Biophysical investigation and conformational analysis of p38α kinase inhibitor doramapimod and its analogues

Article abstract of DOI:10.1039/c6md00262e

Doramapimod (BIRB 796) is a potent inhibitor of p38α mitogen-activated protein kinase. It contains an aryl-pyrazole scaffold as a pharmacophore critical for binding. The aryl-pyrazole scaffold is not planar and adopts an out-of-plane conformation, which is described by the torsion angle θ. In this letter, we report the chemical synthesis and biophysical characterization of different analogues of doramapimod (3-12) exhibiting distinctly different aryl-pyrazole torsion angle θ values. The torsion angle θ values of the synthesized analogues (3-6) were determined by crystal structural analysis and the binding affinities to p38α kinase investigated by microscale thermophoresis. Our results unveil a clear correlation between kinase binding and the torsion angle θ of tested doramapimod analogues, highlighting the importance of inhibitor conformation for protein binding.

Full text of DOI:10.1039/c6md00262e

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RESEARCH ARTICLE
Biophysical investigation and conformational
analysis of p38α kinase inhibitor doramapimod
and its analogues†‡
Cite this: DOI: 10.1039/c6md00262e
Amir H. Nasiri,a Krishna Saxena,ab Jan W. Bats,a
a
ab
*
*
Hamid R. Nasiri and Harald Schwalbe
Doramapimod (BIRB 796) is a potent inhibitor of p38α mitogen-activated protein kinase. It contains an
aryl-pyrazole scaffold as a pharmacophore critical for binding. The aryl-pyrazole scaffold is not planar and
adopts an out-of-plane conformation, which is described by the torsion angle θ. In this letter, we report
the chemical synthesis and biophysical characterization of different analogues of doramapimod (3–12)
exhibiting distinctly different aryl-pyrazole torsion angle θ values. The torsion angle θ values of the synthe-
sized analogues (3–6) were determined by crystal structural analysis and the binding affinities to p38α ki-
nase investigated by microscale thermophoresis. Our results unveil a clear correlation between kinase
binding and the torsion angle θ of tested doramapimod analogues, highlighting the importance of inhibitor
conformation for protein binding.
Received 12th May 2016,
Accepted 23rd May 2016
DOI: 10.1039/c6md00262e
www.rsc.org/medchemcomm
pocket spatially distinct from the ATP-binding site. The co-
crystal structure of the inhibitor 3, an analogue of
Introduction
p38α mitogen-activated protein (MAP) kinase is involved in
stimulated inflammation1 and plays an important role in the
MAP kinase signalling pathway. The vital regulatory behaviour
of protein kinases, their involvement in different biological
pathways and their druggable properties promote them as
major drug targets of the twenty-first century.2 Inhibition of
protein kinases by small molecules appears to be an attractive
therapeutic approach for the treatment of cancer and autoim-
mune diseases.3 There are more than 518 putative protein ki-
nase genes identified in the human genome4 and many ki-
nase inhibitors are currently in clinical development or
already on the market.5 Doramapimod (BIRB 796; 1-(5-tert-
butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)-
naphthalen-1-yl]-urea) (1) (Fig. 1) is a potent type II inhibitor
of p38α kinase with a Kd of 0.1 nM.6 It was developed by
Boehringer Ingelheim Pharmaceuticals, starting from frag-
ment 2,6 in a hit-to-lead program.7 Doramapimod made it to
late-stage clinical trial for treatment of rheumatoid arthritis
and Crohn's disease. Doramapimod utilizes a second binding
doramapimod (1), bound to human p38α kinase revealed that
the binding is predominately defined by hydrogen bonding
and hydrophobic interactions (Fig. 1).7 Similar to
doramapimod (1), compound 3 binds to a conserved region
of protein kinases, the so-called DFG-loop (p38α kinase:
AspIJD)168-PheIJF)169-GlyIJG)170) and stabilizes this loop in a
single conformation, also known as DFG-out conformation.
Inhibitors that preferably bind to the DFG-out conformation
are called “type II” inhibitors in contrast to the “type I” inhib-
itors that bind to the DFG-in conformation. It has been postu-
lated that kinase inhibitors, which bind and stabilize the
DFG-out conformation, provide a better starting point for
follow-up optimization compared to their counterparts that
bind and stabilize p38α kinase in the active DFG-in conforma-
tion. However, a recent study on type II inhibitors unveiled
that type II inhibitors may not be per se more selective than
type I inhibitors.8
In the co-crystal structure, the phenyl part of inhibitor 3 is
buried deeply in a hydrophobic pocket forming additional
interaction to Glu71 via a π-CH2 lipophilic contact.7,9
Based on the interaction between the inhibitor 3 and the
human p38α kinase, Regan and co-workers highlighted the
importance of torsion angle θ in the aryl-pyrazole core for
binding affinity.7 Based on their hypothesis, for an optimal li-
pophilic interaction, the torsion angle θ should be around
54°; and this conformation was referred to as bioactive con-
formation. The twist angle θ of doramapimod is below this
value (46.5°).6 The loss of binding affinity of compound 5,
a Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular
Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University Frankfurt, Max-
von-Laue-Straße 7, D-60438 Frankfurt am Main, Germany.
E-mail: schwalbe@nmr.uni-frankfurt.de, Nasiri@nmr.uni-frankfurt.de
b German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ),
Heidelberg, Germany
† The authors declare no competing interests.
‡ CCDC 1474053–1474059. For crystallographic data in CIF or other electronic
format see DOI: 10.1039/c6md00262e
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crystallography. Microscale thermophoresis (MST) was ap-
plied to determine the corresponding Ki values against
p38α kinase. A clear correlation was found between ligand
conformation and kinase binding affinities.
Results and discussion
Compounds 3–6 were selected in order to test the influence
of aryl-pyrazole ring substituents on the torsion angle θ and
p38α kinase binding.
Co-crystal structures of compounds 2 and 3 bound to
p38α kinase have been reported by Pargellis6 and Regan.7
These structures gave us the opportunity to compare the con-
formation of the protein-bound ligand with the conformation
of the free ligand. Compound 4 represents the original aryl
part of doramapimod with
a
methyl group in the
para-position.
For compounds similar to 5, with a methyl group in the
ortho-position, a decrease in activity was reported.7 The deter-
mination of twist angle θ of both compounds 4 and 5 would
be helpful to rationalize the reported differences in activities.
All compounds were synthesized by the condensation of the
corresponding substituted phenyl-1H-pyrazol-5-amines with
4-chlorophenyl isocyanate.7 The substituted phenyl-1H-
pyrazol-5-amines were prepared from 4,4-dimethyl-3-oxo-
pentanenitrile and substituted phenyl-hydrazines,7 known as
the Knorr pyrazole synthesis (see Scheme 1). The synthesis of
compounds 4–6 has been previously reported.12
Single crystals of doramapimod analogues 3–6 suitable for
X-ray crystal structure analysis were obtained from dimethyl-
sulfoxide or methanol solutions. The values of torsion angles
θ of synthesized analogues were listed in Fig. 2.
Due to the N1-pyrazole basicity and N–H urea hydrogen-
bound donation property, solvated complexes of 3 and 5 were
obtained. In solution, there is a free rotation around the
pyrazole-aryl planes of these molecules, which makes them
quite flexible and allows them to adopt multiple conforma-
tions in solution. In solid state, however, analogues 3 and 4
with a similar aryl-pyrazole part present in doramapimod
have distinct torsion angles between 34.5° and 47°. Compari-
son of these values with the bioactive conformation shows
only a slight deviation. In contrast, the introduction of the
methyl group in ortho-position led to an increase of the tor-
sion angle θ (82.2° and 72.8°). These values are beyond the
bioactive conformation (θ = 54°) and would explain the loss
of activity.
Fig.
1 Key interactions of doramapimod (1) with p38α kinase
according to Regan and co-workers6 (PDB code 1KV2). Constitution of
fragment 2 and doramapimod analogues 3–6 investigated in this study.
with a substituent in the ortho-position was explained by a
possible increase of the torsion angle θ beyond the favoured
54° (Fig. 1).
To test this hypothesis of an inhibitor bioactive confor-
mation and to investigate the influence of the torsion angle
on binding affinity, a conformational analysis was carried
out with the main focus on the torsion angle θ. This analy-
sis was conducted by the chemical modification of
doramapimod (1) within the aryl-pyrazole region (Fig. 1).
Doramapimod (1) has been subjected to different structure–
activity relationship (SAR) studies focussing on the urea-,7
the morpholine part,9 ethoxy morpholino-10 and ethoxy
naphthalene modification.11 For the modification of the
aryl-pyrazole part, derivatives of doramapimod (3–12) were
synthesized. The DFG-out mode of binding of this class of
molecules was confirmed by using nuclear magnetic reso-
nance (NMR) spectroscopy. The torsion angle θ of the ana-
logues (3–6) were characterized by small-molecule X-ray
Scheme 1
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Fig. 2 Perspective view of the X-ray structure of 5 as a methanol sol-
vated complex, showing the displacement ellipsoids drawn at the 50%
probability level with the dihedral angle θ between the pyrazole/σ-tolyl
planes (N1–N2–C–C). Dihedral angle values for derivatives 3–6.
To confirm the DFG-out binding mode of these type II
compounds 2D-NMR technique was used in combination
with selectively 15N-Phe-isotope labelled p38α kinase. The
p38α kinase contains 13 phenylalanine residues (see ESI‡), of
which 12 can be detected and unambiguously assigned in the
1H–15N TROSY spectrum of the apo-form13 (Fig. 3). The miss-
ing signal belongs to Phe169, which is part of the flexible
DFG-loop. As previously reported, the DFG-loop of the p38α
kinase apo-form is involved in a DFG-in/DFG-out conforma-
tional exchange process on an intermediate timescale, caus-
ing a strong line broadening and missing of the Phe169
signal.
Addition of the fragment 2 arrests the DFG-loop of p38α
kinase in the DFG-out conformation, resulting in the detec-
Fig.
3
1H–15N TROSY spectra of 15N-pheylananine-labeled p38α
kinase in the apo-state (in black) and after the addition of fragment 2
(in red). The addition of fragment 2 results in the appearance of a new
peak (indicated by an arrow) in the spectrum.
1
tion of the missing Phe169 signal in the H–15N TROSY spec-
trum (Fig. 3). Similar results were obtained with the other an-
alogues containing the pyrazole-urea scaffold (see ESI‡).
The binding affinities of synthesized analogues to p38α ki-
nase were investigated by microscale thermophoresis (MST).
MST has recently emerged as a powerful technique to investi-
gate ligand-kinase interactions.14 The affinities for the inter-
action is quantified by monitoring the directed movement of
fluorescent molecules in microscopic temperature gradi-
ents.15 All the tested compounds against p38α kinase re-
vealed accurate MST traces with no sign of protein aggrega-
tion or denaturation. As an example the MST thermogram of
compound 3 is shown in Fig. 4. All investigated derivatives
show similar binding curves.
stants were observed, indicating that this type of inhibi-
tors has the same affinity for p38α kinase irrespective of
its phosphorylation state: This finding has already been
reported for doramapimod16 and is in line with our previ-
ous NMR investigations showing the dynamic status of the
DFG-loop in the unphosphorylated and phosphorylated ki-
nase.17 The inhibition constants for analogues 2–6 are
summarized in Fig. 4.
The Ki values for compounds 2 and 4–6 determined by
MST are in good agreement with reported values from bind-
ing studies using an acrylodan-fluorescently labelled p38α
kinase.12
Compounds were tested against the inactive and active
form of the p38α kinase. No differences in inhibition con-
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Fig. 5 Constitution of doramapimod analogues 7–12, with different
substitutions in the para- and ortho-position.
stituent showed no differences in potency when introduced
either in the para-7 or ortho-position 8, compared to the
unsubstituted compound 3.
In contrast, when larger substituents (i.e. chlorine or
methoxy-group) were introduced clear differences in inhibi-
tion were observed. The ortho-substituted analogues (10 and
12) were significantly less active compared to their para-
substituted counterparts (9 and 11).
For fragment 2, compared with doramapimod (1) and the
analogues 3–12, the N-phenyl-1H-pyrazole scaffold, and
thereby the attractive hydrophobic interaction is missing (log
P = 4.3). The lack of this hydrophobic interaction is reflected
in its low binding affinity (Ki = 1.29 μM), as the Ki increases
by thirteen fold compared to compound 3. This is in agree-
ment with the reported data.7 Compound 2 also shows differ-
ent crystallization behaviour. The X-ray structure contains
Fig. 4 MST measurements for the binding of doramapimod analogues
to the inactive p38α kinase. MST thermogram of compound 5 in
concentrations ranging from 1.5 nM to 50 μM against
a fix
concentration of p38α kinase (80 nM) and tracer (25 nM). MST
experimental binding curves of derivatives 3–6. Data are expressed as
the mean of three independent runs. Data were fitted using a variable
slope four parameters fit (R-square >0.98). Ki = inhibition constant,
determined by microscale thermophoresis. Data from competition
experiment against p38α kinase.
Analogues 4 and 6 are three to fivefold more potent com-
pared to analogue 3. This is due to the introduction of the
methyl group which increases the lipophilicity of these com-
pounds as reflected in their calculated log P values (3 log P =
5.6, 4 log P = 6.0 and 6 log P = 6.2) and hence increases the
binding affinity towards the hydrophobic pocket of p38α ki-
nase. In contrast, compound 5, with similar lipophilicity (log
P = 6.2) but with an increase torsion angle value (θ = 77.5°)
revealed a decrease of affinity by sevenfold compared to com-
pound 3. The data show a clear correlation between kinase
binding affinities and the torsion angle θ of the inhibitors.
To further investigate the influence of ligand conforma-
tion on kinase binding, additional compounds (7–12) were
synthesized (Fig. 5).
Pairs of ortho- and para-substituted doramapimod ana-
logues were selected with respect to the spatial requirements
of the substituents. Analogues 7–12 were tested against p38α
kinase by MST (Fig. 6). As expected, the small fluorine sub-
Fig. 6 Inhibition constants of pairs of ortho- and para substituted ana-
logues. Data were generated from competition experiments against
the inactive p38α kinase and expressed as the mean of three indepen-
dent runs. Compound 3 was used as a control.
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the protein-bound conformation. Therefore, the recognition
process presumably follows the conformational selection
mechanism of ligand binding to p38α kinase.
In general, consideration of ligand conformation is impor-
tant for a successful ligand based inhibitor design.
The ability of both determining conformational proper-
ties of small molecule and their effect on target binding is
indispensable for drug discovery as demonstrated for the
cannabinoid CB1 receptor antagonists23 and hepatitis C vi-
rus NS5B RNA polymerase inhibitors.24 In both case studies
conformational characteristics of the interaction of the li-
gand with its respective target were determined. Subse-
quently, conformational-based restrictions of ligand flexible
torsion angles were used to guide the identification of new
inhibitors. As
a result conformational constrained ana-
logues with increased potencies were identified.23,24 In this
study we have analyzed the conformational properties of
doramapimod analogues and revealed a clear correlation
between p38α kinase binding and the torsion angle θ. The
presented study provides strong support for the incorpora-
tion of ligand conformational analysis into the common
SAR studies for conformationally flexible ligands.
Fig. 7 Superposition of the crystal structures of p38α kinase (PDB
code 1KV2), PYK2 (PDB code 3FZS), JNK2 (PDB code 3NPC), EphA3
(PDB code 4TWN) and B-raf (PDB code 4JVG) in complex with
doramapimod (1). The figure was generated using PyMOL program.22
three independent molecules with significantly different con-
formations. Each molecule contains three planar planes. For
the three independent molecules the angles between the
pyrazole–urea, urea–phenyl and pyrazole–phenyl are respec-
tively 30.6/86.3/84.5°, 34.3/30.6/10.5° and 9.3/67.5/84.7°. The
pyrazole–urea, urea–phenyl and pyrazole–phenyl torsion an-
gles for the protein bound conformation 2 are 41.96/41.86
and 69.27.6 This finding underscores the highly flexible na-
ture of fragment 2 even in the solid state.
Experimental part
Chemistry
General remarks. NMR spectra were recorded on Bruker
DPX250 and AVII300 spectrometers operating at a 1H fre-
quency of 250 MHz or 300 MHz and at 13C frequency of 62.9
MHz. Elementary analyses were measured on a Foss Heraeus
CHN-O-RAPID instrument. All reactions were monitored by
thin-layer chromatography (TLC), performed on silica gel
POLYgram® (Macherey-Nagel). Chromatographic purifica-
tions were done with Merck silica gel 60.
5-tert-Butyl-2-(2-fluoro-phenyl)-2H-pyrazol-3-yl-amine, 3-tert-
butyl-1-(4-methoxyphenyl)-1H-pyrazol-5-amine and 3-tert-butyl-
1-(2-methoxyphenyl)-1H-pyrazol-5-amine were purchased from
Fluorochem. 3-tert-butyl-1-(4-fluoro-phenyl)-1H-pyrazol-5-amine,
3-tert-butyl-1-(2-chloro-phenyl)-1H-pyrazol-5-amine, 3-tert-butyl-
1-(4-chloro-phenyl)-1H-pyrazol-5-amine and 4-chlorophenyl iso-
cyanate were purchased from Sigma Aldrich.
1-(5-tert-Butyl-2-methyl-2H-pyrazol-3-yl)-3-(4-chlorophenyl)-
urea (2) and 1-(3-tert-butyl-2-methyl-2H-pyrazol-5-yl)-3-(4-
chlorophenyl)urea (iso-2). A solution of 4,4-dimethyl-3-oxo-
pentanenitrile (18 mmol, 2.25 g) and methyl hydrazine (19
mmol, 0.875 g, 1 mL) in toluene (7.5 mL) was heated to reflux
overnight. Toluene was removed in vacuo. The residue was
dissolved in dichloromethane (DCM) and washed with water
and brine. After concentration in vacuo 2.5 g of crude
amines: 3-tert-butyl-1-methyl-1H-pyrazol-5-yl-amine and 5-tert-
butyl-1-methyl-1H-pyrazol-3-yl-amine were obtained. A mixture
of above crude amines (16 mmol, 2.45 g) and 4-chlorophenyl
isocyanate (15 mmol, 2.3 g) in DCM (28 mL) was stirred over
night at room temperature (see Scheme 1). After removal of
solvent in vacuo, the crude products (2) and its position
For a rational drug discovery approach, doramapimod was
also co-crystalized with c-Jun N-terminal kinase 2 (JNK2) a
closely related mitogen-activated protein kinase.18 Additional
structures of doramapimod in complex with RAF kinase,19
a
member of the serine/threonine protein kinase family, as well
as with tyrosine kinases PYK220 and EphA321 were also
reported. In all these structures, doramapimod stabilizes the
DFG-out conformation of the respective kinases.
A closer look at the dihedral angle θ between the pyrazole/
p-tolyl planes, reveals that doramapimod adopts the same
bioactive conformation with a dihedral angle of θ ∼ 42.3°.
Our findings of the bioactive conformation of doramapimod,
seems to be generalizable to other kinase-doramapimod com-
plexes. The overlay of doramapimod bound to the respective
kinases is highlighted in Fig. 7.
Conclusions
In summary, we showed that the structures of doramapimod
and analogues are highly flexible in solution, whereas in
solid state they adopt a conformation belonging to one of the
local energy minima. This conformation differs slightly from
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isomer (iso-2) were purified by silica gel chromatography
using 50% ethyl acetate in hexane as the eluent.
Rf = 0.15.
moval of solvent in vacuo and crystallization from ethyl ace-
tate and hexane (1 : 1) compound (5) was isolated as a white
powder.
1H-NMR (250.13 MHz, DMSO-d6): δ [ppm] = 9.00 (s, 1H,
N1H), 8.50 (s, 1H, N2H), 7.50 (d, 2H, aromatic,3J = 8.8 Hz),
7.33 (d, 2H, aromatic,3J = 8.8 Hz), 6.05 (s, 1H, C4H), 3.60 (s,
3H, CH3), 1.21 (s, 9H, tert-butyl).
1H-NMR (250.13 MHz, DMSO-d6): δ [ppm] = 9.08 (s, 1H,
N3H), 8.22 (s, 1H, N1H), 7.47–7.30 (m, 8H, aromatic), 6.41 (s,
1H, C4H), 2.04 (s, 3H, CH3), 1.30 (s, 9H, tert-butyl).
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 160.6; 150.9;
138.3; 138.1; 137.0; 136.3; 125.6 32.0 (C), 131.0; 129.1; 128.6;
128.0; 126.7; 119.5; 91.9 (CH), 30.3; 17.0 (CH3).
Anal. calcd. for C21H23ClN4O: C 65.88, H 6.05, N 14.63;
found: C 65.93, H 6.07, N 14.87.
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 158.6; 151.8;
138.4; 136.8; 125.6; 31.8 (C), 128.6; 119.7; 94.0 (CH), 34.9;
30.3 (CH3).
Anal. calcd. for C15H19ClN4O: C 58.73, H 6.24, N 18.26;
found: C 58.67, H 6.12, N 18.04.
MS: m/z 383.18 (MH+).
1-(3-tert-Butyl-2-methyl-2H-pyrazol-5-yl)-3-(4-chlorophenyl)-
1-(5-tert-Butyl-2-m-tolyl-2H-pyrazol-3-yl)-3-(4 chlorophenyl)
urea (6). This compound was prepared from the condensa-
1
urea (iso-2). H-NMR (250.13 MHz, DMSO-d6): δ [ppm] = 9.22
(s, br, 1H, N3H), 8.91 (s, 1H, N1H), 7.50 (d, 2H, aromatic,3J =
8.8 Hz), 7.32 (d, 2H, aromatic,3J = 8.8 Hz), 6.04 (s, 1H, C4H),
3.80 (s, 3H, CH3), 1.32 (s, 9H, tert-butyl).
tion
4-chlorophenyl isocyanate (see Scheme 1).
mixture of above amine (7 mmol, 1.5 g) and
of
5-tert-butyl-2-m-toly-2H-pyrazol-3-yl-amine7
and
A
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 30.9; 93.2;
139.0; 145.3; 151.0; 151.8 (C), 119.7; 128.6; 125.3 (CH), 29.2;
38.6 (CH3).
4-chlorophenyl isocyanate (7 mmol, 1.07 g) in DCM (14 mL)
was stirred over night at room temperature. After removal of
solvent in vacuo and crystalization from ethyl acetate and hex-
ane (1 : 1) compound (6) was isolated as a white powder.
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.6 (s, 1H, N3H),
8.74 (s, 1H, N1H), 7.45–7.13 (m, 8H, aromatic), 6.30 (s, 1H,
C4H), 2.30 (s, 3H, CH3), 1.29 (s, 9H, tert-butyl).
Anal. calcd. for C15H19ClN4O: C 58.73, H 6.24, N 18.26;
found: C 58.66, H 6.21, N 18.35.
1-(5-tert-Butyl-2-phenyl-2H-pyrazol-3-yl)-3-(4-chlorophenyl)-
urea (3). The title compound was prepared according the lit-
erature procedure.7
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 160.9; 152.1;
139.3; 138.9; 138.4; 137.7; 125.9; 32.4 (C), 129.5; 129.1; 128.5;
125.4; 121.9; 120.0; 96.2 (CH), 30.6; 21.4 (CH3).
1H-NMR (400.13 MHz, CDCl3) δ [ppm] = 7.44 (s, 1H, N1H),
7.23–7.00 (m, 9H, aromatic), 6.79 (s, 1H, N2H), 6.23 (s, 1H,
C4H), 1.17 (s, 9H, tert-butyl).
MS: m/z 383.18 (MH+).
13C-NMR (100.6 MHz, CDCl3): δ [ppm] = 162.8; 150.0;
138.0; 136.1; 135.7; 128.0; 32.4 (C), 129.4; 129.1; 127.9; 124.5;
121.5; 97.0 (CH), 30.2 (CH3).
1-(5-tert-Butyl-2-p-fluoro-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (7). This compound was prepared from
the condensation of 3-tert-butyl-1-(4-fluoro-phenyl)-1H-pyrazol-
5-amine and 4-chlorophenyl isocyanate (see Scheme 1).
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.12 (s, 1H,
N3H), 8.41 (s, 1H, N1H), 7.61–7.31 (m, 8H, aromatic), 6.37 (s,
1H, C4H), 1.22 (s, 9H, tert-butyl).
1-(5-tert-Butyl-2-ρ-tolyl-2H-pyrazol-3-yl)-3-(4-chlorophenyl)-
urea (4). This compound was prepared from the condensa-
tion
4-chlorophenyl isocyanate (see Scheme 1).
mixture of above amine (7 mmol, 1.5 g) and
of
5-tert-butyl-2-ρ-toly-2H-pyrazol-3-yl-amine7
and
A
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 163.0; 161.2;
159.7; 152.0; 138.8; 137.6; 1135.4/135.4; 126.1; 32.5 (C), 129.1;
127.1/127.0; 120.1; 116.6/116.3; 96.2 (CH), 30.6 (CH3).
19F-NMR (372.5 MHz, DMSO-d6): δ [ppm] = −114.6.
MS: m/z 387.14 (MH+).
1-(5-tert-Butyl-2-o-fluoro-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (8). This compound was prepared from
the condensation of 5-tert-butyl-2-(2-fluoro-phenyl)-2H-pyrazol-
3-yl-amine and 4-chlorophenyl isocyanate (see Scheme 1).
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.06 (s, 1H,
N3H), 8.45 (s, 1H, N1H), 7.71–7.31 (m, 8H, aromatic), 6.42 (s,
1H, C4H), 1.23 (s, 9H, tert-butyl).
4-chlorophenyl isocyanate (7 mmol, 1.07 g) in DCM (14 mL)
was stirred over night at room temperature. After removal of
solvent in vacuo and crystallization from ethyl acetate and
hexane (1 : 1) compound (4) was isolated as a white powder.
1H-NMR (250.13 MHz, DMSO-d6): δ [ppm] = 9.15 (s, 1H,
N3H), 8.37 (s, 1H, N1H), 7.48–7.25 (m, 8H, aromatic), 6.36 (s,
1H, C4H), 2.38 (s, 3H, CH3), 1.28 (s, 9H, tert-butyl).
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 160.5; 151.4;
138.3; 136.9; 136.7; 136.0; 125.5; 31.9 (C), 129.6; 128.6; 124.3;
119.6; 95.1 (CH), 30.1; 20.5 (CH3).
Anal. calcd. for C21H23ClN4O: C 65.88, H 6.05, N 14.63;
found: C 65.62, H 6.09, N 14.87.
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 158.6; 155.3;
131.2/131.3; 126.3/126.2; 126.1; 125.7/125.6; 117.4/117.1; 32.5
(C), 162.0; 151.4; 139.1/138.6; 130.1; 129.1; 120.0; 93.6 (CH),
30.8 (CH3).
MS: m/z 383.19 (MH+).
1-(5-tert-Butyl-2-σ-tolyl-2H-pyrazol-3-yl)-3-(4 chlorophenyl)
urea (5). This compound was prepared from the condensa-
tion
4-chlorophenyl isocyanate (see Scheme 1).
mixture of above amine (17 mmol, 3.9 g) and
of
5-tert-butyl-2-σ-toly-2H-pyrazol-3-yl-amine7
and
19F-NMR (372.5 MHz, DMSO-d6): δ [ppm] = −121.3.
MS: m/z 387.14 (MH+).
A
1-(5-tert-Butyl-2-p-methoxy-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (9). This compound was prepared from
the condensation of 3-tert-butyl-1-(4-methoxyphenyl)-1H-
4-chlorophenyl isocyanate (14.6 mmol, 2.25 g) in DCM (28
mL) was stirred over night at room temperature. After re-
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pyrazol-5-amine
Scheme 1).
and
4-chlorophenyl
isocyanate
(see
the pETDuet (Novagen, Madison, WI, USA) vector, which is
designed for the co-expression of two target genes.
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.15 (s, 1H,
N3H), 8.34 (s, 1H, N1H), 7.47–7.07 (m, 8H, aromatic), 6.36 (s,
1H, C4H), 3.83 (s, 3H, OCH3), 1.29 (s, 9H, tert-butyl).
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 160.7 ; 158.9;
151.8; 138.9; 137.5; 131.8; 126.0; 32.4 (C), 127.1; 126.4; 119.6;
114.8; 94.9 (CH), 55.9; 30.7 (CH3).
Instrumentation
The MST measurements were performed on a Nanotemper
Monolith NT.115 instrument. A MST power of 40% and a
LED power of 50% with a laser on time of 30 seconds and a
laser off time of 5 seconds were used. Analysis was performed
with NanoTemper Analysis Software and calculated IC50
competition values were converted to Ki values using the Ki
http://sw16.im.med.umich.edu/software/calc_ki/) analysis was
performed with NanoTemper Analysis Software.
MS: m/z 399.16 (MH+).
1-(5-tert-Butyl-2-o-methoxy-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (10). This compound was prepared from the
condensation of 3-tert-butyl-1-(2-methoxyphenyl)-1H-pyrazol-5-
amine and 4-chlorophenyl isocyanate (see Scheme 1).
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.21 (s, 1H,
N3H), 8.16 (s, 1H, N1H), 7.55–7.14 (m, 8H, aromatic), 6.36 (s,
1H, C4H), 3.81 (s, 3H, OCH3), 1.27 (s, 9H, tert-butyl).
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 161.1 ; 154.3;
151.1; 139.0; 138.8; 130.6; 126.0; 32.5 (C), 129.7; 129.1;
127.0121.2; 119.9; 113.3; 92.1 (CH), 56.2; 30.7 (CH3).
MS: m/z 399.16 (MH+).
Microscale thermophoresis (MST)
Competition assay. All compounds were diluted into a
buffer containing 50 mM Tris pH 7,6, 150 mM NaCl, 10 mM
MgCl2 and 0.05% Tween20. The final DMSO concentration
was adjusted to 5% (v/v) in the dilution series. A premix
containing 80 nM (or 160 nM) p38α kinase and 25 nM Kinase
tracer 199 (Life Technologies) was prepared. 10 μl of each
compound concentration was mixed with 10 μl of the premix
containing kinase tracer 199 and p38α kinase.
1-(5-tert-Butyl-2-p-chloro-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (11). This compound was prepared from
the condensation of 3-tert-butyl-1-(2-chloro-phenyl)-1H-pyrazol-
5-amine and 4-chlorophenyl isocyanate (see Scheme 1).
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 9.13 (s, 1H,
N3H), 8.46 (s, 1H, N1H), 7.66 (s, 4H, aromatic), 7.44 (dd, 4H,
aromatic), 6.38 (s, 1H, C4H), 1.29 (s, 9H, tert-butyl).
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 161.6 ; 152.1;
138.8; 137.9; 137.6; 131.9; 32.5 (C), 129.6; 129.1; 126.1; 120.1;
96.9 (CH), 30.5 (CH3).
Acknowledgements
H. S. is member of the DFG-funded cluster of excellence:
macromolecular complexes. K. S. is supported by SFB807. The
work present here has been conducted within the center for
translational cancer research, DKTK. BMRZ is supported by
the state of Hesse. The authors would like to thank Dr. Gerd
Nielsen for her support in running the NMR experiments.
MS: m/z 403.13 (MH+).
1-(5-tert-Butyl-2-o-chloro-phenyl)-2H-pyrazol-3-yl)-3-(4-
chlorophenyl)urea (12). This compound was prepared from
the condensation of 3-tert-butyl-1-(2-chloro-phenyl)-1H-pyrazol-
5-amine and 4-chlorophenyl isocyanate (see Scheme 1).
1H-NMR (300 MHz, DMSO-d6): δ [ppm] = 8.99 (s, 1H,
N3H), 8.32 (s, 1H, N1H), 7.74–7.30 (m, 8H, aromatic), 6.40 (s,
1H, C4H), 1.28 (s, 9H, tert-butyl).
Notes and references
1 J. C. Lee, J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S.
Kumar, D. Green, D. McNulty, M. J. Blumenthal, J. R. Heys,
S. W. Landvatter, J. E. Strickler, M. M. McLaughlin, I. R.
Siemens, S. M. Fisher, G. P. Livi, J. R. White, J. L. Adams
and P. R. Young, Nature, 1994, 372, 739.
2 P. Cohen, Nat. Rev. Drug Discovery, 2002, 1, 309.
3 J. Dumas, Curr. Opin. Drug Discovery Dev., 2002, 5, 718.
4 G. Manning, D. B. Whyte, R. Martinez, T. Hunter and S.
Sudarsanam, Science, 2002, 1912, 298.
13C-NMR (62.9 MHz, DMSO-d6): δ [ppm] = 161.7 ; 151.2;
139.1; 138.6; 136.0; 126.1; 32.5 (C), 132.3; 131.4; 131.1; 130.7;
129.1; 128.3; 120.0; 92.5 (CH), 30.6 (CH3).
MS: m/z 403.13 (MH+).
Crystal structure determinations. Crystallographic data of
compounds (2), (3), (4), (5) and (6) have been deposited with
Cambridge Crystallographic Data Center.
5 R. Roskoski Jr., Pharmacol. Res., 2016, 103, 26.
6 C. Pargellis, L. Tong, L. Churchill, P. F. Cirillo, T. Gilmore,
A. G. Graham, P. M. Grob, E. R. Hickey, N. Moss, S. Pav and
J. Regan, Nat. Struct. Biol., 2002, 9, 268.
7 J. Regan, S. Breitfelder, P. F. Cirillo, T. Gilmore, A. G.
Graham, E. R. Hickey, B. Klaus, J. Madwed, M. Moriak, N.
Moos, C. Pargellis, S. Pav, A. Proto, A. Swinamer, L. Tong
and C. Torcellini, J. Med. Chem., 2002, 45, 2994.
8 Z. Zhao, H. Wu, L. Wang, Y. Liu, S. Knapp, Q. Liu and N. S.
Gray, ACS Chem. Biol., 2014, 6, 1230.
9 J. Regan, A. Capolino, P. F. Cirillo, T. Gilmore, A. G.
Graham, E. R. Hickey, R. R. Kroe, J. Madwed, M. Moriak, R.
Biophysical investigation
Protein production
Murine p38α kinase (2–349) was cloned, expressed and puri-
fied as described in our previous work.11
For expression of phosphorylated active p38α kinase, the
S599D/T603D MKK6/SKK3 activated mutant was co-expressed
with p38α kinase. This activation mutant of MKK6/SKK3 is
needed, since its expression in E. coli results in an inactive
state. For co-expression, both kinase genes were cloned into
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Med. Chem. Commun.

View Article Online
Research Article
Nelson, C. A. Pargellis, A. Swinamer, C. Torcellini, M. Tsang
MedChemComm
15 M. Jerabek-Willemsena, T. Andréa, R. Wannera, H. Marie
Rotha, S. Duhra, P. Baaskea and D. Breitsprechera, J. Mol.
Struct., 2014, 1077, 101.
16 G. J. Zaman, M. M. van der Lee, J. J. Kok, R. L. Nelissen and
E. E. Loomans, Assay Drug Dev. Technol., 2006, 4, 411.
17 G. Nielsen and H. Schwalbe, ChemBioChem, 2011, 17, 2599.
18 A. Kuglstatter, M. Ghate, S. Tsing, A. G. Villaseñor, D. Shaw,
J. W. Barnett and M. F. Browner, Bioorg. Med. Chem. Lett.,
2010, 17, 5217.
19 H. Lavoie, N. Thevakumaran, G. Gavory, J. J. Li, A.
Padeganeh, S. Guiral, J. Duchaine, D. Y. Mao, M. Bouvier, F.
Sicheri and M. Therrien, Nat. Chem. Biol., 2013, 7, 428.
20 S. Han, A. Mistry, J. S. Chang, D. Cunningham, M. Griffor,
P. C. Bonnette, H. Wang, B. A. Chrunyk, G. E. Aspnes, D. P.
Walker, A. D. Brosius and L. Buckbinder, J. Biolumin.
Chemilumin., 2009, 19, 13193.
21 J. Dong, H. Zhao, T. Zhou, D. Spiliotopoulos, C. Rajendran, X. D.
Li, D. Huang and A. Caflisch, ACS Med. Chem. Lett., 2014, 1, 79.
22 W. L. Delano, The PyMOL Molecular Graphics System, 2002.
23 (a) B. F. Thomas, Y. Zhang, M. Brackeen, K. M. Page, S. W.
Mascarella and H. H. Seltzman, AAPS J., 2006, 8, E665; (b)
The cannabinoid receptors, ed. P. Reggio, 2009, p. 95.
24 K. Ikegashira, T. Oka, S. Hirashima, S. Noji, H. Yamanaka,
Y. Hara, T. Adachi, J. Tsuruha, S. Doi, Y. Hase, T. Noguchi, I.
Ando, N. Ogura, S. Ikeda and H. Hashimoto, J. Med. Chem.,
2006, 49, 6950.
and N. Moos, J. Med. Chem., 2003, 46, 4676.
10 (a) N. Moss, S. Breitfelder, R. Betageri, P. F. Cirillo, T. Fadra,
E. R. Hickey, T. Kirrane, R. R. Kroe, J. Madwed, R. M.
Nelson, C. A. Pargellis, K. C. Qian, J. Regan, A. Swinamer
and C. Torcellini, Bioorg. Med. Chem., 2003, 13, 3101; (b)
S. T. Onions, K. Ito, C. E. Charron, R. J. Brown, M. Colucci,
F. Frickel, G. Hardy, K. Joly, J. King-Underwood, Y. Kizawa, I.
Knowles, P. J. Murray, A. Novak, A. Rani, G. Rapeport, A.
Smith, P. Strong, D. M. Taddei and J. G. Williams, J. Med.
Chem., 2016, 5, 1727.
11 (a) J. Regan, C. A. Pargellis, P. F. Cirillo, T. Gilmore, E. R.
Hickey, G. W. Peet, A. Proto, A. Swinamer and N. Moss,
Bioorg. Med. Chem., 2003, 13, 3101; (b) T. Arai, M. Ohno, H.
Inoue, S. Hayashi, T. Aoki, H. Hirokawa, H. Meguro, Y.
Koga, K. Oshida, M. Kainoh, K. Suyama and H. Kawai,
Bioorg. Med. Chem., 2012, 15, 5118.
12 J. R. Simard, M. Getlik, C. Grütter, V. Pawar, S. Wulfert, M.
Rabiller and D. Rauh, J. Am. Chem. Soc., 2009, 37, 13286.
13 M. Vogtherr, K. Saxena, S. Hoelder, S. Grimme, M. Betz, U.
Schieborr, B. Pescatore, M. Robin, L. Delarbre, T. Langer,
K. U. Wendt and H. Schwalbe, Angew Chem., Int. Ed.,
2006, 45, 993.
14 P. Linke, K. Amaning, M. Maschberger, F. Vallee, V. Steier,
P. Baaske, S. Duhr, D. Breitsprecher and A. Rak, J. Biomol.
Screening, 2015, 1–8.
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