Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a Clinical Candidate for the Treatment of IBS

Article abstract of DOI:10.1021/acsmedchemlett.8b00035

Abdominal pain and abnormal bowel habits represent major symptoms for irritable bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our screening effort and subsequent structure-activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small molecule RET kinase inhibitor with a RET IC₅₀ of 0.3 nM and is efficacious in vivo.

Full text of DOI:10.1021/acsmedchemlett.8b00035

Letter
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of a First-in-Class Gut-Restricted RET Kinase Inhibitor as a
Clinical Candidate for the Treatment of IBS
Hilary Schenck Eidam,*,† John Russell,† Kaushik Raha,‡ Michael DeMartino,† Donghui Qin,†
Huiping Amy Guan,§ Zhiliu Zhang,§ Gong Zhen,§ Haiyu Yu,§ Chengde Wu,§ Yan Pan,§ Gerard Joberty,∥
Nico Zinn,∥ Sylvie Laquerre,† Sharon Robinson,⊥ Angela White,# Amanda Giddings,#
Ehsan Mohammadi,∇ Beverly Greenwood-Van Meerveld,∇ Allen Oliff,† Sanjay Kumar,†
and Mui Cheung†
†Virtual Proof of Concept Discovery Performance Unit, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, United States
‡Computational Chemistry, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, United States
§WuXi AppTec, Shanghai, China
∥Cellzome GmbH, a GSK company, Meyerhofstrasse 1, 69117 Heidelberg, Germany
⊥Genetic Toxicology, GlaxoSmithKline, Ware, Hertfordshire, United Kingdom
#Computational Toxicology, GlaxoSmithKline, Ware, Hertfordshire, United Kingdom
∇Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, 1122 NE 13th Street, Oklahoma City,
Oklahoma 73117, United States
S
* Supporting Information
ABSTRACT: Abdominal pain and abnormal bowel habits represent major symptoms for irritable
bowel syndrome (IBS) patients that are not adequately managed. Although the etiology of IBS is not
completely understood, many of the functions of the gastrointestinal (GI) tract are regulated by the
enteric nervous system (ENS). Inflammation or stress-induced expression of growth factors or
cytokines may lead to hyperinnervation of visceral afferent neurons in GI tract and contribute to the
pathophysiology of IBS. Rearranged during transfection (RET) is a neuronal growth factor receptor tyrosine kinase critical for
the development of the ENS as exemplified by Hirschsprung patients who carry RET loss-of-function mutations and lack normal
colonic innervation leading to colonic obstruction. Similarly, RET signaling in the adult ENS maintains neuronal function by
contributing to synaptic formation, signal transmission, and neuronal plasticity. Inhibition of RET in the ENS represents a novel
therapeutic strategy for the normalization of neuronal function and the symptoms of IBS patients. Herein, we describe our
screening effort and subsequent structure−activity relationships (SARs) in optimizing potency, selectivity, and mutagenicity of
the series, which led to the discovery of a first-in-class, gut-restricted RET kinase inhibitor, 2-(4-(4-ethoxy-6-oxo-1,6-
dihydropyridin-3-yl)-2-fluorophenyl)-N-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)acetamide (15, GSK3179106), as a
clinical candidate for the treatment of IBS. GSK3179106 is a potent, selective, and gut-restricted pyridone hinge binder small
molecule RET kinase inhibitor with a RET IC50 of 0.3 nM and is efficacious in vivo.
KEYWORDS: RET kinase, IBS, gut restricted, genotoxicity, AMES
cytokine expression that may mediate neuronal outgrowth or
plasticity in IBS.6−8
rritable bowel syndrome (IBS) is a relatively common
Igastrointestinal illness characterized by a constellation of
clinical symptoms including abdominal pain and discomfort,
abnormal bowel habits, and bloating.1−3 The pathophysiology
of IBS is thought to result from alterations in sensory inputs/
outputs in the peripheral and central nervous system such that a
patient with IBS has a heightened and disproportionate sensory
experience for a given stimulus, i.e., visceral hypersensitivity.4,5
While it is unclear whether an enhancement of central or
peripheral mechanisms underlies visceral hypersensitivity,
sensitization of visceral afferent nociceptors has been
demonstrated to induce visceral hypersensitivity.1 Furthermore,
gastrointestinal (GI) insults, such as infection, can lead to
sensitization of the enteric nervous system (ENS)4 and have
been associated with neurotrophic factors and/or inflammatory
Rearranged during transfection (RET) kinase is a neuronal
growth factor receptor tyrosine kinase that is activated upon
binding to one of four neurotrophic factors glial cell line-
derived neurotrophic factor (GDNF), neurturin, artemin, and
persephin in combination with a GPI-anchored coreceptor
GFRα-1, 2, 3, and 4, respectively.9 RET activity is critical for
the development of the ENS, the kidney, and spermato-
genesis.10 The role of RET kinase in the development of the
ENS has been aided by the study of patients with Hirschsprung
Received: January 23, 2018
Accepted: May 21, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00035
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
disease who frequently suffer from colonic obstruction due to
variable lengths of bowel aganglionosis, often in association
with loss-of-function RET mutations.11−13 Mechanistically,
RET signaling is associated with the maturation of presynaptic
axon terminals through regulating the expression of presynaptic
proteins.14−16 Systemic administration of GDNF in adult
rodents significantly increases submucosal neuron density in
both the small intestine and colon,17 while a conditional
knockout of the RET kinase coreceptor, GFRα3, attenuates
visceral hypersensitivity suggesting that RET kinase plays a role
in the signaling of visceral nociception.18 These observations
led us to hypothesize that antagonizing RET signaling in the
gut may offer a novel therapeutic approach to treat visceral pain
in conditions such as IBS.
enzyme assays. Movement of the methyl-amine (1) to the para-
position of a meta-pyridine (2) showed an improvement in
RET biochemical potency from 22 to 0.7 nM (compound 1 to
compound 2, Table 1). While trying to improve physiochemical
properties of the compounds, we explored pyridones as hinge
binders due to their decreased lipophilicity such as clogP. While
we noted similar RET potency as the methyl-amine pyridines,
we did note an improvement in KDR selectivity (compound 2,
8-fold vs compound 3, 79-fold) and lipophilicity as measured
by clogP (compound 2, 6.18 vs compound 3, 6.01) while
maintaining RET potency. In addition, pyridone hinge binders
(compounds 3 and 4) removed the potential genotoxicity
associated with the amino-pyridine scaffold present in
compounds 1 and 2 in the A ring.
There are many reported orally active RET inhibitors;
however, highly selective gut-restricted RET inhibitors have not
yet been identified.19−21 Yang and colleagues recently published
a report on N-phenyl-7,8-dihydro-6H-pyrimido[5,4-b][1,4]-
oxazin-4-amine derivatives and detailed a list of available RET
inhibitors in the publication and references within.22 In
addition, since chronic dosing is required for the treatment
for IBS, therefore, our initial focus was on identifying a highly
selective and gut-restricted RET inhibitor to improve
tolerability and the safety margin. A high throughput screen
(HTS) campaign was undertaken and resulted in several potent
but kinase nonselective RET inhibitors. The most attractive of
those came from a biphenyl urea series as shown in Table 1.23
To improve KDR selectivity, we sought an understanding of
the structural differences in the RET and KDR kinase domains.
DFG-out cocrystal structures for RET kinase were not available,
which led the team to build homology models based on KDR
and KIT kinase for RET kinase. Based on docking of
compounds into the homology model, it suggested substitution
on the B ring (compound 4, Table 1) could reduce KDR
activity thereby increasing overall kinase selectivity. A figure of
compound 4 docked in the model is provided in the Supporting
Information (Figure S1). The homology model showed key
hinge interactions at Glu805 and Ala807, as well as Glu775 and
Asp892 for the central linker. The model also showed a small
pocket in the hinge, which was optimized with an ethyl ether
on the A ring. Compound 4 with an optimized hinge and F-
substitution on the B ring showed a RET IC50 of 0.1 nM and
KDR IC50 of 20.8 nM, a 208-fold selectivity window.
Table 1. A-Ring (Hinge Binder) Optimization
An optimal compound profile for an IBS indication
necessitated a preclinical candidate devoid of genotoxicity.
Metabolite identification (MET ID) studies and in silico
metabolism predictions showed aniline formation from urea
hydrolysis as a predominant route of elimination. In addition,
there was a risk of aniline synthetic intermediates appearing as
impurities (or degradants), warranting an early genotoxicity risk
assessment of the embedded anilines in the structure. At this
point in the early lead optimization process, owing to the risk of
genotoxicity from anilines from both synthetic intermediates
and potential urea hydrolysis metabolism, we evaluated these
anilines in the standard Ames test for genotoxic liability. Initial
Ames25 testing of both the left-hand and right-hand side
anilines of compound 4 showed positive in the TA98 bacterial
strain, which is typically the most sensitive strain. Additionally,
we tested several structurally similar anilines (see Supporting
Information), and they were also found to be Ames positive.
Due to the lack of Ames negative A/B-rings, we explored urea
isosteres and noted that amides retained biochemical potency
with a pyridone hinge binder. However, compounds were still
not progressible due to the presence of the Ames positive C-
ring aniline; specifically, the anilines with the ethyl piperazine
attached that helps drive RET potency. Therefore, a full
exploration of the C ring was undertaken to find Ames negative
C-ring anilines, while maintaining RET potency and KDR
selectivity as seen in Table 2.
a
b
IC50 is a mean of least two experiments. Value in parentheses for
KDR represent fold selectivity over RET.
While triaging the HTS hits, it became evident that kinase
insert domain receptor (KDR/VEGFR2) selectivity could be
used as a surrogate for overall off-target kinase selectivity for
many of the RET inhibitors, including published RET
inhibitors. Therefore, we used KDR as the off-target selectivity
assay during primary screening. We targeted >100-fold
selectivity over other kinases. One of the most promising
leads (compound 1, Table 1) was a directly linked A and B ring
biaryl urea that was a potent RET inhibitor, with 45-fold
selectivity over KDR and was structurally similar to the known
inhibitor FLT3 inhibitor AST-487 with the ethyl piperazine.24
Both RET and KDR potency were determined in in vitro
Given the relatively low throughput of the Ames assay and
the need for rapid lead optimization, we employed computa-
tional modeling to prioritize anilines for synthesis and testing in
the Ames assay. Multiple computational approaches were
investigated, but ultimately nitrenium ion formation energy26
was found to be most predictive for the RET potent aniline
fragments. The nitrenium ion formation model was validated
B
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ACS Medicinal Chemistry Letters
Letter
kinase. Figures S2−S5 show optimized amino acid interactions
of RET kinase with compound 15 in the homology model.
The highly convergent synthesis of representative amide-
linked RET inhibitors is described in Scheme 1 and has been
Table 2. C-Ring Optimization
Scheme 1. General Synthesis of Pyridone Hinge Binder
a,b
Amides
a
Reagents and conditions: (a) EtONa, THF, 92% yield; (b) NBS,
H2SO4; (c) chromatography, 40% yield; (d) PMBOH, KOH, toluene,
70% yield; (e) NaCN, EtOH, 99% yield; (f) NaOH, MeOH, 92%
yield; (g) H2SO4, H2O, MeOH, 94% yield; (h) Pd(dppf)Cl2, KOAc,
dioxane, 90% yield; (i) Pd(dppf)Cl2, Cs2CO3, 90% yield; (j) LiOH·
H2O, THF, 93% yield; (k) T3P, pyridine, 74% yield; (l) TFA; (m)
b
THF, H2O, 77% yield. Reported yields are for compound 15.
previously published.27 The A-ring is constructed in four
synthetic steps starting from the commercially available 2-
chloro-4-nitropyridine. Nucleophilic aromatic substitution
utilizing sodium ethoxide provides the desired aryl ether in
excellent yield. Subsequent directed bromination followed by
displacement of the 2-chloro functionality with p-methoxyben-
zyl alcohol furnishes the fully elaborated, PMB-protected A-
ring. Synthesis of the B-ring begins with the commercially
available benzyl bromide, which is converted to the
corresponding phenylacetic acid in two synthetic steps and
excellent yield. Esterification of the carboxylic acid followed by
Suzuki coupling with bis(pinacolato) diborane affords the
completed B-ring, which is then coupled to the A-ring under
standard Suzuki conditions. Saponification of the elaborated
methyl ester provides the carboxylic acid necessary for amide
formation utilizing T3P in pyridine. Finally, PMB deprotection
affords the desired RET inhibitor. Experimentals of biaryl urea
RET inhibitors are provided (Supporting Information) and
have previously been published.28
Compound 15 was found to have the best overall profile with
respect to potency and selectivity and was progressed to
additional DMPK studies and in vivo evaluation. Compound 15
(GSK3179106) was a potent RET inhibitor with IC50s of 0.4
and 11 nM in the biochemical assay and cellular assay,
respectively. In addition, it had a clean genotoxic profile with no
embedded genotoxicity liabilities. Compound 15 possessed
good kinase selectivity; only 26 out of a set of >300
recombinant kinases were found to be inhibited at a 1 μM
a
b
IC50 is a mean of least two experiments. IC50 is a mean of one
experiment.
using GSK Ames data on a series of structurally similar anilines.
The model was critical in rapidly identifying readily available
aromatic amines that fitted established RET SAR. Using the
internal data set to validate the model, a nitrenium ion
formation energy of <153 kcal/mol was predictive of a positive
Ames result and an energy of >164 kcal/mol was predictive of a
negative result. Energies between 154 and 163 kcal/mol could
not lead to accurate predictions. Examples listed in the
Supporting Information showed both A/B-ring and C-ring
anilines and their nitrenium ion formation energies alongside
their experimental Ames result.
Table 2 showcases an array of C-ring phenyl substitutions, as
well as heteroaryl replacements optimized for RET potency.
The ortho-fluoro was maintained in the B-ring, as well as an
ethyl ether in the ortho position of the A-ring. Both ortho- and
meta-ethyl ethers displayed similar RET potency. While a wide
range of substitutions with various steric and electronic effects
were tolerated for RET potency, compound triage was now
focused on removing the genotoxic liability, as well as
acceptable PK parameters and low nanomolar cellular potency.
In addition to the RET and KDR biochemical assays,
compounds were evaluated in a cellular assay (TT cell) for
the inhibition of phosphorylation of a constitutively active RET
C
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ACS Medicinal Chemistry Letters
Letter
test concentration (Supporting Information, Table S4). To
further determine the physiologically relevant kinase target(s)
of GSK3179106 in colon tissues isolated from rats, a
chemoproteomics technique identified RET kinase as the
most potent target that was competed for binding to the
A noninflammatory irritation model of colonic hyper-
sensitivity in rats utilizes a low concentration of acetic acid to
induce a transient sensitization of colonic afferents, which, upon
colorectal distension, induces a visceromotor response that can
be monitored visually as abdominal contractions and used to
assess the severity of induced visceral pain.31,32 GSK3179106
dosed orally at 10 mg/kg (n = 7, all groups) for 3.5 days BID
reduced the visceromotor response to colorectal distension in
comparison to rats given an acetic acid enema and dosed with
vehicle (Figure 2, *P < 0.05, ****P < 0.0001 vehicle compared
app
kinobeads by GSK3179106 (pKd = 7.9 or Kd = 0.02 μM;
Supporting Information Table S5).29,30 However, two other
app
kinases, DDR1 (pKd = 7.6 or Kd = 0.04 μM) and DDR2
app
(pKd = 7.3 or Kd = 0.09 μM) were also competed by
GSK3179106 with comparable potency and may represent
additional targets of the compound. Compound 15 has
relatively poor solubility with the kinetic chemiluminescent
nitrogen detection (CLND) solubility of 21 μM and 4 h fasted
state simulated intestinal fluid (FaSSIF) solubility of 0.4 μg/
mL.
Single dose IV (bolus, 0.06 mg/kg) PK in male Sprague−
Dawley rats of compound 15 formulated as 0.04 mg/mL in
DMSO/6% HP-beta-CD = 5:95 with a pH of 7 as a clear
solution showed low exposure with an AUC of 102 ng·h/mL
(Figure 1, full details in the Supporting Information). Oral PK
Figure 2. Colonic hypersensitivity induced by acute irritation stress is
ameliorated by RET kinase inhibition with compound 15.
to GSK3179106 treatment, repeated-measure two-way
ANOVA, Bonferroni post-test). Treatment with tegaserod, a
5-HT4 agonist used as a positive control for the model,
exhibited a decrease in observed abdominal contractions as
expected (Supporting Information, Figure S6).33,34
Compound 15 (GSK3179106) was further progressed to
rodent safety studies and found to have a favorable safety
profile to support clinical testing.35
In summary, a selective, gut-restricted RET kinase inhibitor
has been developed for the treatment of IBS in a clinical setting.
The calculation of nitrenium ion formation energies greatly
aided the deprioritization of anilines with a genotoxic risk. In
due course, the clinical study of GSK3179106 will be
presented.35
Figure 1. Rat IV PK (0.06 mg/kg) of compound 15.
was evaluated (Supporting Information) with the same dosing
regimen as the in vivo colonic hypersensitivity model, seven
doses of 10 mg/kg given over 3.5 days. Full gut PK
measurements were also taken to help understand the PK/
PD relationship.
A full gut pharmacokinetics analysis revealed high concen-
trations of compound 15 in the colon contents, jejunum,
duodenum, and ileum, over that in plasma (Table 3). Upon
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00035.
■
S
Table 3. Satellite Full Gut PK for 10 mg/kg 3.5 days BID
p.o. Dosing of Compound 15 in Rats
Cmax (ng/mL)
Tmax (h)
colon contents
duodenum
jejunum
ileum
287500
15713
12800
5520
3358
40
7
0
1
2
7
4
Compound synthesis and spectroscopic characterization
of compound 15, along with nitrenium ion formation
energy calculations and Ames data, as well as homology
model details and images. Enzyme and cell-based assay
methods, kinase selectivity, chemoproteomics, pharma-
cokinetics, and in vivo models methods and standards
(PDF)
colon
plasma
further investigation, pharmacokinetic exposures of most
compounds in the chemical series were predominantly gut
restricted when administered orally. Specifically, following 10
mg/kg given seven doses in 3.5 days, plasma concentrations
were only 40 ng/mL at Cmax (Table 3), while colon tissue
homogenate concentrations were 3358 ng/mL at Cmax. The full
PK curve data is available in the Supporting Information
(Figure S5).
AUTHOR INFORMATION
Corresponding Author
*E-mail: hilary.s.eidam@gsk.com.
ORCID
Hilary Schenck Eidam: 0000-0003-0939-298X
Mui Cheung: 0000-0002-3794-2937
■
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ACS Medicinal Chemistry Letters
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Notes
The authors declare the following competing financial
interest(s): H.S.E., J.R., K.R., M.D., D.Q., G.J., N.Z., S.L.,
S.R., A.W., A.G., A.O., S.K., and M.C. are current or past
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GlaxoSmithKline.
ACKNOWLEDGMENTS
■
The authors thank Karl Tyler (University of Oklahoma), as well
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animals were conducted after review by the GlaxoSmithKline
(GSK) Institutional Animal Care and Use Committee and in
accordance with the GSK Policy on the Care, Welfare and
Treatment of Laboratory Animals.
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ABBREVIATIONS
■
CLND, chemiluminescent nitrogen detection; FaSSIF, fasted
state simulated intestinal fluid; IBS, irritable bowel syndrome;
KDR, kinase insert domain receptor; RET, rearranged during
transfection; VEGFR2, vascular endothelial growth factor
receptor type 2; THF, tetrahydrofuran; NBS, N-bromosuccini-
mide; PMBOH, 4-methoxybenzel alcohol; Pd(dppf)Cl2, [1,1′-
Bis(diphenylphosphino)ferrocene]dichloropalladium(II); T3P,
propylphosphonic anhydride; TFA, trifluoroacetic acid
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