Rational Design, Synthesis, and Pharmacological Characterization of Novel Ghrelin Receptor Inverse Agonists as Potential Treatment against Obesity-Related Metabolic Diseases

Article abstract of DOI:10.1021/acs.jmedchem.8b00794

A new chemotype of ghrelin inverse agonists was discovered through chimeric design based on molecular scaffolds known as growth-hormone secretagogue receptor (GHSR) modulators but with divergent pharmacodynamic and pharmacokinetic properties. The structur

Full text of DOI:10.1021/acs.jmedchem.8b00794

Subscriber access provided by UNIVERSITY OF TOLEDO LIBRARIES
Article
Rational Design, Synthesis and Pharmacological Characterization
of Novel Ghrelin Receptor Inverse Agonists as Potential
Treatment against Obesity-related Metabolic Diseases
Antoine Daina, claudio giuliano, Claudio Pietra, Junbo Wang, Yushi Chi, Zack Zou, Fugang
Li, Zhonghua Yan, Yifan Zhou, Angelo Guainazzi, Silvina Garcia Rubio, and Vincent Zoete
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00794 • Publication Date (Web): 28 Sep 2018
Downloaded from http://pubs.acs.org on September 29, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Rational Design, Synthesis and Pharmacological
Characterization of Novel Ghrelin Receptor Inverse
Agonists as Potential Treatment against Obesityꢀ
related Metabolic Diseases.
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
1
,2
,2
3
3
3
Antoine Daina , Claudio Giuliano* , Claudio Pietra* , Junbo Wang , Yushi Chi , Zack Zou ,
4
4
4
5
5,#
Fugang Li , Zhonghua Yan , Yifan Zhou , Angelo Guainazzi , Silvina Garcia Rubio , Vincent
,
1,6
Zoete*
1
Molecular Modeling Group, SIB Swiss Institute of Bioinformatics, CHꢀ1015 Lausanne,
Switzerland
2
2
Research and Preclinical Development Department, Helsinn Healthcare, CHꢀ6912 Lugano,
Switzerland
3
Department of Medicinal Chemistry, Pharmacokinetics and Metabolism, Sundia MediTech, 388
Jialilue Road, Zhangjiang HiꢀTech Park, Shanghai 201203, China
4
Department of Discovery Biology, HD Biosciences, 590 Ruiqing Road
Zhangjiang East Campus, Shanghai 201201, China
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
Research and Development Department, Helsinn Therapeutics (US), Inc., Iselin, NJ 08830,
UnitedꢀStates
6
Department of Fundamental Oncology, Lausanne University, Ludwig Institute for Cancer
Research, Route de la Corniche 9A, 1066 Epalinges, Switzerland
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
#
th
Present address: Bridge Medicines, Belfer Research Building, 413 East 69 Street, New York,
NY 10021, UnitedꢀStates
ACS Paragon Plus Environment
2

Page 3 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
KEYWORDS
Ghrelin, GHSR, ghrelin receptor, inverse agonist, obesity, diabetes, metabolic diseases
ABSTRACT
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
A new chemotype of ghrelin inverse agonists was discovered through chimeric design based on
molecular scaffolds known as growthꢀhormone secretagogue receptor (GHSR) modulators but
with divergent pharmacodynamic and pharmacokinetic properties. The structureꢀ
activities/properties exploration led to compound 47, which displayed potent human GHSR
antagonism and inverse agonism in cellular assays (IC = 68 nM, EC = 29 nM), moderate oral
5
0
50
bioavailability and notable brain penetration in rat (F = 27 %, B/P ratio = 1.9). First in vivo
studies demonstrated effective reduction of food intake after oral or parenteral administration to
mouse (78% at 1 h and 38% at 8 h, respectively). Further preclinical studies are needed to
evaluate the most suited mode of administration with the aim of promoting a first centralꢀacting
ghrelin inverse agonist molecule to development, which would represent a significant step
towards therapeutic agents to treat metabolic disorders related to obesity, such as typeꢀ2 diabetes
mellitus.
INTRODUCTION
The growth hormone secretagogue receptor (GHSR, Uniprot ID Q92847) is a sevenꢀ
1
transmembrane Gꢀprotein coupled receptor (GPCR) discovered in 1996. While mainly found in
the pituitary gland, GHSR is widely distributed across tissues, including pancreas and regions of
2
the brain linked with energy homeostasis and feeding. A remarkable quality of GHSR is its
ability to adopt an active conformation without the need of an agonistic ligand. Accordingly, a
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
high constitutive activity can be measured on the receptor, autonomous from any ligand binding,
3
of about half of its maximum signaling activity.
Ghrelin was first described in 1999. This hormone is a 28ꢀresidue peptide, whose serine at
position 3 requires an octanoylation for ghrelin to develop as the effective endogenous agonistic
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
4
ligand of GHSR. The first five residues at the Nꢀterminus of the peptide (sequence: FLSPE)
5
mostly explains molecular recognition by and activation of GHSR. Ghrelin, sometime called the
‘hunger hormone’, is essentially implicated in the control of food intake and energy
6
homeostasis. It is secreted by endocrine cells in the stomach during fasting and prior to meals,
7
,8
while circulating concentrations are lowered by feeding or glucose intake. Ghrelin was
7
,9
described to foster food intake and adiposity both in rodent and human. When administrated to
10,11
human or rodent, ghrelin decreases insulin release and leads to insulin resistance.
Also
ghrelinꢀdeficient mice were observed to secret more insulin with higher protection against
1
1
glucose intolerance induced by fat diet. In the liver, ghrelin promotes the expression of specific
1
2
gluconeogenic enzymes and thus downꢀregulates insulin signaling. Finally, ghrelin induces
13,14
enzymatic lipogenic activity and inhibits lipid oxidation in white adipose tissue.
In principle, the ghrelin pathway offers great therapeutic opportunities to treat a broad
spectrum of metabolic disorders. In particular, the high constitutive activity of the receptor lays
the biological rational for an investigation of ghrelin receptor inverse agonists (GHSRꢀIA) with
anticipated higher in vivo efficacy than pure antagonists against obesityꢀrelated metabolic
1
5
diseases.
In 2003, Holst et al. described an engineered substance P with potent inverse ghrelin effect,
3
16
but the first small molecule GHSRꢀIA series was reported by Merck in 2009. Later other
1
7ꢀ22
groups from industry and academia published GHSRꢀIA of diverse chemotypes.
As shown in
ACS Paragon Plus Environment
4

Page 5 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 1, these molecules tend to be large (MW > 500 g/mol), positively charged and to bear
2
3
numerous polar moieties. The fairly extended topological polar surface areas (TPSA) reflect
the difficulty for these compounds to permeate through the bloodꢀbrain barrier (BBB). Moreover
all compounds except 1 are predicted substrates of the Pꢀglycoprotein 1 (Pꢀgp) efflux pump by a
support vector machine (SVM) classification model available through the swissADME web
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
24
tool. In fact, only compounds 3, 6 and 7 have been measured in sufficient concentration in the
brain when administrated at high dose in rodent to make them interesting preclinical central
agents.
Some physiological effects of ghrelin and other GHSR agonists were shown to be peripherally
2
5
mediated, however, the transport through BBB proven for the acetylated ghrelin together with
26ꢀ28
the high level of GHSR expression in the brain argue for significant CNS role of ghrelin.
By
comparing in vivo effect of compound 7 and a brain nonꢀpermeant (more polar) analog of the
2
same series (MW: 549.02 g/mol; TPSA: 110.81 Å ), it was concluded that a peripheral inverse
agonist can achieve reduction of food intake but weightꢀloss requires a CNS penetrant GHSRꢀ
22
29
IA. Recently, Abegg et al. performed a battery of in vitro, in cellulo and in vivo experiments
to investigate thoroughly two undisclosed GHSRꢀIA molecules. At cellular level,
immunohistochemical studies established a clear relationship between ghrelin inverse activity
and reduced cꢀFos response in mouse brain neurons after oral or subcutaneous administration.
2
9
Further in the same article, the complete beneficial impact of GHSRꢀIA on energy balance
and glucose homeostasis was confirmed by two different animal models.
All GHSRꢀIAs disclosed so far do not display sufficient access to the CNS to be promoted as
centrally acting experimental drugs. In fact, the only ghrelin antagonist/inverse agonist, which
has completed preclinical investigations to enter clinical phases is compound 4, a Pfizer nonꢀ
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
30
CNS investigational drug tagged PFꢀ05190457, whose pharmacokinetic optimization rational
1
9
was said to target peripheral GHSR. PFꢀ05190457 was shown to increase glucoseꢀstimulated
insulin secretion in human isolated pancreatic islets and thus is anticipated in the diabetes
3
1
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
mellitus therapeutic area. Recently, a study in healthy individuals reported high tolerability and
pharmacokinetic data compatible with oral administration of PFꢀ05190457 but also unexpected
(
reversible) pharmacological effects, i.e. tachycardia and somnolence. While the former can be
attributed to a probable peripheral effect, the latter requires further investigation to be linked to
32
the brain impairment of PFꢀ05190457.
In the present publication, we describe the structureꢀbased rational design, synthesis details and
pharmacological characterization of a novel GHSRꢀIA chemotype. The most promising
compound 47 shows significant brain exposure and may represent an important step towards
therapeutic agents to treat metabolic disorders related to obesity, such as typeꢀ2 diabetes
mellitus.
Figure 1. Representative structures of chemical series reported as ghrelin inverse agonists
ACS Paragon Plus Environment
6

Page 7 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
GHSRꢀIA); in brackets are the name of the company, the seminal reference, and when found, an
2
4
identifier. Parameters calculated with the SwissADME web tool; MW: molecular weight;
3
3
WLOGP: inꢀhouse implementation of Wildman and Crippen log P; TPSA: topological surface
2
3
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
area.
RESULTS AND DISCUSSION
Our very first step in crafting a novel chemotype able to inversely agonize GHSR was to
compare the structure of the most advanced GHSRꢀIA, i.e. compound 4 (PFꢀ05190457, Figure
1
9
34
2
) with bioactive modulators of GHSR from Helsinn’s proprietary collection. Compound 8
(H0700, Figure 2) was selected based on its potent purely competitive antagonist activity in
FLIPR assay and on its favorable properties for absorption, distribution, metabolism and
excretion (ADME) and for pharmacokinetic (PK) profile.
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Competitive GHSR antagonist (8, H0700) with favorable ADME and PK profile and
stateꢀofꢀtheꢀart GHSRꢀIA (4, PFꢀ5190457) used as template structures for the chimeric design.
IC (FLIPR) measures antagonism, EC (FLIPR) measures agonism, EC (inositol phosphate
5
0
50
50
accumulation assay, IPꢀ1) measures inverse agonism. CL is for clearance, B/P ratio for
brain/plasm concentration ratio and PPB for plasma protein binding (refer to Experimental
Section for details).
Generation of structure-based hypothesis. Superimposition of 4 and 8 was achieved by
predicting their most probable binding mode in a theoretical tridimensional structure of human
GHSR retrieved from the ModBase repository (https://modbase.compbio.ucsf.edu/). In silico
35
docking was performed with the Attracting Cavities (AC) method , which not only was proven
a highꢀperforming and broadly applicable docking engine but also allows for flexibility of the
ACS Paragon Plus Environment
8

Page 9 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
binding site residues, thus potentially fixing structural uncertainties imputable to homology
models. The inner channel of GHSR to study was defined as an extended search volume
encompassed in a rectangular parallelepiped of 24 x 20 x 33 Å, with Phe309 side chain as center
and the longer side of the box in the main orientation of the receptor (refer the Experimental
Section for more details).
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
As depicted in Figure 3A, our computer modeling investigation predicted the acidic residue
Glu124 as chief anchor point that drives the recognition of 4 and 8 by GHSR. The saltꢀbridge
counterparts in both ligands are basic nitrogen atoms allowing highly stabilizing ionic
interactions. This is in accordance with mutagenesis studies evidencing the critical importance of
Glu124 to generate an agonistic response by recognizing ghrelin or other ligands, without
36,37
impacting the ligandꢀindependent activity of GHSR.
Furthermore, Glu124 is involved in
ionic interactions with a variety of positively charged peptide and nonꢀpeptide GHSR
3
8,39
modulators.
This pharmacophoric basic moiety is located differently in compounds 4 and 8.
Indeed, the methylpiperidine is at one end of molecule 8, whereas the spiroazetidine is central in
the structure of 4. Little rotational adaption of Glu124 side chain is observed upon binding
(Figure 3B), making both basic nitrogens not exactly superimposed in the binding site. More
drastic conformational changes are required for GHSR to accommodate the large dihydroindene‐
methylpyrimidine terminal moiety of 4. An additional cavity is made accessible deeper inside the
receptor structure (in translucent pink in Figure 3A) mainly through motions of Trp276, Phe279,
Phe312 and Phe309 bulky side chains (refer to Figure 3B). Thanks to these conformational
rearrangements, and other more modest geometrical changes, 4 can make favorable interactions
with amino acids deeper inside GHSR, either aromatic or hydrogenꢀbonding. The most
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
remarkable interactions include the indane moiety in sandwich piꢀstacking between Phe279 and
Phe312, as well as the methylpyrimidine making aromatic interaction with Trp276, with both
nitrogen heteroatoms accepting hydrogenꢀbond from Thr127 and Ser315, respectively. Phe279,
4
0
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
Phe312 and Phe309 are part of the aromatic cluster, which was described by Holst et al. as an
41
important motif for the binding of (agonists or antagonists) substanceꢀP analogues on GHSR.
Further supporting our predictive computer model, the opening of the deeper additional cavity is
4
2
reported as possible binding mode of carboxyamidated wFwLL peptides. This study strongly
suggests that ligands binding deep in the GHSR cavity show inverse agonist effect by preventing
the motion of extracellular part of two transmembrane domains (TMꢀVI and VII, bearing the
aromatic cluster), which is mandatory for activation. On the contrary, agonistic ligands are
expected to show a superficial binding mode. The chemical series from which H0700 (molecule
4
3
8
) belongs comprises many GHSR agonists with very similar chemical structure. It is thus
inferred that compounds of this chemotype have very similar binding modes, regardless being
agonist or antagonist.
ACS Paragon Plus Environment
10

Page 11 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Predicted binding modes of compounds 4 (inverse agonist, PFꢀ05190457, carbon
atoms in magenta) and 8 (antagonist, H0700, carbon atom in turquoise) in GHSR (white cartoon,
extracellular compartment on the top). Nonꢀpolar hydrogens are hidden for clarity. (A) General
view with the major anchor point Glu124 making ionic interactions (dashed green lines) with the
basic nitrogen of each molecule. An additional deeper cavity (in translucent pink) is accessible to
accommodate the large dihydroindene‐methylpyrimidine terminal moiety of 4. (B) Zoom into the
binding site showing significant motions of key residues upon docking of 4, which open the
deeper cavity and/or make additional intermolecular interactions (polar in dashed green lines, or
aromatic). Carbon atoms of the ligand (ballsꢀandꢀsticks) and of the residues (thin sticks) are of
the same color for a given calculated binding mode.
.
Three families of chimeric compounds. The robust structural hypothesis generated by the in
situ superimposition of a central GHSR antagonist (8) with a peripheral GHSRꢀIA (4) enabled
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the design of chimeric molecules. Our aim is the discovery a novel GHSR modulator chemotype
displaying different pharmacokinetic and pharmacodynamic properties compared to the stateꢀofꢀ
theꢀart.
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
Three families of chimeric structures were designed as depicted in Figure 4 and evaluated for
their GHSRꢀIA potential. The “spiro” molecules (9 and 10 in Table 1) were designed by merging
the
whole
leftꢀhand
2‐[5‐(6‐methylpyrimidin‐4‐yl)‐2,3‐dihydro‐1H‐inden‐1‐yl]‐2,7‐
diazaspiro[3.5]nonane‐7‐carbaldehyde part from 4 with the urea function of 8; the “piperidine”
molecules (11 and 12 in Table 1) are resulting from connecting the leftꢀhand 4‐(2,3‐dihydro‐1H‐
inden‐5‐yl)‐6‐methylpyrimidine end from 4 directly on the piperidine of 8; and the
“imidazothiazol” (13 and 14 in Table 1, for challenging the “inverseꢀagonistꢀdeeperꢀbinding”
hypothesis) simply replaces the rightꢀhand aromatic rings of 8 by the methylimidazothiazol
system of 4.
ACS Paragon Plus Environment
12

Page 13 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 4. Chimeric design from template molecules 4 and 8 to generate “spiro”, “piperidine” and
“imidothiazol” families of structures. The parts of the crossover structures are boxed in colors
corresponding to the molecule they are originated from (pink for the inverse agonist and
turquoise for the antagonist; grey is for overlay regions).
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 1. Evaluation of three families of chimeric molecules as GHSR modulators
FLIPR
FLIPR IC50
(nM)
c,d
c,d
IPꢀ1 EC50 (nM)
a
a
a
EC (nM)
50
Cmpd
family
R1
R2
inverse agonism
agonism
> 30000
> 30000
> 30000
antagonism
1210
N
N
9
spiro
Me
Me
Me
483
1
1
0
1
spiro
H
H
4010
inactive
194
N
N
piperidine
369
1
1
2
3
4
piperidine
Me
Me
H
> 30000
> 30000
> 30000
2330
inactive
inactive
inactive
b
imidazothiazol
n/a
n/a
> 30000
> 30000
1
imidazothiazol
a
b
c
refer to Figure 4; not applicable; mean of at least two independent measurements done in
d
triplicates; SEM was less than 25% for all values.
None of the molecules tested in FLIPR assay (9ꢀ14) displayed agonistic activity. Both
compounds belonging to the “imidazothiazol” family (13 and 14) were found inactive in all three
FLIPR and IPꢀ1 assays. This result verifies an early SAR observation made from Helsinn’s
asymmetric urea scaffold, which suggested that GHSR modulation requires a benzylic moiety on
3
3,43
the rightꢀhand side of the ligand structure.
Accordingly, all molecules of the “spiro” (9 and
1
0) and of the “piperidine” (11 and 12) families were found bioactive on the GHSR. In both
families, the pyrazine substitution in R1 increased antagonism potency in FLIPR by a factor of 3
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 6 for the “spiro” and “piperidine” compounds, respectively. Nevertheless, unsubstituted
compounds in R1, 10 and 12, retained weak GHSR antagonistic activity with IC of 4.0 µM and
5
0
3
4
2.3 µM, respectively. This is in agreement with previous SAR. Most gratifyingly, the pyrazine
substituted compounds displayed subꢀmicromolar GHSR inverse agonism activity in IPꢀ1 assay,
with EC50 of 483 nM and 194 nM for 9 and 11 respectively. Because both “piperidine”
compounds showed higher potency, this family of chimeric structures was selected as a
molecular scaffold for further optimization. Chemical variations were first undertaken on the pꢀ
substitution of the dichlorobenzyl rightꢀhand part (R1 in Tables 1 and 2).
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 2. Exploration of right-hand dichlorobenzyl p-substitution of the “piperidine”
scaffold: potency and metabolic stability.
Clearance
dog
microsom
e
(µL/min/
mg)
FLIPR
Clearance
human
Clearance
mouse
IPꢀ1 EC
IC50
50
b,c
Clearance rat
b,c
(nM)
(nM)
Cmpd R1
microsome microsome microsome
(µL/min/mg (µL/min/mg (µL/min/mg)
)
inverse
agonism
antagonis
m
)
N
N
a
11
369
171
194
67
n/d
n/d
n/d
n/d
1
5
OMe
94.2
122
69.5
241.1
1
1
6
7
COOMe
COOH
303
96
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
> 30000
inactive
18
NH
2
571
121
n/d
n/d
n/d
n/d
ACS Paragon Plus Environment
14

Page 15 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
2
9
0
ethynyl
156
10.6
298
165.1
n/d
157.2
n/d
65.6
n/d
136.9
n/d
cyclopropyl
2870
2
1
80
51
53.9
42.6
19.2
62.3
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
a
b
c
not determined; mean of at least two independent measurements done in triplicate; SEM was
less than 25% for all values.
Chemical explorations on the “piperidine” family. Table 2 shows the potency and (for
selected ones) the metabolic stability of compounds featuring eight different pꢀsubstituents of the
dichlorophenyl moiety instead of the pyrazine as in 11. The only fully inactive molecule was the
benzoic acid derivative (17), whereas its methylbenzoate analog (16) showed increased potency
in both antagonism and inverse agonism assays compared to 11. This indicates that a negative
charge at that position is not tolerated, contrarily to a polar group. A decrease of activity on
GHSR compared to 11 is observed for the cyclopropyl substituted molecule (20), mainly in
FLIPR, but also in IPꢀ1. The aniline analog (18) turned out to be a slightly more potent inverse
agonist, but with less antagonistic behavior than 11. The most significant increases of inverse
agonism activity were measured for the dichlorobenzyl pꢀmethoxy (15) pꢀethynylcyclopropane
(
21) and pꢀethynyl analogues (19). As a first evaluation for metabolic stability of the latter three
molecules, clearance was measured in liver microsomes from different sources (human, mouse,
rat and dog). Remarkably, the most hydrophobic ethynylcyclopropane substituent led to
molecule 21, which is clearly more stable in all four species compared to 15 and 19. This is
fundamental for further preclinical evaluations and offers good prospects for BBB permeation
ability, with the aim of obtaining a centrally acting GHSRꢀIA. Besides, 21 showed a potent
GHSR antagonism in FLIPR assay. According to these initial results, compound 21 represents
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
already an interesting candidate to be promoted for further preclinical investigation. However,
for the sake of possible physicochemical optimization towards drugꢀlikeness, further
modifications on the “piperidine” family were performed to broaden the chemical space
explored.
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 3 shows the exploration on the benzylic position at the rightꢀhand side of the
“piperidine” scaffold (R2) together with some variations on the pꢀsubstituents of the
dichlorobenzyl (R1), based on previous results (as in Tables 2).
Table 3. Exploration of right-hand benzylic position of the “piperidine” family.
FLIPR
IC50
a,b
a,b
IPꢀ1 EC50 (nM)
inverse agonism
(nM)
Cmpd
R1
H
R2
H
antagonism
2700
2
2
2
3
inactive
inactive
H
1130
2
2
4
5
ethyl
192
306
N
N
CF
8080
2164
3
ACS Paragon Plus Environment
16

Page 17 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
N
N
2
6
4540
484
970
27
H
5400
1800
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
2
8
OMe
439
a
b
mean of at least two independent measurements done in triplicates; SEM was less than 25% for
all values.
An unsubstituted benzyl position (R2) annihilates inverse agonism of “piperidine” molecules
with the dichlorobenzyl para position (R1) substituted (23) or not (22). Also, comparison with
methylated counterparts (21 et 12, respectively) shows that the antagonism effect of 23 and 22 is
lower. Following previous SAR, we selected a small set of wellꢀtolerated alkyl substituents that
34
retained antagonism when placed in the benzylic position in a previous series. Replacing
methyl (21) by ethyl (24) at the benzylic position decreased the inverse agonism potency 6ꢀfold
and the antagonism potency 2.5ꢀfold. The decrease was even more important when
trifluoromethyl or a bulky group such as cyclopropylmethylene is at the benzylic position (see
2
5, 26 compared to 11, and 28 compared to 15). As a consequence, in this chemical context, a
methylated benzylic position appears optimal for GHSR modulation and thus no further
explorations nor analyses regarding this position were pursued.
Attention was then given to the leftꢀhand side of the “piperidine” chimeric structures. Table 4
shows the SAR of fourteen derivatives focusing on the leftꢀhand indane substitution in position 5
(R3) together with some variations on the pꢀsubstituents of the rightꢀhand dichlorobenzyl (R1),
based on previous results (as in Tables 2 and 3).
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Exploration of left-hand indane substitution in position 5 of the “piperidine”
scaffold: potency and metabolic stability.
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
Clearance
rat
microsome
FLIPR IC
IPꢀ1 EC50
(nM)
inverse
Clearance
human
microsome
(µL/min/mg)
Clearance
mouse
microsome
(µL/min/mg)
Clearance
dog
microsome
(µL/min/mg)
5
0
b,c
b,c
(nM)
Cmpd
R1
R3
(µL/min/mg
antagonism agonism
)
N
a
29
30
798
85.9
282
919
634
84.8
117
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
N
N
n/d
n/d
N
3
1
Me
H
3
3
2
3
inactive
73
n/d
n/d
n/d
n/d
OMe
Me
Cl
329
315
80.3
216.8
143.1
168.5
3
3
4
5
OMe
OMe
OMe
OMe
40.7
241.2
n/d
387.5
n/d
270.8
n/d
n/d
n/d
n/d
n/d
OMe 120
85.7
36
37
OH
NH
1300
inactive
inactive
87.1
47.1
117.3
70.4
n/d
1510
n/d
2
N
N
N
38
OMe
240
70
79.8
244
n/d
n/d
ACS Paragon Plus Environment
18

Page 19 of 75
Journal of Medicinal Chemistry
1
2
3
3
4
4
9
0
1
OH
Me
Me
1166
106
189
n/d
n/d
n/d
nd
n/d
n/d
n/d
n/d
n/d
n/d
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
etynyl
inactive
inactive
n/d
n/d
cyclopropyl Me
2367
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
42
Me
2005
inactive
n/d
n/d
n/d
n/d
Si
a
b
c
not determined ; mean of at least two independent measurements done in triplicates; SEM was
less than 25% for all values.
Exchanging 6‐methylpyrimidine (21 in Table 2) in position 5 of the dihydroindene by
pyrimidine reduced the antagonism and the inverse agonism effects to very different extent with
respect to isomers (refer to 29 and 30 in Table 4). This indicates the sensitivity of this
pharmacophoric zone, in agreement with the localized hydrogenꢀbonding interactions involving
both azaheteroatoms of 4 with Thr127 and Ser315 in the deeper cavity of GHSR and
hypothesized related to inverse agonism (refer to Figure 1B).
Whereas a nonꢀsubstituted dihydroindene at position 5 annihilates inverse agonism (32), a
methyl substitution (31) leaves some hope of significantly reducing the size of the molecule
(
potentially more druglike and BBB permeant) yet keeping the inverse agonistic capability. This
was partly achieved by 33, bearing a paraꢀmethoxy on the dichlorophenyl (EC of 73 nM in IPꢀ
5
0
1). However, 33 was unstable in liver microsomes, especially in rodents and dog, which could
hamper further development. Unfortunately, other 5ꢀmethyldihydroindene analogues with
variation in para of dichlorophenyl did not show inverse agonism activity on GHSR (39, 40, 41,
4
2). All other attempts of including diverse small substituents in position 5 of the dihydroindene
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
while
keeping pꢀmethoxydichlorophenyl on the rightꢀhand side failed at producing
metabolically stable and potent GHSRꢀIA (34, 35, 36, 37, 38).
Finally, Table 5 lists the result of a very limited number of variations on the central
3
3,43
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
“piperidine” based on the experience gained in asymmetric urea GHSR effectors
. Compound
2
1 was modified by applying changes able to retain agonistic or antagonistic activity in previous
series.
Table 5. Chemical variation on the central “piperidine” core.
FLIPR
IC50
a,b
a,b
IPꢀ1 EC50 (nM)
inverse agonism
(nM)
Cmpd
core
antagonism
148
4
4
3
4
inactive
363
530
4
5
2920
937
a
b
mean of at least two independent measurements done in triplicates; SEM was less than 25%
for all values.
ACS Paragon Plus Environment
20

Page 21 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
While the pyrrolidine compound (43) did not show inverse agonism activity, other two
piperidine molecules (44, 45) confirmed the GHSR inverse agonism ability of this chemotype,
with antagonism effect as well. However, these changes on the piperidine core (i.e. gemꢀ
dimethyl 44 and ethyl urea 45) did not improve the potency, failing to decrease IC /EC below
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
5
0
50
1
00 nM neither in FLIPR nor IPꢀ1 assays. Consequently, they were not considered for further
exploration.
As a result of the presented SAR investigations, one of the first examples of the promising new
“piperidine” scaffold, i.e. the diastereomeric mixture 21, showed the best potential for further
investigation. Chiral HPLC enabled separation at the asymmetric center on the indane moiety of
21 (the benzylic asymmetric carbon is kept in S as a known determinant for GHSR activity in all
34,44
tested asymmetric urea series)
. As shown in Table 6, a drastic drop of antagonist and inverse
agonist potency can be observed for the Sꢀenantiomer (46), whereas the Rꢀenantiomer (47) show
an increase of activity in FLIPR and more significantly in IPꢀ1, compared to 21.
Table 6. Exploration of compound 21 chirality.
IPꢀ1
EC50
(nM)
inverse
agonism
Clearance
human
Clearance
mouse
Clearance
Clearance rat dog
FLIPR
IC50
b,c
Cmpd Chirality of *
b,c
microsomes microsomes microsomes microsomes
(µL/min/mg (µL/min/mg (µL/min/mg) (µL/min/mg
)
(nM)
)
)
antagonis
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
m
2
4
1
6
racemate
80
51
53.9
42.6
n/d
19.2
n/d
62.3
a
S
1276
1276
n/d
n/d
4
7
R
68.2
29.4
74.7
<10
57.8
52.2
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
b
c
not determined ; mean of at least two independent measurements done in triplicates; SEM was
less than 25% for all values.
Interestingly, the differences in in vitro metabolic stability for compounds 47 and 21 depended
very much on the species. Whereas the clearance in human and rat microsomes was higher for
47, the clearance in dog microsomes was measured lower. The most significant variation is seen
in mouse liver microsomes where 47 was very stable. This latter result is of crucial importance
for preclinical assessments in this species. Therefore, the most active stereoisomer of the most
promising compound (i.e. compound 47) was promoted for further evaluation as a GHSRꢀIA
potential preclinical candidate.
Pharmacodynamics and binding mode of 47. Molecule 47 (structure in Figure 5A) reached
high level of GHSR inverse agonism in IPꢀ1 assay (EC = 29.4 nM) as well as antagonism in
50
FLIPR test (IC = 68.2 nM). The binding mode of 47 in GHSR was predicted through molecular
5
0
docking following the exact same protocol as for the template molecules 4 and 8. Figure 5B
shows how inverse agonist 47 is suitably accommodated deep inside the GHSR cavity. Similarly
to modeled Pfizer’s molecules 4, residues Phe279, Phe312 and Phe309 moved in order to open
the deeper side pocket and to make aromatic interactions with the ligand. In the case of 47, an
extra aromatic interaction with Phe221 is predicted. Besides, the most favorable positioning
shows a rotated 4ꢀmethylpyrimidine ring compared to 4. This favors a distinct hydrogenꢀbonding
network involving both azaheteroatoms with Thr130 and Trp276, whereas the methyl substituent
ACS Paragon Plus Environment
22

Page 23 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
makes purely LennardꢀJones contacts with the backbone of TM2. Other hydrophobic interactions
can be observed with the side chains of Cys126 and Ile92. Apart from the chief anchoring saltꢀ
bridge between the basic nitrogen and Glu124, the piperidine moiety is in contact with Gln120
and Arg283 side chains. The latter are two important residues for GHSR intrinsic activity, and
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
42
for the binding of agonistic and inverse agonistic ligands as well. As described for the binding
3
7
of various ghrelin receptor agonists, the arginine is predicted to make not very specific
intermolecular interactions, but an extended part of 47 ureaꢀpiperidine central core is “leaned” on
the side chain. This extended contact area by itself may explain the importance of Arg283 for the
binding of such molecular structures. Gln120 is predicted to point towards both chlorine atoms,
which may indicate some electronic interplay in that region of the binding site. The
dichlorophenyl ring is principally involved in a cationꢀpi interaction with the guanidine of
Arg199, which was found as an essential residue for the effect of pioneer growth hormone
3
,45
secretagogue Lꢀ692,429, for instance.
Another important amino acid for ligand binding,
3
7
Phe286,
is predicted in aromatic interaction with dichlorophenyl moiety. The pꢀ
ethynylcyclopropane substituent is in hydrophobic contacts with Arg102 and Leu103. The
cyclopropane ring can be part of more specific interactions with Tyr106 side chain, possibly with
the aromatic and/or as a weak hydrogenꢀbond acceptor.
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Potential preclinical candidate GHSRꢀIA, compound 47. (A) Chemical structure and
parameters (physicochemical, PD and PK). (B) Predicted binding modes (ballsꢀandꢀsticks) in
GHSR (white cartoon, and interacting residues in thin sticks). All carbon atoms are in orange,
while nonꢀpolar hydrogens are hidden for clarity. The model shows how 47 is saltꢀbridged to
Glu124 (dashed green line) and how it interacts specifically with residues deep in the cavity, e.g.
accepting hydrogenꢀbond from Thr130 and Trp276 (dashed green lines) or making aromatic
interactions with Phe221, Phe279, Phe309 and Phe312. Other important interactions to
rationalize binding include Arg199, Arg283 and Phe286. Finally, the ligand is in contact with
Ile92, Arg102, Leu103 Gly120 and Cys126. (C) The Bioavailability Radar generated with
2
4
SwissADME shows suitable polarity, flexibility and saturation for 47 but suboptimal
lipophilicity, size and solubility, which can explain moderate oral bioavailability in rat (F =
2
7.2%).
ACS Paragon Plus Environment
24

Page 25 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ADME and pharmacokinetic parameters of 47. Molecule 47 was found metabolically stable
in vitro showing a very low intrinsic clearance in mouse liver microsomes. In microsomes from
other species (human, rat and dog), the intrinsic clearance was higher, albeit inferior compared to
the less lipophilic compounds of the same structural class (for racemate 21 vs. 15 and 19, refer to
Table 2). Despite the relative liability in vitro in rat liver microsomes, compound 47, after
intravenous administration to rat (3 mg/kg), showed low plasma clearance (0.91 L/h/kg), long
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
halfꢀlife (t1/2 = 6.49 h), large volume of distribution (V = 8.48 L/kg) and remarkable brain
d
penetration with a brain/plasma (B/P) ratio of 1.86 (at 2h). After oral administration (3 mg/kg),
compound 47 showed moderate bioavailability (F = 27%), a long absorption phase (Tmax = 6.7 h)
and longer halfꢀlife (t1/2 = 15.85 h) than after intravenous administration.
The main parameter affecting bioavailability of 47 is certainly the poor permeability as
determined in MadinꢀDarby Canine kidney cells expressing multidrug resistance protein 1
(MDR1ꢀMDCK). Apparent permeability of 47 (when inhibiting MDR1 with ciclosporine) was
ꢀ6
measured as low as PA→B = 2.4x10 cm/s. The Bioavailability Radar of molecule 47 generated
2
4
through the SwissADME Web tool and plotted in Figure 5C indicates size (602.60 g/mol), high
4
6
lipophilicity (WLOGP = 6.36) and poor solubility (predicted log S = ꢀ7.75) as the main
properties responsible for the moderate oral bioavailability. Unfortunately, all attempts made to
correct these suboptimal properties failed at producing “piperidine” core molecules with potent
GHSR inverse agonistic activity. Moreover, 47 was determined highly bound to plasma protein
(99.99% in human, rat and dog and 99.98% in mouse). However, a big and lipophilic molecule
like 47 was found metabolically stable in vivo and notably in significant CNS concentration in
2
rat’s brain probably thanks to its moderate apparent polarity (TPSA 61.36 Å ) although active
2
4
transport into the brain cannot be excluded. Finally, 47 was predicted (by SwissADME ) and
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
afterward experimentally confirmed in MDR1ꢀMDCK cells not to be a substrate of the Pꢀgp
ꢀ6
efflux pump (PA→B = 3.9x10 cm/s, when MDR1 not inhibited). These results rewarded our
effort in designing a central GHSRꢀIA and thus compound 47 entered first assessments for
reduction of food intake in rodents.
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
Pharmacological characterization of 47. Preliminary in vivo efficacy was encouraging. A
first experiment was designed to evaluate the ghrelin inverse agonist activity on an animal
model. To this aim, we have measured the ability of molecule 47 to counteract the increase of
food intake induced by a ghrelin agonist, i.e. anamorelin. Anamorelin was used as a reference
standard GHSR agonist compound, for which the ability to increase food intake and body weight
4
7
has been well characterized in rodent models, as well as in human. Fed mice were given 47
either 10 mg/kg i.p. (n=6) or 30 mg/kg p.o. (n=6), and 10 min later, anamorelin 30 mg/kg p.o.
The total amount of food intake measured at 1, 2, 4, 8 and 24 h. after dosing are shown in Table
7.
Table 7. Increase of food intake in mice counterbalanced by compound 47.
VEH+ANA+Cmpd 47
a
VEH+ANA+Cmpd 47
T
(
b
c
VEH
VEH+ANA
h)
(10 mg/kg i.p.)
(30 mg/kg p.o.)
1
2
0.24 ± 0.02
.45 ± 0.03
0.63 ± 0.04
1.37 ± 0.05
0.13 ± 0.02*
0.56 ± 0.03*
0.22 ± 0.02*
0.86 ± 0.20
0
4
8
0.79 ± 0.02
1.38 ± 0.05
7.23 ± 0.3
2.09 ± 0.08
2.97 ± 0.09
8.31 ± 0.2
1.66 ± 0.08
2.78 ± 0.05
7.63 ± 0.8
1.45 ± 0.15
1.80 ± 0.08*
7.42 ± 0.5
2
4
ACS Paragon Plus Environment
26

Page 27 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
b
Cumulative food intake (in g) as the mean values ± SEM (n=6); time after administration; VEH:
c
vehicle; ANA: anamorelin (30 mg/kg p.o.); *p<0.05 vs. VEH+ANA (ANOVA plus Dunnett t
test)
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
Compound 47 inhibited food intake, induced by anamorelin, by 38.2% at 8 h. after 10 mg/kg
intraperitoneal administration with activity observed up to 8 hours. Moreover, inhibition of food
intake reached 78.2% at 1 hour after 30 mg/kg oral administration with activity observed up to 2
hours. Although interspecies translation regarding the brain penetration of compound 47 cannot
be proven at this stage, these in vivo results represent an additional strong indication of this
chemotype potential for central action on obesityꢀrelated metabolic disease. Additional
preclinical evaluation experiments are required for confirmation.
Furthermore, in vitro inhibition of hERG was measured at IC =3.16 µM. Whereas this
5
0
preliminary evaluation should not be considered as a severe warning, cardiovascular safety
issues cannot be ruled out. Further experimental assessments on offꢀtarget activities are
necessary at later preclinical stage, first in vitro on other ion channels and generally on a diverse
panel of biotargets. Final evaluation will require in vivo monitoring of cardiovascular parameters
in different animal species, when the dose can be anticipated.
SAR, optimization of physicochemical properties and initial in vivo data allowed to identify 47
as a suitable candidate for further preclinical investigations as centrally acting GHSRꢀIA.
Synthesis. Chemical syntheses of various intermediates and the corresponding derivatives are
depicted in Schemes 1ꢀ8.
Heterocyclic substituted 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀones were
4
8
synthesized starting from 5ꢀbromoꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone by Suzuki coupling (9-6, 29-1,
2
3
0-1) or Buchwald amination (38-1) (refer to Scheme 1). tertꢀButyl (1ꢀoxoꢀ2,3ꢀdihydroꢀ1Hꢀ
indenꢀ5ꢀyl)carbamate (37-5) was prepared by CO insertion reaction followed by hydrolysis and
4
9
Curtis rearrangement with DPPA/tꢀBuOH .
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 75
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. Synthesis of substituted 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀone derivatives
O
O
O
a
b or c
O
B
R
R =
N
, 9-6
N
N
9
-5
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
,
29-1
O
N
Br
N
N
d
O
N
N
N
,
30-1
38-1
O
O
O
O
O
O
g
e
f
BocHN
HO
3
7-3
37-4
37-5
Reagents and conditions: (a) Pd(PPh )Cl , 4 ,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane), K OAc, dioxane, 29%;
3
2
(
b) Pd(PPh ) , 4-chloro-6-methylpyrimidine or 5-bromopyrimidine, Na CO , dioxane/H O, reflux, overnight, 64% for 9-5 and
3
4
2
3
2
o
4
7.6% for 30-1; (c) Pd(dppf)Cl , Na CO , DME, 95 C, overnight, 41% for 29-1; (d) triazole, Fe(acac) , CuO, K CO , DMF,
2
2
3
3
2
3
o
80oC, overnight, 21%; (e) Pd(dppf) Cl , CO, TEA, MeOH/DMF, 80 C, overnight, 92%; (f) LiOH.H O, MeOH/H O, rt, 5h, 89%;
2
2
2
2
o
(g) DPPA/TEA, t-BuOH, 90 C, overnight, 32%.
As shown in Scheme 2, intermediates of substituted Nꢀmethylꢀpiperidinꢀ4ꢀamine derivatives
(15-4, 33-2, 34-3, 36-2) were prepared by reductive amination of 2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀones
with benzyl methyl(piperidinꢀ4ꢀyl)carbamate (15-2) followed by removal of Cbz group with
5
0
TMSI . The hydroxyl substituted intermediate (36-3) was prepared from demethylation of
methoxy substituted intermediate (36-2) in the presence of BBr and DCM.
3
Scheme 2. Synthesis of substituted Nꢀmethylꢀpiperidinꢀ4ꢀamine derivatives
ACS Paragon Plus Environment
28

Page 29 of 75
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Starting from the iodide 8-1, several substituted benzylic amines were prepared by Stille
coupling (9-4), Suzuki coupling (20-2) or Sonogashira coupling (21-2) followed by removal of
Boc group in the presence of acidic conditions (as in Scheme 3). The methoxy substituted
benzylic amine (15-9) was obtained by chiral separation of the corresponding racemate (15-8),
4
4
which was prepared from 2,3ꢀdichlorophenol in three steps .
Scheme 3. Synthesis of substituted benzylic amine derivatives
ACS Paragon Plus Environment
29