Discovery of Pyrazolocarboxamides as Potent and Selective Receptor Interacting Protein 2 (RIP2) Kinase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00141

Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

Full text of DOI:10.1021/acsmedchemlett.9b00141

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of Pyrazolocarboxamides as Potent and Selective
Receptor Interacting Protein 2 (RIP2) Kinase Inhibitors
§
†
‡
Curt D. Haffner,† Adam K. Charnley,*,† Christopher J. Aquino, Linda Casillas, Maire A. Convery,
Julie A. Cox,† Mark A. Elban,† Nicole C. Goodwin,† Peter J. Gough,† Pamela A. Haile,†
Terry V. Hughes,∥ Beth Knapp-Reed,† Constantine Kreatsoulas,† Ami S. Lakdawala,† Huijie Li,†
Yiqian Lian,† David Lipshutz,† John F. Mehlmann,† Michael Ouellette,† Joseph Romano,†
Lisa Shewchuk,† Arthur Shu,† Bartholomew J. Votta,† Huiqiang Zhou,† John Bertin,†
and Robert W. Marquis†
́
†GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426, United States
‡GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, U.K.
S
* Supporting Information
ABSTRACT: Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of
receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the
inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1
and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic
pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of
RIP2 inhibition as a therapeutic target for autoinflammatory disorders.
KEYWORDS: Fragment based drug design (FBDD), receptor interacting protein 2 kinase, RIPK2, RIP2K, RIP2,
nucleotide-binding oligomerization domain 1, NOD1, nucleotide-binding oligomerization domain 2, NOD2, ALK5, VEGFR2, LCK,
structure based drug design (SBDD)
he NOD-like receptors NOD1 and NOD2 (nucleotide-
tion of NOD/RIP2-dependent signaling has been linked to
Tbinding oligomerization domain-containing proteins 1 human disease, including asthma,13 early onset inflammatory
bowel disease,14 sarcoidosis,15 and Crohn’s disease (CD).16,17
and 2) are cytosolic pattern recognition receptors (PRRs) that
Furthermore, activating mutations in the central nucleotide
binding (NACHT) domain of NOD2 have been reported to
cause Blau syndrome, an ultrarare autoinflammatory disease,18
and increased expression of the activated/phosphorylated RIP2
kinase has been observed in intestinal biopsies taken from
pediatric CD and ulcerative colitis (UC) patients with active
disease.19−22 Synergistic interactions of the NOD and TLR
signaling pathways23 expand the implications of modulating
the NOD/RIP2 pathways.
serve as bacterial sensors and mediate the host immune
response. NOD1 and NOD2 bind distinct bacterial peptido-
glycan components diaminopimelic acid (DAP) and muramyl
dipeptide (MDP), respectively.1−3 Upon binding their
respective ligands, NOD1 or NOD2 recruit the serine/
threonine kinase receptor interacting protein 2 kinase (RIP2
kinase), also known as RIPK2, CARD3, RICK, CARDIAK.4−6
RIP2 kinase subsequently undergoes both autophosphoryla-
tion and polyubiquitination.7−9 These modifications facilitate
assembly of a signaling complex, activation of the canonical
NF-kB and mitogen-activated protein kinase (MAPK) signal-
ing pathways, and ultimately, production of multiple
proinflammatory cytokines and chemokines.10−12 Dysregula-
Received: March 29, 2019
Accepted: September 26, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00141
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Recently, groups including ours have reported that small
molecule RIP2 inhibitors block proinflammatory signaling and
reduce inflammation in preclinical models.24−29 Together,
these data support the notion that the NOD/RIP2 signaling
pathways likely play a significant role in the pathogenesis of
inflammatory diseases and that specific targeting of this
pathway may have broad therapeutic value.
In an effort to identify structurally diverse inhibitors of RIP2,
we embarked on a fragment based screening and design
program.30 The FBDD effort began by screening a GSK
fragment set designed around chemical motifs expected to
interact in the ATP binding site of kinases. Screening roughly
1000 compounds through a fluorescence polarization binding
assay31 at 400 μM afforded 49 compounds with >50%
inhibition. These were progressed into full curve analysis
which gave IC50’s ranging from 500−5 μM. All 49 fragments
were progressed into a thermal melt stabilization assay to
validate binding to RIP2. Gratifyingly, 30 of the hits confirmed
binding in the thermal shift assay. From these, 27 fragments
were progressed into crystallography experiments yielding 20
RIP2-fragment cocrystal structures. Careful analysis of the
resultant cocrystal structures enabled clustering the fragments
by key binding interactions and potential growth vectors. Hit-
to-lead chemistry was initiated on five fragment series
representing four different binding clusters. Ultimately,
chemistry efforts were narrowed to a single fragment series
derived from commercially available pyrazolocarboxamide 1a
(Figure 1).32
Figure 2. Overlay of pyrazolecarboxamides 1a (plum) and 2a (cyan)
illustrating the two pyrazolocarboxamide binding modes.
To investigate the two pyrazolocarboxamide binding modes,
we synthesized several sets of matched pairs that swapped the
C3 and N1 substitutions. As shown in Table 1, the C3 aryl
Table 1. In Vitro Potency Data for Compounds 2a−f and
a
3a−f
Figure 1. RIP2 inhibitor 1a and data mining hit 2a.
Fragment-to-lead efforts on the pyrazolocarboxamides began
with a crystal structure of 1a, which showed that the C4-amide
makes two hinge binding H-bonds, the C5 amine (C5 N)
interacts with the gatekeeper side-chain, and the N1-phenyl
group projects to the kinase back pocket (Figure 2). Additional
examination of the crystal structure revealed that the C3
position of the pyrazole offered a growth vector toward Ser25,
a key glycine rich loop residue which makes interactions with
other known RIP2 inhibitors and presents potential selectivity-
driving interactions.33 We envisioned growing off of this
position of the fragment to incorporate an appropriately placed
hydrogen bond acceptor in order to engage Ser25. Mining the
GSK compound collection for related molecules with C3
substitution led to the identification of 2a. This pyrazolo-
carboxamide compound contained aryl substitution at the C3
position with OMe as a potential Ser25-engaging group, and a
bulky tert-butyl group at N1 as a putative replacement for
phenyl back pocket binder. When we obtained a cocrystal
structure of 2a, we observed this compound had flipped in the
pocket relative to our original fragment hit (Figure 2), with the
C3 aryl occupying the back pocket and reversing the hinge and
gatekeeper interactions.
RIP2 FP IC50
R1
C3 aryl
N1 aryl
3-methoxyphenyl
2a
1.6
2b
0.08
2c
5.0
2d
0.12
2e
1.2
2f
0.06
3a
32
3b
20
3c
25
3d
>10
3e
25
3f
50
b
c
4-chloro-3-methoxyphenyl
m-tolyl
b
c
c
c
3,4-dichlorophenyl
1H-indazol-5-yl
2-naphthalenyl
b
c
c
c
b
c
a
b
c
IC50 values in μM. n ≥ 6. n = 2.
B
DOI: 10.1021/acsmedchemlett.9b00141
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ACS Medicinal Chemistry Letters
Letter
compounds (see 2a−f) were significantly more potent
compared to their N1 aryl counterparts (3a−f), even though
the molecules can engage the same residues along the hinge
and place an aryl group in the back pocket, regardless of
orientation of the pyrazole ring. We hypothesize that these
potency differences can be attributed to electronic differences
in the interactions between the pyrazole C5 amine and C4
amide with Met98, Glu96, and Thr95. Subtle differences in the
placement of the back pocket aryl group between the two
motifs also likely contribute to the potency differences. Among
the C3 aryl substituents, we were able to increase potency by
filling the back pocket with substitution at the 3-position (2c),
3,4-disubstitution (2d), and fused bicycles (2e−f). Of these
inhibitors, 2f was the most potent but suffered from poor
solubility. The crystal structures of the C3 aryls suggested that
an appropriately placed hydrogen bond acceptor could engage
with the backbone NH of Asp164. With this in mind, we
designed a set of heterocyclic back pocket groups that would
test this hypothesis and hopefully improve solubility. Gratify-
ingly, benzothiazole 2k offered a nice balance of potency and
improved physicochemical properties (Table 2).
Table 3. Lead Optimization SAR on N1/C5 N
Substitution
a
24
,
GSK′583
2k
4
5
b
b
b
f
c
e
c
c
d
b
RIP2 FP IC50
hWB IC50
ALK5 IC50
VEGFR2 IC50
LCK IC50
0.00524
0.23724
0.04
0.54
0.32
0.25
0.25
0.05
0.64
0.32
0.40
1.00
0.06
0.12
0.79
2.51
b
e
b
3.16
>5.024
b
b
b
b
12.6
3.98
a
Known RIP2 inhibitor GSK′583 is included for reference. IC50
b
c
d
e
f
values in μM. n ≥ 6. n = 2. n = 3. n = 4. n = 5.
selectivity.35,36 Importantly, 5 also possessed a suitable oral
pharmacokinetic profile in rat (data not shown). The activity
in hWB combined with oral exposure made 5 the first
progressible molecule from the pyrazolocarboxamide class, and
it was profiled in a seven day rat safety assessment study.
Unfortunately, substantial toxicity findings precluded further
progression of this compound.
a
Table 2. In Vitro Potency Data for Compounds 2g−k
With the goal to identify a clinical candidate from the
pyrazolecarboxamide series, the team set out to identify an
inhibitor that demonstrated both improved selectivity and
potency, setting a goal of at least 100-fold biochemical
selectivity against the sentinel kinases (ALK5, VEGFR2,
LCK) and whole blood activity <100 nM.
RIP2 FP IC50
R1
C3 aryl
Leveraging our in-house crystallography to generate
numerous RIP2 cocrystal structures with our pyrazolocarbox-
amide inhibitors,24,25,33 we set out to rationally improve the
RIP2 affinity and selectivity of our molecules. One approach
we employed was to install a covalent linkage between the N1
and C5 substitutions. This would also enable further
substitution as dictated by the biochemical data. Therefore,
three N1−C5 cyclized analogs were synthesized to probe the
SAR spanning six to eight membered rings (Table 4). The six-
benzo[d][1,3]dioxol-5-yl
2g
0.79
2h
0.79
2i
3.2
2j
0.12
b
benzo[c][1,2,5]oxadiazol-5-yl
1H-benzo[d]imidazol-5-yl
1H-indol-6-yl
d
c
c
benzo[d]thiazol-5-yl
2k
0.04
b
a
b
c
d
Table 4. In Vitro Potency and Selectivity Data for
Compounds 5−9
IC50 values in μM. n ≥ 6. n = 2. n = 4.
a
With the discovery of 2k, the program transitioned from a
hit-to-lead effort to a lead optimization program. A primary
point of optimization was inhibition activity in human whole
blood (hWB) and kinase selectivity. The team chose to fix the
back pocket group as benzothiazole and explore the impact of
N1/C5 N substitution (Table 3). Converting the tert-butyl to
an ethyl group, maintained RIP2 binding activity (compound
4) but had reduced human whole blood (hWB) activity.34 The
addition of a methyl group onto the exocyclic nitrogen
attached to C5 afforded 5 and a substantial improvement in
whole blood activity. Additionally, selectivity against three key
sentinel kinases (activin receptor-like kinase 5 - ALK5, vascular
endothelial growth factor receptor 2 - VEGFR2, and
lymphocyte-specific protein tyrosine kinase - LCK) was
improved relative to 2k and 4.
compound
5
6
7
8
9
n
1
0.25
1.15
3.16
7.94
2
0.06
0.70
3.98
2.51
3.16
3
b
b
b
b
b
f
b
d
f
d
d
d
RIP2 FP IC50
RIP2 hWB IC50
ALK5 IC50
VEGFR2 IC50
LCK IC50
0.06
0.12
0.79
2.51
3.98
0.025
0.37
1.26
3.2
10.0
0.05
0.29
>10.0
5.01
7.94
b
d
d
c
d
f
f
f
f
e
d
d
d
This profile identified 5, and more broadly C5 N alkyls, as
an attractive inhibitor series. Screening of activity against a
broad panel of kinases suggested reasonable off-target kinase
10.0
a
b
c
d
e
f
IC50 values in μM. n ≥ 6. n = 1. n = 2. n = 3. n = 4.
C
DOI: 10.1021/acsmedchemlett.9b00141
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ACS Medicinal Chemistry Letters
Letter
membered ring compound 6 was found to be less potent than
compound 5 (FP IC50 = 250 nM), and the selectivity window
to ALK5, VEGFR2, and LCK remained the same. Interestingly,
the 7-membered ring compound 7 retained the same RIP2
biochemical potency as compound 5 (IC50 = 60 nM) but
became 5-fold more selective against ALK5 (no significant
change was observed against VEGFR2 and LCK). Finally, the
8-membered ring analog, compound 8, demonstrated the best
biochemical activity with an IC50 = 25 nM versus RIP2.
Importantly, the kinase selectivity window also widened with
the best improvement seen against LCK. A cocrystal structure
was solved with compound 6 (data not shown) which was used
to design additional interactions with the protein via proper
placement of substituents off of the tether. In particular, by
projecting out of the plane of the bicyclic ring, hydrophobic
interactions with Leu24 and Leu153 may be realized.
Accordingly, a bridged bicyclic analog, 9, was designed and
synthesized. This compound was found to have similar
biochemical potency to compound 7 (Table 4). The most
striking piece of data was the loss of ALK5 activity. The
selectivity window against all three kinases was now ≥100-fold.
However, the desired whole blood potency was still lacking.
Given the kinase selectivity success achieved with the
bridged bicyclic analog 9, we expanded on this SAR using
guidance from our cocrystal structure of 6. In particular, we
targeted a hydrogen bonding interaction with Ser25 via
strategically placed hydroxyl groups on the ethano bridge.
Supporting this strategy, only three known kinases have a
serine in this position,37 and the bridging saturated ring sits
beneath the loop containing Ser25. We hypothesized that
installation of this functionality would improve potency
through an additional interaction with the protein and further
widen the kinase selectivity. To investigate this SAR, four
analogs were synthesized containing a hydroxyl moiety on the
ethano bridge (Table 5). Compound 10 was found to have
similar potency to 9, and no activity was observed at the
highest concentration against all three sentinel kinases. The
hWB IC50 for this compound was comparable to compound 9.
Compound 11, the enantiomer of 10, was the most potent
within the series (IC50 = 30 nM) and, like 10, showed very
good kinase selectivity. Interestingly, the hWB potency was
also improved (IC50 = 50 nM). Compound 13 was less active
than either 10 or 11 but was again quite selective, and the least
active compound was 12 (enantiomer of 13). All hydroxyl
compounds demonstrated good selectivity. A crystal structure
of compound 11 was obtained (Figure 3), yet examination of
Figure 3. Cocrystal structure of RIP2 with bridged bicyclic inhibitor
11.
this structure revealed that the hydroxyl residue is directed
toward solvent and makes no key interactions with the protein.
Interestingly, the hydroxyl moiety on 13 was within 2.6 Å of
Ser25, as designed (a cocrystal structure was solved),
suggesting that this interaction was not critical in driving
RIP2 affinity, yet impacting the whole blood activity of the
molecule.
Given both 11 and 12 direct their hydroxyl groups toward
solvent (12 based on modeling), the roughly 5-fold
biochemical potency difference between 11 and 12 could be
due to the increased hydrophobic interactions of the ethano
versus methano bridge with the hydrophobic loop of the
protein. The hWB potency for compound 11, as mentioned
above, was substantially better than the other three
compounds, yet it is unclear what is driving this difference.
Importantly, compound 11 achieved both the potency and
selectivity criteria established for a follow up to 5.
Table 5. In Vitro Potency and Selectivity Data for
a
Compounds 10−13
In conclusion, fragment-based screening and drug design
delivered an efficient lead series of RIP2 inhibitors (LE = 0.43
for 2a, 0.47 for 5, and 0.41 for 11).32 By employing fragment
evolution principles, robust crystallography, and structure-
based design, we converted hit 1 to bridged bicyclic
pyrazolcarboxamide 11. This compound combines excellent
biochemical and whole blood activity with kinase selectivity38
and promises to enable investigation of RIP2 inhibition as a
viable modality for the treatment of inflammatory indications.
ASSOCIATED CONTENT
■
10
11
12
13
S
* Supporting Information
d
b
d
e
RIP2 FP IC50
hWB IC50
ALK5 IC50
VEGFR2 IC50
LCK IC50
0.06
0.28
0.03
0.05
0.16
0.22
0.08
0.19
d
b
d
d
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00141.
f
b
d
f
b
b
d
>10.0
>10.0
>31.0
>10.0
>10.0
>10.0
>31.0
>10.0
>10.0
>31.0
f
f
7.94
31.6
c
d
d
Experimentals procedures for the preparation of all
compounds PDF)
a
b
c
d
e
f
IC50 values in μM. n ≥ 6. n = 1. n = 2. n = 3. n = 4.
D
DOI: 10.1021/acsmedchemlett.9b00141
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ACS Medicinal Chemistry Letters
Letter
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: adam.k.charnley@gsk.com.
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Adam K. Charnley: 0000-0003-1188-1715
Nicole C. Goodwin: 0000-0002-6093-7175
Pamela A. Haile: 0000-0003-3950-4890
Present Addresses
§(C.J.A.) CJAMedChem, LLC, 1600 W. Bromfield Dr.,
Hillsborough, NC 27599, United States.
∥(T.V.H.) Janssen Research & Development LLC, P.O. Box
776, Spring House, PA 19477, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Karl Erhard for the assistance in
optimizing the purification of compound 11 and the Protein &
Cellular Sciences, MST, for protein production.
ABBREVIATIONS
■
RIP2, receptor interacting protein 2 kinase; NOD1, nucleo-
tide-binding oligomerization domain 1; NOD2, nucleotide-
binding oligomerization domain 2; ALK5, activin-like kinase 5;
VEGFR2, vascular endothelial growth factor receptor 2; LCK,
lymphocyte-specific protein tyrosine kinase
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