Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2-Phenylindole Privileged Structure Scaffold

Article abstract of DOI:10.1021/acs.jmedchem.5b01254

G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1-3). Herein, we present the first structure-activity relationship study for the 2-arylindole antagonist 3, comprising the design, synthesis, and pharmacological evaluation of a focused library of 3-substituted 2-arylindoles. In a FRET-based inositol monophosphate (IP₁) assay we identified compounds 7, 13e, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date.

Full text of DOI:10.1021/acs.jmedchem.5b01254

Subscriber access provided by UNIV OF OTTAWA
Article
Selective allosteric antagonists for the G protein-coupled receptor
GPRC6A based on the 2-phenylindole privileged structure scaffold
Henrik Johansson, Michael Worch Boesgaard, Lenea Nørskov-Lauritsen, Inna Larsen,
Sebastiaan Kuhne, David E. Gloriam, Hans Bräuner-Osborne, and Daniel Sejer Pedersen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01254 • Publication Date (Web): 30 Oct 2015
Downloaded from http://pubs.acs.org on November 8, 2015
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Selective allosteric antagonists for the G protein-coupled receptor GPRC6A
based on the 2-phenylindole privileged structure scaffold
1
1
†,‡
‡
§
Henrik Johansson, Michael Worch Boesgaard, Lenea Nørskov-Lauritsen, Inna
12
1
14
15
¶
||
#
#
Larsen, Sebastiaan Kuhne, David E. Gloriam, Hans Bräuner-Osborne and Daniel
#
,
Sejer Pedersen *
16
17
18
19
2
2
2
23
Department of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Jagtvej 162, 2100, Copenhagen, Denmark
24
25
26
27
28
Abstract.
29
30
31
3
3
34
35
36
37
38
39
40
41
42
G protein-coupled receptors (GPCRs) represent a biological target class of
fundamental importance in drug therapy. The GPRC6A receptor is a newly
deorphanized class C GPCR that we recently reported the first allosteric antagonists
for based on the 2-arylindole privileged structure scaffold (e.g. 1-3). Herein, we
present the first structure-activity relationship study for the 2-arylindole antagonist 3,
comprising the design, synthesis and pharmacological evaluation of a focused library
4
4
of 3-substituted 2-arylindoles. In a FRET-based inositol mono-phosphate (IP ) assay
1
45
4
47
48
49
50
51
52
53
we identified compounds 7, 13e and 34b as antagonists at the GPRC6A receptor in
the low micromolar range and show that 7 and 34b display >9 fold selectivity for the
GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and
selective antagonists for the GPRC6A receptor reported to date.
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 54
1
2
3
4
5
6
7
8
9
Introduction
G protein-coupled receptors (GPCRs), constitute the largest superfamily of
1
,2
membrane-bound proteins encoded in the human genome, and represent the most
1
1
3-5
common target class in drug therapy. Based on phylogenetic analyses, the human
12
1
14
15
GPCRs have been divided into a number of classes of which class A (rhodopsin
family) is by far the most numerous, diverse with respect to endogenous ligands, and
16
17
18
1
,5,6
common as drug target.
The Class C (Glutamate family) is a significantly smaller
19
2
2
group of dimeric GPCRs comprising eight metabotropic glutamate (mGlu) receptors,
2
23
three taste 1 (T1R1-3) receptors, two γ-aminobutyric acid type B (GABA ) receptors,
B
24
25
the calcium-sensing (CaSR) receptor, the G protein-coupled receptor class C, group 6,
26
2
28
29
30
31
3
3
7
-9
subtype A (GPRC6A) and seven orphan receptors. Class C GPCRs are generally
activated by nutrients (e.g. amino acids, cations and sugar molecules), and display
characteristic topological features such as a large extracellular and ligand-binding N-
terminal domain (‘Venus flytrap domain’) in addition to the generic transmembrane
34
35
3
37
9
helical domain present in all GPCRs. Furthermore, class C GPCRs have proven
38
3
40
viable as drug targets, exemplified by two marketed drugs; the CaSR positive
41
42
allosteric modulator cinacalcet and the GABA receptor agonist baclofen used in the
B
4
4
1
0
treatment of primary hyperparathyroidism and spasticity, respectively.
45
46
47
48
49
In 2004 our research group reported the first cloning, expression and sequencing of
50
51
52
53
1
1
the GPRC6A receptor. Subsequently the receptor was deorphanized as a
promiscuous L-amino acid-sensing receptor with preference for basic amino acids
5
5
56
(e.g. arginine, lysine and ornithine), and displaying wide tissue expression in
57
5
1
2,13
humans.
Efforts by our group and others to elucidate the physiological roles of the
59
60
GPRC6A receptor using knock-out mouse models have yielded partly contradictory
ACS Paragon Plus Environment

Page 3 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14-18
19
findings,
but generally link the receptor to metabolism and energy homeostasis.
In an attempt to shed new light on the physiological functions of the GPRC6A
receptor, we initiated a medicinal chemistry program aimed at identifying new
pharmacological tool compounds for studying the GPRC6A receptor. Recently, we
reported the chemogenomic discovery of the first allosteric antagonists with modest
14
15
16
2
0
preference for the GPRC6A receptor. In a focused screening based on the GPCR
17
18
2
1,22
privileged structure
2-phenylindole, we identified allosteric antagonists 1-3 (Fig.
19
2
2
2
23
24
25
26
27
1) binding in the transmembrane domain of the mouse GPRC6A receptor and
displaying inhibitory activity in the micromolar range in an inositol phosphate
turnover assay. The 2-phenylindole scaffold of allosteric antagonists 1-3 were
proposed to bind to the receptor with hydrogen bonding, hydrophobic and aromatic-
aromatic interactions in a hydrophobic pocket, and the binding mode was validated by
28
29
30
31
3
2
0
receptor mutagenesis and ligand modifications.
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
Figure 1. 3-Substituted 2-phenylindoles 1-3 acting as allosteric antagonists at the
51
52
53
2
0
GPRC6A receptor.
5
5
56
5
58
59
The identification of indoles 1-3 as the first allosteric antagonists targeting the
GPRC6A receptor provided a good starting point for ligand optimization. However,
GPCR privileged structure scaffolds are inherently promiscuous GPCR binders, and if
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 54
1
2
3
4
5
6
7
8
9
not equipped with unique extensions, ligands containing such structural elements may
consequently display activity at a range of GPCRs. Indeed, besides the GPRC6A
receptor ligands 2 and 3 were found to also antagonize the class A muscarinic
acetylcholine M and M receptors and the metabotropic glutamate receptor subtype 5
1
1
1
3
12
1
14
15
16
17
18
(mGluR5). However, we were encouraged by the fact that ligand 1 displayed
selectivity for the GPRC6A receptor over six related GPCRs that all contain the 2-
arylindole allosteric binding motif, demonstrating that this class of privileged
19
2
2
2
0
structures can indeed be made selective by appropriate modification. Compounds 1
2
23
and 2 contained a potentially labile ester moiety and displayed significantly lower
24
25
2
0
aqueous solubility than compound 3 (5-10 fold). Despite initial attempts to address
26
2
28
29
30
31
3
3
34
35
36
37
2
3
these issues, such as replacing the ester of compound 2 with an amide, the
compounds generally suffered from poor aqueous solubility. Consequently,
compounds 1 and 2 were deselected for further ligand development at this stage.
Instead, indole 3 was chosen as our lead structure and we set out to improve the
selectivity for the GPRC6A receptor over other GPCRs, in particular the muscarinic
38
3
40
41
42
4
4
45
2
0
acetylcholine receptors that had previously proven problematic. By an empirical
approach, we envisioned that a focused structure-activity relationship study based on
indole 3, in combination with a selectivity screening over two related class C (CaSR
and mGluR5) and two distant class A (M and serotonin 5-HT ) GPCRs would
46
47
3
2C
48
49
50
51
52
53
5
5
56
57
58
59
reveal structural determinants for receptor potency and selectivity, and thereby
provide a structural basis for further ligand optimization. We chose four different
structural regions of indole 3 as target for our chemical modifications: 1) the indole
scaffold, 2) the 2-phenyl substituent, 3) the 3-carbonyl moiety and 4) the side chain in
the indole-3-position (Fig. 2). Only the 3- and the 5-positions of the 2-arylindole were
substituted in the present study. These represent the most frequently substituted
60
ACS Paragon Plus Environment

Page 5 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
positions among known 2-arylindole GPCR ligands, and have access to adjacent
2
0
binding cavity sub sites in our previously reported GPRC6A model. Furthermore,
hydrogen bonding between the indole nitrogen and the receptor has been validated as
1
1
20
a determinant receptor-ligand interaction, and therefore no substitutions were
12
1
14
carried out at this position in this study.
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
Figure 2. The four structural regions of indole 3 subject to chemical modifications are
highlighted in dashed shapes and exemplified with target molecules 4 (indole
scaffold, blue), 5 (2-phenyl substituent, green), 6 (3-carbonyl moiety, red) and 7 (side
chain in the indole-3-position, yellow).
49
50
51
52
5
5
5
56
We have previously published the synthesis of antagonist 3 via a synthetic route based
on the alkylation of a secondary amine with chloro-acetylated 2-phenylindole 8 that in
57
58
2
0,23
turn is obtained by acylation of 2-phenylindole 9 (Scheme 1).
We envisioned that
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 54
1
2
3
4
5
6
7
8
9
this route could be applied to a broader range of substituted indoles and amines to
obtain a series of analogs of antagonist 3, i.e. indoles 10 (Scheme 1).
1
1
Scheme 1. The optimized synthesis route to antagonist 3 goes via acylation of 2-
12
1
14
15
16
17
18
2
0,23
phenylindole 9 followed by alkylation of a secondary amine.
general route to obtain analogs of 3 bearing various substituent on the indole scaffold
R and R ), and the amine functionality (R and R ).
It was proposed as a
(
1
2
3
4
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
Herein, we report the design, synthesis and pharmacological evaluation of a focused
library of 3-substituted 2-arylindole analogs of antagonist 3 targeting the GPRC6A
receptor, as well as the first structure-activity relationship (SAR) study conducted for
this receptor.
45
46
47
48
4
50
Results and Discussion.
51
52
53
5
5
56
57
58
Ligand synthesis. We first set out to explore the impact on ligand binding by
introducing substituents at the 5-position of the indole scaffold and by varying the
substituent in the indole-2-position e.g. using electron-withdrawing and donating
groups, alkyl and hydroxyalkyl substituents and heterocycles. As part of our
medicinal chemistry program targeting GPCRs we have previously reported the
59
60
ACS Paragon Plus Environment

Page 7 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
24
synthesis of a number of 3-acylated 2-arylindole building blocks e.g. 11a-f that we
2
0
employed in the present study for alkylation of secondary amine 12 (Scheme 2).
1
1
Scheme 2. Synthesis of analogs 4, 5, 13b, and 13d-f via alkylation of secondary
12
1
14
a
amine 12 using indole building blocks 11a-f.
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
a
Reagents and conditions: (a) NaI, NaHCO , MeCN or acetone, 60 °C.
3
30
31
3
3
3
35
36
37
38
39
40
41
42
4
4
45
46
47
48
The alkylation of secondary amine 12 was generally uncomplicated, giving analogs of
antagonist 3 lacking the 2-phenyl group (5), bearing electron-withdrawing
substituents (4 and 13d), electron-donating substituents (13b and 13e) and a
heterocycle (13f) in moderate to good yields. Lower yields were generally associated
with the generation of small amounts of side-products that were challenging to
remove by column chromatography (e.g. 13d and 13f). With these analogs in hand we
went on to investigate the impact of introducing larger or more polar substituents onto
the indole scaffold; substituents that would challenge the hypothesis of the 2-
arylindole scaffold binding to a relatively small, hydrophobic pocket in the
49
50
51
52
53
5
5
56
57
58
59
60
2
0
transmembrane domain of the GPRC6A receptor. The 5-carboxamidomethyl-
substituted analogue 18 was selected as a target molecule with new hydrogen bonding
opportunities (Scheme 3).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 54
1
2
3
4
5
6
7
8
9
Scheme 3. Synthesis of 5-carboxamidomethyl-substituted analog 18 from carboxylic
a
acid 14.
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
a
Reagents and conditions: (a) (COCl) , DMF, CH Cl , 0 °C – rt; (b) 7.9 M aqueous
2
2
2
35
36
37
Me NH, THF, 0 °C – rt; (c) Pd/C, H , EtOH, 0 °C – rt; (d) acetophenone, 3Å MS,
2
2
38
3
PhMe, rt – 75 °C; (e) n-Bu NBr, Pd(OAc) , O , DMSO, 60 °C; (f) chloroacetyl
4 2 2
40
41
42
chloride, pyridine, PhMe, 1,4-dioxane, CH Cl , rt – 65 °C; (g) 12, NaI, NaHCO ,
2
2
3
4
4
MeCN, 60 °C.
45
46
47
48
49
50
51
Aniline 15 was obtained from carboxylic acid 14 in three steps without purification of
intermediates. Condensation of 15 with acetophenone gave an imine that underwent
52
5
5
25
Pd(II)-catalyzed oxidative cyclization under the conditions reported by Wei et al.
5
56
57
58
59
60
Indole 16 was acylated in the indole-3-position using chloroacetyl chloride and
pyridine to give alkyl chloride 17, and subsequent alkylation of secondary amine 12
gave the target molecule in good yield.
ACS Paragon Plus Environment

Page 9 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
Finally, we decided to complete the targeted set of analogs bearing substituents on the
indole scaffold by replacing the aryl group in the indole 2-position. We replaced the
aryl group with a polar substituent (hydroxymethyl group) to explore the effect on the
ligands’ physicochemical properties and potency. Moreover, our previous
mutagenesis study suggests that the indole-NH forms a crucial hydrogen bonding
14
15
16
17
18
2
0
interaction to the receptor. Thus, the introduction of a hydroxy-group in this position
19
20
2
may give rise to an additional stabilizing hydrogen bonding interaction. Starting from
2
23
2
-carboxyindole 19, 2-hydroxymethyl-substituted analog 25 was synthesized as
24
25
outlined in Scheme 4.
26
27
28
2
30
a
Scheme 4. Synthesis of 2-hydromethyl-substituted analog 25 from indole 19.
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
a
Reagents and conditions: (a) SOCl , MeOH, 0 °C – reflux; (b) LiAlH , THF, 0 °C;
2
4
48
49
50
(
c) Ac O, pyridine, 0 °C – rt; (d) TBDMS-Cl, imidazole, DMF, 0 °C – rt; (e) AcOH,
2
51
5
53
chloroacetyl chloride, Ac O, 60 °C; (f) 12, NaI, NaHCO , MeCN, 60 °C; (g) TBAF,
2 3
5
5
THF, 0 °C.
56
57
58
5
60
Esterification and reduction of carboxylic acid 19 provided alcohol 21 in good yield
followed by acetylation to give 22a. However, the following acylation at the indole 3-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 54
1
2
3
4
5
6
7
8
9
position to give indole 23a was complicated. Chloroacetylation under basic conditions
using chloroacetyl chloride and pyridine in toluene only gave complex product
mixtures, and attempts of using Lewis acid catalysis with SnCl and chloroacetyl
4
1
1
chloride in dichloromethane and nitromethane according to the method of Ottoni et
12
1
14
15
16
17
18
19
20
2
2
23
24
25
2
6
al. failed to produce the desired product. We suspected that the acetyl-protection
group was causing problems during the acetylation. Thus, tert-butyldimethylsilyl-
protected (TBDMS) indole 22b was synthesized but again, acylation under basic
conditions only gave trace amounts of product and Lewis acidic conditions made no
improvement. Ultimately, 3-chloroacetylation of indole 22b was achieved using a
mixture of chloroacetyl chloride and acetic acid in acetic acid anhydride, inspired by
26
2
27
the method of Slätt et al. The acidic conditions produced significant amounts of
side-products, but allowed isolation of the required acyl chloride 23b. In order to
improve the stability of the starting material TBDMS was replaced with the less acid
labile tert-butyldiphenylsilyl-protection group but to no avail. Alkylation of
secondary amine 12 using alkyl chloride 23b and subsequent silyl ether deprotection
produced the desired analog 25 in good yield.
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
Next, we turned our attention to modification of the ketone moiety of antagonist 3. As
45
4
47
2
0
mentioned above, hydrogen bonding of the ligand indole-NH is required for activity.
48
49
50
51
52
53
5
5
We hypothesized that the ketone at the 3-position might play a critical role through
direct interactions with the receptor (e.g. hydrogen bonding), or indirectly by electron
delocalization from the indole nitrogen over the carbonyl. To address this we decided
to include analogs devoid of the ketone moiety or with significantly altered electronic
properties. In a previous communication we have reported the synthesis of analogs 6
56
57
58
59
60
2
3
and 28 (Scheme 5). Analog 28 is a truncated amide analogue of antagonist 3, and in
ACS Paragon Plus Environment

Page 11 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
analog 6 the carbonyl has been replaced by a methylene unit. In addition, secondary
alcohol 26 was synthesized by direct reduction of antagonist 3 (Scheme 5).
1
1
a
Scheme 5. Reduction of antagonist 3 gave analogue 26 and cyclized side-product 27.
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
3
a
Reagents and conditions: (a) NaBH , CeCl × 7H O, MeOH, 0 °C.
4
3
2
38
39
40
41
42
Initial attempts at reducing ketone 3 using NaBH in MeOH did not go to completion,
4
4
4
45
2
8,29
however by switching to Luche conditions (CeCl and NaBH in MeOH)
the
3
4
46
47
48
49
50
51
52
53
desired alcohol 26 was obtained in good yield. The reduction also gave rise to
morpholine side product 27 that was isolated in a low yield. The formation of 27 is
rationalized by cyclization of the formed alcohol with the amide carbonyl, expulsion
of morpholine and subsequent reduction.
5
5
56
57
58
59
Being a privileged scaffold, 2-arylindoles display activities at a number of biological
6
20,30,31
targets,
and receptor potency and selectivity often arises from variation in the
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
32
structure of the substituent in the indole 3-position. Consequently, the final
structural modifications of antagonist 3 undertaken in this study was the alteration of
the substituent in the indole 3-position, trying to identify structural determinants for
receptor potency and selectivity. We modified the tertiary amine, the amide moiety,
and the length and shape/flexibility of the 3-substiutent, implementing a synthesis
route based on alkylation of suitable amines 30, 32 and 33 with alkyl chloride 8
14
15
16
17
18
(Schemes 6 and 7). While amine 33 was purchased, 30 and 32 were synthesized from
19
2
2
the corresponding N-Boc-protected amino acid via amide coupling and subsequent
2
23
Boc-deprotection (Scheme 6).
24
25
26
2
28
a
Scheme 6. Synthesis of amines 30 and 32 from carboxylic acids 29 and 31.
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
4
a
Reagents and conditions: (a) Boc O, 3.8 M aq NaOH, THF, 0 °C; (b) morpholine,
2
47
48
49
N,N-diisopropylethylamine, EDCI × HCl, DMAP, CH Cl , rt; (c) 4 M aq HCl, 1,4-
2
2
50
51
dioxane, rt.
52
53
5
5
56
Scheme 7. Synthesis of analogs 7, 34a and 34b via alkylation of secondary amines
57
5
a
3
0, 32 and 33 using alkyl chloride 8. R = H or alkyl; R = alkyl.
1 2
59
60
ACS Paragon Plus Environment

Page 13 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
a
Reagents and conditions: (a) NaI, NaHCO , MeCN, 60 °C.
3
26
27
28
29
3
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
The synthesis and isolation of analogs 7, 34a, and 34b proved challenging due to their
high polarity and closely eluting side products. However, ultimately all three ligands
were isolated in high purity in low to moderate yield. In addition, to analogs 7, 34a
and 34b, two commercially available analogs 35 and 36 (Scheme 7, >95% purity)
were purchased to deliver a focused library of 16 structurally similar analogs of
antagonist 3, covering chemical modifications in four structural regions; the indole
scaffold (4, 13b, 18), the 2-phenyl substituent (5, 13d-f, 25), the 3-carbonyl moiety
46
47
(6, 26, 28), and the substituent in the indole 3-position (7, 34a, 34b, 35, 36; Table 1).
48
49
50
51
52
Pharmacology. All compounds were tested on the mouse GPRC6A receptor using a
53
5
recently described time-resolved Förster resonance energy transfer (FRET) based
5
5
57
3
3
inositol monophosphate assay protocol. The reason for choosing the mouse rather
58
59
than the human GPRC6A ortholog was that the latter is not cell surface expressed or
60
11-13
functional in recombinant cell lines.
The Ile759 residue, shown to be important
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
20
for binding of compound 3, is conserved in both human, mouse, rat and goldfish
1
3
GPRC6A orthologs indicating the compound series will be active across species.
The compounds were tested up to concentrations of 400 µM, with exception of 13e,
which was limited by its solubility to 100 µM. Compounds 7, 13e and 34b, were
found to be more potent than 3 at the GPRC6A receptor (Table 1 and Fig. 3) and
compound 13a to be equipotent. Notably, compound 5, 18, 25, 26 and 28 were found
to posses none to little inhibitory activity at 400 µM. To examine the selectivity of the
new potent compounds, 7, 13e and 34b were tested on recombinant rat CaSR, human
2
23
5
-HT , native human M receptors expressed in Human Embryonic Kidney 293T
2C 3
24
25
(HEK-293T) cells and recombinant rat mGluR5 in Chinese Hamster Ovary (CHO)
26
2
28
cells (Table 2). Choosing these receptor subtypes and species enabled comparison
2
30
20
with our previous results on the compound 3 series.
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
1
00
50
0
compound 3
compound 34b
4
49
50
-7
-6
-5
-4
-3
log [Compound] (M)
51
52
53
5
5
56
57
58
59
Figure 3. Representative concentration-response curves showing the inhibitory effect
of compound 3 and 34b on the response elicited by 500 µM L-ornithine on HEK-
293T transfected with mouse GPRC6A receptor and GqG66D. The data was obtained
via a FRET-based inositol monophosphate assay. The response for each concentration
has been determined in triplicate and is shown as the mean±SD.
60
ACS Paragon Plus Environment

Page 15 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Pharmacological evaluation of lead compound 3 and the focused library for
in vitro antagonistic effect at the mouse GPRC6A receptor, in a FRET-based inositol
monophosphate assay.
1
1
12
13
14
15
16
17
18
19
2
2
2
23
24
25
26
27
28
29
30
31
3
3
3
35
36
37
38
39
40
41
42
4
4
45
46
Cmpd
Structure
pIC50
IC50
Relative
n
7
3
2
4
5
3
3
a
±
SEM
(µM) potency (%)
3
4
4.66 ±
21.9
39.8
>400
132
100
55
0
.05
4.40 ±
0
.06
-
c
5
< 6
17
b
6
3.88 ±
0.08
7
5.21 ±
6.17
66.1
21.3
355
33
0
.01
4.18 ±
.09
4.67 ±
.05
b
1
3b
0
47
48
4
50
51
52
13d
103
0
53
5
5
56
57
1
3e
5.03 ±
.15
9.33
235
4
0
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
2
2
2
23
24
25
26
27
28
2
30
31
3
3
34
35
36
37
b
1
3f
3.61 ±
248
9
4
2
0
.07
-
c
1
8
5
>400
< 6
c
2
-
-
-
>400
>400
>400
256
< 6
< 6
< 6
9
2
2
2
3
5
3
3
c
c
26
2
8
b
3
4a
3.59 ±
.04
5.26 ±
.03
4.24 ±
.04
3.94 ±
.06
0
3
4b
5.50
57.3
116
398
38
0
38
3
40
41
42
4
4
45
b
35
0
b
36
19
46
47
0
48
49
a
n = Number of independent experiments. The relative antagonistic potency of the
50
51
52
5
5
analogs in relation to lead compound 3 was calculated as IC (3)/IC (cmpd) × 100.
5
0
50
b
Due to low solubility/potency, the fitted concentration-response curve was obtained
5
56
57
58
c
by restraining lower-plateau to buffer control values. Little to no inhibition seen at
compound concentrations of 40 µM and 400 µM.
59
60
ACS Paragon Plus Environment

Page 17 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 2. Activity of compound 3, 7, 13e and 34b at the mouse GPRC6A receptor and
human 5-HT , rat CaSR, human M and rat mGluR5. All the receptors couple via a
2C
3
G mediated signaling pathway. A FRET-based assay was used to measure the
q
1
1
generation of accumulated inositol monophosphate from receptor activation, in the
12
1
14
2
+
presence of the following agonists: 500 µM L-ornithine, 1 µM serotonin, 20 mM Ca ,
15
16
1
mM carbachol and 100 µM L-glutamate for the mouse GPRC6A, human 5-HT , rat
2C
17
18
CaSR, human M and rat mGluR5 receptors, respectively.
3
19
20
2
2
23
24
2
Receptor
mouse
human
5-HT2C
rat
human
M3
rat
GPRC6A
CaSR
mGluR5
26
27
28
29
30
31
3
3
34
35
36
3
38
39
40
41
42
4
4
45
46
47
48
4
50
Cmpd
3
pIC50±SEM/IC50 (µM)/Independent experiments (n)
a
a
a
a
a
a
a
a
a
a
a
a
a
4.66±0.05
3.67±0.11
3.50±0.04
4.56±0.03
3.65±0.06
2
7
1.9
214
316
27.5
224
3
3
3
3
a
7
5.21±0.01
3.57±0.07
3.46±0.03
4.24±0.03
3.57±0.04
6
5
.17
269
347
58
269
3
3
3
3
1
3e
5.03±0.15
4.29±0.07
4.25±0.06
5.38±0.13
4.38±0.03
9
4
.33
51.3
56.2
4.17
41.7
3
3
7
3
5
52
a
3
4b
5.26±0.03
3.68±0.03
3.62±0.05
4.29±0.04
3.58±0.02
53
5
5
56
5
5
.50
209
3
240
3
51.3
3
263
3
57
58
59
a
Due to low solubility and potency, the bottom half of the concentration-response
60
curve was obtained by restraining lower-plateau to buffer level.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
Structure Activity Relationship. The data in Table 1 shows that the substitution pattern
on the indole scaffold has a large impact on receptor inhibition. Introducing
substituents in the indole 5-position causes 2-fold and 3-fold loss of activity for
analogs 4 and 13b respectively, with the bulkiest analog 18 being completely inactive
at the GPRC6A receptor. However, introducing substituents on the 2-phenyl group
appears advantageous for activity. 2-(4-Fluorophenyl)indole 13d retains the activity
and the electron-donating and bulkier methoxy-substituted analog 13e showed a 2-
fold increase in activity compared to 3. Switching to the smaller 2-furanyl-substituent
2
23
(13f) caused significant loss of activity and removal of the 2-substituent (5) or
24
25
introducing the polar hydroxymethyl group (25) rendered the analogs completely
26
2
28
inactive at the GPRC6A receptor. These findings support the previously proposed
2
30
20,32
hypothesis
that the privileged 2-phenylindole part of the ligands bind to a
31
3
3
34
35
36
37
38
relatively small and hydrophobic pocket in the GPCR transmembrane domain, clearly
demonstrates the importance of the 2-phenyl group as a prerequisite for ligand
activity, and provides incentives for exploring the effects of substitution on the 2-
phenylindole scaffold further.
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
Analogs 6, 26, and 28 were synthesized in order to evaluate the role of the ketone in
the indole 3-position. The delocalization of electrons from the indole nitrogen over
the carbonyl group in the indole 3-position is absent in analogs 6 and 26, which may
influence ligand hydrogen bonding at the indole nitrogen. Furthermore, analog 28 can
act as a hydrogen bond acceptor at the carbonyl group, whereas analog 26 features
both hydrogen bond donor and acceptor abilities and analog 6 lacks such abilities.
Noteworthy, reduction of 3 to give alcohol 26 or replacement with the shorter amide-
analog 28 results in complete loss of activity at the GPRC6A receptor while the 3-
alkylsubsituted analog 6 shows some activity (6-fold less than 3). These findings
57
58
59
60
ACS Paragon Plus Environment

Page 19 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
suggest that the ketone does not merely function as a hydrogen bond acceptor but that
delocalization of electrons from the indole nitrogen over the ketone carbonyl may also
be an important feature of the pharmacophore.
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
The last SAR sub study was to alter the substituent in the indole 3-position. Removal
of the N-methyl group (34a) resulted in more than a 10-fold loss of activity, while
rigidifying and extending the side chain by a single methylene (34b), or entirely
removing the amide carbonyl group (7) resulted in more than a 3-fold gain in potency.
Analogue 36, which lacks the morpholine oxygen atom, displays approximately 6-
fold lower potency at the GPRC6A receptor, some of which could be regained for the
cyclized analog 35 (3-fold less than 3). These results show that small structural
changes in the side chain at the indole 3-position can have a major influence on ligand
potency. The morpholine ring appears to be important for binding, possibly by
hydrogen bonding interactions with the receptor and we speculate that the structural
changes in analogs 7 and 34b could allow the morpholine moiety to be positioned
more favorably. In the case of ligand 7 removal of the amide carbonyl leads to
38
3
40
protonation of the nitrogen (morpholine pK ~ 8) and produces a more flexible side
a
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
chain, while structural constraints restricts ligand 34b into what appears to be a
biologically relevant conformation. Low energy conformations were indeed found in
which the 34b and 3 amide carbonyls overlay very well (data not shown). The
reduced analog 7 and the constrained analog 34b represent the two most potent leads,
and new hybrid ligands also incorporating other heterocycles will be explored in
future optimization. Importantly, it should be noted that ligand 34b was tested as a
racemic mixture and that likely there will be a difference in the potency between the
two enantiomers. Furthermore, in a follow-up study we have initiated work on a new
GPRC6A model based on the first Class C crystal structures, the metabotropic
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
34
35
mGluR1 and mGlu5 receptors, which will form the basis for a mutagenesis study
to further elucidate the molecular requirements for allosteric antagonistic activity.
The pharmacological evaluation of the focused set of analogs presented in Table 1,
identified 7, 13e, and 34b as the most potent antagonists at the GPRC6A receptor
reported to date. Antagonist 3 has previously been reported to display activity at the
muscarinic acetylcholine M and M receptors (class A GPCRs) and the mGluR5
14
15
16
1
3
17
18
2
0
receptor (a class C GPCR) in addition to the GPRC6A activity. This was confirmed
19
2
2
for the M receptor in the present study. Improving the selectivity of ligand 3 for the
3
2
23
24
25
26
27
28
29
GPRC6A receptor is critical for developing a useful pharmacological tool compound,
and it was the main objective of the present study to do so while also improving
potency. Consequently, the three most potent antagonists discovered in the present
study, 7, 13e and 34b, were tested for activity at the class A GPCRs 5-
30
31
3
hydroxytryptamine receptor 5-HT2C and muscarinic acetylcholine receptor M , as
3
3
34
35
well as the related class C GPCRs CaSR and mGluR5 receptor (Table 2). As
36
37
previously reported ligand 3 displays highest potency at M amongst these receptors,
3
38
3
40
which in the present study is close to equipotent with the GPRC6A receptor (Table 2).
41
42
Ligand 13e in which the side chain in the indole 3-position is unaltered displays even
4
4
poorer selectivity than ligand 3 being equipotent at M and GPRC6A and showing
3
45
4
47
moderate and similar potencies at the three remaining receptors. Gratifyingly, the two
48
49
remaining ligands 7 and 34b show a significantly improved selectivity profile. Both
50
5
ligand 7 and 34b display 9-fold selectivity for GPRC6A over the M receptor and
3
52
53
5
5
56
57
58
only show weak potencies at the remaining three receptors (>38-fold selectivity). To
assess the prospects of obtaining selectivity over the off-targets, we identified the
most similar ligands for the off-target receptors (5-HT , CaSR, M , and mGluR ), as
2
C
3
5
59
60
available in ChEMBL (SI, Table S1). Only the 5-HT ligand displays a complete 2-
2
C
ACS Paragon Plus Environment

Page 21 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
phenylindole scaffold and a high structural similarity (Tanimoto Similarity of 0.78).
However, it lacks the morpholinoethan-1-one moiety, which could explain the
selectivity we observe for GPRC6A. Given the privileged structure scaffold it is
evident that the antagonists could have activities at other GPCRs than those tested
here. Going forward it is thus our aim to first develop even more potent compounds
with nanomolar potency, and subsequently test them on a much broader set of
GPCRs.
14
15
16
17
18
19
20
2
2
23
Conclusion
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
We recently identified 3-substituted 2-phenylindoles 1-3 as the first allosteric
antagonists with preference for the GPRC6A receptor, and a medicinal chemistry
program was initiated aimed at developing potent and selective inhibitors for this
receptor. Such inhibitors would constitute valuable pharmacological tool compounds
for elucidating the physiological functions of the GPRC6A receptor. Antagonist 3 had
the best physicochemical profile and was selected as our lead compound for further
optimization. Herein, we report a profiling of the pharmacophore and a preliminary
structure-activity relationship study to improve the ligands selectivity towards the
GPRC6A receptor. Pharmacological evaluation of antagonistic effect at the GPRC6A
receptor was carried out for 16 new ligands that each had been modified
systematically in one of the four regions of the pharmacophore. We show that
substituted aryl groups in the indole 2-position and modifications in the structure of
the substituent in the indole 3-position improve the ligand antagonistic effect and
report antagonists 7, 13e and 34b as the most potent allosteric antagonists at the
GPRC6A receptor to date. Moreover, antagonists 7 and 34b display an improved
selectivity profile for the GPRC6A receptor over related class A and C GPCRs (>9-
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 54
1
2
3
4
5
6
7
8
9
1
1
12
13
fold). Thus, it is clear from the present study that it is indeed possible to improve the
selectivity for the GPRC6A receptor over related receptors that bear the same 2-
arylindole binding motif. Moreover, the present study has demonstrated that potency
can be increased by modifying the 2-arylindole scaffold (e.g. 13e) and that receptor
selectivity can be achieved by modification of the side chain at the indole 3-position
14
15
16
(e.g. 7 and 34b). These findings provide an important structural basis for further
17
18
19
20
ligand optimization and hold promise that antagonists for the GPRC6A receptor with
even higher pharmacological utility can be obtained in the near future.
2
2
23
24
25
Experimental section.
26
27
28
General procedure for amine alkylation (GP-Alkylation)
29
30
A microwave vial was charged with an alkyl chloride (1.0 equiv), NaHCO (1.8 to 3.0
3
31
3
3
34
35
36
37
38
39
40
41
42
4
4
equiv), NaI (0.2 to 0.3 equiv) and a free amine or amine hydrochloric salt (1.0 equiv).
Anhydrous MeCN was added and the vial was sealed and stirred at 60 °C in a heating
block until TLC indicated full conversion of the alkyl chloride (2-7.5 h). The solids
were removed by filtration, washed with MeCN and the combined organic phases
were concentrated in vacuo to afford the crude tertiary amine. Purification by column
chromatography (MeOH, aqueous NH in CH Cl or n-heptane in EtOAc) typically
3
2
2
45
4
47
gave a colorless film that was dissolved in CH Cl or Et O and concentrated in vacuo
2 2 2
48
49
50
51
to give an amorphous solid.
-(5-Fluoro-2-phenyl-1H-indol-3-yl)-2-(methyl(2-morpholino-2-
1
52
53
5
oxoethyl)amino)ethanone (4)
5
56
Synthesized according to the general procedure GP-Alkylation using indole 11a
57
5
(0.101 g, 0.35 mmol), NaI (0.010 g, 0.07 mmol), NaHCO (0.088 g, 1.05 mmol),
3
59
60
amine hydrochloride 12 (0.068 g, 0.35 mmol) and anhydrous MeCN (1.75 mL).
ACS Paragon Plus Environment

Page 23 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Stirred at 60 °C for 2 h 15 min. Purification by column chromatography (3% MeOH,
0
.2% aqueous NH in CH Cl , v/v) gave indole 4 (0.114 g, 80%) as a white,
3 2 2
amorphous solid. TLC R = 0.35 (3% MeOH, 0.2% aqueous NH in CH Cl , v/v);
f
3
2
2
1
1
1
HPLC t = 6.17 min (Method A); IR (neat) ν = 3196, 2856, 1630, 1446, 1114; H
R
max
12
1
14
3
4
NMR (400 MHz, DMSO-d ): δ 12.22 (br s, 1H; NH), 7.82 (dd, J = 10.5 Hz, J
=
6
HF
HH
15
16
2
.5 Hz, 1H; indole-H4), 7.64-7.60 (m, 2H; PhH), 7.58-7.55 (m, 3H; PhH), 7.43 (dd,
17
18
3
4
3
3
4
JHH = 9 Hz, J = 5 Hz, 1H; indole-H7), 7.09 (ddd, J = 9 Hz, J = 9 Hz, J =
HH
HF
HF
HH
19
2
2
2
23
2.5 Hz, 1H; indole-H6), 3.48-3.31 (m, 10H; 4 × morpholine-CH , Ar(C=O)CH ), 3.12
2 2
13
(
s, 2H; CH (C=O)N), 2.12 (s, 3H; CH ); C NMR (151 MHz, DMSO-d ): δ 194.2
2 3 6
24
2
1
(
ketone-C=O), 167.9 (amide-C=O), 158.5 (d, J = 233 Hz; CF), 145.9 (indole-C2),
CF
26
2
28
132.4 (Ph-C1), 132.1 (indole-C7a), 129.7 (Ph-C), 129.5 (Ph-C4), 128.5 (Ph-C), 127.5
2
30
3
4
3
(
d, J = 11 Hz, indole-C3a), 113.0 (d, J = 4 Hz; indole-C3), 112.9 (d, J = 10
CF CF CF
31
3
2
2
Hz; indole-C7), 111.0 (d, J = 26 Hz; indole-C6), 106.3 (d, J = 25 Hz; indole-
CF
CF
3
34
35
C4), 66.2, 66.1 ((CH ) O), 64.4 (Ar(C=O)CH ), 58.8 (CH (C=O)N), 45.3
2
2
2
2
3
37
+
(
(C=O)NCH ), 42.3 (CH ), 41.5 ((C=O)NCH ); HRMS (ESI+) m/z [M+H] Calcd for
2 3 2
38
3
40
+
C H FN O 410.1874, found 410.1857.
23 25 3 3
41
42
2
-((2-(1H-Indol-3-yl)-2-oxoethyl)(methyl)amino)-1-morpholinoethanone (5)
4
4
A microwave vial was charged with indole 11c (0.062 g, 0.32 mmol), NaI (0.010 g,
45
4
47
0.06 mmol), NaHCO (0.081 g, 0.96 mmol), amine hydrochloride 12 (0.062 g, 0.32
3
48
49
50
51
52
53
5
5
mmol), and acetone (1.5 mL). The vial was capped and heated at 60 °C for 2 h, then
allowed to cool. The mixture was filtered and the solids were washed with acetone.
The combined organic fractions were concentrated in vacuo to afford the crude
product as an orange film. Purification by column chromatography (4% MeOH, 0.3%
aqueous NH in CH Cl , v/v) gave indole 5 (0.063 g, 62%) as an off-white,
56
57
58
3
2
2
59
60
amorphous solid. TLC R = 0.3 (4% MeOH, 0.3% aqueous NH in CH Cl , v/v);
f
3
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 54
1
2
3
4
5
6
7
8
9
1
HPLC t = 5.34 min (A); IR (neat) ν = 3213, 2855, 1638, 1432; H NMR (400
R
max
MHz, DMSO-d ): δ 11.91 (br s, 1H; NH), 8.52 (d, J = 3 Hz, 1H; indole-H2), 8.18-
6
8
.16 (m, 1H; indole-H4), 7.48-7.45 (m, 1H; indole-H7), 7.23-7.15 (m, 2H; indole-H6,
1
1
H5), 3.72 (s, 2H; Ar(C=O)CH ), 3.61-3.59 (m, 2H; (C=O)NCH ) 3.56-3.53 (m, 4H;
2
2
12
1
14
15
(CH ) O), 3.45-3.43 (m, 2H; (C=O)NCH ), 3.37 (s, 2H; CH (C=O)N), 2.32 (s, 3H;
2 2 2 2
13
CH ); C NMR (100 MHz, DMSO-d ): δ 192.9 (ketone-C=O), 168.2 (amide-C=O),
3
6
16
17
18
1
36.2 (indole-C7a), 134.3 (indole-C2), 125.5 (indole-C3a), 122.7 (indole-C6), 121.7
19
20
2
(indole-C5), 121.2 (indole-C4), 114.9 (indole-C3), 112.0 (indole-C7), 66.3, 66.2
2
23
(
(CH ) O), 63.8 (Ar(C=O)CH ), 59.2 (CH (C=O)N), 45.5 ((C=O)NCH ), 42.6 (CH ),
2 2 2 2 2 3
24
2
+
+
4
1.6 ((C=O)NCH ); HRMS (ESI+) m/z [M+H] Calcd for C H N O 316.1656,
2 17 22 3 3
26
2
28
found 316.1667.
29
30
2
-(Methyl(2-morpholinoethyl)amino)-1-(2-phenyl-1H-indol-3-yl)ethan-1-one (7)
31
3
3
34
35
36
37
38
Synthesized according to GP-Alkylation using indole 8 (0.300 g, 1.11 mmol),
NaHCO3 (0.168 g, 2.00 mmol), NaI (0.045 g, 0.3 mmol), N-methyl-2-
morpholinoethan-1-amine 33 (0.160 g, 1.11 mmol) and MeCN (8 mL). The mixture
was stirred at 60 °C for 5 h. Purification by column chromatography (2.5% MeOH,
39
40
41
42
0
.2% aqueous NH in CH Cl , v/v) gave indole 7 (0.075 g, 18%) as an orange-colored
3 2 2
4
4
film. TLC R = 0.15 (4% MeOH, 0.3% aqueous NH in CH Cl , v/v); HPLC t = 5.85
f
3
2
2
R
45
4
47
1
min (A); IR (neat) νmax = 3177, 3062, 2954, 2805, 1637, 1449, 1115; H NMR (600
48
49
MHz, CDCl ): δ 8.43 (br s, 1H; NH), 8.31-8.29 (m, 1H; indole-H4), 7.57-7.55 (m,
3
50
51
52
53
2
7
H; Ph-H2, H2’), 7.52-7.49 (m, 3H; Ph-H3, H3’, H4), 7.40-7.37 (m, 1H; indole-H7),
.31-7.28 (m, 2H; indole-H6, H5), 3.62 (t, J = 5 Hz, 4H; ((CH ) O), 3.44 (s, 2H;
2
2
5
5
56
(
C=O)CH ), 2.56-2.54 (m, 2H; NCH CH ), 2.36-2.34 (m, 6H; (CH N(CH CH ) O),
2 2 2 2 2 2 2
57
5
13
2.25 (s, 3H; CH ); C NMR (DEPTQ) (150 MHz, CDCl ): δ 195.7 (C=O), 143.5
3 3
59
60
(indole-C2), 135.3 (indole-C7a), 133.1 (Ph-C1), 129.9 (Ph-C4), 129.7 (Ph-C2, C2’),
ACS Paragon Plus Environment

Page 25 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
29.0 (Ph-C3, C3’), 127.5 (indole-C3a), 123.8 (indole-C6), 122.8 (indole-C5), 122.5
(indole-C4), 114.6 (indole-C3), 110.9 (indole-C7), 67.0 ((CH ) O), 66.0 ((C=O)CH ),
2 2 2
5
7.0 (NCH CH ), 54.13 ((CH CH ) O), 54.07 (NCH CH ), 42.9 (CH ); HRMS
2 2 2 2 2 2 2 3
1
1
+
+
(
ESI+) m/z [M+H] Calcd for C H N NaO 400.1995, found 400.2005.
23 27 3 2
12
1
14
1-(5-Methoxy-2-phenyl-1H-indol-3-yl)-2-(methyl(2-morpholino-2-
15
16
oxoethyl)amino)ethanone (13b)
17
18
Synthesized according to the general procedure GP-Alkylation using indole 11b
19
2
2
(0.105 g, 0.35 mmol), NaI (0.010 g, 0.07 mmol), NaHCO (0.088 g, 1.05 mmol),
3
2
23
amine hydrochloride 12 (0.068 g, 0.35 mmol) and MeCN (1.75 mL). Stirred at 60 °C
24
2
for 3 h. Purification by column chromatography (3.5% MeOH, 0.3% aqueous NH in
3
26
2
28
CH Cl , v/v) gave indole 13b (0.058 g, 39%) as a light-yellow, amorphous solid.
2
2
29
30
TLC R = 0.2 (4% MeOH, 0.2% aqueous NH in CH Cl , v/v); HPLC t = 6.05 min
f
3
2
2
R
31
3
1
(
A); IR (neat) νmax = 3217, 3054, 2855, 1626, 1443, 1114; H NMR (400 MHz,
3
34
35
DMSO-d ): δ 11.97 (br s, 1H; NH), 7.63 (d, J = 2.5 Hz, 1H; indole-H4), 7.61-7.54 (m,
6
36
37
5
H; PhH), 7.32 (d, J = 9 Hz, 1H; indole-H7), 6.87 (dd, J = 9, 2.5 Hz, 1H; indole-H6),
38
39
40
41
3.80 (s, 3H; OCH ), 3.48-3.33 (m, 10H; 4 × morpholine-CH , Ar(C=O)CH ), 3.13 (s,
3
2
2
1
3
2
H; CH (C=O)N), 2.12 (s, 3H; NCH ); C NMR (150 MHz, DMSO-d ): δ 194.3
2 3 6
42
4
4
(ketone-C=O), 167.9 (amide-C=O), 155.3 (indole-C5), 144.6 (indole-C2), 132.9 (Ph-
45
4
47
48
49
C1), 130.4 (indole-C7a), 129.7 (Ph-C), 129.2 (Ph-C4), 128.5 (Ph-C), 127.8 (indole-
C3a), 112.8 (indole-C3), 112.6 (indole-C6), 112.4 (indole-C7), 103.2 (indole-C4),
50
5
6
6.2, 66.1 ((CH ) O), 64.4 (Ar(C=O)CH ), 58.9 (CH (C=O)N), 55.3 (OCH ), 45.3
2 2
2
2
3
52
53
5
+
(
(C=O)NCH ), 42.3 (NCH ), 41.5 ((C=O)NCH ); HRMS (ESI+) m/z [M+H] Calcd
2
3
2
5
56
+
for C H N O 422.2074, found 422.2070.
2
4
28
3
4
57
5
1-(2-(4-Fluorophenyl)-1H-indol-3-yl)-2-(methyl(2-morpholino-2-
59
60
oxoethyl)amino)ethan-1-one (13d)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 54
1
2
3
4
5
6
7
8
9
Synthesized according to GP-Alkylation using indole 11d (0.150 g, 0.50 mmol),
amine hydrochloride 12 (0.100 g, 0.52 mmol), NaHCO (0.11 g, 1.3 mmol), NaI
3
(0.020 g, 0.13 mmol) and MeCN (3 mL). The mixture was heated at 60 °C for 7.5 h.
1
1
12
13
14
15
16
17
DMSO (0.5 mL) was added and the mixture was stirred for an additional 30 minutes,
then allowed to cool to room temperature. The solids were removed by filtration and
washed with EtOAc (3 × 5 mL). The solution was washed with saturated aqueous
NaHCO (25 mL), brine (15 mL), dried over Na SO , filtered and concentrated in
3
2
4
18
19
2
2
vacuo to give the crude product as a yellow oil. Purification by column
2
23
chromatography (1% MeOH, 0.1% aqueous NH in EtOAc, v/v) gave indole 13d
3
24
2
(
0.055 g, 26%) as a white, amorphous solid. TLC R = 0.3 (1% MeOH, 0.1% aqueous
f
26
2
28
1
NH in EtOAc, v/v). HPLC t = 6.11 min (A); IR (neat) ν = 3215, 1639, 1439; H
3
R
max
29
30
NMR (400 MHz, DMSO-d ): δ 12.11 (s, 1H; NH), 8.09-8.06 (m, 1H; indole-H4),
6
31
3
7
.70-7.66 (m, 2H; Ph-H2, H2’), 7.44-7.37 (m, 3H; indole-H7, Ph-H3, H3’), 7.25-7.18
3
34
35
(
m, 2H; indole-H6, H5), 3.48-3.43 (m, 10H; N(CH CH ) O, Ar(C=O)CH ), 3.16 (s,
2
2 2
2
3
37
13
2
H; CH (C=O)N), 2.17 (s, 3H; CH ); C NMR (100 MHz, DMSO-d ) δ 194.2
2 3 6
38
3
40
41
1
(ketone-C=O), 167.9 (amide-C=O), 162.6 (d, J = 245 Hz; Ph-C4), 143.1 (indole-
CF
3
C2), 135.4 (indole-C7a), 132.0 (d, J = 8 Hz; Ph-C2, C2’), 129.1 (Ph-C1), 126.7
CF
42
4
4
2
(
indole-C3a), 122.8 (indole-C6), 121.7 (indole-C5), 121.3 (indole-C4), 115.4 (d, J
CF
45
4
47
= 22 Hz; Ph-C3, C3’), 112.9 (indole-C3), 111.7 (indole-C7), 66.16, 66.12 ((CH ) O),
2 2
48
49
6
4.8 (Ar(C=O)CH ), 58.9 (CH (C=O)N), 45.4 ((C=O)NCH ), 42.4 (CH ), 41.5
2 2 2 3
50
5
+
+
(
(C=O)NCH ). HRMS (ESI+) m/z [M+H] Calcd for C H FN O 410.1874, found
2 23 25 3 3
52
53
5
5
56
4
1
10.1862.
-(2-(4-Methoxyphenyl)-1H-indol-3-yl)-2-(methyl(2-morpholino-2-
57
5
oxoethyl)amino)ethan-1-one (13e)
59
60
Synthesized according to GP-Alkylation using indole 11e (0.060 g, 0.20 mmol),
ACS Paragon Plus Environment

Page 27 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
NaHCO (0.050 g, 0.60 mmol), NaI (0.006 g, 0.04 mmol), amine hydrochloride 12
3
(0.039 g, 0.20 mmol) and MeCN (2 mL). Stirred at 60 °C for 3.5 h. Purification by
column chromatography (5% MeOH in CH Cl , v/v) gave indole 13e as a white solid.
2
2
1
1
12
13
Drying on a freeze-dryer caused contamination and partial protonation of the amine,
but basic work-up using CH Cl (75 mL), saturated aqueous NaHCO (50 mL) and
2
2
3
14
15
16
drying over Na SO , filtration and concentration in vacuo gave the free amine as a
2
4
17
18
white solid. Dispersion and sonication of the solid in diethyl ether removed residual
19
2
2
CH Cl and gave indole 13e (0.035 g, 42%) as a white solid. TLC R = 0.35 (5%
2
2
f
2
23
MeOH in CH Cl , v/v); HPLC t = 6.11 min (A); IR (neat) ν = 3237, 2870, 1631,
2
2
R
max
24
2
1
1
434; H NMR (600 MHz, DMSO-d ): δ 11.98 (s, 1H; NH), 8.09-8.06 (m, 1H;
6
26
2
28
29
30
indole-H4), 7.57-7.54 (m, 2H; Ph-H2, H2’), 7.42-7.40 (m, 1H; indole-H7), 7.22-7.17
m, 2H; indole-H6, H5), 7.13-7.09 (m, 2H; Ph-H3, H3’), 3.85 (s, 3H; OCH ), 3.47-
(
3
31
3
3
.33 (m, 10H; 4 × morpholine-CH , Ar(C=O)CH ), 3.16 (s, 2H; CH (C=O)N), 2.15
2 2 2
3
34
35
13
(
s, 3H; NCH3); C NMR (150 MHz, DMSO-d ): δ 194.5 (ketone-C=O), 167.9
6
36
37
(amide-C=O), 160.1 (Ph-C4), 144.3 (indole-C2), 135.4 (indole-C7a), 131.1 (Ph-C2,
38
3
40
C2’), 127.0 (indole-C3a), 124.7 (Ph-C1), 122.6 (indole-C6), 121.6 (indole-C5), 121.2
41
42
4
4
(
indole-C4), 113.9 (Ph-C3, C3’), 112.6 (indole-C3), 111.5 (indole-C7), 66.2, 66.1
(CH ) O), 64.6 (Ar(C=O)CH ), 59.0 (CH (C=O)N), 55.3 (OCH ), 45.3
(
2
2
2
2
3
45
4
47
+
((C=O)NCH ), 42.3 (NCH ), 41.5 ((C=O)NCH ); HRMS (ESI+) m/z [M+H] Calcd
2 3 2
4
49
+
for C H N O 422.2074, found 422.2072.
2
4
28
3
4
50
51
1
-(2-(Furan-2-yl)-1H-indol-3-yl)-2-(methyl(2-morpholino-2-
52
53
5
oxoethyl)amino)ethanone (13f)
5
56
Synthesized according to the general procedure GP-Alkylation using indole 11f
57
5
(0.085 g, 0.33 mmol), NaI (0.010 g, 0.065 mmol), NaHCO (0.082 g, 0.98 mmol),
3
59
60
amine hydrochloride 12 (0.064 g, 0.33 mmol) and anhydrous MeCN (2.0 mL). Stirred
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 54
1
2
3
4
5
6
7
8
9
at 60 °C for 2 h. Purification by column chromatography (2% MeOH, 0.2% aqueous
NH to 4% MeOH, 0.3% aqueous NH in CH Cl , v/v) gave indole 13f (0.031 g, 25%)
3
3
2
2
as a light-brown, amorphous solid. TLC R = 0.2 (2% MeOH, 0.2% aqueous NH in
f
3
1
1
1
CH Cl , v/v); HPLC t = 5.83 min (A); IR (neat) ν = 3225, 1630, 1420, 1113; H
2
2
R
max
12
1
14
NMR (600 MHz, DMSO-d ): δ 12.30 (br s, 1H; NH), 7.97 (dd, J = 2, 0.5 Hz, 1H;
6
15
16
17
18
19
20
2
2
furan-H5), 7.91 (d, J = 8 Hz, 1H; indole-H4), 7.58 (dd, J = 3.5, 0.5 Hz, 1H; furan-
H3), 7.50 (ddd, J = 8, 1, 0.5 Hz, 1H; indole-H7), 7.24 (ddd, J = 8, 6.5, 1 Hz, 1H;
indole-H6), 7.20 (ddd, J = 8, 6.5, 1 Hz, 1H; indole-H5), 6.74 (dd, J = 3, 2 Hz, 1H;
furan-H4), 3.87 (s, 2H; Ar(C=O)CH ); 3.48-3.45 (m, 4H; (C=O)NCH , CH O), 3.37-
2
2
2
23
24
2
3
.36 (m, 4H; (C=O)NCH , CH O), 3.30 (br s, 2H; CH (C=O)N), 2.32 (s, 3H; CH );
2 2 2 3
26
2
1
3
C NMR (150 MHz, DMSO-d ): δ 193.8 (ketone-C=O), 168.0 (amide-C=O), 145.6
6
28
29
30
(furan-C2), 144.4 (furan-C5), 135.6 (indole-C7a), 132.5 (indole-C2), 125.9 (indole-
31
3
3
34
C3a), 123.0 (indole-C6), 121.7 (indole-C5), 121.2 (indole-C4), 113.6 (furan-C3),
12.4 (furan-C4), 112.1 (indole-C7), 111.9 (indole-C3), 66.2, 66.1 ((CH ) O), 65.8
1
2
2
35
36
37
(
Ar(C=O)CH ), 59.3 CH (C=O)N), 45.4 ((C=O)NCH ), 42.4 (CH ), 41.5
2 2 2 3
38
3
40
+
+
((C=O)NCH ); HRMS (ESI+) m/z [M+H] Calcd for C H N O 382.1761, found
2 21 24 3 4
41
42
4
4
3
82.1749.
-(4-Aminophenyl)-N,N-dimethylacetamide (15)
2
45
4
47
Acid chloride formation: 2-(4-Nitrophenyl)-acetic acid (3.00 g, 16.6 mmol) was
48
49
dispersed in anhydrous CH Cl (55 mL). The mixture was cooled to 0 °C using an
2
2
50
51
52
53
5
5
56
57
ice-water bath and oxalyl chloride (1.42 mL, 21.5 mmol) was added followed by 5
drops of DMF. After 5 min the cooling bath was removed and the mixture was stirred
at ambient temperature for 2 h 15 min. The solvent was removed under reduced
pressure and the residue was re-dissolved in CH Cl and concentrated in vacuo once
58
2
2
59
60
ACS Paragon Plus Environment

Page 29 of 54
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
more to afford the crude acid chloride as a yellow oil. The crude product was used
without further purification.
Amide formation: The crude acid chloride was dissolved in anhydrous THF (50 mL)
and the mixture was cooled to 0 °C using an ice-water bath. Commercial 7.9 M
aqueous dimethylamine (5.2 mL, 41.1 mmol) was added in portions over 5 min and
the mixture was stirred for another 5 min. The cooling bath was removed and the
mixture was stirred at ambient temperature over night. After stirring for 16 h in total
the mixture was concentrated in vacuo and the resulting crude orange solid was taken
up in sulfate buffer (150 mL) and EtOAc (50 mL) and transferred to a separation
funnel. The phases were separated and the aqueous phase was extracted with EtOAc
(3 × 30 mL). The combined organic phases were washed with saturated aqueous
NaHCO (2 × 60 mL), brine (60 mL), dried (Na SO ), filtered and concentrated in
2
23
24
25
26
27
28
29
3
2
4
30
31
3
3
34
vacuo to give the crude amide (2.15 g) as a light-yellow solid. The crude amide was
used without further purification.
35
36
37
38
39
Reduction: The crude amide (2.15 g, 10.3 mmol) was dissolved in anhydrous EtOH
(50 mL) under nitrogen and the mixture was cooled to 0 °C using an ice-water bath.
40
41
42
1
0 wt% Pd/C (1.10 g, 1.03 mmol Pd) was added and the flask was pump-filled with
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
hydrogen gas. The cooling bath was removed and the mixture was stirred vigorously
under hydrogen for 3 h. The reaction mixture was filtered through a plug of Celite and
the solids were washed with MeOH (75 mL). The combined organic phases were
concentrated in vacuo to afford the crude product as an orange solid. EtOAc (40 mL)
was added and the mixture was heated to reflux. The hot solution was filtered through
a filter paper to remove insoluble solids, and the solution was concentrated in vacuo.
Recrystallization of the afforded solid from EtOAc/n-heptane gave aniline 15 (1.50 g,
59
60
5
1% over 3 steps) as white needles in two crops. TLC R = 0.1 (20% n-heptane in
f
ACS Paragon Plus Environment