 A1.
16. Prepared via Sonogashira cross-coupling according to Tibiletti, F.;
Simonetti, M.; Nicholas, K.M.; Palmisano, G.; Parravicini, M.;
Imbesi, F.; Tollari, S.; Penoni, A. Tetrahedron 2010, 66, 1280.
17. The yield of the cyclocondensation reaction of 9 and 10 could
neither be improved by changing the stoichiometry, nor by
prolonged heating (48 hours) in the presence of ZnCl2, Et3N or
DBU nor by microwave heating at 160 °C in the presence of
MgCl2 or AlCl3.
18. Yang, S.H.; Lee, D.J.; Brimble, M.A. Org. Lett. 2011, 13, 5604.
19. (a) Perola, E. J. Med. Chem. 2010, 53, 2986; (b) Tarcsay, A.;
Nyíri, K.; Keserű, G.M. J. Med. Chem. 2012, 55, 1252.
20. (a) Laufer, S.A.; Wagner, G.K.; Kotschenreuther, D.A.; Albrecht,
W. J. Med. Chem. 2003, 46, 3230; (b) Laufer, S.; Striegel, H.G.;
Albrecht, W.; Tollmann, K. WO03/09673A1; (c) Liverton, N.J.;
Butcher, J.W.; Claiborne, C.F.; Claremon, D.A.; Libby, B.E.;
Nguyen, K.T.; Pitzenberger, S.M.; Selnick, H.G.; Smith, G.R.;
Tebben, A. J. Med. Chem. 1999, 42, 2180.
21. Selection criteria: IC50 < 1 µM, LLE > 5, LogS > -4, rule of 3/75:
cLogP < 3, TPSA > 75Å2, Mol Wt < 500.
22. (a) Radziszewski, B. Ber. 1882, 15, 1493; (b) Wolkenberg, S.E.;
Wisnoski, D.D.; Leister, W.H.; Wang, Y.; Zhao, Z.; Lindsley,
C.W. Org. Lett. 2004, 6, 1453; (c) Magnus, N.A.; Diseroad, W.D.;
Nevill, C.R.; Wepsiec, J.P. Org. Proc. Res. Dev. 2006, 10, 556;
(d) Arduengo, A.J.; Gentry, F.P.; Taverkere, P.K.; Simmons, H.E.
US6177575.
23. Bonjouklian, R.; Hamdouchi, C.; Shih, C.; De Dios, A.; del Prado,
M.F.; Aguado, C.J.; Kotiyan, P.; Mader, M.M.; Selgas, S.P.;
Sanchez-Martinez, C., US7582652.
24. Demko, Z.P.; Sharpless, K.B. J. Org. Chem. 2001, 66, 7945.
25. For recent applications of endothelial-cell specific delivery of
siRNA with SAINT-O-Somes and background information see:
(a) Kowalski, P. S.; Zwiers, P.J.; Morselt, H. W. M.; Kuldo, J.M.;
Leus, N. G. J.; Ruiters, M. H. J.; Molema, G.; Kamps, J. A. A. M.
J.Controlled Release 2014, 176, 64; (b) Kowalski, P. S.;
Lintermans, L. L.; Morselt, H. W. M.; Leus, N. G. J.; Ruiters, M.
H. J.; Molema, G.; Kamps, J. A. A. M. Mol. Pharmaceutics 2013,
10, 3033; (c) Adrian, J.E.; Morselt, H.W.; Süss, R.; Barnert, S.;
Kok, J.W.; Asgeirsdóttir, S.A.; Ruiters, M.H.; Molema, G.;
Kamps, J.A. J. Controlled Release 2010, 144, 341; (d) Kuldo,
J.M.; Westra, J.; Asgeirsdóttir, S.A.; Kok, R.J.; Oosterhuis, K.;
Rots, M.G.; Schouten, J.P.; Limburg, P.C.; Molema, G. Am. J.
Physiol. Cell Physiol. 2005, 289, C1229; (e) Kuldo, J.M.;
Ogawara, K.I.; Werner, N.; Asgeirsdóttir, S.A.; Kamps, J.A.; Kok,
R.J.; Molema, G. Curr. Vasc. Pharmacol. 2005, 3, 11.
26. Hicken, E.J; Marmsater, F.P.; Munson, M.C.; Schlachter, S.T.;
Robinson, J.E.; Allen, S.; Burgess, L.E.; DeLisle, R.K.; Rizzi,J.P.;
Topalov,G.T.; Zhao, Q.; Hicks, J.M.; Kallan, N.C.; Tarlton, E.;
Allen, A.; Callejo, M.; Cox, A.; Rana, S.; Klopfenstein, N.;
Woessner, R.; Lyssikatos, J.P. ACS Med. Chem. Lett., 2014, 5 ,
78.
27. (a) Ghosh, B.; Jones, L.H. Med. Chem. Commun., 2014, 5, 24; (b)
Kalesh, K.A.; Shi, H.; Ge, J.; Yao, S.Q. Org. Biomol. Chem.,
2010, 8, 1749.