Discovery of PF-5190457, a potent, selective, and orally bioavailable ghrelin receptor inverse agonist clinical candidate

Article abstract of DOI:10.1021/ml400473x

The identification of potent, highly selective orally bioavailable ghrelin receptor inverse agonists from a spiro-azetidino-piperidine series is described. Examples from this series have promising in vivo pharmacokinetics and increase glucose-stimulated insulin secretion in human whole and dispersed islets. A physicochemistry-based strategy to increase lipophilic efficiency for ghrelin receptor potency and retain low clearance and satisfactory permeability while reducing off-target pharmacology led to the discovery of 16h. Compound 16h has a superior balance of ghrelin receptor pharmacology and off-target selectivity. On the basis of its promising pharmacological and safety profile, 16h was advanced to human clinical trials.

Full text of DOI:10.1021/ml400473x

Letter
pubs.acs.org/acsmedchemlett
Discovery of PF-5190457, a Potent, Selective, and Orally Bioavailable
Ghrelin Receptor Inverse Agonist Clinical Candidate
Samit K. Bhattacharya,*,† Kim Andrews,‡ Ramsay Beveridge,† Kimberly O. Cameron,† Chiliu Chen,†
Matthew Dunn,† Dilinie Fernando,† Hua Gao,† David Hepworth,† V. Margaret Jackson,‡ Vishal Khot,†
Jimmy Kong,‡ Rachel E. Kosa,§ Kimberly Lapham,§ Paula M. Loria,∥ Allyn T. Londregan,†
Kim F. McClure,† Suvi T. M. Orr,† Jigna Patel,⊥ Colin Rose,† James Saenz,⊥ Ingrid A. Stock,∥
Gregory Storer,† Maria VanVolkenburg,‡ Derek Vrieze,† Guoqiang Wang,† Jun Xiao,† and Yingxin Zhang†
‡
§
†Worldwide Medicinal Chemistry, Cardiovascular and Metabolic Research Unit, Pharmacokinetics, Dynamics, and Metabolism,
∥Primary Pharmacology Group, and Pharmaceutical Sciences, Pfizer Global Research and Development, 620 Memorial Drive,
⊥
Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: The identification of potent, highly selective orally bioavailable ghrelin receptor
inverse agonists from a spiro-azetidino-piperidine series is described. Examples from this series have
promising in vivo pharmacokinetics and increase glucose-stimulated insulin secretion in human
whole and dispersed islets. A physicochemistry-based strategy to increase lipophilic efficiency for
ghrelin receptor potency and retain low clearance and satisfactory permeability while reducing off-
target pharmacology led to the discovery of 16h. Compound 16h has a superior balance of ghrelin
receptor pharmacology and off-target selectivity. On the basis of its promising pharmacological and
safety profile, 16h was advanced to human clinical trials.
KEYWORDS: Ghrelin, ghrelin receptor inverse agonist, ghrelin receptor antagonist, diabetes, PF-5190457
led to increases in appetite, fasting glucose, HbA1c levels, and
ype 2 diabetes mellitus (T2DM) is a rapidly expanding
Tpublic health problem affecting over 285 million people insulin resistance. Thus, ghrelin receptor antagonists/inverse
worldwide.1 The disease is characterized by elevated fasting
agonists are anticipated to improve glucose homeostasis and
insulin sensitivity. Described herein is the discovery of PF-
5190457, a potent and selective ghrelin receptor inverse agonist
that increases glucose-stimulated insulin secretion (GSIS) in
human whole and dispersed islets.
plasma glucose, insulin resistance, abnormally elevated hepatic
glucose production, and reduced glucose-stimulated insulin
secretion.2 Uncontrolled glucose levels can lead to severe
downstream complications, such as elevated risks of cardiovas-
cular disease, retinopathy, and nephropathy. While several
classes of antidiabetic therapy are available for clinical use, there
still remains a significant need for new therapies with improved
efficacy, safety, and tolerability to help diabetes patients achieve
their treatment goals and avoid long-term complications.3
Ghrelin, an agonist of the ghrelin receptor (earlier names for
the receptor that have been used are GHS-R1a, GRLN, etc.),
stimulates release of growth hormone (GH) from the pituitary
gland and increases food intake.4−8 Through infusion studies in
man, ghrelin has been shown to suppress both glucose-
dependent insulin secretion and insulin sensitivity.9−13 The
ghrelin receptor is expressed in pancreatic islets, and ghrelin is
released into the pancreatic microcirculation. Islet-derived
ghrelin has been reported to play an important role in the
regulation of insulin release in rodents.14 Although ghrelin has
been shown to cross the blood−brain barrier,15 several effects
of ghrelin appear to be peripherally mediated as shown by the
clinical results from the brain-impaired ghrelin receptor agonist
capromorelin (CP-424391).16,17 Treatment with capromorelin
A number of small molecule ghrelin receptor antagonists
have been described in the literature (see Figure 1).18−26 The
Figure 1. Examples of ghrelin receptor antagonists.
Received: November 18, 2013
Accepted: February 18, 2014
Published: February 24, 2014
© 2014 American Chemical Society
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vast majority of these efforts have focused on centrally acting
agents targeted toward the treatment of obesity. We previously
reported on a spiro-azetidino-piperidine series that was
identified from high-throughput screening (HTS) of our
corporate file.27 In a later report, we described efforts to
optimize this series to achieve high central receptor occupancy
targeting an obesity indication. Thus, the HTS-hit (10) in the
aforesaid series was transformed to a centrally acting ghrelin
receptor inverse agonist lead (11, Table 1).28
Emerging ghrelin biology suggests that blocking ghrelin
receptor signaling has the potential to ameliorate diabetes
through direct action on the pancreas.29 To test this hypothesis,
lead 11 was assessed in a human dispersed islet cell assay. As
anticipated, 11 induced insulin secretion in a glucose-
dependent manner (Supporting Information, Figure S1).
These data, in conjunction with other published work linking
ghrelin receptor inhibition to improved insulin sensitivity,
caused us to redirect our research efforts toward diabetes
(T2DM) as the primary indication, and we targeted the
peripheral compartment as the key site of action.
potency while increasing the overall polarity of the inverse
agonists. This physicochemistry-based strategy should provide
the added benefit of decreasing CNS penetration, leading to a
reduction in CNS-based side effects.
Scaffold 13 provided a modular framework for exploring
structure−activity relationship (SAR), and earlier work
established the preference for the R-enantiomer at C(5) as
providing robust ghrelin receptor activity. A one-pot sequence
reacting chiral amine 12a with chloroaldehyde 12b in the
presence of NaCNBH3 was developed and used to deliver bulk
quantities of 13 (with retention of stereochemistry at the C(5)
position), a key chiral intermediate for flexible incorporation of
a variety of R1 and R2 groups onto scaffold 16.28 Suzuki
coupling to incorporate R1 followed by in situ deprotection
(Scheme 1) under acidic conditions unmasked the piperidine
Scheme 1. Synthesis of Analogues 16a−h
The spiro-azetidino-piperidine series was an attractive
starting point for optimization for this indication. It provided
a rigid core scaffold with high fractional sp3 content (for leads
in series, Fsp = 0.41−0.53)30 and two point vectors for
3
introducing orthogonal diversity using simple coupling method-
ologies. A significant factor in the selection of this series was
consistent inverse agonism of ghrelin receptor. Functional switching
(between antagonism and partial agonism) has been observed
by us and others and has demonstrated impact on the in vivo
performance of compounds.26,31 We believed that by focusing
on a series where receptor functionality was not an issue, we
could accelerate our drug discovery program by eliminating one
dimension from our lead optimization efforts and, at the same
time, streamline our in vitro pharmacology testing funnel.
Furthermore, because of the high constitutive activity of the
ghrelin receptor, inverse agonism of ghrelin receptor may
provide a greater opportunity for in vivo efficacy through
reductions in basal receptor firing.32
Although lead 11 showed encouraging insulin secretion
effects in islet studies, more extensive profiling highlighted
issues that prevented its further development. In preclinical CV
safety testing, 11 showed inadequate safety margins to allow
progression. The discovery of the indane amine moiety in 11
(built as a less lipophilic surrogate of an ortho-chloro benzylic
amine) had allowed improvements in selectivity within the
spiro-azetidino-piperidine series.28 However, while selectivity
over muscarinic receptors (in particular M2) was improved
with the desired chiral indane, in wide-ligand promiscuity
profiling (CEREP; tested at a dose of 10 μM, Figure 2) 11 was
active at a number of targets including adrenergic α2c and α2a,
dopamine D2s, D3, and D4, and histamine H1. Compound 11
Reagents and conditions: (a) (i) HOAc, MeOH, 50 °C, 2 h; (ii)
NaCNBH3, 70 °C; 99%; (b) bis(pinacolato)diboron, Pd(dppf)Cl2 (1.6
mol %), KOAc, dioxane, 110 °C, 1 h, >99% conversion; (c) R1-Cl or
R1-Br, Pd(dppf)Cl2 (2.5 mol %), K2CO3 (aq), dioxane, 110 °C, 3 h,
90% conversion; (d) HCl; (e) TFA; (f) R2CH2CO2H (14), HBTU or
CDI, DIEA or TEA, 50−99% (over 2 steps).
nitrogen (15b) for incorporation of the R2 amide. Amide
coupling with a variety of carboxylic acids was accomplished
with HBTU or CDI as the preferred coupling agent.
We identified two R2 moieties, pyridyl-3-methoxy (16a) and
the bicyclic imidazothiazole (16b),27 that maintained ghrelin
receptor potency at lower logD with retention of other
properties, such as low clearance and satisfactory permeability.
Unfortunately, when combined with 2-pyrimidinyl at R1,
neither compound had a positive effect on selectivity over
M2 (Table 1). As anticipated, these changes, which were
designed to lower lipophilicity, had the effect of increasing
efflux ratios in an in vitro MDR assay (b-a/a-b efflux ratio of
>2.5 being generally indicative of reduced CNS exposure; Table
1).38
̀
also showed human ether-a-go-go-related gene (hERG) activity
(IC50 = 2.9 μM). Thus, the overall off-target pharmacology
profile, coupled with the physicochemical properties of 11
(Table 1), was of concern. An increased risk of adverse safety
findings for compounds with higher clogP and lower TPSA has
been described.33 In addition, several groups report that off-
target promiscuity leads to elevated safety risk.34,35 Therefore,
our primary objective was to identify a compound in this series
with reduced off-target pharmacology in order to achieve broad
safety margins to allow advancement to the clinic. Our strategy
to reduce off-target pharmacology was to increase the lipophilic
efficiency (LipE)36,37 and retain or improve ghrelin receptor
In an attempt to improve M2 selectivity, we switched
attention to modification of R1. Attempts to further increase
polarity, as exemplified by carboxamide 16c, had no effect on
selectivity (Table 1). Thus, to complement the property-based
design strategy, we investigated the effect of more subtle
structural changes on M2 potency. Small changes around the
pyrimidinyl group (as exemplified by 16d, 16e, and 16f) did
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ACS Medicinal Chemistry Letters
Letter
Table 1. Data for Spiro-azetidino-piperidine Analogues 10, 11, and 16a−h
a
b
elogD = measured logD at pH 7.4 [calculated logD (ACD Laboratories program v12) in parentheses]. TPSA = topological polar surface area.
c
d
Geometric mean
standard error of ≥3 measurements (unless noted otherwise). Human ghrelin receptor SPA receptor binding assay as
published.27 Human ghrelin receptor agonist/antagonist/inverse agonist GTP-γ-S functional assay as published.27 Minimum percent effect in
e
f
g
parentheses; a negative value indicates an inverse agonist. Muscarinic M2 β-Arrestin PathHunter assay from Discoverx. Kb for antagonists
determined in the presence of EC80 concentration of agonist, oxotremorine.39 Ratio of M2 Kb over human ghrelin receptor functional Ki values.
h
i
j
Human liver microsome (HLM) apparent intrinsic clearance, mL/min/kg, uncorrected for fraction unbound. Passive permeability (Papp in 10−6
cm/s) measured using low-efflux MDCKII cells as described.40 MDR BA/AB efflux ratio using MDCK cell line transfected with human MDR1.41
k
l
Less than three independent replicates.
not provide significant improvement with the exception of the
6-methyl-4-pyrimidinyl group (as in 16g), which showed
improved ghrelin receptor potency, as compared to 16a, and
a marked enhancement in M2 selectivity. Gratifyingly,
combination of the 6-methyl-4-pyrimidinyl group at R1 with
the more polar imidazothiazole moiety at R2, provided 16h,
which demonstrated significant improvements in both func-
tional potency and selectivity against M2 relative to 11 (Table
1). Indeed, compound 16h has one of the highest lipophilic
efficiencies (LipEelogD = 6.9) in this series, which is a desirable
predictor for lowered promiscuity-related developmental
attrition risk.34,36 Compound 16h maintained a moderate
clearance in human liver microsomes (HLM). In addition, the
reductions in logP achieved in moving to 16h had the
anticipated and desired effects on MDR efflux ratio, suggesting
a high probability of reduced CNS exposure. (Incidentally, 16h
was 11-fold impaired in rat brain after chronic dosing for 14
days.)
Given its promising in vitro pharmacology and ADMET
parameters, compound 16h was profiled in more detail.
Compound 16h showed reduced off-target activity, as assessed
by the CEREP panel (screened at 10 μM, Figure 2), with
serotonin 5-HT2B (IC50 = 3700 nM) being the only target
inhibited to >50%. In follow-up screening, 16h did not
demonstrate any agonist (or antagonist) functional effects at
this receptor.
Compound 16h was advanced to a number of ex vivo and in
vivo studies. The full pharmacology profile for 16h (GTP-γ-S
functional profile in Supporting Information, Figure S2),
Figure 2. Comparative CEREP profile of 11 (bottom) and 16h (top).
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ACS Medicinal Chemistry Letters
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dosing interval (52.4 nM free). Thus, the likelihood of
achieving drug exposures in the clinic that would effect QTc
prolongation was considered low.
With the primary design objectives of delivering a
peripherally acting ghrelin receptor inverse agonist with
improved selectivity satisfied, the nonclinical safety profile of
16h was assessed in both rats and dogs in 1-month toxicology
studies and in safety pharmacology studies. Gratifyingly, the
safety profile of 16h demonstrated sufficient safety margins
above the projected Ceff in humans and supported continued
development of the compound.
In summary, we set out to transform a centrally acting
ghrelin receptor inverse agonist indane lead (11) to a
peripherally restricted one, by pursuing a physicochemistry-
based strategy to increase LipE for ghrelin receptor potency
and reduce off-target pharmacology while retaining low
clearance and satisfactory permeability. The addition of the 6-
methyl-4-pyrimidinyl indane in the R configuration and the
imidazothiazole group to the spiro-azetidino-piperidine core led
to the discovery of 16h. Compound 16h is a potent inverse
agonist with excellent selectivity and demonstrated robust
increases in glucose-stimulated insulin secretion in human
islets. Human pharmacokinetic predictions project a dose of 35
mg bid to achieve 10 × Kd at trough concentrations in the
clinic. On the basis of the pharmacological profile and safety
results, 16h (PF-5190457) was advanced to human clinical
trials, and results from these trials will be reported in due
course.
Figure 3. Glucose-stimulated insulin secretion in human whole islet
static culture following incubation with 16h at 1 μM. **p < 0.001; *p
< 0.05. Measurement data are expressed as the arithmetic mean
standard error.
including a biomarker for target engagement, will be described
separately.42 A human islet assay was used in order to gain
confidence in the ability of 16h to increase insulin secretion in
humans (Figure 3). Human whole islets in static culture were
incubated at both low (2.8 mM) and high (11.2 mM) glucose
concentrations and demonstrated that the islets were glucose
responsive.43 The sulfonylurea, glibenclamide (glyburide), was
tested as a positive control. Compound 16h (1 μM)
significantly increased insulin secretion above the 11.2 mM
glucose control.
To predict human pharmacokinetics and enable clinical dose
setting, the pharmacokinetics of 16h were evaluated preclini-
cally in three species (Supporting Information, Table S1). Rat
pharmacokinetics revealed both high plasma clearance and
volume of distribution (consistent with the high clearance value
derived from rat liver microsomal in vitro assay). Dog and
monkey plasma clearance and volume of distribution were
moderate. Urinary and biliary elimination of 16h contributed
minimally to the overall clearance in the species investigated.
Because of its high in vivo rat clearance, 16h was tested in
portal vein cannulated rats and demonstrated excellent
absorption (Fa = 100%) as anticipated given its high solubility
and moderate passive permeability.44 The human plasma
clearance of 16h was predicted by scaling human liver
microsomal data (human hepatocyte data was consistent with
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures of analogue preparation and descrip-
tion of biological assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.K.B.) Tel: 617-551-3177. E-mail: samit.k.bhattacharya@
pfizer.com.
■
Notes
The authors declare no competing financial interest.
microsomal data). When corrected for microsomal (fu,mic
=
ABBREVIATIONS
■
0.8) and plasma binding (fu = 0.15), human plasma clearance
was predicted to be low (3.3 mL/min/kg). Using preclinical
data and physiologically based pharmacokinetic (PBPK)
models,45 16h was predicted to have moderate to high
human absorption (86%) along with moderate measures of
bioavailability (67%), Vdss (1.79 L/kg), and half-life (6.3 h).
Human free drug concentrations (Ceff,u) needed to achieve
efficacy were based on the equilibrium binding constant (Kd) of
16h, which was determined using the Motulsky kinetics
TPSA, topological polar surface area; dppf, 1,1′-bis-
(diphenylphosphino)ferrocene; HBTU, O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate; CDI,
1,1′-carbonyldiimidazole; GHS-R1a, growth hormone secreta-
gogue receptor; GSIS, glucose stimulated insulin secretion;
QTc, heart rate-corrected QT interval; PK, pharmacokinetic
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