Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase

Article abstract of DOI:10.1021/acs.jmedchem.9b00318

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

Full text of DOI:10.1021/acs.jmedchem.9b00318

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Article
Discovery and Lead-optimization of 4,5-Dihydropyrazoles as
Mono-Kinase Selective, Orally Bioavailable and Efficacious
Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase
Philip A. Harris, Nicolas Faucher, Nicolas George, Patrick M Eidam, Bryan W. King, Gemma White, Niall
A Anderson, Deepak Bandyopadhyay, Allison M. Beal, Veronique BENETON, Scott B. Berger, Nino
Campobasso, Sebastien Campos, Carol A. Capriotti, Julie A Cox, Alain DAUGAN, Frederic DONCHE, Marie-
Helene Fouchet, Joshua N. Finger, brad geddes, Peter J. Gough, Pascal Grondin, Bonnie Hoffman, Sandra
J Hoffman, Susan Hutchinson, Jae U Jeong, Emilie JIGOREL, Pauline Lamoureux, Lara K Leister, John
D Lich, Mukesh K. Mahajan, Jamel Meslamani, Julie E. Mosley, Rakesh Nagilla, Pamela Nassau, Sze-
Ling Ng, Michael T. Ouellette, Kishore Pasikanti, Florent POTVAIN, Michael A. Reilly, Elizabeth J. Rivera,
stephane SAUTET, Michelle C Schaeffer, Clark A. Sehon, Helen H. Sun, James H. Thorpe, Rachel D Totoritis,
Paris Ward, Natalie Wellaway, David D Wisnoski, James M. Woolven, John Bertin, and Robert W Marquis
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00318 • Publication Date (Web): 23 Apr 2019
Downloaded from http://pubs.acs.org on April 23, 2019
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Rivera, Elizabeth; GlaxoSmithKline USA
SAUTET, stephane; Oncodesign Biotechnology,
Schaeffer, Michelle; GlaxoSmithKline USA
Sehon, Clark; GlaxoSmithKline USA
Sun, Helen; GlaxoSmithKline USA
Thorpe, James; GlaxoSmithKline Research and Development
Totoritis, Rachel; GlaxoSmithKline USA
Ward, Paris; GlaxoSmithKline USA
Wellaway, Natalie; GlaxoSmithKline Research and Development
Wisnoski, David; GlaxoSmithKline USA
Woolven, James; GlaxoSmithKline Research and Development
Bertin, John; GlaxoSmithKline USA
Marquis, Robert; GlaxoSmithKline USA
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Discovery and Lead-optimization of 4,5-
Dihydropyrazoles as Mono-Kinase Selective, Orally
Bioavailable and Efficacious Inhibitors of Receptor
Interacting Protein 1 (RIP1) Kinase
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Philip A. Harris, Nicolas Faucher, Nicolas George, Patrick M. Eidam, Bryan W. King,
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Gemma V. White, Niall A. Anderson, Deepak Bandyopadhyay, Allison M. Beal, Veronique
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Beneton, Scott B. Berger, Nino Campobasso, Sebastien Campos, Carol A. Capriotti, Julie A.
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Cox, Alain Daugan, Frederic Donche, Marie-Hélène Fouchet, Joshua N. Finger,
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Hutchinson, Jae U. Jeong, Emilie Jigorel, Pauline Lamoureux, Lara K. Leister, John D. Lich,
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Mukesh K. Mahajan, Jamel Meslamani, Julie E Mosley, Rakesh Nagilla, Pamela M Nassau,
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Sze-Ling Ng, Michael T. Ouellette, Kishore K. Pasikanti, Florent Potvain, Michael A. Reilly,
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Elizabeth J. Rivera, Stéphane Sautet, Michelle C. Schaeffer, Clark A. Sehon, Helen Sun, James
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H. Thorpe, Rachel D. Totoritis, Paris Ward, Natalie Wellaway, David D. Wisnoski, James M.
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Woolven, John Bertin and Robert W. Marquis
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Pattern Recognition Receptor DPU and Platform Technology & Science, GlaxoSmithKline,
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Collegeville Road, Collegeville, Pennsylvania 19426, United States. Flexible Discovery unit,
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GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. Flexible
Discovery unit, GlaxoSmithKline, 25-27 avenue du Québec, 91951, Les Ulis cedex, France.
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ABSTRACT
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing
to a variety of human pathologies, including inflammatory and neurological diseases. Currently
RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory
diseases such as psoriasis, rheumatoid arthritis and ulcerative colitis, and neuro-logical diseases
such as amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease. In this paper we report on
the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting
from a high-throughput screen and the lead-optimization of this series from a lead with minimal
rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles
in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a
valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of
disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis
pigmentosa.
INTRODUCTION
Over the past decade, Receptor Interacting Protein 1 (RIP1) kinase activity has emerged as the
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master regulator of a newly identified form of cell death termed necroptosis. Necroptosis, like
apoptosis, is a programmed and highly orchestrated form of cell death which is important for
organismal development and homeostasis.2,3 However, unlike apoptosis which is largely
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quiescent, necroptosis results in a highly pro-inflammatory response which is thought to underpin
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multiple human pathologies. Although RIP1 kinase driven necroptosis was originally identified
downstream of TNFR1 activation, work over the last few years has shown that RIP1 kinase activity
can also be activated in response to various other cellular signals including activation of other
death receptors (TRAIL, FAS), toll-like receptors (TLR3, TLR4) and some forms of cellular
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stress. Furthermore, although RIP1 kinase activity was originally shown to be critical for driving
necroptosis, additional work has now shown that RIP1 kinase enzymatic activity can also be a
direct regulator of pathogenic forms of apoptosis and pro-inflammatory cytokine production.8,9
Based on the multiple signaling path-ways that can drive RIP1 kinase activity and the potential for
multiple inflammatory outputs, inhibitors of this RIP1 kinase pathway have the potential for a
broad therapeutic footprint to treat multiple inflammatory diseases.
Degterev et al. identified several series of RIP1 kinase inhibitors using a phenotypic cell screen
that measured their ability to block necrotic death induced by treatment with a combination of TNF
and the caspase inhibitor zVAD.fmk.10,11 The most studied inhibitor, indole-hydantoin 1 (known
as Nec-1s or 7-Cl-O-Nec-1, see Figure 1), was subsequently co-crystallized in the RIP1 kinase
domain showing it to occupy an allosteric lipophilic pocket at the back of the ATP binding site.12
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This allosteric type III binding mode resulted in an excellent kinase selectivity profile. Our screen
of a GSK library of kinase inhibitors collected from prior kinase programs identified a number of
potent RIP1 kinase inhibitors belonging to the type II class, which are known to target a conserved
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“DFG-out” inactive conformation of the kinase, as exemplified by 2. This conserved DFG
sequence is located immediately before the kinase activation loop and in RIP1 this sequence is the
more rarely observed DLG. In parallel, a separate DNA-encoded library screen led to the
identification of a novel and highly kinase selective benzoxazepinone RIP1 kinase inhibitor
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template, exemplified by 3. The co-crystal structure of 3 in the RIP1 kinase domain showed it
occupied the same allosteric binding pocket as the indole-hydantoin 1. A favorable developability
profile and complete kinase specificity for RIP1 led us to select this series for lead-optimization.
This effort resulted in selection of development candidate 4, known as GSK2982772, which is
currently under evaluation in phase 2a clinical trials in psoriasis, rheumatoid arthritis and
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ulcerative colitis patients. Recently, Yoshikawa et al. have disclosed a benzoxazepinone analog
with a higher brain penetration and efficacy in a mouse experimental autoimmune
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encephalomyelitis (EAE) model of multiple sclerosis.17
Figure 1. Structure of RIP1 kinase inhibitors
RESULTS AND DISCUSSION
RIP1 kinase type III inhibitors, exemplified by both indole-hydantoin 1 and benzoxazepinone 3,
offer clear advantages as starting points for drug discovery in terms of their RIP1 kinase specificity,
combined with more favorable physicochemical profiles, such as solubility, lipophilicity and
protein binding, compared to type II inhibitors such as 2. To identify additional novel chemotypes
targeting this allosteric pocket type in RIP1 kinase we initiated a high-throughput screen (HTS) of
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the GSK compound collection. From this HTS, two hits, a dihydropyrazole urea 6 and a pyrrolidine
pivalate 7, were of particular interest, as they possessed sub-micromolar activity in both a RIP1
kinase fluorescence polarization (FP) binding assay and a cellular assay of necrotic human U937
cell death (Table 1). Inhibitors 6 and 7 share a common acyl benzylamine pharmacophore and a
hybrid dihydropyrazole (DHP) pivalate 8 was prepared in an effort to bridge these two series. This
hybrid DHP 8 showed a significant increase in both RIP1 kinase FP activity (12-fold) and U937
cellular efficacy (40-fold). As the potency of 8 was approaching the lower limit of sensitivity in
the RIP1 kinase FP binding assay, the biochemical activity was profiled in an ADP-Glo tight
binding assay revealing its potency to be in the low nanomolar range. A clear stereochemical
dependence was observed as the (S) enantiomer 9 was active, compared to the inactive (R)
enantiomer 10. Consistent with the type III binding mode, DHP 9 showed no additional activity
when profiled against 339 other human kinases in a P33 radiolabeled assay screen at Reaction
Biology Corporation at 10 M (see Supporting Information).
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Table 1. RIP1 kinase HTS Screening Hits and Hybrid
Cpd RIP1 FPa,b
ADP-Gloa
U937 cella
LEc
IC50 (nM) IC50 (nM) IC50 (nM)
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Assay protocols are described in Supporting Information; IC50 values are the average of at least
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two determinations. Lower limit of sensitivity is ca. 10 nM. Ligand efficiency (LE) based on
RIP1 FP (6,7) and ADP-Glo potency (8,9) and is derived from LE = 1.4(pIC50)/N, where N is the
number of non-hydrogen atoms.
In general, the DHP inhibitors such as the pivalate 8 were about 10-fold more potent than their
pyrrolidine analogs, such as pivalate 7. The DHP ring of 8 has less conformational flexibility than
the saturated pyrrolidine of 7 and the additional nitrogen atom acts to restrict the torsional angle
around the carbonyl group, thus holding the inhibitor in conformations that are likely more
favorable for binding to RIP1. Although we did not progress the pyrrolidine series, we were able
to successfully crystallize the meta-fluorophenyl pyrrolidine pivalate 11 in the RIP1 kinase domain
confirming this pharmacophore occupies the same allosteric pocket in the back of the ATP pocket
as indole-hydantoin 1 (Figure 2a). The inhibitor makes no interaction with the kinase hinge but the
pivaloyl carbonyl of 11 makes a H-bond to the backbone of Asp156 (Figure 2b).
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Figure 2. a) Co-crystal structure of RIP1 (1−294, C34A, C127A, C233A, and C240A) and
pyrrolidine pivalate 11. The inhibitor 11, represented as spheres with color coding: carbon
(
yellow), nitrogen (blue), oxygen (red), fluorine (green), occupies an allosteric region distinct from
the ATP binding pocket, which is shaded in pink. b) Close-up view of 11 bound in the allosteric
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pocket; the amide carbonyl of 11 makes a direct hydrogen-bond interaction with the backbone
amide NH of Asp156. Coordinates and structure factors for this cocrystal structure have been
deposited in the Protein Data Bank with the accession numbers 6OCQ.
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The excellent potency and kinase selectivity of the RIP1 kinase inhibitor 9, combined with its
inactive enantiomer as a negative control, made it an excellent in vitro tool for evaluating the in
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vitro biology of RIP1 kinase. The main goal of the initial lead optimization was to improve the
pharmacokinetic profile of this series as 9 possessed minimal oral exposure in rats. Additionally,
despite being a highly efficient ligand (LE = 0.73), inhibitor 9 exhibited a high log P of 5.9
resulting in a suboptimal lipophilic ligand efficiency (pIC50 – log P) of 2.9 and thus reducing
lipophilicity was also desirable.
We previously reported that type III RIP1 kinase inhibitors such as 3 and 4 demonstrate a species
selectivity for inhibition of primate RIP1 kinase compared to non-primate RIP1 kinase.15
Consistent with this observation, inhibitors from this DHP series, as exemplified by 9 in Table 2,
exhibited approximately equivalent RIP1 kinase FP potency against human and monkey RIP1
kinase, but were significantly less potent against rodent, minipig, rabbit and dog RIP1 kinase. This
species selectivity was not observed with any of the type II “DFG-out” RIP1 kinase inhibitors we
previously profiled (for example 2) but appears to be reserved for this type III binding mode. As
detailed in our prior report, selectively mutating key amino acids in the mouse RIP1 kinase
activation loop, where non-primate differed from primate, increases the potency of
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benzoxazepinone 3 in mouse cells to that approaching human. This suggests the murine RIP1
kinase activation loop lacks the flexibility to adopt the preferred conformation required for optimal
binding of type III inhibitors.
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Table 2. RIP1 kinase Species Selectivity of Compound 9
RIP1 FP
Species
Fold
IC50 (nM)a
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a
b
Data reported for side-by-side experiments (n=4, except mouse n=2). Values at the lower limit
of sensitivity (ca. 10 nM).
Route of clearance studies in rat for DHP 9 demonstrated a high systemic clearance, greater than
liver blood flow, but no excretion via renal or biliary routes. The oral exposure in rat at 2 mg/kg
was below the limit of detection. The observed clearance was attributed to metabolism, with
hydroxylation at the 3-position of the DHP ring detected in rat blood, bile and urine, along with
other hydroxylated regioisomers of unconfirmed identity. This was consistent with high in vitro
metabolism observed in rat liver microsomes as evidenced by short half-lives (Table 3). In vitro
metabolite identification in rat, dog, monkey and human liver microsomes confirmed that the
parent was extensively metabolized, as detailed in Figure 3. The major metabolite (M246d) in rat
and dog resulted from hydroxylation on the DHP ring. The 3-hydroxylated metabolite (M246a)
was formed only in low levels. In human liver microsomes the major metabolite was hydroxylation
at the tert-butyl group (M246c). No human unique metabolites where observed for DHP 9. A small
amount of metabolite (M246b) from oxidation at the phenyl ring occurred and no pyrazole
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metabolite (14) was detected in any species. Bis-hydroxylation metabolites (M262a,b,c) were also
observed in all species, most predominantly in monkey, along with a trihydroxylation/reduction
metabolite (M276).
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 3. Metabolite identification of DHP 9 in rat, dog, monkey, human liver microsomes.
Hydroxylated metabolites are labeled in green, bis-hydroxylation metabolites in blue and a
trihydroxylation/reduction metabolite in red. Structural assignments for metabolites were made
tentatively based upon mass spectral fragmentation pattern.
Our initial SAR investigations around modifying or replacing the DHP heterocycles met with
limited success, as shown in Table 3. Addition of methyl groups to the ring (12, 13) or
aromatization of the ring (14) led to reduction in potency and no improvement in intrinsic
clearances. Replacement of the DHP ring with 6-membered ring analogs (15-18) or a thiazoline
(19) were significantly less active in our assays; however, the smaller azetidine replacement (20)
retained moderate activity. Zhang et al. have recently disclosed an analogous series of RIP1 kinase
19
inhibitors with a variety of heterocyclic modifications of the core ring. The restricted SAR with
regards to alterations to the heterocycle support the observations in the co-crystal structure of 11
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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
locating the inhibitor in a narrow lipophilic pocket. Changes in this central ring that result in
alterations in the conformation of the aryl and amide groups were less optimal for inhibitor binding.
Table 3. Replacements or Modifications of the DHP Heterocycle
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
Cpd RIP1 FPa cell U937a
Microsome/hepatocyte
CHI b
c
IC50 (nM) IC50 (nM) Log D
t1/2 (min.)
rat
human
20
9
16
4
5.8
6.4
6.5
8.3
6.7
4.7
2.5
4.9
5.6
5.0
3.5
2.5
12
1
2
3
4
5
6
7
8
9
0
126
159
37
1
1
1
1
1
1
1
200
400
27
>10,000
>10,000
>10,000
>10,000
>10,000
3,980
>10,000
>10,000
>1,000
>10,000
>10,000
>1,000
126
3.2
30
18
2
79
>165
a
Assay protocols are described in Supporting Information; IC50 values are the average of at least
b
two determinations. CHI (chromatographic hydrophobicity index) log D at pH 7.4 was calculated
from the retention time (t
c
R
) observed in a fast gradientreverse-phase HPLC. Microsome half-lives
reported for 9, 13 and 19, hepatocyte half-lives for 12 and 20.
We next examined changes around the aryl ring. Small lipophilc groups such as methyl and
fluoro (e.g. 21-25) retained potency comparable to 8, albeit with no improvements in intrinsic
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clearance (Table 4). Bulkier substitutions, such as tert-butyl (e.g. 27) or more polar groups (e.g.
2
8) were less tolerated. Replacement of the phenyl ring with more polar heterocycles such as
pyridines (29-31), pyrazole (32), imidazole (33) and oxazole (34) were detrimental to potency.
Despite their drop in potency it was encouraging that the pyridine (29, 30) and oxazole analog 34
did show significantly improved stability in microsomes. Nonpolar cyclohexyl (35) or cyclopentyl
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
(36) replacements had moderate potency; whereas more polar aliphatic heterocycles such as
tetrahydropyran (37) and N-methylpiperidine (38) were inactive in our assays.
Table 4. Substitution or Replacements of the Phenyl Ring
Cpd FP/ADP-Gloa,b cell U937a
CHI
microsome
t1/2 (min.)
c
IC50 (nM)
IC50 (nM) Log D
rat
human
8
4.8
0.5
2
8
4
5.8
5.9
6.6
6.3
6.4
6.6
6.6
8.0
2
1
2
3
4
5
6
7
2
2
2
2
2
2
2.5
6.4
4
2.6
16
1.3
6.3
6.3
9
2.3
8.7
3.0
22
26
13
63
8.3
3,980
>1,000
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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
2
2
3
3
3
3
3
3
3
3
8
9
0
1
2
3
4
5
6
7
8
2,000
316
>1,000
4.1
3.5
3.2
3.2
2.7
1.7
3.0
7.3
6.7
4.1
1.4
398
126
79
>90
>90
159
>90
5,012
3,980
3,980
794
>1,000
>1,000
5,012
1000
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
>90
<1.4
2.6
>90
5.8
5.9
126
794
200
794
>10,000
>10,000
>1,000
>1,000
3
a
Assay protocols are described in Supporting Information; IC50 values are the average of at least
b
two determinations. RIP1 FP binding assay was used to assess the potency of inhibitors with an
IC50 ca. > 10 nM; whereas for more potent inhibitors where the limit of sensitivity FP assay was
reached, the ADP-Glo tight binding analyses were employed to more accurately determine
potency. CHI (chromatographic hydrophobicity index) log D at pH 7.4 was calculated from the
retention time (t ) observed in a fast gradient reverse-phase HPLC.
c
R
The inhibitor 11 bound in the RIP1 kinase allosteric binding pocket showed the pivalate group
orientated towards the opening of the pocket suggesting this area would have more space for
optimization. Our initial SAR exploration around this region is summarized in Table 5. Reducing
the size of the tert-butyl group or adding heteroatoms reduced potency as exemplified by DHPs
39-41. Cyclizing the aliphatic group led to potent analogs (42-44) but did not improve the
microsomal stability. The heterocycles tetrahydropyran and tetrahydrofuran (45, 46) were less
active; however, 45 showed encouragingly improved intrinsic stability. Replacing the tert-butyl
moiety with aryl groups represented by DHPs 47-50 was less favorable regarding potency. The N-
methylpiperidine 51 lost activity in our assays, indicating that a protonatable amine in this region,
as with analog 41, is not favorable with the lipophilic environment. However, reduction of the
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nitrogen basicity, as seen with the N-phenylpiperidine 52 and N-acetylpiperidine 53 analogs, led
to recovery of the potency. The N-acetylpiperidine active S-enantiomer 54 was prepared and
proved to be both potent and stable in rat and human liver microsomes.
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
Table 5. Substitution or Replacements of the tert-Butyl
Cpd FP/ADP-Gloa,b cell U937a
CHI
microsome
c
IC50 (nM)
IC50 (nM) Log D
t
1/2 (min )
.
rat
human
8
4.8
80
8
159
5.8
4.4
3.4
1.4
5.4
5.9
4.7
3.1
3.0
4.6
2.7
2.7
2.4
3
9
0
1
2
3
4
5
6
7
8
9
0
80
55
>90
>90
4
4
4
4
4
4
4
4
4
4
5
631
501
>10,000
16
>10,000
63
12
5
68
37
1.6
13
126
200
35
>90
83
316
794
>90
1,000
398
>1,000
794
2,000
2,512
>10,000
2,000
2,000
>10,000
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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
5
5
5
5
1
2
3
4
>10,000
13
7,940
1.1
6.3
2.5
2.5
79
15
>90
126
501
126
35
>165
>165
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
a
Assay protocols are described in Supporting Information; IC50 values are the average of at least
b
two determinations. RIP1 kinase FP binding assay was used to assess the potency of inhibitors
with an IC50 ca. > 10 nM; whereas for more potent inhibitors where the limit of sensitivity FP
assay was reached, the ADP-Glo tight binding analyses were employed to more accurately
determine potency. CHI (chromatographic hydrophobicity index) log D at pH 7.4 was calculated
from the retention time (t ) observed in a fast gradient reverse-phase HPLC.
c
R
One clear trend that emerged is that decreased lipophilicity of the DHPs correlated to reduced
microsomal intrinsic clearances, as demonstrated in Figure 4. Only DHPs 30, 34, 45 and 54, each
with a log D under 3.5, have greater than 90 min. half-lives in rat microsomes. The oral rat
pharmacokinetic profile of these four DHPs is summarized in Table 6. Compared to DHP 9, which
had undetectable rat oral exposure, these all showed improved exposures, particularly DHP 54,
albeit still with a relatively short duration. From this initial round of SAR investigation, DHP 54
possessed a good combination of RIP1 potency, low intrinsic clearance and rat oral exposure to be
evaluated in vivo. DHP 54 was also highly soluble as indicated by fasted state simulated intestinal
fluid (FaSSIF) solubility of the crystalline form being 7.4 mg/mL after 4 h at 37 °C.
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Figure 4. Comparison of chromatographic log D with rat liver microsome half-lives for DHPs in
Tables 3-5.
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
Table 6. Oral Pharmacokinetic Profiles of DHPS in Rata
Cmax
(ng/mL)
Tmax
(min.)
AUC0−∞
μg·h/mL)
0.26
Cpd
(
3
3
4
5
0
4
5
4
360
345
95
5
5
0.20
0.044
1.6
5
795
30
aDosed at 2 mg/kg in 5% DMSO/6% Cavitron™ solution.
Despite the lower non-primate RIP1 activity characteristic of these type III inhibitors, we had
sufficient murine cellular potency and exposure to examine this series in an acute in vivo mouse
model evaluating protection from TNF induced lethal shock. DHP 54 exhibited moderate cellular
activity against murine L929 (IC50 6.3 M) in blockage of necrotic death induced by TNF plus the
caspase inhibitor zVAD.fmk. In this in vivo model, injection of TNF plus zVAD.fmk leads to a
systemic inflammatory response, characterized by hypotension, hepatitis, hypothermia and bowel
2
0
necrosis. Inhibitor 54 was dosed orally, at doses of 1.4, 14 and 140 mg/kg, 5 minutes prior to
TNF/zVAD.fmk injection and showed 22, 43 and 96% protection from body temperature loss
over 3 hours, respectively, compared to TNF/ zVAD.fmk alone (Figure 5). Assuming the efficacy
is Cmax driven, the calculated IC50 of 1.3 M from this in vivo study correlated reasonably well
with the observed in vitro murine cellular L929 IC50 of 6.3 M.
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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
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 5. Evaluation of DHP 54 in the TNF/zVAD induced lethal shock mouse model at doses of
1
.4, 14 and 140 mg/kg, measuring reduction in body temperature loss over time. Data are presented
as the mean +/- SEM, *p<0.05.
We next focused on modifying the N-acyl piperidine region of DHP 54 to further improve the
profile as summarized in Table 7. Replacement of the methyl with trifluoromethyl (55) retained
potency but diminished intrinsic stability; whereas larger or cycloalkyl groups reduced potency
(56, 57). Phenyl replacement (58) of the methyl was more potent but did not have microsomal
stability. A series of heterocycles were also evaluated from which the most notable were the
oxazoles (61, 62) for their excellent cellular potency and the 4-pyrazole (64) and 1,2,3-triazole
(65) for good intrinsic clearances. The rat IV pharmacokinetic profiles of the most promising DHPs
are summarized in Table 8. These inhibitors show moderate volumes of distribution, moderate to
high clearances and short half-lives in rat and therefore did not represent an improvement over
DHP 54. The good stability in rat microsomes for DHPs 54, 62, 64 and 65 did not translate into
longer half-lives in rat. We therefore switched from evaluating intrinsic clearances in microsomes
to hepatocytes, which, by the inclusion of phase 2 metabolic pathways, may provide an improved
correlation.
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Table 7. N-Acylpiperidine DHPs
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
Cpd FP/ADP-Gloa,b cell U937a
CHI b
Microsome t1/2 (min )
.
IC50 (nM)
IC50 (nM)
Log D
rat
human
54
55
56
57
35
126
2.5
4.9
3.2
5.4
4.4
2.6
3.1
2.9
2.7
2.4
2.1
2.0
3.0
>165
>165
16
126
794
10
160
316
2511
79
29
64
>90
>165
5
5
6
6
6
6
6
6
8
9
0
1
2
3
4
5
6
5
103
159
2.5
6.3
2.5
25
316
316
7.9
32
>180
19
>165
>180
>165
>165
>165
>180
10
100
251
159
16
79
>165
>165
39
32
6
20
a
Assay protocols are described in Supporting Information; IC50 values are the average of at least
b
two determinations. RIP1 FP binding assay was used to assess the potency of inhibitors with an
IC50 ca. > 10 nM; whereas for more potent inhibitors where the limit of sensitivity FP assay was
reached, the ADP-Glo tight binding analyses were employed to more accurately determine
b
potency. CHI (chromatographic hydrophobicity index) log D at pH 7.4 was calculated from the
retention time (t
R
) observed in a fast gradient reverse-phase HPLC.
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2
3
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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 8. Rat IV Pharmacokinetic Profiles of DHPsa
Cl
Vss
t
1/2
AUC0−∞
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
Cpd
54
(
mL/min/Kg) (L/Kg) (min.) (μg·h/mL)
44
58
42
46
37
1.7
0.98
2.1
51
12
28
25
25
0.42
0.29
0.40
0.36
0.45
61
62
64
1.6
65
1.3
aDosed at 1 mg/kg in 5% DMSO/20% Cavitron™ solution.
With no significant improvement over DHP 54 observed with N-acyl piperidine substitutions,
we next explored replacement of the acyl group with an aryl or heterocyclic ring. The N-phenyl
piperidine 67 showed an improvement in potency compared to 54 but this was accompanied by a
significant increase in lipophilicity. Attachment of nitrogen heterocycles such as pyridine,
pyrimidine and pyridazine (68-71) maintained the potency, reduced lipophilicity but lacked
stability in rat hepatocytes. However, replacement with 5-methyl-1,3,4-oxazdiazole led to inhibitor
7
2 with good in vitro potency and improved stability in rat hepatocytes. The rat pharmacokinetic
profile of 72 compared to 54 was significantly improved with a lower clearance and higher
exposure, as shown in Table 10.
A consistent trend was that addition of 3,5-difluoro substitution to the aryl ring resulted in
modest (~ 2-fold) improvement in potency in U937 cellular activity. Figure 6 shows this trend in
a head-to-head comparison of unsubstituted phenyl-DHPs with their 3,5-difluorophenyl analogs.
The fluorines are most likely providing additional Van der Waals contacts in the rear of the
allosteric pocket where the aryl ring binds. A series of cyano substituted nitrogen heterocycles, as
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represented by DHPs 73-76, were also evaluated which showed excellent cellular potencies. Their
rat intrinsic clearances were not as good as DHP 72, which translated into higher rat in vivo
clearances, as shown in Table 10 for DHPs 74 and 76. Combining the more stable 5-methyl-1,3,4-
oxazdiazole 72 with the 3,5-di-fluoro aryl substitution to improve cell potency led to DHP 77. This
DHP had good potency combined with full stability in both rat and human hepatocytes, which
translated into a favorable rat pharmacokinetic profile with a low clearance, good exposure and
good oral bioavailability.
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
Table 9. N-Aryl-Piperidine Substitutions
Cpd FP/ADP-Gloa,b cell U937a
CHI
Hepatocyte
c
IC50 (nM)
IC50 (nM) Log D
t
1/2 (min )
.
rat
>187
human
>187
5
6
6
6
7
7
4
7
8
9
0
1
35
16
2.5
1.6
5
126
25
16
10
32
50
2.5
6.3
5.1
4.7
3.2
3.4
3.8
8.2
>187
>187
198
<24
<31
20
139
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9
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2
2
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2
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7
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2
3
4
5
6
7
50
7.9
2.0
2.5
1.0
20
126
25
3.2
4.8
5.2
5.1
5.0
3.6
173
125
107
55
198
>480
198
126
198
198
7.9
2.0
4.0
63
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
69
198
a
Assay protocols are described in Supporting Information; IC50 values are the average of at least
b
two determinations. RIP1 FP binding assay was used to assess the potency of inhibitors with IC50
10 nM; whereas for more potent inhibitors where the limit of sensitivity FP assay was reached,
>
c
the ADP-Glo tight binding analyses were employed to more accurately determine potency. CHI
chromatographic hydrophobicity index) log D at pH 7.4 was calculated from the retention time
(t ) observed in a fast gradient reverse-phase HPLC.
(
R
Figure 6. Comparison of U937 cellular potencies of phenyl-DHPs compared to their 3,5-
difluorophenyl-DHP analogs.
Table 10. Rat Pharmacokinetic Profiles of DHPs
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IV parametersa
PO parametersb
Cl
Vss
t
1/2
AUC0−∞
C
max
T
max
F
(%)
210
Cpd
54
(
mL/min/Kg) (L/Kg) (min.) (μg·h/mL)
(ng/mL)
795
(min.)
30
c
44
18
47
46
14
1.7
1.6
6.1
4.6
1.3
51
60
0.42
0.80
0.40
0.41
1.3
7
7
7
7
2
4
6
7
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
114
99
73
38
30
25
99
88
215
102
1100
130
a
b
IV arm dosed at 1 mg/kg in 5% DMSO/20% Cavitron™ aqueous solution. Oral arm dosed at
2 mg/kg in 5% DMSO/6% Cavitron™ aqueous solution. High bioavailability calculated in a non-
crossover fashion using average exposures from separate oral and iv studies.
c
The FaSSIF solubility of DHP 77 was in a reasonable range of 0.25 mg/mL after 4 h at 37 °C.
In addition to good stability in rat and human hepatocytes, inhibitor 77 was also stable in mouse,
minipig, dog and monkey hepatocytes with half-lives in the first three over 3 hours, and 92 minutes
in the fourth. The pharmacokinetic profile of 77 in mouse, mini-pig and monkey, as detailed in
Table 11, showed a similar profile to rat (Table 10) with low clearances, good exposures and good
oral bioavailability. DHP 77 displayed a high free fraction in blood in mouse (45%), rat (41%),
monkey (55%) and human (47%). Allometric scaling and in vitro to in vivo extrapolations with
and without effects of protein binding were used to generate predictions of the human
pharmacokinetic parameters of 77 (Table 12). These different methodologies showed a high level
of correlation and predicted 77 to have moderate bioavailability, moderate to low clearance with a
moderate volume and a terminal half-life in the order of 4 hours in humans. A human whole blood
assay in which the necroptosis pathway is activated through stimulation with TNF, co-incubated
2
1
with the caspase inhibitor zVAD.fmk and the SMAC mimetic RMT 5265, was employed to
establish the target concentration. In this assay DHP 77 was also shown to have good potency as
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5
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5
5
6
measured by inhibition of cytokine MIP-1 (IC50 = 8.5 nM). The average clearance and volume
values were then used to reconstruct a mono-exponential predicted human blood concentration
time profile. As shown in Figure 7, a dose of 175 mg twice daily is predicted to maintain blood
concentrations above the human whole blood inhibition IC90 level over 24 hours.
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
Table 11. IV/PO Pharmacokinetic Profiles of DHP 77
IV parameters
PO parameters
Cl
t
1/2
Vss
AUC0−∞
C
max
T
max
F
Species
Mousea
(
mL/min/Kg) (min.) (L/Kg) (μg·h/mL) (ng/mL) (min.) (%)
34
13
32
78
1.8
1.3
1.1
1.1
1.3
3.1
5414
576
30
80
Minipig b
65
40
Monkey b
5.5
144
480
120
a
IV arm dosed at 2 mg/kg in 5% DMSO/20% Cavitron™ aqueous solution. Oral arm dosed at
b
3
0 mg/kg in 1% aqueous methylcellulose as a suspension. IV arm dosed at 1 mg/kg in 5%
DMSO/20% Cavitron™ aqueous solution. Oral arm dosed at 2 mg/kg in 5% DMSO/6%
Cavitron™ aqueous solution.
Table 12. Average Predicted Human PK Profile of DHP 77 Based on Allometry and In Vitro
to In Vivo Extrapolation.
Avera 95%
-95%
C.I.
Parameter
a
a
ge
C.I.
Clearance (mL/min/kg) 7.6
9.1
1.4
4.3
80
6.2
1.1
3.3
68
Volume (L/kg)
Half life(h)b
1.3
3.8
74
Bioavailability (%)
a
The ± 95% confidence intervals represent variability in parameter estimates from different
b
methodologies. Half-life determined from the terminal phase of the predicted human PK profile
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0
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9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Predicted human blood concentration time profile of DHP 77 dosed at 175 mg twice
daily overlaid with the human whole blood inhibition IC90 and IC50 concentrations.
To confirm the binding mode, DHP 77 was crystallized in the RIP1 kinase domain which showed
it bound to the allosteric region in the back of the ATP pocket similar to the pyrrolidine pivalate
1
1 (Figure 8a). Although inhibitor 77 does not occupy the same space as the adenine ring of ATP,
the piperidine-oxadiazole portion does occupy space where the ATP phosphates would reside and
the inhibitor shows competitive inhibition with respect to ATP. The pyrazole carbonyl of 77
accepts a hydrogen bond from the backbone nitrogen of Asp156, with the chair conformation of
the piperidine demonstrating a complementary shape to the pocket geometry and providing a
vector for the oxadiazole to access the solvation front at the entrance to the active site pocket
between Lys45 and Leu157 (Figure 8b).
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0
1
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9
0
1
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8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. (a) Co-crystal structure of RIP1 (1−294, C34A, C127A, C233A, and C240A) and DHP
77. (b) Location of oxadiazole of 77 at the solvation front at the entrance to the active site pocket.
Coordinates and structure factors for this cocrystal structure have been deposited in the Protein
Data Bank with the accession numbers 6R5F.
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A long-standing goal of ours has been to evaluate RIP1 kinase inhibitors in rodent chronic
models of disease. This has been hampered by the reduced non-primate RIP1 kinase potency we
have observed for all type III inhibitors that target the allosteric pocket of RIP1 kinase. For
example, for DHP 77 the cellular potency in prevention of necrotic death in murine fibrosarcoma
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
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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
L929 cells (IC50 = 3.2 M) represents a 50-fold reduction compared to human U937 cells (IC50
63 nM). However, DHP 76 had significantly improved mouse cell L929 cellular efficacy (IC50
=
=
3
2 nM). The increased RIP1 kinase murine potency of 76 relative to other DHPs may be due to
the cyano-pyrimidine making hydrogen bond interactions with the mouse glycine-rich loop, which
is predicted to have less conformational flexibility than the human glycine-rich loop (see
Supporting Information). The superior mouse potency of DHP 76 afforded us the opportunity to
utilize this RIP1 kinase inhibitor as an in vivo tool compound in murine chronic models of disease.
Administration of DHP 76 in mouse chow at doses of 10 and 100 mg/kg/day were able to maintain
concentrations above the L929 IC50 and IC90, respectively, over a 24h period (Figure 9). During 6
weeks of dosing at the top dose of 100 mg/kg/day, serum concentrations of 76 were consistently
maintained above the L929 ~IC90 and were well tolerated, as shown in figure 10. Another option
would be to administer DHP 76 via oral dosing twice daily, but for these chronic studies lasting
up to 6 weeks duration food-based delivery was chosen for convenience. A higher dose of 300
mg/kg/day was also evaluated via food-based delivery, but this was not optimal since a decrease
in food consumption accompanied by weight loss was observed, presumably because this higher
concentration of drug was affecting the food’s taste.
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 9. DHP 76 dosed in mouse chow maintains concentrations above the murine L929 IC90
over 24 hours. Concentrations decrease during daytime when mice are least active and feeding
less. The 24 hour time point is extrapolated with the assumption that the profile returns to the 0
hour time point level and this is indicated by dotted lines from 18-24 hour timepoints.
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Figure 10. DHP 76 dosed in mouse chow maintains concentrations above the murine L929 IC90
over 24 hours. Sampling was done early morning after nocturnal feeding when drug concentrations
should be at their maximum, and late afternoon when concentrations should approach their lowest.
1
1
1
1
1
1
1
1
1
1
2
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2
2
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0
1
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0
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0
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9
0
Inhibition of RIP1 kinase has been implicated in protection against the experimental
autoimmune encephalomyelitis (EAE) mouse model of human multiple sclerosis.17,22 To evaluate
the efficacy of RIP1 kinase inhibitor 76 in this chronic model we elected to administer the inhibitor
via mouse food to maintain serum concentrations above either estimated IC90 and IC50 L929
inhibition levels over the course of the study. Mice were pre-dosed with DHP 76 in mouse chow
one day prior to EAE induction, such that mice (15 mice per group) received on average 96
mg/kg/day or 9.6 mg/kg/day of 76 in diet, or control diet, as shown in Figure 11. At both of these
doses, the concentration of DHP 76 in mouse brain was determined to be close to parity (0.9-1.0)
compared to blood levels indicating high murine CNS penetration of this inhibitor. The free
fraction of DHP 76 in mouse blood and brain homogenate were 15 and 37%, respectively, leading
to a brain-to-blood unbound ratio (Kp,uu) of 2.5. EAE induction was achieved as described in the
supporting information. Mice were monitored daily until 35 days post-induction and scored for
clinical signs of disease (see supporting information for details on scoring). DHP 76 showed
protection in this EAE model at the dose maintaining concentration over the cellular L929
estimated IC90 level, exhibiting a delay in the disease onset and reduced clinical severity. The dose
group targeting sustained L929 IC50 inhibition level was not efficacious. This may be useful when
considering doses in future clinical trials. The efficacy of DHP 76 in this EAE model is comparable
to that observed by Yoshikawa et al. with benzoxazepinone 5 over a shorter dosing duration of 26
days.17
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