Identification and SAR of Glycine Benzamides as Potent Agonists for the GPR139 Receptor

Article abstract of DOI:10.1021/acsmedchemlett.5b00247

A focused high throughput screening for GPR139 was completed for a select 100K compounds, and new agonist leads were identified. Subsequent analysis and structure-activity relationship studies identified (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl

Full text of DOI:10.1021/acsmedchemlett.5b00247

Letter
pubs.acs.org/acsmedchemlett
Identification and SAR of Glycine Benzamides as Potent Agonists for
the GPR139 Receptor
Curt A. Dvorak,* Heather Coate, Diane Nepomuceno, Michelle Wennerholm, Chester Kuei, Brian Lord,
David Woody, Pascal Bonaventure, Changlu Liu, Timothy Lovenberg, and Nicholas I. Carruthers
Janssen Research & Development, LLC, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: A focused high throughput screening for GPR139 was completed for a select 100K
compounds, and new agonist leads were identified. Subsequent analysis and structure−activity
relationship studies identified (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl)benzamide 7c
as a potent and selective agonist of hGPR139 with an EC50 = 16 nM. The compound was found to
cross the blood−brain barrier and have good drug-like properties amenable for oral dosing in rat.
KEYWORDS: Orphan receptors, GPR139, agonists, glycine amides
G-protein coupled receptors (GPCRs) make for compelling
drug targets within discovery research. Indeed, nearly one-third
of all approved drugs on the market today target this type of
receptor. The sequencing of the human genome has revealed
thousands of genes, many of which encode G-protein coupled
receptors, offering many new opportunities for novel therapies
for unmet medical needs. However, these new GPCRs were
identified based solely on their sequence homology to known
GPCRs and their corresponding ligands, and thus the biological
relevance of these new signaling pathways remains to be
determined. Without the ligands for these orphan receptors, it
is difficult to understand their physiological role in mammalian
systems.
expression in the brain and high degree of sequence homology
across many different species suggests that it could play an
important role in vertebrate physiology.
In searching for exogenous ligands to explore the function of
GPR139, Shi et al.7 have reported on a series of
benzohydrazides as potent and selective agonists for the
receptor. They were able to elegantly demonstrate that removal
of one of the hydrogen bond donors found in 1, thereby
reducing the overall TPSA and lowering the MDCK ratio,
resulted in an improved brain to plasma ratio for 2 (b/p 0.03 vs
2.8). In addition, Hu et al.8 have also described surrogate
ligands, both agonists and antagonists, for GPR139. Isberg et
al.6 utilized a pharmacophore model of 1 to identify and screen
a set of aromatic dipeptides as potential ligands for GPR139.
More recently, Wang et al.9 have reportedly identified several
new GPR139 antagonists after screening a 16K set of diverse
small molecules.
Our own interest to better understand the pharmacology and
physiology of GPR139 led to a focused screening campaign of
our 100K diversity set to identify potential small molecule
ligands for the receptor. The agonist hit that was selected to
investigate is shown in Scheme 1. The hit displayed some very
similar functional group characteristics to the known agonist 1.
Two carbonyls were present in a 1,4 relationship, separated by
two atoms, accompanied by a large flanking hydrophobic
surface. In analyzing the structure of 3 the sulfur atom was
considered to be a liability. Not only is the sulfur easily
oxidized, but the carbonyl of the thioester is reactive toward
nucleophilic attack and therefore replaced with the more stable
corresponding amide seen in 4 and 7h. Both enantiomers of
alpha methyl benzyl amine are readily available, and
consequently, it was straightforward to establish if there was a
Figure 1. Structure of GPR139 agonists.
GPR139 is an orphan G-protein coupled receptor that is
predominantly expressed in the brain.1−3 Human expression of
GPR139 can be found in the habenula, lateral caudate nucleus,
and parts of the hypothalamus. The receptor is coupled with
Gq signaling and appears to be constitutively active when
recombinantly expressed in mammalian cells.3 The closest
homologue to GPR139 is GPR142, which is highly expressed in
the pancreas and a potential target for the treatment of type II
diabetes. Yu et al.4 have reported on a series of phenylalanine
base agonists for GPR142 and specified that L-Trp has been
identified as the corresponding endogenous ligand. There have
also been reports of L-Trp and L-Phe being able to activate
GPR139 thus suggesting that putative physiological ligands for
GPR139 may have been identified.5,6 GPR139s’ predominant
Received: June 17, 2015
Accepted: July 20, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00247
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ACS Medicinal Chemistry Letters
Letter
Scheme 1. High Throughput Screening Lead and Initial
Optimization
Table 1. Effect of Linker on Human GPR139 Potency
a
b
Cmpd
linker
EC50 (nM)
33
Emax (%)
7h
10
11
12
13
Gly
7
146
143
88
L-Ala
D-Ala
L-Ser
L-Phe
66 17
440 34
283 46
10 000
107
ND
stereochemical preference in the observed activity. Making
these simple changes and incorporating the known key binding
element of a 3-methoxy into the benzoyl moiety provided 7h,
representing a suitable launching point for chemistry to explore
these glycine amides. In the present report we wish to describe
the synthesis, structure−activity relationship (SAR), and
characterization of a new series of glycine benzamides as
potent agonists for the GPR139 receptor.
a
Data are reported as mean SEM from at least three experiments.
The efficacy of L-Trp was set to 100%.
b
molecule constant. The glycine linker was found to provide
excellent potency at hGPR139 with an EC50 = 33 nM.
Following with that of L-alanine suggested small groups may be
allowed in this region of the template. D-Alanine was found to
be 4-fold less potent then L-alanine and a preference for the
natural configuration was observed. L-Serine was about as
potent as D-Ala suggesting that neither linker could achieve the
preferred conformation required for efficient binding. The use
of L-Phe in the central linker, as found in select GPR142
agonists, was not tolerated. Larger groups in this area gave a
pronounced reduction in potency at GPR139.
Compounds 7−9 were prepared as depicted in Scheme 2.
Commercially available (S)-alpha methyl benzyl amine was
a
Scheme 2. Synthesis of Compounds 7−9
Table 2 contains SARs obtained by exploring substituents
around the aryl of the benzamide while holding glycine and (S)
Table 2. Effect of R1 on Human GPR139 Potency
a
Reagents and conditions: (a) Boc-Gly-OH, EDCI, HOBt, iPr2NEt,
a
b
DCM; (b) 4 M HCl, dioxane; (c) R1ArCOCl, Et3N, DCM; (d) Boc-
NHCH2CHO, NaBH(OAc)3, DCM; (e) 4 M HCl, dioxane; (f) 3-
chlorobenzoyl chloride, Et3N, DCM; (g) 3-chlorobenzaldehyde,
NaBH(OAc)3, DCM.
Cmpd
R1
EC50 (nM)
Emax (%)
7a
7b
7c
7d
7e
7f
H
162 30
150 23
130
120
138
117
137
129
118
146
134
129
113
106
139
119
124
113
149
ND
ND
125
2-Cl
3-Cl
4-Cl
16
118 49
24
2
coupled to Boc glycine, using EDCI and Hunig’s base with
added HOBt. The protecting group on the amino acid nitrogen
was then removed with 4 M HCl in dioxane to provide key
intermediate 6.10 Treatment of 6 with the corresponding acid
chlorides in DCM with triethylamine as the base thus provided
compounds 7a−q.
In addition, intermediate 6 was treated with 3-chlorobenzal-
dehyde and sodium triacetoxyborohydride via reductive
amination11 to give compound 9 with the benzamide carbonyl
removed. (S)-Alpha methyl benzyl amine was also treated with
N-Boc-2-aminoacetaldehyde and sodium triacetoxyborohydride
to give the two carbon extension with the distal nitrogen
protected. The Boc group was then removed with 4 M HCl in
dioxane and the resulting amine coupled with 3-chlorobenzoyl
chloride in DCM with triethylamine to obtain compound 8
with the glycine carbonyl removed. Compounds 10−13 found
in Table 1 were prepared in a similar fashion to compounds
7a−q shown in Scheme 2. These modifications to the linker
portion of the template were carried out by starting with the
corresponding Boc-protected amino acids alanine, serine, and
phenyl glycine, respectively (only glycine shown).
3-Me
5
3-CN
59 13
54 11
7g
7h
7i
3-F
3-MeO
3-CF3
33
7
52 17
46 22
52 21
193 76
40 14
7j
3-OCF3
2,3-diCl
3,4-diCl
2,5-diCl
3,5-diCl
3-Cl,5-F
3-Br, 5-Cl
3,5-diMeO
7k
7l
7m
7n
7o
7p
7q
8
46
4
24 22
78
32
8
7
>10 000
>10 000
43
9
1
a
Data are reported as mean SEM from at least three experiments.
b
The efficacy of L-Trp was set to 100%.
alpha methyl benzyl amine constant. A scan at the ortho, meta,
and para positions of the aryl benzamide revealed that the
addition of a chloro atom anywhere on the ring (7b−d)
resulted in an increase or comparable activity to that of the
parent phenyl 7a. A clear preference was observed for a chloro
The central linker was explored with a small select set of
amino acids: Gly, Ala, Ser, and Phe. Table 1 displays the
potency at the human GPR139 receptor of these modifications
while holding the 3-methoxy benzamide portion of the
B
DOI: 10.1021/acsmedchemlett.5b00247
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Table 3. In Vitro ADME Assessment of Compounds 7h and 7c
a
b
c
d
e
f
Cmpd
hER
rER
MDCK Papp (1 × 10−6 cm/s)
MDCK ratio (BA/AB)
hPPB (%free)
rPPB (%free)
rBB (%free)
7h
7c
0.3
0.3
0.8
16
32
5.5
1.7
14
10
36
25
16
9
0.4
a
b
Human extraction ratio. Rat extraction ratio. Extraction ratios are calculated as in vitro CL/Q where Q is species specific hepatic blood flow.
c
d
e
f
MDR1-MDCK from Madin Darby canine kidney cells. Human plasma protein binding. Rat plasma protein binding. Rat brain tissue binding.
Table 4. Pharmacokinetics of Compound 7c in the Rat
a
b
c
d
Cmpd
%F
Cmax (ng/mL)
Cl (mL/min/kg)
t1/2 (h)
Vss (L/kg)
AUC (h·ng/mL)
b/p (rat)
7c
49
317
53
2.5
2.1
683
1.2
a
b
c
d
Clearance. The iv half-life. Volume of distribution at steady state. Ratio of brain to plasma conc.
positioned meta (7c) to the carbonyl, which gave excellent
potency with a hGPR139 EC50 = 16 nM. The meta position
was then explored further with both electron withdrawing and
electron donating groups. All of the groups examined were
found to be tolerated, and there was a clear preference for
lipophilic groups. The methyl 7e was found to be nearly as
potent as the chloro analogue 7c (EC50 16 nM vs 24 nM) and
the trifluoromethoxy 7j slightly less active at hGPR139. The
results of this screen led to the discovery of (S)-3-chloro-N-(2-
oxo-2-((1-phenylethyl)amino)ethyl)benzamide (7c), which to
our knowledge is one of the most potent compounds assayed
for the GPR139 receptor to date.
any extent providing for a selective GPR139 agonist. We then
examined the pharmacokinetics of compound 7c in the rat to
determine if observed in vitro properties translated in vivo. The
parameters from the study (1 mg/kg iv; 5 mg/kg po) are
shown in Table 4. The IV clearance (53 mL/min/kg) was
found to be higher than was predicted in vitro, which was
attributed to the lack of amidases in the microsomal assay. Very
good systemic exposure was achieved with an oral dose giving
an observed Cmax of 317 ng/mL (∼1 μM). More importantly,
the compound was able to cross the blood−brain barrier (BBB)
with a brain to plasma ratio (b/p) of 1.2, thus providing
excellent coverage into the brain compartment.
The impact of removing either carbonyl group was also
examined. The effects on GPR139 binding suggest that each
carbonyl engages in hydrogen bond interactions with the
receptor protein. Indeed, deletions of either carbonyl
completely abolished GPR139 potency (8 and 9). The
corresponding enantiomer of 7c was also prepared and found
to be significantly less active at human GPR139 (R enantiomer
EC50 = 2.1 μM; structure not shown).
Compound 7c was then profiled in more detail. A
comparison of the in vitro ADME data from the initial lead
(7h) and compound 7c is shown in Table 3. Both compounds
were found to have good extraction ratios in human
microsomes. Extraction ratios are a measure of first pass
metabolism representing the fraction of compound that is
eliminated from blood as it travels through the liver.
Compound 7h was found to have a much higher extraction
ratio in rat microsomes therefore suggesting that it is
metabolically less stable in this species. Both compounds
displayed good permeability in the MDCK assay, but
compound 7h was found to have a potential efflux issue as
determined by the higher basal to apical ratio (5.5 vs 1.7) as
compared to 7c. Compounds 7h and 7c were found to be
highly soluble at both pH 2 and pH 7 in aqueous media (>35
μM) with no CYP450 inhibition, suggesting no potential drug−
drug interaction (DDI) issues. Low protein binding provided
for very large drug free fractions in both the plasma and brain
for each compound. The initial lead displayed fairly good
physical properties representing an excellent starting point for a
medicinal chemistry program. Subsequently, it was discovered
that by replacing the 3-OMe found in compound 7h with 3-Cl
not only improved the rat extraction ratio (rER 0.8 vs rER 0.4)
but also lowered the efflux potential.
Figure 2. Total and non-specific binding of [3H]-7c.
Compound 7p was then utilized to prepare [3H]-7c (24.7
Ci/mmol) via reduction of the bromide with tritium through a
contract with Moravek Biochemicals (Brea, CA). The radio-
chemical purity of [3H]-7c was determined to be 99.1% by
HPLC analysis with radioactive flow detection. Total binding of
[3H]-7c (@10 nM) was carried out in CHO-TRex cells stably
expressing the human GPR139 receptor. Nonspecific binding
was obtained by blocking with 10 μM 1. The compound was
found to have good specific binding in CHO-TRex cells
providing an additional tool for exploring the GPR139 receptor.
We have identified a new series glycine benzamides as
agonists for GPR139. The SAR studies that were carried out led
to the discovery of compound 7c, JNJ-63533054, with an EC50
= 16 nM (138% of max). Furthermore, the compound was
found to have good drug-like properties. Compound 7c exhibits
good stability in both human and rat microsomes and high
solubility in aqueous media, and no DDI potential was found.
Additionally, MDCK assay results indicated good cellular
permeability with no efflux potential. This is especially desirable
since the receptor is located in the CNS. These physical
chemical properties then provided for very good pharmacoki-
netics in the rat. Oral dosing in rat (49%F) provided excellent
Compound 7c was found to be clean of any cross reactivity
as judged by an external selectivity panel (Eurofins Scientific) of
50 known GPCRs, ion channels, and transporters as well as our
own internal whole cell lead generation biology selectivity
panel. In addition, compound 7c does not activate rGPR142 to
C
DOI: 10.1021/acsmedchemlett.5b00247
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ACS Medicinal Chemistry Letters
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exposure both systemically and within the CNS. We also
prepared [3H]-7c to enable the development of binding and
occupancy assays providing very useful tools for exploring
GPR139. Further studies to deepen our understanding of the
function and pharmacology of GPR139 are ongoing.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the synthesis of all compounds,
compound characterization, description of the GPR139 assay,
and ADME/PK protocols. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acsmedchemlett.5b00247.
AUTHOR INFORMATION
Corresponding Author
*E-mail: cdvorak@its.jnj.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Kevin Coe and Discovery
Sciences for ADME support and Mark Seierstad for CADD and
informatics support. C.A.D. would also like to thank Christine
Gelin for help in the editing of this manuscript.
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D
DOI: 10.1021/acsmedchemlett.5b00247
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX