Fast Iterative Synthetic Approach toward Identification of Novel Highly Selective p38 MAP Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01227

p38 mitogen-activated protein kinases are key mediators of environmental stress response and are promising targets for treatment of inflammatory diseases and cancer. Numerous efforts have led to the discovery of several potent inhibitors; however, so far no highly selective type-II inhibitors have been reported. We previously identified VPC-00628 as a potent and selective type-II inhibitor of p38α/β with few off-targets. Here we analyzed the chemical building blocks of VPC-00628 that played a key role in achieving potency and selectivity through targeting an inactive state of the kinases induced by a unique folded P-loop conformation. Using a rapid, systematic combinatorial synthetic approach, we identified compound 93 (SR-318) with excellent potency and selectivity for p38α/β, which potently inhibited the TNF-α release in whole blood. SR-318 therefore presents a potent and selective type-II inhibitor of p38α/β that can be used as a chemical probe for targeting this particular inactive state of these two p38 isoforms.

Full text of DOI:10.1021/acs.jmedchem.9b01227

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Article
A fast iterative synthetic approach towards the identification
of novel highly selective p38 MAP kinase inhibitors
Sandra Röhm, Benedict-Tilman Berger, Martin Schröder, Apirat Chaikuad, Rob
Winkel, Koen F.W. Hekking, Jorg J.C. Benningshof, Gerhard Mueller, Roberta
Tesch, Mark Kudolo, Michael Forster, Stefan A. Laufer, and Stefan Knapp
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01227 • Publication Date (Web): 08 Nov 2019
Downloaded from pubs.acs.org on November 10, 2019
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A fast iterative synthetic approach towards the
identification of novel highly selective p38 MAP
kinase inhibitors
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Sandra Röhm,†,‡ Benedict-Tilman Berger,†,‡ Martin Schröder,†,‡ Apirat Chaikuad,†,‡ Rob
§
§
§
ɷ
Winkel, Koen F.W. Hekking, Jorg J.C. Benningshof, Gerhard Müller, Roberta
Tesch,†,‡ Mark Kudolo, Michael Forster, Stefan Laufer, Stefan Knapp*
ɸ
ɸ
ɸ
,†,‡
†Johann Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry, Max-von-Laue-Str. 9, D-60438
Frankfurt am Main, Germany
‡Johann Wolfgang Goethe-University, Structure Genomics Consortium (SGC), Buchmann Institute for Life Sciences,
Max-von-Laue-Str. 15, D-60438 Frankfurt am Main, Germany
§Mercachem BV, Kerkenbos 1013, 6546 BB, Nijmegen, The Netherlands
ɷGotham Therapeutics, 430 East 29th Street, Alexandria Center, New York, NY 10016, USA
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ɸDepartment of Pharmaceutical / Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8,
72076 Tübingen, Germany
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Correspondence: Stefan Knapp, e-mail: knapp@pharmchem.uni-frankfurt.de
ABSTRACT: p38 mitogen-activated protein kinases are key mediators of environmental
stress response and are promising targets for treatment of inflammatory diseases and
cancer. Numerous efforts have led to the discovery of several potent inhibitors, however,
so far no highly selective type-II inhibitors have been reported. We previously identified
VPC-00628 as a potent and selective type-II inhibitor of p38α/β with few off-targets. Here
we analyzed the chemical building blocks of VPC-00628 that played a key role in
achieving potency and selectivity through targeting an inactive state of the kinases
induced by a unique folded P-loop conformation. Using a rapid, systematic combinatorial
synthetic approach, we identified compound 93 (SR-318) with excellent potency and
selectivity for p38α/β which potently inhibited the TNF-α release in whole blood. SR-318
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presents therefore a potent and selective type-II inhibitor of p38α/β, that can be used as
a chemical probe for targeting this particular inactive state of these two p38 isoforms.
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INTRODUCTION
The p38α mitogen-activated protein kinase is a Ser/Thr kinase that is activated through
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stress stimuli such as heat shock or hypoxia. In the nucleus, p38α modulates the activity
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of other kinases as well as transcription factors, a process that regulates the expression
of specific target genes, most importantly pro-inflammatory cytokines including the tumor
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necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1 β). Dysregulated p38α
therefore plays a major role in the development of inflammatory diseases such as
rheumatoid arthritis (RA), inflammatory bowel diseases, chronic obstructive pulmonary
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disease (COPD) and autoimmune diseases. However, despite many years of intensive
research and clinical efforts, so far no p38 small molecule inhibitors have been approved
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yet, in many cases due to insufficient selectivity profiles leading to side effects as well as
activation of compensatory pathways.6-8 The p38 MAPK family consists of four isoforms,
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α, β, δ and γ, with diverse physiological roles. For example, several studies reported that
p38α, but not p38β, stimulates TNF-α release and other inflammatory cytokines.10 In
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addition, inhibition of p38β may induce apoptosis and stimulate the glycogen synthase
pathway, potentially leading to undesirable side effects of p38α/β dual inhibitors.12
Therefore, an isoform selective inhibition of p38α would be preferable. Many studies have
demonstrated several inhibitor design strategies that can lead to >100-fold selective
compounds for α/β over the γ and δ isoforms, nevertheless achieving such selectivity
among the two structurally most related α and β isoform remains a challenging task.13, 14
This is due to their high sequence identity of 97% with the only difference in the ATP
binding pocket to be the substitution of His107 in the hinge region of the α-isoform to a
threonine in the β counterpart. Recently, Wrobleski et al. reported the type-I 3-amino-4-
methyl-N-cyclopropylbenzamide inhibitors that exhibited surprisingly high potencies for
p38α with nearly 150-fold selectivity over the β isoform.15
Dynamic changes in the hinge backbone have been suggested as structural
mechanisms of inhibitor and isoform selectivity (Figure 1). In particular, the presence of
a glycine residue in the hinge region (G110), which is a rare sequence variation in protein
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kinases, has been utilized for the development of highly selective type-I inhibitors such
as Skepinone-L whereas reorientation of the Leu108-Met109 peptide backbone has been
discussed to contribute to p38α/β isoform selectivity.15-17
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Targeting an inactive DFG-out conformation of the kinases offers another strategy for
the development of potent type-II inhibitors, exemplified for example by the BCR/ABL-
inhibitor Imatinib.18, 19 Benefits of type-II inhibitors often include high potencies with a
prolonged resident time and potential targeting of non-catalytic functions of the kinases.20-
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Nevertheless, achieving selectivity with type-II binders is a proven difficulty as an
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inactive DFG-out conformation can be adopted by many kinases. This is exemplified by
the p38 type-II inhibitor BIRB-796 that exhibits picomolar on-target affinity, but only a poor
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kinome wide selectivity. A selective type-II chemical probe for p38α/β that would allow
targeting of the inactive kinase is still lacking. To fill this gap for this well studied target we
developed a potent and selective type-II inhibitor which is also able to induce a highly
distorted inactive state. We developed this chemical probe by a combinatorial synthetic
approach of a promising type-II hit identified by a large DNA encoded library screen.25
Selectivity for the p38α and p38β isoform was monitored during the optimization process
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which identified several inhibitors with favorable isoform selectivity. The best compound
of this series, 93 (SR-318) displayed good kinome wide selectivity and cellular potency
and can therefore by utilized as a chemical probe for p38α and p38β for target validation
and for a better understanding of the role of p38 inactive state in signaling and for the
treatment of diseases. In addition, targeting different conformational states, such as an
inactive form, may provide further understanding of non-catalytic functions of kinases as
well as potential different phenotypic responses.
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Figure 1. Binding mode of type-I and type-II p38 MAPK inhibitors. A) Canonical type-I binding mode of SB203580 (PDB:
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GCP) 26, B) an induced glycine flipped conformation observed upon the binding of selective type-I inhibitor Skepinone-
L (PDB: 3QUE) 17, C) the binding mode of a recently reported alpha isoform selective type-I inhibitor (1) inducing a
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hinge backbone flip at L108 (PDB: 4KIN) 15. CH---O interaction (green dash), hydrogen bonds (red dash), D) Schematic
illustration of the P-loop folded binding mode of type-II inhibitor VPC-00628 (PDB:5LAR).25 Highlighted are the building
blocks P1 (green), P2 (wheat), P3 (blue). E) VPC-00628 in complex with p38α (PDB: 5LAR), folded P-loop (yellow),
hinge region (green), DFG motif (pink), αC helix (blue), water molecule (red), hydrogen bonds (black). Insert:
stabilization of P-loop, hydrogen bonds (black dash), distance measurements (green dashes). Hydrophobic region I
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(HRI), hydrophobic region II (HRII).
Previously we have reported the potent and selective type-II p38 inhibitor VPC-0062825
(Figure 1D, 1E), that in addition to targeting the DFG-out conformation, also induces a
folded P-loop conformation, providing a structural mechanism for its remarkably improved
kinome wide selectivity compared to other well-known type-II p38 inhibitors like BIRB-
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96.24, 27 To understand structure-based activity relationship and a basis for selectivity,
we analyzed the chemical building blocks of VPC-00628 through the use of a fast-iterative
and systematic synthetic approach varying the different vectors P1, P2 and P3 followed
by biological screening methods like enzyme kinetic as well as temperature shift assays
(Figure 1D).
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CHEMISTRY
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Exploiting commercial availability of the P1, P2, P3 building blocks containing an
accessible amine group, we employed an amide coupling reaction for fast synthesis of
compounds 9-16 (Scheme 1). In brief, the P1 building block 5-amino-1-phenyl-1H-
pyrazole-4-carboxylic acid (2) was linked to 3a or 3b, followed by cleavage of the ester
group in the products 4 and 5 under basic conditions. The resulting acids 6 and 7 were
further reacted with the P3 building blocks (amines 8a-d) respectively, using a second
amide coupling reaction.
Scheme 1. Synthetic route using chemical approach A) for the preparation of P1 fixed
compoundsa
O
O
H2N
N
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N
N
H
N
H
O
OH
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O
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N
O
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III
I
final compounds
-16
OH
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H2N
H2N
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H2N
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H
H
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H N
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O
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OH
NH2
NH2
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NH2
3
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b
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N
H2N
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O
S
H2N
8d
3
O
a
Reagents and conditions: (I) EDC-HCl, HOAt, Et N, DCM, rt; (II) LiOH, THF/H O, rt;
3
2
(
III) HATU, Et N, DMF, rt; For final compounds 9 – 16 see Table 1.
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For the synthesis of compounds 27-34 a different synthetic approach B) was
established. The pyrazole heterocycle 19 was built up from phenylhydrazine (17) and (E)-
ethyl 2-cyano-3-ethoxyacrylate (18). Subsequently, the free amine of 19 was bis-
protected with benzyl protecting groups and in the next step the ester 20 was cleaved
under basic conditions. The resulting compound 21 formed the P1 building block and was
fused with 22a and 22b in an Einhorn type Schotten-Baumann reaction to generate 23
and 24. The products 25 and 26 of the ester hydrolysis of 23 and 24 were subsequently
fused with 8a-d following a standard amide coupling protocol to obtain the final
compounds 27-34 (Scheme 2).
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Scheme 2. Synthetic route using chemical approach B) for the preparation of P1 fixed
compoundsa
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NH
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CN 18
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c
aReagents and conditions: (I) EtOH, reflux; (II) NaH, BnBr, DMF, rt; (III) LiOH,
MeOH/H O, 80°C; (IV) a) SOCl , b) amine 22a or 22b, pyridine, DCM; (V) a) Pd/C, H ,
2
2
2
MeOH, rt, b) LiOH, MeOH/H O, rt; (VI) a) CF SO H, DCM, rt, b) LiOH, MeOH/H O, 60°C;
2
3
3
2
(VII) HATU, Et N, DMF, rt; For final compounds 27 – 34 see Table 1.
3
Compounds 45-60 with a fixed methyl 4-(aminomethyl)benzoate P2 building block were
synthesized by initially coupling the EDC-HCl/HOAt activated acids 36a-d with the
corresponding amine 35. The ester groups of the resulting intermediates 37-40 were then
cleaved under basic conditions to yield the acid compounds 41-44, which were
subsequently linked with 8a-d using an amide coupling reaction (Scheme 3).
Scheme 3. Synthetic route for the preparation of P2 invariant compoundsa
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O
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OH
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H
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3
8
42
H3N
Cl
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
I
II
III
-
final compounds
5-60
O
4
35
O
O
O
F3C
O
F3C
O
H2N
8a
OH
N
H
N
H
NH2
N
N
N
N
O
36a
N
N
N
N
39
43
O
H2N
8b
NH2
OH
O
36b
N
N
H
O
O
H
F3C
N
NH
O
NH
N
O
N
OH
40
44
H2N
NH2
36c
8c
N
OH
NH 36d
H2N
N
8d
a
Reagents and conditions: (I) EDC-HCl, HOAt, Et N, DCM, rt; (II) LiOH, THF/H O, rt;
3
2
(
III) HATU, Et N, DMF, rt; For final compounds 45 - 60 see Table 2.
3
To obtain the final compounds 71-86 with the fixed (S)-2-amino-4-
cyclohexylbutanamide building block P3, the amino acid derivative 61 was reacted in an
amide coupling reaction with HATU and the different acids 62a-d. The Boc or Cbz
protecting group was cleaved afterwards under acidic conditions and the free amine was
reacted with the hetero aromatic acids 36a-d in a second amide coupling reaction
(Scheme 4).
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4. Synthetic route for the preparation of P3 fixed compoundsa
Cbz NH
N
O
H2N
N
O
H
H
N
N
S
NH2
S
NH2
63
O
O
67
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Boc
HN
O
H2N
O
H
S
O
N
S
NH2
HN
NH2
O
64
68
O
H2N
NH2
I
IIa/b
III
final compounds
1-86
7
61
O
O
Cbz NH
N
O
O
H
H
Boc
N
N
N
OH
OH
62a
S
NH2
H2N
NH2
N
N
H
36a
O
O
O
69
65
Boc
HN
OH
OH
S
N
N
62b
O
36b
O
O
Boc
N
F3C
Boc
OH
OH
O
H N
O
2
N
H
H
H
OH
H
N
N
62c
O
NH2
NH2
N
N
N
36c
O
O
70
66
HN
OH
Boc
NH
36d
62d
O
a
Reagents and conditions: (I) HATU, Et N, DMF, rt; (IIa) TFA, DCM, rt for Boc
3
deprotection; (IIb) 33% HBr in AcOH for Cbz deprotection; (III) HATU, Et N, DMF, rt; For
3
final compounds 71-86 see Table 3.
For the synthesis of the P3 substituted compounds 89-103, the building block 2 was
fused with methyl 4-(aminomethyl)benzoate hydrochloride (35) via an amide coupling
reaction with HATU under basic conditions. The ester group of the intermediate 87 was
hydrolyzed with lithium hydroxide, resulting in the free acid 88, which was then coupled
with the amines P3 building blocks in a second amide coupling reaction with HATU (Table
4). An exception was noted for 103 where the starting intermediate 88 was first activated
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to the acid chloride using thionyl chloride prior to linking with the corresponding amine
(Scheme 5).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 5. Synthetic route for the preparation of P3 substituted compoundsa
O
O
O
H2N
N
H2N
N
H2N
N
I
II
IIIa/b
OH
N
N
final compounds
89-103, VPC-00628
H
H
O
OH
N
N
N
RNH2
H3N
2
Cl
-
87
O
88
O
O
35
O
a
Reagents and conditions: (I) EDC-HCl, HOAt, Et N, DCM, rt; (II) LiOH, MeOH/H O, rt;
3
2
(
IIIa) HATU, Et N, DMF, rt; (IIIb) for compound 103 a) SOCl , rt b) RNH , DCM/Pyridine;
3
2
2
For residues R see Table 4.
Compound 1 and the P3 substituted compounds 107-121 were synthesized using an
amide coupling reaction of 5-(2-chlorophenyl)thiophene-2-carboxylic acid (104) with
methyl 3-amino-4-methylbenzoate (22a) using the coupling reagents EDC-HCl and HOAt
under basic conditions. The ester intermediate 105 was cleaved with aqueous lithium
hydroxide solution in methanol. The resulting free acid 106 was subsequently reacted
with various amine P3 building blocks using HATU and trimethylamine in N,N’-
dimethylformamide to yield 107-121 (Table 6). For 121, the free acid was treated with
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
thionyl chloride, from which the resulting acid chloride was then linked with the amine P3
building block (Scheme 6).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Synthetic route for the preparation of 1 and P3 substituted compounds 1a
O
I
O
II
O
Cl
Cl
Cl
IIIa/b
O
OH
final compounds
1, 107-121
S
OH
S
N
S
N
H
H
O
O
RNH2
104
O
105
106
H2N
22a
O
a
Reagents and conditions: (I) EDC-HCl, HOAt, Et N, DCM, rt; (II) LiOH, MeOH/H O, rt;
3
2
(IIIa) HATU, Et N, DMF, rt; (IIIb) for compound 121: a) SOCl , rt; b) RNH , DCM/Pyridine;
3
2
2
For residues R see Table 5.
For the negative compound 126, 1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic
acid (122) was reacted with methyl 4-(aminomethyl)benzoate hydrochloride (35) in an
EDC-HCl mediated amide coupling reaction. The ester intermediate 123 was
subsequently cleaved under basic conditions and the resulting acid 124 was further
activated with HATU and reacted with 3-cyclohexylpropan-1-amine (125) (Scheme 7).
Scheme 7. Synthetic route for the preparation of 126a
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8
9
O
O
F3C
N
F3C
N
O
F3C
N
I
N
II
N
H
III
final compound
126
OH
H
O
OH
N
N
N
H3N
Cl-
O
O
122
O
123
124
H2N
35
O
125
a
Reagents and conditions: (I) EDC-HCl, HOBt, Et N, DCM, rt; (II) LiOH, MeOH/H O, rt;
3
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(III) HATU, DIPEA, DMF, rt.
RESULTS AND DISCUSSION
The screening hit VPC-00628 induces not only a DFG-out conformation but also a
folded P-loop conformation. Both of these induced inactive conformations may contribute
to the favorable selectivity profile of this compound. We dissected the chemical moieties
of the inhibitor into three parts (P1, P2 and P3) and exploited a facile and variable
synthetic approach combining a high diversity of chemical building blocks to generate a
comprehensive compound set. This allowed us to study the structure-activity relationships
(
SARs) for this unique binding mechanism, as well as to design an inhibitor with even
improved selectivity and potency. The 78 synthesized derivatives were initially
characterized for their ability to bind to p38α and p38β in a temperature shift (Differencial
Scanning Fluorimetry (DSF)) assay28 and for their inhibitory potency in an orthogonal
radiometric activity-based assay.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
For the first series, we explored the potential of targeting the hydrophobic region I and
the DFG-out pocket through the P2 and P3 building blocks, respectively, while retaining
the hinge binding 5-amino-1-phenyl-1H-pyrazole P1 moiety (Table 1). The results
showed that all derivatives with a meta-substituted tolyl linker in P2 position demonstrated
low nanomolar affinities (compounds 27-30). Although this “magic methyl” decoration is
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18
frequently used in type-II kinase inhibitors such as Imatinib , we assumed that the most
potent compound in this series, 30 with IC s of 2 nM and 10 nM for p38α and p38β,
50
respectively, might bind in a canonical type-I mode due to its high similarity to 1. However,
the presence of a pyrazole instead of a thiophene hinge binding group might lead to the
lack of α-isoform selectivity of 30 in comparison to 1. In order to protect the accessible
methylene bridge towards rapid metabolic degradation, we introduced a methyl group at
the benzyl linker in P2 of the compounds 9-12. Combining this P2 moiety with the bulky
P3 moieties either (S)-3-cyclohexyl-2-(methylamino)propanamide (8b) or the
cyclopropanamide (8d) resulted also in inhibitors 10 and 12 of nanomolar potency,
respectively. Interestingly, compound 10 showed a surprisingly high selectivity for the α-
isoform with a ratio of 40.8 in enzymatic assays. The combination with 2-methyl-2-
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(
methylamino)propanamide (8a) as P3 building block was the less suitable moiety
compared to the other P3 building blocks (8b, 8c and 8d) as exemplified by compounds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
, 13, 27 and 31. Thiazole was observed to also be a favorable P2 group demonstrated
by nanomolar potencies observed for 13-16. However, this was strongly dependent on
the P3 moiety as 13 lost its binding ability to the kinases completely. Similarly, the total
loss of binding affinities for 31-34 suggested that the thiophene heterocycle (22b) was a
sub-optimal P2 building block.
Table 1. Potency of P1-fixed compounds for p38α/β, determined by DSF- and enzyme
kinetic assays for p38α/β inhibition.
P1
P2
P3
H N
O
2
N
H
O
N
N
H
N
NH2
O
∆
Tma [°C]
∆Tma [°C]
IC50b
[µM]
IC50b
[µM]
ratioc
[fold]
comp
.
P2
P3
± SD
± SD
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
p38α
p38β
p38α
p38β
p38α/β
O
H
N
VPC-
NH2
N
H
0062
13.0 ± 0.7
12.5± 0.6
0.039
0.25
6.46
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
O
O
N
H
H
N
9
1.2 ± 0.1
4.1 ± 0.6
2.5 ± 0.3
0.7 ± 0.2
1.6 ± 0.2
1.2 ± 0.1
> 0.5
0.21
4.8
8.7
n.d.
40.78
n.d.
NH2
NH2
O
O
O
O
H
N
N
H
10
11
12
O
H
N
N
H
NH2
> 0.5
> 10
H
N
H
N
2.9 ± 0.4
0.6 ± 0.2
8.6 ± 0.9
1.6 ± 0.2
0.4 ± 0.3
9.1 ± 0.5
0.4
7.3
18.37
n.d.
O
O
N
H
N
H
N
> 0.5
0.045
> 10
0.37
S
O
NH2
NH2
1
3
4
H
N
N
H
N
1
8.28
S
O
O
H
N
N
NH2
H
N
15
3.6 ± 0.5
3.9 ± 0.3
0.12
1.7
14.66
S
O
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9
H
N
N
H
N
1
6
7
9.8 ± 0.8
2.7 ± 0.4
10.9 ± 0.8
5.6 ± 0.7
14.8 ± 0.4
0.9 ± 0.1
1.1 ± 0.2
0.8 ± 0.2
1.4 ± 0.2
9.6 ± 0.5
0.009
0.044
0.014
0.053
0.002
> 0.5
> 0.5
> 0.5
> 0.5
0.059
0.96
6.35
21.60
6.93
13.77
6.09
n.d.
S
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
H
N
2
N
H
2.1± 0.3
12.5 ± 0.8
7.0 ± 0.6
14.0 ± 0.7
0.0 ± 0.1
0.5 ± 0.1
0.1 ± 0.3
0.4 ± 0.3
NH2
NH2
O
O
O
O
H
N
28
29
N
H
0.099
0.736
0.010
> 10
O
H
N
NH2
N
H
H
N
30
31
32
33
34
N
H
O
O
H
N
S
O
O
O
O
N
NH2
H
H
N
NH2
S
S
S
> 10
n.d.
N
H
O
H
N
NH2
> 10
n.d.
N
H
H
N
> 10
n.d.
N
H
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
∆
T
m
average derived from seven replicates for p38α and six replicates for p38β at a compound concentration of
1
0 µM compound; b IC50 values determined by a radiometric protein kinase assay; c calcd. from IC50 values; Standard
deviation (SD) is given when n>2; n.d. = not determined.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, we evaluated the combinatorial effect of the hinge binding P1 group and the P3
substituent that targets the DFG-out hydrophobic pocket. Using the benzyl linker, which
is present in VPC-00628, as a fixed P2 moiety, 16 compounds (45-60) were synthesized
and were tested for their binding to p38α and p38β (Table 2). Within this series, we also
aimed to investigate the influence of the primary amine decoration of the pyrazole ring in
VPC-00628, which was shown to participate in the previously observed three layer π-
stacking interaction with the folded P-loop Tyr35 and the DFG-out Phe169 (Figure 1E).25
Therefore, the amine group of the pyrazole was removed in compounds 45-52 or
exchanged with a trifluoromethyl group in compounds 53-56. In addition, a differently
substituted pyrazole isomer with a free hydrogen donor group and without an amine (36d)
was used in compounds 57-60. Interestingly, regardless of the P3 moieties, all of these
introduced modifications (36a, 36b, 36c, 36d) of the P1 pyrazole led to the loss of binding
of almost all compounds to the kinases as demonstrated by no significant Tm shift in the
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DSF-assay and IC values larger than 0.5 µM or 10 µM for p38α and p38β, respectively.
50
This consistent trend suggested therefore the importance of the free amine substituent at
the pyrazole hinge binder of VPC-00628. Nevertheless, we observed surprisingly two
outliers within this series, compound 50 and 52, which retained moderate binding affinities
to p38α (IC ~0.26-0.28 μM). Both compounds comprised similarly the amine-free
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
pyrazole (36b), but differed in the P3 groups 8b and 8d. Analyses of the chemical
components revealed a closer similarity of 50 to VPC-00628 and 52 to 1, suggesting the
former compound to display type-II inhibitor characteristics. The retention of the binding
ability of 50 to p38α led to our next hypothesis that with an optimal P3 group for targeting
the hydrophobic DFG-out pocket the combination of the P1 and P2 moieties could play a
crucial role in dictating the type-II binding of the inhibitors. To test this hypothesis, we
generated another series of compounds by fixing cyclohexyl P3 group, which was used
in VPC-00628 while varying the combination of the P1 and P2 moieties.
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Enzymatic activity of compounds with invariant P2 moiety for p38α/β, determined
by DSF- and enzyme kinetic assays for p38α/β inhibition.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
P1
P2
P3
H N
O
2
N
H
O
N
N
H
N
NH2
O
∆
Tma [°C]
± SD
∆Tma [°C]
± SD
IC50b
[µM]
p38α
IC50b
[µM]
p38β
ratioc
comp
.
P1
P3
[fold]
p38α
p38β
p38α/β
O
O
O
O
H
N
4
5
6
0.0 ± 0.1
1.5 ± 0.3
0.0 ± 0.3
0.5 ± 0.3
> 0.5
> 0.5
> 10
> 10
n.d.
n.d.
N
N
NH2
NH2
H
N
4
N
N
O
H
N
O
NH2
47
0.6 ± 0.2
1.7 ± 0.2
0.5 ± 0.2
0.1 ± 0.3
0.0 ± 0.2
0.2 ± 0.2
> 0.5
> 0.5
> 0.5
> 10
> 10
> 10
n.d.
n.d.
n.d.
N
N
O
O
H
N
4
8
9
N
N
O
H
N
4
N
N
NH2
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2
3
4
5
6
7
8
9
O
O
H
N
O
O
O
NH2
5
0
1
N
N
N
3.6 ± 0.4
1.4 ± 0.2
3.0 ± 0.4
0.0 ± 0.1
0.3 ± 0.1
0.0 ± 0.2
0.4 ± 0.2
0.2 ± 0.1
1.4 ± 0.3
2.5 ± 0.3
0.26
> 0.5
0.28
1.96
> 10
2.18
> 10
> 10
> 10
> 10
> 10
> 10
7.40
n.d.
7.64
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
N
N
N
H
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NH2
5
0.7 ± 0.2
2.2 ± 0.3
-0.3 ± 0.2
-0.1 ± 0.2
-0.2 ± 0.3
0.1 ± 0.4
-0.1 ± 0.2
0.5 ± 0.4
H
N
52
53
O
F3C
N
O
O
H
N
> 0.5
> 0.5
> 0.5
> 0.5
> 0.5
> 0.5
NH2
NH2
N
H
N
O
F3C
N
54
55
56
57
58
N
O
H
N
O
O
O
F3C
N
NH2
N
F3C
N
H
N
N
O
O
H
N
NH2
NH
N
H
N
O
NH2
NH
N
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Journal of Medicinal Chemistry
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
H
O
N
NH2
5
9
0
0.6 ± 0.1
0.3 ± 0.1
-0.1 ± 0.2
> 0.5
> 0.5
> 10
> 10
n.d.
NH
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
H
N
6
0.0 ± 0.3
n.d.
NH
N
a
average derived from seven measurements for p38α and six measurements for p38β, 10 µM compound; b IC50
∆T
m
values determined by a radiometric protein kinase assay; c calcd. from IC50 values; Standard deviation (SD) is given
when n>2; n.d. not determined.
Systematic combination of four P2 groups and four hinge binding P1 motifs, including
1
-phenylpyrazole (71-74), (o-tolyl)pyrazole (75-78), phenyl-5-(trifluoromethyl)pyrazole
(
79-82) and 3-phenyl-1H-pyrazole (83-86), led to 16 new derivatives (Table 3). Initial
screening results revealed indeed that the optimal P2 group is of major importance for
inhibitor binding as demonstrated by the loss of binding affinities of 72, 76, 80 and 84, all
of which harbored the P2 thiophene. In addition, we observed that the optimal P2 o-tolyl
group could rescue the potency of a sub-optimal P1 motif to some extent, such as phenyl-
5
-(trifluoromethyl)pyrazole (36c) and 3-phenyl-1H-pyrazole (36d), as seen in the case for
8
1 and 85. Nevertheless, we observed also a trend of low nanomolar binding upon the
combination of three classes of P2 moieties (62a, 62c, 62d) and P1 1-phenylpyrazole
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(
36a) in compounds 71, 73 and 74 as well as P1 (o-tolyl)pyrazole (36b) in compounds 75,
7
7 and 78. Together with the observation for 50, this supported our hypothesis that a right
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
combination of P1 and P2 could be important for the type-II inhibitor binding.
As observed previously in the crystal structure of p38α-VPC-00628 complex (Figure
1
E), the pyrazole amino group in the P1 moiety most likely played an important role
inducing, as well as stabilizing the P-loop in a folded, inactive conformation. To further
our understanding of the role of this moiety inducing a folded P-loop conformation, we
determined the structures of p38α in complex with 77 and 81, where the pyrazole group
was deaminated or substituted by a trifluoromethyl group, respectively. We observed that
the binding mode of 77 was remarkably similar to VPC-00628, including the folded P-loop
conformation despite the lack of the amine that stacked onto the aromatic ring of Y35 in
the VPC-00628 complex. Interestingly, the introduction of the trifluoromethyl group at the
pyrazole resulted in the stabilization of an active P-loop con formation with the P-loop
residue Y35 moved away from the ATP binding site. In addition, the DFG phenylalanine
(F169) side chain flipped into an “out conformation, resulting in an unstructured activation
segment (Figure 2).
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Journal of Medicinal Chemistry
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Comparison of the binding modes of 77 (PDB: 6SFJ) and 81 (PDB: 6SFK). Substitution of the pyrazole amino
3
group by an CF -group results in the induction of an active P-loop conformation. The aromatic sidechain of DFG
phenylalanine F169 rotates away from the ATP side resulting also in an unstructured activation segment conformation.
In general, the potency of compounds 71-78 for p38α/β was largely improved compared
to 45-52, although sharing the same P1 pyrazole hinge binder. The most optimal
combination was the use of a tolyl P2 group (62c) as a linker (73, 77, 81, 85), showing
collectively the lowest IC50 with every hinge binder used throughout this study.
Interestingly, all compounds were slightly selective for the p38α isoform. Compound 77
showed the highest temperature shift (∆T p38α/p38β = 14.8 / 15.3 °C). The use of a
m
thiophene linker P2 (62b) generally resulted in a loss of affinity, similar to compounds 31-
34 (Table 1). We assumed that compounds with a thiophene linker adopted an
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9
unfavorable conformation which prohibited the binding of the P3 building block to the
DFG-out pocket and therefore destabilizing the protein ligand interaction. With the
exchange of the C3-carbon atom of the thiophene heterocycle to a nitrogen atom and the
introduction of an additional methylene spacer (62a), a part of the binding affinity can be
retained. Compound 75 represented the best combination with this P2 thiazole linker
group (∆T p38α/p38β =12.2 / 13.0 °C, IC p38α/p38β [nM] = 26 / 191). The binding
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
m
50
mode of this inhibitor is conserved when compared with VPC-00628 (Figure 3).
Figure 3. Comparison of the binding mode of VPC-00628 (PDB: 5LAR) with 75 (PDB: 6SFI). Structural comparison
revealed conservation of the binding mode of the P2 thiazole linker. Structural superimposition reveals also similar
conformation of both ligands.
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
However, combination of the (o-tolyl)pyrazole hinge binding motive in P1 (36b) and the
tolyl linker (62c) in P2, resulted in a downward oriented methyl group in P1 for compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
77 (Figure 2), whereas this hinge binding moiety combined with the P2 thiazole linker
group (62a) led to a 180 degree rotated orientation of the (o-tolyl)pyrazole methyl group
(Figure 3). It seems that the hydrogen acceptor nitrogen was involved in the putative
intramolecular stabilizing of the compound conformation via the neighboring amide NH
group. These results also demonstrate the important contribution of the DFG-out binding
cyclohexyl moiety P3 to the binding affinity. Overall, the isoform selectivity for compound
74 was improved with an α/β ratio of 18 which was unfortunately also associated with an
affinity loss (IC p38α/p38β [µM] = 0.28 / 5.05).
50
Table 3. Potency of P3 fixed compounds 71-86 for p38α/β, determined by DSF- and
enzyme kinetic assays for p38α/β inhibition.
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4
5
6
7
8
9
P1
P2
P3
H N
O
2
N
H
O
N
N
H
N
NH2
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
∆
Tma [°C]
± SD
∆Tma [°C]
± SD
IC50b
[µM]
p38α
IC50b
[µM]
p38β
ratioc
[fold]
comp
.
P1
P2
p38α
p38β
p38α/β
O
O
O
N
H
9
.8 ± 0.6
10.6 ± 0.6
-1.0 ± 0.7
12.0 ± 1.2d
0.031
> 0.5
0.013
0.328
> 10
12.16
n.d.
N
N
S
O
N
7
1
2
7
S
O
-0.1 ± 0.7
N
N
N
H
N
N
73
N
H
12.6 ± 0.7d
0.209
16.31
O
O
N
H
2.5 ± 0.4d
13.0 ± 0.7
-0.9 ± 0.5
74
75
76
4.2 ± 0.8
12.2 ± 0.6
0.1 ± 0.9
0.28
0.026
> 0.5
5.05
0.191
> 10
18.08
7.24
n.d.
N
N
O
O
N
H
N
N
S
O
N
O
N
S
O
N
N
H
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