Identification and Pharmacological Profile of an Indane Based Series of Ghrelin Receptor Full Agonists

Article abstract of DOI:10.1021/acs.jmedchem.8b00322

Cachexia and muscle wasting are very common among patients suffering from cancer, chronic obstructive pulmonary disease, and other chronic diseases. Ghrelin stimulates growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGF-1 plasma levels. The activation of the GH/IGF-1 axis leads to an increase of muscle mass and functional capacity. Ghrelin further acts on inflammation, appetite, and adipogenesis and for this reason was considered an important target to address catabolic conditions. We report the synthesis and properties of an indane based series of ghrelin receptor full agonists; they have been shown to generate a sustained increase of IGF-1 levels in dog and have been thoroughly investigated with respect to their functional activity.

Full text of DOI:10.1021/acs.jmedchem.8b00322

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Article
Identification and pharmacological profile of an
indane based series of ghrelin receptor full agonists
Cristina Gardelli, Hiroki Wada, Asim Ray, Moya Caffrey, Antonio Llinas, Igor Shamovsky, Joakim
Tholander, Joakim Larsson, Ulf Sivars, Leif Hultin, Ulf Andersson, Hitesh J. Sanganee, Kristina
Stenvall, Brith Leidvik, Karin Gedda, Lisa Jinton, Marie Ryden Landergren, and Kostas Karabelas
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00322 • Publication Date (Web): 17 Jun 2018
Downloaded from http://pubs.acs.org on June 18, 2018
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Identification and pharmacological profile of an
indane based series of ghrelin receptor full agonists
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#,*
#,†
#
#
ꢀ
Cristina Gardelli, Hiroki Wada, Asim Ray, Moya Caffrey, Antonio Llinas, Igor
#
ꢁ
,†
#,†
ꢂ
ꢃ
Shamovsky, Joakim Tholander,
Joakim Larsson,
Ulf Sivars, Leif Hultin, Ulf
ǂ
§
ǁ
¤,†
¤
Andersson, Hitesh J. Sanganee, Kristina Stenvall, Brith Leidvik, Karin Gedda, Lisa
ꢂ
#
ǁ,†
Jinton, Marie Rydén Landergren, Kostas Karabelas
#
ꢀ
ꢂ
ǁ
Medicinal Chemistry Department, DMPK Department, Bioscience Department, Projects
Department Respiratory, Inflammation and Autoimmunity IMED Biotech Unit, AstraZeneca
ꢁ
Gothenburg, 43183 Mölndal, Sweden; Medicinal Chemistry Department, Cardiovascular and
Metabolic Diseases IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden;
ꢃ
Precision Medicine Laboratories, Precision Medicine and Genomics IMED Biotech Unit,
ǂ
AstraZeneca Gothenburg, 43183 Mölndal, Sweden; Drug Safety and Metabolism IMED Biotech
§
Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden; Scientific Partnering & Alliances
¤
IMED Biotech Unit, AstraZeneca, SK10 4TF Cambridge, United Kingdom; Discovery Sciences
IMED Biotech Unit, AstraZeneca Gothenburg, 43183 Mölndal, Sweden.
ABSTRACT. Cachexia and muscle wasting are very common among patients suffering from
cancer, chronic obstructive pulmonary disease and other chronic diseases. Ghrelin stimulates
growth hormone secretion via the ghrelin receptor, which subsequently leads to increase of IGFꢀ
1 plasma levels. The activation of the GH/IGFꢀ1 axis leads to an increase of muscle mass and
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functional capacity. Ghrelin further acts on inflammation, appetite and adipogenesis, and for this
reason was considered an important target to address catabolic conditions. We report the
synthesis and properties of an indane based series of ghrelin receptor full agonists; they have
been shown to generate a sustained increase of IGFꢀ1 levels in dog and have been thoroughly
investigated with respect to their functional activity.
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INTRODUCTION
Ghrelin is a 28 amino acid peptidic hormone, characterized by a postꢀtranslational nꢀ
octanoylation on the Serine 3 residue. It is synthesized and secreted by X/Aꢀlike cells localized
1
within the oxyntic glands of the mucosa of the gastric fundus. It is highly conserved across
2
several species indicating its physiological importance. Its pharmacological properties are
mediated by the type 1a growth hormone secretagogue receptor (GHSꢀR1a), a seven
transmembrane Gꢀproteinꢀcoupled receptor (GPCR) expressed predominantly in the
hypothalamus and in the pituitary gland. Ghrelin exhibits several biological activities: it is an
orexigenic agent, stimulates secretion of growth hormone (GH, an anabolic hormone that
stimulates body growth), enhances gastric motility and acts on the cardiovascular system to
increase cardiac output. With this wide range of biological activities, the ghrelin receptor
represents an interesting target for drug discovery. We were particularly interested in the
capability of ghrelin to increase endogenous GH and to elevate the plasma level of a second
hormone called insulinꢀlike growth factorꢀ1 (IGFꢀ1); consequently, it could have exciting
applications in diseases where skeletal muscles are impaired as in cachexia which is associated
with many chronic diseases such as cancer, chronic obstructive pulmonary disease (COPD),
chronic heart failure, HIV and many others. The use of ghrelin to reduce cachexia and to increase
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functional capacity in cachectic patients with COPD has clinical precedence. First, Nagaya et al.
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demonstrated, in an openꢀlabel pilot study, that intravenous administration of human ghrelin for
three weeks to cachectic patients with COPD markedly increased growth hormone secretion,
food intake, body weight, lean body mass, peripheral and respiratory muscle strength, the
Karnofsky performance status score (a marker for functional capacity), as well as distance
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walked in six minutes. Later SUN11031, a synthetic human ghrelin form, was brought into a
Phase 2 study where 224 cachectic patients with COPD were treated to determine whether it
could enhance body weight, lean body mass and physical performance. Treatment resulted in a
rapid and significant increase in body weight and lean body mass although without significant
improvement in functional performance measures. However, subgroup analysis revealed a
statistically significant increase in functional performance in subjects with more advanced
cachexia. Studies in other chronic disease conditions showed reduced muscle wasting and
5
improved exercise capacity after ghrelin treatment in patients with chronic heart failure.
Employing exogenous ghrelin administration shows that the halfꢀlife of total ghrelin is between
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1
0 and 31 minutes in the mammalian bloodstream; therefore a number of programs were
dedicated to identifying long acting growth hormone secretagogues (GHS) with the potential to
7
ꢀ8
increase 24 hours IGFꢀ1 levels. A number of short acting GHSs have been previously
described as full agonists of the ghrelin receptor; a few of them have reached advanced clinical
9ꢀ15
stages for cancer cachexia, frailty in the elderly and gastrointestinal diseases.
A highꢀthroughput screening (HTS) campaign of AstraZeneca´s proprietary collection to
identify ghrelin agonists, followed by a hit to lead program, led to the discovery of a series of
indane diamides (compounds 1-3) with submicromolar potency (Figure 1, Table 1). This lead
series showed partial agonism expressed by the effect of the compounds as a percentage of
maximal effect of growth hormoneꢀreleasing peptide 6 (GHRPꢀ6). We then embarked on a lead
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optimization project to switch from partial to full agonism by structural alterations as previously
16,17
reported for other GPCRs.
Furthermore, we optimized against a poor selectivity of the
chemical series towards the hERG channel.
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Herein, we report our efforts and SAR analysis leading to the identification of compound 39,
showing the required pharmacological profile for in vivo studies to predict GH/IGFꢀ1 activity in
humans.
1
2
3
39
Figure 1. Initial leads in the indane series 1-3 and optimized compound 39.
ꢄ CHEMISTRY
Indane 1,2 diaminoamides 4, 6, 8-21, 34 and 51 were synthesized in seven steps in overall
yield of around 50% from the commercially available (1S, 2R) and (1R, 2R)ꢀaminoindanols
1
8
following Scheme 1. 1ꢀAminoꢀ2ꢀhydroxyindane 44 and 52 were Boc protected and an azide
group was inserted through mesylation and displacement with NaN to give intermediates 47 and
3
5
4. Hydrogenation with Pd/C provided amines 48 and 55 which were coupled with the diverse
carboxylic acids to provide intermediates 49 and 56. Boc deprotection with tꢀ
butyldimethylsilyltriflate and a second amide coupling provided the desired analogs 4, 6, 8-21,
3
4 and 51.
These overall sequences were also applied to provide enantiomers 5 and 7 starting from the
opposite enantiomers of 1ꢀaminoꢀ2ꢀhydroxyindanes.
a
Scheme 1. Synthesis of indane diamino derivatives 4, 6, 8-21, 34 and 51.
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4, 8-21, 34, 51
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a
Reagents and conditions: (a) Boc O, Et N, DCM, r.t., o/n; (b) MsCl, Et N, DCM, 0º to r.t., 3h;
2
3
3
(
c) NaN , dry DMF, 90ºC, o/n. (d) H , 10% Pd/C, o/n; (e) RCOOH or 5ꢀmethylꢀ1Hꢀindoleꢀ2ꢀ
3
2
carboxylic acid, DIPEA, HOBT, DCM, EDCI, r.t.; (f) TBDMSOTf, DCM, 6 h, r.t.; (g)
R COOH, HATU, Et N, DMF.
1
3
Compounds reported in Table 3 were obtained from compound 51 or analogs. As an example, 51
was submitted to Boc deprotection of the corresponding morpholine to give compound 32 and
methylated through reductive amination to give 23, Scheme 2. Homomorpholines, piperidine,
homopiperidine and pyrrolidine derivatives were treated similarly to yield compounds 22-33 and
3
5-38. Compound 34 was obtained by acetylation of 32 with acetic anhydride.
a
Scheme 2. Synthesis of compound 23 (as representative of the synthesis of compounds 22-33
and 35-38) and 34.
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R = Me, 23
R = Ac, 34
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Reagents and conditions: (a) TBDMSOTf, DCM, 6 h, r.t.; (b) HCHO, MeOH, NaBH CN; (c)
3
Ac O.
2
The reverse amide 39-43 were synthesized from the commercially available ketoester 58,
Scheme 3. Condensation of compound 58 with NH OAc gave enamine 59 that was reduced with
4
NaBH and TFA in THF (caution, exothermic reaction). Boc protection yielded beta amino
4
protected ester 60, in a cis:trans ratio of 1:1. The mixture, was submitted to hydrolysis to obtain
acid 61 as a cis:trans 1:6.3 ratio. After HPLC, the pure trans acid 62 was submitted to amide
coupling with diverse amines and Boc deprotected. HPLC separation gave the pure enantiomers
6
3a and 63b. The desired enantiomer 63b was then coupled with a variety of carboxylic acids to
give final compounds 39-43, Table 4.
a
Scheme 3. Synthesis of βꢀamino acids derivatives 39-43.
a
b,c
d
58
60, 1:1
trans:cis
5
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e
h,i
f,g
6
2, pure
trans
6
1, 6.3:1
trans:cis
39-43
6
3a and 63b
a
Reagents and conditions: (a) NH OAc, MeOH, 50°; (b) TFA, NaBH , THF; (c) Boc O; (d)
MeONa, H O, MeOH; (e) HPLC; (f) N ,N ꢀdimethylethaneꢀ1,2ꢀdiamine or NH R, HATU, Et N,
DMFꢀDCM; (g) HCl, dioxane; (h) chiral HPLC; (i) R COOH, HATU, Et N, DMFꢀDCM.
4
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Absolute configuration of key compound 39 and its enantiomer 40 was assigned by vibrational
circular dichroism (VCD) analyses in an indirect way. Direct VCD analysis of the final
compounds 39 and 40 gave inconclusive results and therefore smaller analog intermediates 65a
and 65b were prepared for VCD analyses, Scheme 4. The pure trans racemic ester 62 was
submitted to chiral separation to obtain the two enantiomers 62a and 62b. Further manipulation
of these two enantiomers led to N,N dimethyl amides 65a and 65b whose absolute configuration
was assigned by VCD. Once the absolute configuration of the analogs 65a and 65b was
assigned, the absolute configuration of the Boc protected acids 62a and 62b was revealed as
well. Compounds 39 and 40 were resynthesized starting from these intermediates and their
respective 1S,2S and 1R,2R absolute configuration was assigned. For VCD spectra see
Supplementary Material.
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Scheme 4. Synthesis of the intermediates 65a and 65b for the elucidation of the absolute
configuration of the indane compounds 39-40 through VCD.
a
6
2 pure
racemic trans
62a
64a
62b
b
c
6
2a
65a
65b
40
b
c
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64b
39
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Reagents and conditions: (a) chiral HPLC; (b) NHMe , DIPEA, HATU, PyBOP, dry DMF; (c)
2
HCl 4M in dioxane.
RESULTS AND DISCUSSION
The binding activity of the compounds to the GHSꢀR1a receptor was measured in a
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competition experiment with [ I]ꢀghrelin in membranes prepared from HEK293 cell line. The
agonistic activity of the compounds was assessed by measuring intracellular myoꢀInositol 1
phosphate (IP1). DꢀmyoꢀInositol 1ꢀphosphate as a surrogate of Dꢀmyoꢀinositol 1,4,5ꢀtriphosphate
serves to monitor G proteinꢀcoupled receptor activation. As a comparison, ghrelin, Capromorelin
and Ibutamoren data are reported as well.
The indane lead series showed pEC50 between 6.8 and 7.5 in the functional assay and a
moderate metabolic stability in human liver microsomes. The compounds were characterized by
a low agonism and a lack of human ERG selectivity. The initial medicinal chemistry work
focused on extending the chemical diversity of the series. It confirmed the required
stereochemistry in positions 1 and 2 of the indane core and assessed the characteristics of the
pharmacophoric features for further optimization.
N
O
N
O
N
O
N
O
N H
N H
N H
N H
H
N
H
N
H
N
H
N
O
N
H
O
N
H
O
N
H
O
N
H
4
5
6
7
Figure 2. Compounds 4-7 made to assess the desired stereochemistry.
Table 1. Potency in binding and functional assay, agonism and potency in hERG assay of
compounds 1-7, ghrelin, Capromorelin and Ibutamoren.
[
a]
[b]
Binding
Functional assay
hERG
pIC50
Entry
[d]
[
c]
pIC50 IC (ꢀM) pEC and agonism (%) EC (ꢀM)
50
50
50
1
6.4
0.43
6.8 (75)
0.160
5.8
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7.0
6.7
7.8
6.0
7.2
5.9
8.05
0.098
0.189
0.016
0.93
7.5 (52)
7.3 (61)
7.7 (54)
6.6 (40)
7.0 (84)
5.8 (101)
0.035
0.047
0.021
0.226
0.10
5.4
< 5.0
5.1
5.6
4.6
5.2
ꢀ
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0.069
1.26
1.5
3
7.7 (82%)
ghrelin
0.009
0.021
n= 40
9
9.04 (87%)
Capromorelin
7.7
8.7
0.022
0.002
0.0009
0.0004
ꢀ
ꢀ
n= 9
11
9.4 (90%)
Ibutamoren
n= 42
[
a]
In vitro assay. Binding assay to detect compounds binding to the GHSꢀR1a expressed in
HEK293 cells, by competing with [ I]ꢀghrelin. Values are the mean of at least 2 independent
determinations; for reference compounds n ≥ 8.
ghrelin receptor results in a production of IP1. IP1 is accumulated in the cell in presence of LiCl
and measured by time resolved fluorescence. Values are the mean of at least 2 independent
determinations. The assay validation resulted in a CIR value of +/ꢀ 0.07 and the MDR value of
0.1 (on a log scale) when tested at 2 separate occasions. For reference compounds n ≥ 9. Effect
of the compounds on the voltageꢀdependent potassium channel encoded by the human etherꢀaꢀ
goꢀgoꢀrelated gene (hERG).
1
25
[b,c]
In vitro assay. Agonist activation of the
[
d]
The preferred stereochemistry was assessed through the synthesis of the four diastereoisomers 4-
7
, Figure 2. The (1S,2S) trans enantiomer 4 proved to have higher affinity than its enantiomer 5
with a pIC of 7.8 vs 6.0 in the binding assay. The cis diastereoisomers 6 and 7 showed higher
5
0
agonism but lower potency.
The preferred conformations of nine ghrelin full agonists reported in the literature including SMꢀ
1
9
20
21
22
130686, MKꢀ677 (Ibutamoren), ONOꢀ7643 (Anamorelin), CPꢀ424391 (Capromorelin),
7
23
24
25
CPꢀ464709, SB791016 and GSK894490A, Lꢀ163540, and aryl sulphonamide were
2
6
superimposed with those of our leads, using the software Phase (Schrödinger LLC). The
calculations indicate three pharmacophoric features, a basic (A), an aromatic residue (B) and a
Hꢀbond acceptor (C) which are likely relevant for the interaction with the ghrelin receptor,
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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
Figure 3. These features are consistent with the putative binding site proposed by a published
homology model of the ghrelin receptor that was built based on (1) the Xꢀray structure of the
2
7
inactive state of rhodopsin and (2) results of siteꢀdirected mutagenesis studies. To understand
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
the origin for the observed structureꢀactivity relationships, the identified pharmacophore features
28
and established mutational hits have to be linked to a putative binding mode of the compounds
in the binding site of the receptor. The basic center A likely forms a saltꢀbridge to the acidic
residue Gluꢀ124 of the receptor, a very important mutational hit, which is used as a major
anchoring site for agonists, antagonists and inverse agonists in many published attempts to derive
2
8,29,30
potency and efficacy from binding mode.
The aromatic moiety B forms π−π and/or
hydrophobic interactions with the Pheꢀ309 residue and the Hꢀbond acceptor C forms a chargeꢀ
reinforced Hꢀbond donor with the basic residue, Argꢀ283, two other important mutational hits.
Figure 3. Conformation of 2 mapped into the pharmacophore model. Color spheres illustrate the
optimal locations of the three points pharmacophore features: A (basic, red), B (aromatic, amber)
and C (Hꢀbond acceptor, green). Only polar hydrogens are illustrated.
In the course of the optimization we then identified compound 8, reasonably potent and, for the
first time, showing a 93% maximal response associated with pEC50 of 7.8, Table 2. This
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7
8
9
encouraging result led to further docking studies in a homology model now based on the newly
available Xꢀray crystallographic structure of A2a agonist bound to the human adenosine A2a
3
1
receptor (Brookhaven PDB access code 3QAK), guiding the design of further molecules.
Compound 8 was studied in detail in respect of the bicyclic heteroaryl moiety; indole showed
to be one of the best groups and its substitution pattern was quickly investigated, Table 2. The
presence of the aminopyridine usually led to compounds with a high percentage of agonism as in
compounds 14-17 and 19-21; only compound 18 being an outlier.
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
R =
9
10
11
12
13
8
all pIC < 6
5
0
Figure 4. SAR around the aminopyridine moiety of compound 8.
The SAR around the 4ꢀamino pyridine moiety was very narrow. Figure 4 shows several analogs
with replacements of the amino pyridine ring (9-13), all with pIC < 6.
5
0
The interactions of the amino pyridine moiety with residues Glnꢀ120, Pheꢀ121 and Gluꢀ124 seem
to be crucial for full agonism at the ghrelin receptor and minor structural alterations lead to
interconversion of full agonists to partial agonists or antagonists as has been previously observed
16
with other GPCRs, Figure 5. Consistent with previous observations, the agonists under study
form a superficial binding mode with no interactions with a more deeply located mutational hit
Aspꢀ99, which are thought to be an interaction site for more profoundly bound antagonists and
28,29,30
inverse agonists.
Residue Pheꢀ286, which has been shown to be a mutational hit for more
bulky peptidic agonists and inverse agonists are located far from the putative binding mode of
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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
the compact indaneꢀbased agonists, consistent with suggested binding modes for other smallꢀ
2
7
molecule agonists.
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
N
O
NH2
NH
H
N
N
O
8
Figure 5. Interactions of compound 8 with the homology model of ghrelin receptor as structural
drivers for full agonism. The binding mode of 8 was obtained by energy minimization of the
32
complex using the OPLS3 force field.
Table 2. Potency in binding, functional assay, agonism and logD of compounds 8, 14-21.
N
N H 2
O
N H
H
N
R
O
[
a]
[b]
Binding
Functional assay
[
d]
Entry
R
LogD
[c]
pIC50 IC (ꢀM) pEC and agonism (%) EC (ꢀM)
50
50
50
7
.8 (93)
8
6.6
0.246
0.015
4.1
(n = 80)
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3
4
5
6
7
8
9
1
4
5
7.1
7.9
0.089
0.014
7.6 (74)
8.6 (88)
0.023
0.003
2.4
3.1
1
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
1
6
7
7.9
7.5
0.013
0.035
8.0 (82)
7.9 (73)
0.003
0.014
2.9
3.0
CN
1
1
2
8
9
0
6.7
7.3
ꢀ
0.187
0.049
ꢀ
7.4 (65)
7.6 (93)
6.9 (84)
0.044
0.025
0.135
2.1
2.4
3.1
N
H
21
ꢀ
ꢀ
8.1 (83)
0.007
4.0
[
a,b,c]
[d]
See footnotes in Table 1. Distribution coefficient expressing partitioning of compounds
between 1ꢀoctanol and aqueous buffer at pH 7.4.
However the aminopyridine based derivatives, although full agonists, were quite lipophilic and
during the work to explore the influence of individual residues on ghrelin receptor agonism, we
designed the morpholine compound 23. As shown in Figure 6, the interaction with Glnꢀ120 is
now engaged by the morpholine oxygen atom. Both epimers at the morpholine ring stereocenter
were investigated (compounds 23 and 24), Table 3. Compound 23 with S configuration at the
morpholine ring maintained a potency with pEC of 7.7 in the functional assay and showed the
5
0
desired full agonism (91), while the epimer 24 showed a drop both in pEC (6.6) and in agonism
50
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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
(
65%). To challenge the role of the morpholine oxygen, piperidine analogs were prepared
(compounds 25 and 26) and they showed lower agonism, 70 and 69 % respectively. The more
potent analog 22 with higher lipophilicity and metabolic instability was not further elaborated.
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
F-121
F-121
Q-120
Q-120
E-124
E-124
F-312
F-279
8
F-312
F-309
23
F-309
F-279
R-283
R-283
Figure 6. Residues of human ghrelin receptor within the homology model that interact with
compounds 8 and 23. Ionic, polar, chargeꢀreinforced Hꢀbonding and hydrophobic interactions are
shown in red, amber, purple and green, respectively.
Table 3. Potency in binding, functional assay, agonism and logD of compounds 22-38.
[a]
[b]
Binding
Functional assay
[d]
Entry
R
R1
LogD
IC50
pEC and
EC50
(ꢀM)
50
pIC50
7.9
[c]
(
ꢀM) agonism (%)
8
.9 (81)
2
2
2
3
0.014
0.0014
0.021
3.7
2.6
(
n = 5)
7.7 (91)
6.6
0.241
>2.9
(n = 80)
2
4
<5.5
6.6 (65)
0.274
2.4
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
2
5
6
6.3
6.3
7.0
6.7
7.2
0.528
0.556
0.107
0.189
0.071
6.8 (70)
7.6 (69)
7.4 (97)
7.1 (79)
8.0 (91)
0.170
0.028
0.036
0.076
0.01
1.9
2.1
2.1
1.6
2.3
2
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
27
28
29
30
a
a
<6.0
>1.0
6.6 (83)
7.7 (72)
7.3 (92)
6.9 (55)
< 5.0 (ꢀ)
0.262
0.021
0.052
0.137
>7.7
2.3
3.7
1.8
2.6
2.2
3
3
3
3
1
2
3
4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
3
3
5
6
7
6.5
6.5
6.7
0.288
0.343
0.184
6.9 (61)
6.6 (65)
0.129
0.25
1.4
1.4
1.8
< 6.1 (84)
>0.74
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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
3
8
7.5
0.029
7.7 (57)
0.019
1.7
[a,b,c,d]
See footnotes in Table 2.
Increase of lipophilicity, in order to fill the hydrophobic pocket, with a homopiperidine ring as in
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 27 gave the desired agonism of 97%. The (S)ꢀconfigured homomorpholine derivative
2
9 showed a pEC50 of 8 and 91% agonism while the epimer 30 was lower in potency and
agonism. The position of the oxygen in this ring is relevant as shown by the loss in agonism of
the regioisomeric compound 31 and the corresponding demethylated compound 33. Acetylation
of the basic nitrogen led to the inactive acetyl derivative 34. Pyrrolidine derivatives 35-38 did not
offer any advantage in terms of potency or agonism.
Unfortunately, the morpholine derivative 23, although being a potent full agonist, was not a
candidate to progress. The compound showed a significant amount of reactive metabolites
formed in human microsomes and rat hepatocytes. Metabolite identification using KCN and
MeONH as trapping agents confirmed the formation of iminium ions and aldehyde adducts on
2
the morpholine ring in both the parent and the demethylated morpholine 32 (main metabolite),
(
hydroxylation of the morpholine ring and dehydration result in the formation of iminium ions
that are unstable, but can be trapped by forming cyanide stable adducts which can be detected by
LCꢀMS/MS). The mechanism of formation of iminium intermediates and aldehydes by oxidative
3
3ꢀ35
dealkylation on morpholine groups has been previously described,
more recently in the case
36
of the bioactivation of Foretinib. Moreover, all the morpholine derivatives showed quite
significant brain penetration, which according to our hypothesis at that point of time, was
unnecessary to achieve the desired effect (see below), but could cause undesired side effects.
Brain penetration was measured as Kpu,u, which is the ratio between the compound free
37
concentration in brain and its free concentration in plasma.
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8
9
Looking for a morpholine bioisoster, we designed the reverse amide 39 where the carbonyl
directly attached to the indane might engage in the same interaction with Glnꢀ120 as the
morpholine oxygen to maintain full agonism (Figure 7). At the same time, we hoped that
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
reducing permeability and increasing efflux, would keep these compounds out of the brain.
Fꢀ121
Qꢀ120
Eꢀ124
Fꢀ121
Eꢀ124
Qꢀ120
23
39
Figure 7. Morpholine diamide 23 and reverse amide 39.
Table 4 shows a series of reverse amides. The stereochemistry was confirmed with the
enantiomer 40 and the isomer cis (1R,2S) 42 being less potent. By increasing lipophilicity (as
with compound 41), a very high potency could be obtained, pIC50 of 10 but at the cost of
metabolic instability. Methylation of the NH of the reverse amide as in compound 43 was
possible, but the resulting compound was less metabolically stable.
Table 4. Potency in binding, functional assay, agonism, logD and metabolic stability of
compounds 39-43.
[
a]
[b]
Binding
Functional assay
[
f]
[d]
Clint
HLM
hERG
Entry
R
LogD
[e]
IC50
pEC and
EC50
(ꢀM)
50
pIC50
pIC50
[c]
(ꢀM) Agonism (%)
39
8.8 (89)
8
6
.4
.3
0.004
0.002
2.4
2.3
9
3
4.5
4.9
(1S, 2S)
(n = 9)
4
0
0.56
ꢀ
6.2 (85)
10 (96)
0.63
(1R, 2R)
3
0%inh.
@ 11ꢀM
4
1
ꢀ
0.0001
3.0
132
(
1S, 2S)
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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
4
2
4
@
2%inh.
11ꢀM
50%inh.
11ꢀM
cis
ꢀ
ꢀ
6.6 (85)
8.3 (84)
0.236
0.005
2.3
2.5
ꢀ
(1R, 2S)
4
3
7
.9
0.014
26
(
1S, 2S)
@
[a,b,c,d]
[e]
See footnotes in Table 2. In vitro clearance in human liver microsomes (ꢁL/min/mg).
See footnote [d] in Table 1.
[
f]
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 39 (MW 404, LogD 2.4, PSA 81, solubility 100 ꢀM) without significant inhibition
of the hERG channel, (pEC 4.5) showed a promising profile. Its plasma protein binding is 4, 8
50
and 12% in rat, dog and human respectively. The intrinsic metabolic clearance is variable
depending on the species. Compound 39 is quite stable in vitro with low hepatic intrinsic
clearance in human (<4 ꢀL/min/mg) but medium in dog (9 ꢀL/min/mg) and high in rat (48
ꢀ
L/min/mg). 39 did not inhibit the major CyP450 enzymes with pIC < 4.7 in CyP 3A4, 2D6,
50
1
A2, 2C9 and 2C19. No time dependent inhibition was observed in human liver microsomes
suggesting a low risk of drugꢀdrug interactions. Compound 39, despite showing a very high
clearance (above hepatic blood flow) in rat (170 ml/min/Kg), showed an acceptable in vivo
pharmacokinetic profile in dog with a clearance of 18.8 ml/min/Kg, a volume of distribution of
4
.8 L/Kg and an oral half life of 16.6 hours. The oral bioavailability was 13% with absorbed
fraction 20%.
The in vivo clearance in dog was well predicted from the dog in vitro hepatic Clint, indicating
an elimination pathway dominated by hepatic CyPꢀmetabolism. Bile and urine elimination
studies confirmed that only around 2% of the dose was eliminated via urine in dog, but active
secretion was taking place in rat since the renal clearance (Fe > 5%) was measured to be far
above (~10 fold higher) the filtration clearance. This extrahepatic clearance mechanism, together
with a significant bile clearance explained the underprediction of clearance in vivo (rather than a
misleading low rat hepatic clint in hepatocytes due to the low permeability and high efflux in rat
hepatocytes). Metabolite identification in vivo in urine, bile and plasma showed that the main
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metabolite was not the Nꢀdemethylation of the tertiary amine as expected, but the hydroxylation
of the 5ꢀmethyl of the indole ring, first to the aldehyde and finally to the carboxylic acid. Cyp
Reaction Phenotyping showed that hydroxylation of the methyl group of the indole was
catalysed mainly by CYP 2C19.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
Based on its properties, 39 was considered an appropriate tool compound to be studied in a dog
pharmacokinetic/pharmacodynamic model to understand the relationship between the plasma
concentration of the drug and its effect on GH and IGFꢀ1 after a single dose of compound. We
further were interested in probing the importance of CNS penetration for the efficacy of a ghrelin
agonist. Literature has discordant hypotheses about the relevance of CNS penetration for
efficacy. Compound 39 was designed for low CNS penetration. It presented a low intrinsic
ꢀ6
permeability (0.64 10 cm/s) and a high efflux (67.2), sufficient to keep the compound
effectively out of the brain (Kpu,u < 0.020) while still getting an adequate absorption in dog,
Table 5. As a comparator, compound 22 with higher CNS penetration was added to the study.
CNS penetration is expressed as Brainunbound / Plasmaunbound ratio, which was 0.292 for compound
2
2 and < 0.020 for compound 39. Compound 22 with a short half life (1.2 h in dog) and high
CNS penetration and compound 39 with an oral half life of 13.2 h and low CNS penetration were
profiled against ghrelin, Figure 8.
Table 5. Dog pharmacokinetics data and CNS penetration value of compounds 39 and 22.
Cacoꢀ2
Cacoꢀ2
Cacoꢀ2
Perm.AtB
pH=6.5
[
a]
[b]
[b]
Cacoꢀ2
Efflux
Ratio
[e]
Dog PK
Cl, Vd, t , F
P AtB
P BtA
[g]
app
app
Entry
CNS
pH=7.4
1Eꢀ6 cm/s) (1Eꢀ6 cm/s)
pH=7.4
[b]
1
/2
(
(1Eꢀ6 cm/s)
[
c]
[c]
[c]
2
2
28, 2.1, 1.2, 39
26, 5.8, 13.2, 9
15.3
7.5
0.5
44.4
0.292
[
d]
[d]
[d]
[f]
39
0.6
17.7
67.2
<2.4
< 0.020
[a]
Pharmacokinetic study in dogs: clearance, volume of distribution, terminal half life and
[
b]
bioavailability, n=4 p.o; n=4 i.v.; Apparent permeability across Cacoꢀ2 cell monolayers:
P AtB = apparent permeability coefficient in apical to basolateral direction; P BtA = apparent
app
app
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2
2
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2
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permeability coefficient in basolateral to apical direction; Efflux ratio = P BtA/ P AtB;
app
app
[c]
[d]
[e]
n=4; n=8; Apparent intrinsic permeability across Cacoꢀ2 cell monolayers in presence of a
38
cocktail
n=2¸ Brainunbound/Plasma
of
inhibitors
toward
ratio.
the
major
efflux
transporters
[
f]
[g]
unbound
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
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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
Fig. 8. Increase in IGFꢀ1 plasma levels (mean ±SEM) from baseline after oral administration of
mg/kg of 39, 4 mg/kg of 22 and 30 µg/kg (iv) of ghrelin, four dogs per group. All compounds
8
caused a significant (p<0.05) increase in IGFꢀ1 compared to vehicle, four and seven hours after
administration. 39 caused a significant IGFꢀ1 increase 24h after administration.
Fig. 9. Plasma concentration from baseline after oral administration of compound 39, 8 mg/kg
and 22, 4 mg/kg, n= 4.
Dogs were dosed with compounds 39 and 22 orally at 8 and 4 mg/Kg, respectively. Results are
reported in Figure 8; compounds 39 and 22 both showed a sustained IGFꢀ1 response.
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The comparison of the two compounds supports the hypothesis that compounds with low
unbound brain/plasma ratios can show efficacy, indicating that CNS penetration is not a
requirement for affecting GH/IGFꢀI axis.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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4
4
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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
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7
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
In spite of all positive intrinsic properties, in vitro and in vivo pharmacokinetics and efficacy in
our dog model, compound 39 unfortunately showed activity on the ꢀ1ꢀopioid receptor both in a
binding assay (pIC 7.1) and in a cell based assay evaluating its agonistic activity (pIC 6.63).
5
0
50
The primary µꢀopioid agonism risk is constipation by reducing gut motility, which is a wellꢀ
known side effect of opioid use; this will be of particular concern of the intended patient
population which includes primarily elderly people. In addition, µꢀopioid agonists are likely to
become highly addictive with resulting prescription limitations on the label. These considerations
led stopping this compound from further progression. Work to overcome this off target effect is
ongoing and will be reported in due course.
ꢄ CONCLUSION
This study describes a new series of ghrelin receptor full agonists based on an indane scaffold.
The chemical evolution from partial to full agonists was possible due to the identification of the
structural features that drive full agonism. The optimized compounds show limited brain/plasma
levels, long half life and increase IGFꢀ1 secretion in dog. This last effect may be beneficial in a
variety of clinical conditions where skeletal muscles are impaired as in cachexia which is
associated with many chronic diseases. We have identified a drugꢀlike potent ghrelin full agonist,
compound 39, with beneficial in vivo efficacy and acceptable pharmacokinetic profile. This
molecule suffers from off target activities that forced us to stop the development of this series.
Further studies are needed to address the specificity of the compound and further development
will be disclosed in due course.
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6
ꢄ EXPERIMENTAL SECTION
General Chemistry. All chemicals purchased from commercial suppliers where used as
received. All reactions involving airꢀ or moistureꢀsensitive reagents were performed under a
nitrogen atmosphere using dry solvents. All compounds were purified to ≥95% purity as judged
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
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7
8
9
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1
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5
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8
9
0
1
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4
5
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0
1
13
by HPLC with UV and MS analysis. Proton ( H) and carbon ( C) NMR spectra were recorded at
room temperature, unless otherwise specified. Solutions were typically prepared in deuterated
dimethyl sulfoxide (DMSO ), with chemical shifts referenced to solvent as an internal standard.
d6
1
H NMR data are reported indicating the chemical shift (δ), the multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; p, quintet; m, multiplet; br, broad; dd, doublet of doublets; etc.), the
coupling constant (J) in Hz, and the integration (e.g., 1H). HRMS experiments were performed
on a Waters Acquity, Waters 2777, or Waters 2700 UV−HPLC system and a Waters Xevo G2
TOF, Waters LCT Premiere, Waters LCT, or Waters QTOFmicro mass spectrometer.
Compounds were named using ACD/Labs 2015 IUPAC Name.
Representative chemistry is reported for the synthesis of compound 23. Similar procedure was
applied to the synthesis of compounds 4-38.
1
-Methyl-N-[(1S,2S)-1-{[(2S)-4-methylmorpholine-2-carbonyl]amino}-2,3-dihydro-1H-
inden-2-yl]-1H-indole-5-carboxamide (23). To a solution of 1ꢀmethylꢀNꢀ((1S,2S)ꢀ1ꢀ((S)ꢀ
morpholineꢀ2ꢀcarboxamido)ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ2ꢀyl)ꢀ 1Hꢀindoleꢀ5ꢀcarboxamide 32 (245 mg,
0
.59 mmol) in MeOH (10mL) was added HCHO (53 mg, 1.76 mmol, 3.0 eq.), AcOH (5 drops).
The mixture was stirred at r.t. for 1 h. Then NaBH CN (110 mg, 1.76 mmol) was added. The
3
reaction mixture was stirred at r.t. for 15 h. The mixture was concentrated and the residue was
subjected to prepꢀHPLC for purification to afford compound 23 (30 mg, 12% yield) as a white
1
solid. H NMR (500 MHz, DMSO ) δ 8.64 (d, J = 8.38 Hz, 1H), 8.18 (d, J = 8.91 Hz, 1H), 8.14
d6
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(
d, J = 1.28 Hz, 1H), 7.70 (dd, J = 1.61 and 8.63 Hz, 1H), 7.49 (d, J = 8.66 Hz, 1H), 7.41 (d, J =
3
.09 Hz, 1H), 7.31ꢀ7.14 (m, 3H), 7.10ꢀ6.97 (m, 1H), 6.54 (d, J = 3.01 Hz, 1H), 5.56 (t, J = 8.78
Hz, 1H), 4.80 (p, J = 8.70 and 8.71 Hz, 1H), 3.95 (dd, J = 2.66 and 10.02 Hz, 1H), 3.90ꢀ3.78 (m,
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
3
H), 3.82 (s, 3H), 3.55 (td, J = 2.39 and 11.11 Hz, 1H), 3.23 (dd, J = 8.26 and 15.47 Hz, 1H),
.01ꢀ2.77 (m, 2H), 2.57 (d, J = 10.58 Hz, 1H), 2.19 (s, 3H), 2.08ꢀ1.86 (m, 2H). Mass Spectrum
+
(ESI) m/z 433 (M+H). HRMS (ESI) m/z calculated for C H N O + H [M+H]: 433.2240.
25
28
4
3
Found: 433.2227.
1
-Methyl-N-((1S,2S)-1-((S)-morpholine-2-carboxamido)-2,3-dihydro-1H-inden-2-yl)- 1H-
indole-5-carboxamide (32). To a solution of tertꢀbutyl (2S)ꢀ2ꢀ({(1S,2S)ꢀ2ꢀ[(1ꢀmethylꢀ1Hꢀ
indoleꢀ5ꢀcarbonyl)amino]ꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl}carbamoyl)morpholineꢀ4ꢀcarboxylate 51
(308 mg, 0.59 mmol, 1.0 eq.) in DCM (15 mL) was added TBDMSOTf (165 mg, 0.62 mmol,
1
.05 eq.) dropwise. The reaction mixture was stirred at r.t. for 6 h. Then Et N (3 mL) was added,
3
the mixture was extracted with DCM and saturated NaHCO aq. 3 times. The combined organic
3
layer was dried over Na SO , filtered and concentrated in vacuum to afford the crude title
2
4
compound that was subjected to prepꢀHPLC for purification to afford compound 32 (245 mg) as
1
a white solid. H NMR (500 MHz, DMSO ) δ ppm 8.63 (d, J = 8.36 Hz, 1H), 8.13 (d, J = 1.34
d6
Hz, 1H), 8.09 (d, J = 8.96 Hz, 1H), 7.70 (dd, J = 1.63 and 8.62 Hz, 1H), 7.49 (d, J = 8.66 Hz,
1
H), 7.41 (d, J = 3.09 Hz, 1H), 7.28ꢀ7.15 (m, 3H), 7.04 (d, J = 5.62 Hz, 1H), 6.54 (d, J = 3.08
Hz, 1H), 5.55 (t, J = 8.78 Hz, 1H), 4.82 (p, J = 8.70 and 8.67 Hz, 1H), 3.85 (dd, J = 2.76 and 9.94
Hz, 1H), 3.82 (s, 3H), 3.78 (d, J = 10.99 Hz, 1H), 3.47 (td, J = 3.21, 10.80 and 10.81 Hz, 1H),
3
.22 (dd, J = 8.22 and 15.46 Hz, 1H), 3.04 (dd, J = 2.60 and 12.32 Hz, 1H), 2.88 (dd, J = 9.15
and 15.42 Hz, 1H), 2.73ꢀ2.54 (m, 3H). Mass Spectrum (ESI) m/e = 419 (M+1). HRMS (ESI)
+
m/z calculated for C H N O + H [M+H]: 419.2083. Found:419.2102.
2
4
26
4
3
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N-((1S, 2S)-1-(2-(dimethylamino)ethylcarbamoyl)-2,3-dihydro-1H-inden-2-yl)-1H-indole-2-
carboxamide (39). 1S,2Sꢀ2ꢀAminoꢀNꢀ(2ꢀ(dimethylamino)ethyl)ꢀ2,3ꢀdihydroꢀ1Hꢀindeneꢀ1ꢀ
carboxamide 63b (194 mg, 0.78 mmol), 5ꢀmethylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (151 mg, 0.86
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
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1
2
3
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0
1
2
3
4
5
6
7
8
9
0
mmol),
hexafluorophosphate (V) (HATU) (388 mg, 1.18 mmol) were taken up in dry DMF (8 ml). DIEA
0.9 ml, 668 mg, 5.17 mmol) was added and the mixture was stirred overnight at r.t. It was
evaporated and the residue was purified by HPLC (xꢀbridge, Cꢀ18, CH CNꢀH O 10:90 to 45:55,
and
2ꢀ(3Hꢀ[1,2,3]triazolo[4,5ꢀb]pyridinꢀ3ꢀyl)ꢀ1,1,3,3ꢀtetramethylisouronium
(
3
2
2
5 min gradient with 0.2% formic acid on aq. solvent, fraction size 40 ml). The pure fractions
were combined and freezeꢀdried to compound 39 as a light brown solid (152 mg, as formic acid
1
salt, e.e. 99.58%). H NMR (500 MHz, DMSO ) δ ppm 11.42 (s, 1H), 8.66 (d, J = 7.70 Hz, 1H),
d6
8
1
.17 (t, J = 5.61 Hz, 1H), 7.36 (bs, 1H), 7.31 (d, J = 8.37 Hz, 1H), 7.28ꢀ7.15 (m, 4H), 7.06 (d, J =
.45 Hz, 1H), 7.00 (dd, J = 1.40 and 8.45 Hz, 1H), 4.97 (p, J = 7.01 and 7.06 Hz, 1H), 4.05 (d, J
= 6.39 Hz, 1H), 3.40ꢀ3.22 (m, 2H), 3.13 (dq, J = 6.44, 6.58 and 13.30 Hz, 1H), 2.99 (dd, J = 6.82
1
3
and 16.04 Hz, 1H), 2.35 (s, 3H), 2.37ꢀ2.25 (m, 2H), 2.14 (s, 6H). C NMR (125 MHz, DMSO )
d6
δ ppm 171.32, 160.94, 141.43, 141.21, 134.80, 131.62, 128.19, 127.26, 127.20, 126.46, 125.10,
1
24.40, 124.01, 120.69, 111.98, 102.25, 58.16, 57.05, 52.97, 45.11, 38.26, 36.93, 21.12. Mass
+
Spectrum (ESI) m/e = 405 (M+1). HRMS (ESI) m/z calculated for C H N O + H [M+H]:
2
4
28
4
2
20
4
05.2290. Found: 405.2308. Optical rotation in MeOH [α] D= +112.
N-((1R, 2R)-1-(2-(dimethylamino)ethylcarbamoyl)-2,3-dihydro-1H-inden-2-yl)-1H-indole-
-carboxamide (40). 1R,2Rꢀ2ꢀAminoꢀNꢀ(2ꢀ(dimethylamino)ethyl)ꢀ2,3ꢀdihydroꢀ1Hꢀindeneꢀ1ꢀ
carboxamide 63a (224 mg, 0.91 mmol), 5ꢀmethylꢀ1Hꢀindoleꢀ2ꢀcarboxylic acid (175 mg, 1.0
mmol), 2ꢀ(3Hꢀ[1,2,3]triazolo[4,5ꢀb]pyridinꢀ3ꢀyl)ꢀ1,1,3,3ꢀtetramethylisouronium
2
hexafluorophosphate(V) (HATU) (448 mg, 1.18 mmol) were put in dry DMF (8 ml) and DIPEA
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(
1.0 ml, 5.74 mmol) was added. The mixture was stirred overnight at r.t. It was evaporated and
the residue, taken up in DMSO, was purified by HPLC (xꢀbridge, Cꢀ18, CH CNꢀH O from 10:90
3
2
to 45:55, 25 min gradient with 0.2% formic acid on aq. solvent). The pure fractions were
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
combined and freezeꢀdried to compound 40 as a light brown solid (isolated yield: 98 mg, as
1
formic acid salt, e.e. 99.58%). H NMR (500 MHz, DMSO ) δ ppm 11.42 (s, 1H), 8.66 (d, J =
d6
7.70 Hz, 1H), 8.17 (t, J = 5.61 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J = 8.37 Hz, 1H), 7.28ꢀ7.15 (m,
4
4
H), 7.06 (d, J = 1.42 Hz, 1H), 7.00 (dd, J = 1.40 and 8.45 Hz, 1H), 4.98 (p, J = 7.00 Hz, 1H),
.05 (d, J = 6.39 Hz, 1H), 3.45ꢀ3.21 (m, 2H), 3.13 (dq, J = 6.58, 6.50 and 13.31 Hz, 1H), 2.99
(dd, J = 6.82 and 16.05 Hz, 1H), 2.35 (s, 3H), 2.30 (tt, J = 6.01 and 11.99 Hz, 2H), 2.14 (s, 6H).
+
Mass Spectrum (ESI) m/e = 405 (M+1). HRMS (ESI) m/z calculated for C H N O + H
2
4
28
4
2
[
M+H]: 405.2290. Found: 405.2321.
2
0
Optical rotation in MeOH [α] D= ꢀ113.
tert-Butyl [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]carbamate (45). To a solution of
the commercially available (1S,2R)ꢀ1ꢀaminoꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ2ꢀol (21 g, 141 mmol, 1.0
eq.) in anhydrous DCM (500 mL), Et N (28 g, 282 mmol, 2.0 eq.) and Boc O (36.9 g, 169 mmol,
3
2
1
.2 eq.) were added sequentially. The mixture was stirred at r.t. overnight. The reaction mixture
was concentrated and the residue was purified by column chromatography with 0ꢀ25% EtOAc in
1
PE to afford compound 45 as a white solid (32 g, 91.2% yield). H NMR (400 MHz, DMSO ) δ
d6
7
1
.24ꢀ7.12 (m, 4H), 6.35 (d, J = 8.4 Hz, 1H), 4.97 (d, J = 4.8 Hz, 1H), 4.86 (dd, J = 8.6, 4.6 Hz,
H), 2.99 (dd, J = 16.0, 5.2 Hz, 1H), 2.77 (dd, J = 16.0, 1.0 Hz, 1H), 1.43 (s, 9H).
(1S,2R)-1-[(tert-Butoxycarbonyl)amino]-2,3-dihydro-1H-inden-2-yl
methanesulfonate
(46). To a mixture of tertꢀbutyl [(1S,2R)ꢀ2ꢀhydroxyꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl]carbamate 45 (32
g, 129 mmol, 1.0 eq.) and Et N (26.1 g, 258 mmol, 2.0 eq.) in dry DCM (400 mL) at 0ºC a
3
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solution of methanesulfonyl chloride (22.1 g, 193 mmol, 1.5 eq.) in DCM (100mL) was added
slowly. The resulting mixture was stirred at r.t. for 3 h. Water was added to the mixture, then the
aqueous phase was extracted with DCM 3 times. The combined organic layers were dried over
1
0
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Na SO , filtered and concentrated to afford the intermediate mesylate 46 (41 g, 97.2%). H NMR
2
4
(
400 MHz, DMSO ) δ 7.41 (d, J = 8.8 Hz, 1H), 7.30ꢀ7.20 (m, 4H), 5.32ꢀ5.28 (m, 1H), 5.20 (dd,
d6
J = 8.8, 5.2 Hz, 1H), 3.24 (dd, J = 17.2, 4.8 Hz, 1H), 3.14 (s, 3H), 3.11ꢀ3.03 (m, 1H), 1.42 (s,
H).
tert-Butyl [(1S,2S)-2-amino-2,3-dihydro-1H-inden-1-yl]carbamate (48). To a solution of
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the mesylate 46 (41 g, 125 mmol, 1.0 eq.) in DMF (500 mL), sodium azide (9.8 g, 151 mmol, 1.2
º
eq.) was added and the mixture was stirred at 90 C overnight. After cooling to r.t., the mixture
was diluted with EtOAc (1 L), washed with H O (3x200 mL), dried over Na SO and filtered to
2
2
4
obtain the crude compound 47. To this crude, 10% Pd/C (4 g, wet) was added and the mixture
was stirred under a H atmosphere overnight. The reaction mixture was then filtered through
2
celite and concentrated, the residue was purified by column chromatography with 50ꢀ100%
1
EtOAc in PE as eluent to afford compound 48 (22 g, 71%). H NMR (400 MHz, DMSO ) δ
d6
7
.18ꢀ7.10 (m, 4H), 7.05 (d, J = 4.0 Hz, 2H), 4.52 (t, J = 8.2 Hz, 1H), 3.35ꢀ3.26 (m, 1H), 2.97 (dd,
J = 17.4, 7.4 Hz, 1H), 2.52 (dd, J = 17.4, 9.2 Hz, 1H), 1.81ꢀ1.71 (bs, 2H), 1.43 (s, 9H).
tert-Butyl {(1S,2S)-2-[(1-methyl-1H-indole-5-carbonyl)amino]-2,3-dihydro-1H-inden-1-
yl}carbamate (49, RCOOH = 1-methyl-1H-indole-5-carboxylic acid). To a solution of 1ꢀ
methylꢀ1Hꢀindoleꢀ5ꢀcarboxylic acid (374 mg, 2.1 mmol, 1.1 eq.) in DCM (20 mL) was added
HOBt (288 mg, 2.1 mmol, 1.1 eq.), EDCI (410 mg, 2.1 mmol, 1.1 eq.) and DIEA (501 mg, 3.8
mmol, 2.0 eq.) sequentially. The resulting solution was stirred at r.t. for 30 min. Then tertꢀbutyl
(1S,2S)ꢀ2ꢀaminoꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ1ꢀyl carbamate 48 (482 mg, 1.9 mmol, 1.0 eq.) was
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added. The reaction mixture was stirred at r.t. overnight. The mixture was diluted with DCM and
washed with aq. NaOH three times. The combined organic phase was concentrated to afford the
1
title compound 49 (724 mg, 92% yield) as a white solid. H NMR (400 MHz, DMSO ) δ 8.62
d6
1
1
1
1
1
1
1
1
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1
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(d, J = 8.0 Hz, 1H), 8.13 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.48 (d, J = 4.8 Hz, 1H), 7.41ꢀ7.36 (m,
2
H), 7.21 (d, J = 8.4 Hz, 3H), 7.12 (d, J = 4.0 Hz, 1H), 6.52 (d, J = 3.2 Hz, 1H), 5.20 (t, J = 8.8
Hz, 1H), 4.67ꢀ4.58 (m, 1H), 3.81 (s, 3H), 3.19ꢀ3.13 (m, 1H), 2.84ꢀ2.78 (m, 1H), 1.38 (s, 9H).
N-((1S,2S)-1-Amino-2,3-dihydro-1H-inden-2-yl)-1-methyl-1H-indole-5-carboxamide (50).
To a solution of tertꢀbutyl {(1S,2S)ꢀ2ꢀ[(1ꢀmethylꢀ1Hꢀindoleꢀ5ꢀcarbonyl)amino]ꢀ2,3ꢀdihydroꢀ1Hꢀ
indenꢀ1ꢀyl}carbamate 49 (200 mg, 0.49 mmol, 1.0 eq.), in DCM (7 mL) was added TBDMSOTf
(137 mg, 0.52 mmol, 1.05 eq.) dropwise. The reaction mixture was stirred at r.t. for 6 h. Then
Et N (2 mL) was added, the mixture was extracted with DCM and saturated aq. NaHCO for 3
3
3
times. The combined organic layers were dried over Na SO , filtered and concentrated in
2
4
vacuum to afford title compound 50 (145 mg, 96% yield) as a yellow solid. Mass Spectrum (ESI)
m/e = 306 (M+1).
tert-Butyl
(2S)-2-({(1S,2S)-2-[(1-methyl-1H-indole-5-carbonyl)amino]-2,3-dihydro-1H-
inden-1-yl}carbamoyl)morpholine-4-carboxylate (51). To a solution of (S)ꢀ4ꢀ(tertꢀ
butoxycarbonyl)morpholineꢀ2ꢀcarboxylic acid (158 mg, 0.68 mmol, 1.1 eq.) in DMF (10 mL)
was added HATU (260 mg, 0.68 mmol, 1.1 eq.) and Et N (126 mg, 1.24 mmol, 2.0 eq.)
3
sequentially. The resulting solution was stirred at r.t. for 0.5 h. Then Nꢀ((1S,2S)ꢀ1ꢀaminoꢀ2,3ꢀ
dihydroꢀ1Hꢀindenꢀ2ꢀyl)ꢀ1ꢀmethylꢀ1Hꢀindoleꢀ5ꢀcarboxamide 50 (190 mg, 0.62 mmol, 1.0 eq.)
was added. The reaction mixture was stirred at r.t. overnight. The mixture was concentrated and
extracted with DCM and NaOH aq. (0.5 mol/L) for 3 times. The combined organic phase was
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concentrated to afford the title compound 51 (308 mg, 95% yield) as a yellow solid. Mass
Spectrum (ESI) m/z 519 (M+H).
Methyl 2-amino-1H-indene-3-carboxylate (59). A 3ꢀneck round bottom flask was charged
with the commercially available keto ester 58 (5.00 g, 26.29 mmol), ammonium acetate (6.08 g,
0
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8.88 mmol) was added, followed by the addition of MeOH (50 ml). The reaction mixture was
heated to 55°C, and vacuum was gradually applied until MeOH started to distill over. Pressure
and oil bath temperature were adjusted to make sure that T was not far from 45°C. After 18 h, all
solvent had distilled over and MeOH (50 mL) was added to the solid in the flask and the
temperature in the oil bath was adjusted to ca 50°C for two more h. The reaction mixture was
allowed to slowly reach r.t. and it was stirred overnight.The precipitate was filtered off, rinsed
with MeOH (15 mL) to give a sandꢀcoloured solid (3.03 g). The filtrate, in which a second crop
of crystals had begun to precipitate, was concentrated to dryness to yield a dark brown/red semiꢀ
solid that was taken up in DCM (60 mL) and water (30 mL). The organic phase was dried over
MgSO and concentrated to yield a dark solid (1.83 g) that was recrystallized from MeOH to
4
1
obtain compound 59 as a mustardꢀcoloured powder (0.632 g) for an overall yield of 73.6%. H
NMR (400 MHz, DMSO ) δ 7.88 (bs, 1H), 7.46 (d, J= 7.62 Hz, 1H), 7.45 (bs, 1H), 7.16 (d,
d6
J=7.24 Hz, 1H), 7.09 (t, J=7.55 Hz, 1H), 6.85 (td, J=7.41 and 1 Hz, 1H), 3.74 (s, 3H), 3.55 (s,
2
H).
Methyl 2-(tert-butoxycarbonylamino)-2,3-dihydro-1H-indene-1-carboxylate (60). An
ovenꢀdried 2ꢀneck round bottom flask under nitrogen was charged with the NaBH (0.75 g, 26.29
4
mmol) and THF (15 ml). The mixture was cooled down to ꢀ17°C. At that T, the addition of
trifluoroacetic acid (8.14g, 71.35 mmol) was initiated at such a rate that the T was maintained
below ꢀ10°C. (Caution, exothermic reaction). Temperature was kept below ꢀ10°C for 1 h, then
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the enaminoester 59 (1.50 g, 7.93 mmol) was added in 6 rapid portions; T hardly rose
(Caution!). Immediately after the addition the flask was moved to an oil bath set to +10 °C and
stirred overnight. The reaction T was lowered to 2 °C and the mixture was quenched by the
addition of water (15 mL) via a syringe pump during 1 h (15 mL/h). Caution, Immediate gas
evolution. Tmax seemed to end up at around +10°C. Upon completion of the addition, the
reaction mixture was stirred at 7°C overnight. To the crude reaction mixture at 7°C, NaOH (aq.,
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0 % w/w, 2.15 mL) was added dropwise at such a rate that T was kept < 10°C. The pH was
measured to ca 8. Boc O (2.265 g, 10.38 mmol) was added in one portion. More NaOH (aq.,
2
5
0% w/w, 0.40 mL) was added. Some gas evolution could be observed. The reaction mixture
was stirred at r.t. overnight. The reaction mixture was taken up with iPrOAc (30 mL), THF (15
mL) and water (15 mL). The aqueous phase was extracted with iPrOAc (15 mL), dried over
MgSO and concentrated to give compound 60, a 1:1 cis:trans mixture, as a purple viscous oil
4
(3.82 g).
trans-2-[(tert-Butoxycarbonyl)amino]-2,3-dihydro-1H-indene-1-carboxylic acid (62). A
1
:1 mixture of cis and trans methyl 2ꢀ(tertꢀbutoxycarbonylamino)ꢀ2,3ꢀdihydroꢀ1Hꢀindeneꢀ1ꢀ
carboxylate 60 (1.65 g, 5.66 mmol) was dissolved in MeOH:water (20 ml, 4:1) and sodium
methoxide (0.521 g, 9.63 mmol) was added in five portions carefully monitoring the reaction
until full conversion. The reaction was taken up in MTBE (50 ml) and water (50 ml). The
aqueous phase was acidified with KHSO (aq.sol. 1M) to pH 3 and extracted with MTBE. The
4
organic phase was dried over MgSO and evaporated to give compound 61 as a mixture of trans
4
and cis (1.467 g) compounds in ratio 6.3:1. The mixture was separated by HPLC to give the pure
1
racemic trans diastereoisomer 62. H NMR (500 MHz, DMSO ) δ 12.54 (bs, 1H), 7.26 (d, 1H),
d6
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