Radiosynthesis and characterisation of a potent and selective GPR139 agonist radioligand

Article abstract of DOI:10.1039/c5ra21326f

Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC₅₀ = 39 nM). In this study, we describe the synthesis, radiolabelling and in vitro evaluation of [³H]-1 for the characterisation of GPR139 and its spatial expression in the brain using autoradiography. Two different synthesis routes for the radiolabelling of 1 based on a reductive debromination strategy were investigated using deuterium (D₂, g). The route based on reductive debromination of the bromonaphthyl precursor 5 proved superior over arylbromide 4 and was employed for the radiolabelling experiments. Reductive debromination of precursor 5 was accomplished using ³H₂, Pd/C and triethylamine in DMF at ambient temperature to give target molecule [³H]-1 with a specific activity of 19.3 Ci mmol^-1 and a radiochemical purity of ≥95%. By application of autoradiography and binding studies, it was not possible to discriminate [³H]-1 binding to wildtype mice brains from GPR139 knockout mice brains and total binding from non-specific binding in CHO-k1 cells stably expressing human GPR139 receptor. Based on these experiments we conclude that [³H]-1 is not a suitable radioligand for the characterisation of GPR139.

Full text of DOI:10.1039/c5ra21326f

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Radiosynthesis and characterisation of a potent
and selective GPR139 agonist radioligand†
Cite this: RSC Adv., 2016, 6, 947
Sebastiaan Kuhne,‡ab Anne Cathrine Nøhr,‡a Ales Marek,c Tomas Elbert,c
ˇ
´ˇ
Anders Bue Klein,a Hans Brauner-Osborne,a Petrine Wellendorpha
¨
a
*
and Daniel Sejer Pedersen
Compound 1 is a selective and potent agonist of the G protein-coupled receptor GPR139 (EC50 ¼ 39 nM). In
this study, we describe the synthesis, radiolabelling and in vitro evaluation of [3H]-1 for the characterisation
of GPR139 and its spatial expression in the brain using autoradiography. Two different synthesis routes for
the radiolabelling of 1 based on a reductive debromination strategy were investigated using deuterium (D2,
g). The route based on reductive debromination of the bromonaphthyl precursor 5 proved superior over
arylbromide 4 and was employed for the radiolabelling experiments. Reductive debromination of
precursor 5 was accomplished using 3H2, Pd/C and triethylamine in DMF at ambient temperature to give
target molecule [3H]-1 with a specific activity of 19.3 Ci mmolꢀ1 and a radiochemical purity of $95%. By
application of autoradiography and binding studies, it was not possible to discriminate [3H]-1 binding to
wildtype mice brains from GPR139 knockout mice brains and total binding from non-specific binding in
CHO-k1 cells stably expressing human GPR139 receptor. Based on these experiments we conclude that
[3H]-1 is not a suitable radioligand for the characterisation of GPR139.
Received 14th October 2015
Accepted 10th December 2015
DOI: 10.1039/c5ra21326f
www.rsc.org/advances
et al.7 followed up by a structure-activity study identied 1 as
a potent GPR139 agonist. In addition, an agonist (LP-360924)
Introduction
and two antagonists (LP-471756 and LP-114958) were identi-
ed in a high-throughput screen by Hu et al.8 Recently, we have
suggested that L-tryptophan (L-Trp) and L-phenylalanine (L-Phe)
may be the endogenous agonists for GPR139,9 using a pharma-
cophore model based on the ligands described by Shi et al.7
During the preparation of this manuscript, scientists at Janssen
R&D conrmed our hypothesis that L-Phe and L-Trp are poten-
tial endogenous agonists and a surrogate agonist JNJ-63533054
and other analogues were disclosed.4,10 Moreover, Wang et al.
recently reported ve antagonists obtained from a screening of
16 000 compounds.11
In an effort to further characterise GPR139 and to deter-
mine its anatomical distribution a suitable radioligand was
required. Binding experiments with a radioligand together
with site-directed mutagenesis studies can be used to reveal
key amino acid residues involved in ligand binding, identify
novel receptor ligands and to determine binding kinetics.
Moreover, autoradiography studies can reveal the spatial
expression of proteins (e.g. GPR139) in the brain, which can
give indications of receptor physiology and function. Ligand 1
is a potent GPR139 agonist that has been screened against
a panel of 90 different receptors, enzymes, transporters and
ion channels with no observed cross reactivity.7 Based on the
high potency and in particular, excellent selectivity, 1 was
selected as our candidate for development of a GPR139
radioligand.
G protein-coupled receptors (GPCRs) constitute one of the
largest protein families in the human proteome,1 targeted by
approximately 30% of currently marketed drugs.2 GPCRs have
been the focus of extensive studies for decades, but still many
GPCRs remain for which the endogenous ligands are unknown
(orphan receptors). The unravelling and characterisation of
orphan receptors is of utmost importance to determine their
physiological role and evaluate their potential as targets for
drug development. GPR139 is an orphan class A GPCR.3 GPR139
mRNA is exclusively expressed in the CNS in particular in
striatum and hypothalamus, and it has been suggested that
GPR139 plays a role in the modulation of locomotor activity or
food intake.4–6 Several ligands with different chemotypes have
been reported for GPR139 (Fig. 1). A screening campaign by Shi
aDepartment of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Denmark. E-mail: daniel.pedersen@sund.ku.dk
bAmsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of
Medicinal Chemistry, Faculty of Science, VU University Amsterdam, De Boelelaan
1083, 1081 HV Amsterdam, The Netherlands
cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech
Republic, 16610, Prague 6, Czech Republic
† Electronic supplementary information (ESI) available: Reaction progress of
reductive debromination reactions based on HPLC. Synthesis and
characterisation of all compounds. Detailed description of the pharmacological
assays, clog P and log S calculations. See DOI: 10.1039/c5ra21326f
‡ These authors contributed equally.
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Scheme 1 Synthetic routes considered for the synthesis of a GPR139
radioligand. Synthesis of mono-bromo precursors 4 and 5 followed by
reductive debromination to give mono-tritiated radioligands 6 and
[3H]-1, respectively.
the formation of undesired side products, and the reaction rate.
Due to the poor solubility of substrates 4 and 5, it was necessary
to perform the reductive debromination step in DMF. The
initial reductive debromination reactions were performed on
a 30 mg scale under identical conditions. Deuteration of
substrate 4 using deuterium gas and palladium on carbon gave
a mixture of compounds [2H]-9 and 1 in a ꢁ4 : 1 ratio, whereas
substrate 5 gave a mixture of compound [2H]-1 and 1 in a ꢁ1 : 1
ratio (determined by 1H NMR). The formation of side products
during both reductive debromination reactions was minor and
comparable according to HPLC (see ESI† for details). Although
deuterium incorporation was more efficient for substrate 4, the
reaction time to obtain >95% conversion was much longer than
for substrate 5 (>21 vs. 1 hour(s), respectively), thus the route
employing substrate 5 was favoured. To conrm the result the
Fig. 1 Selected ligands targeting GPR139. 1,7 LP-360924, LP-471756,
LP-114958,8 and JNJ-63533054.4,10
Results and discussion
Ligand synthesis and radiosynthesis
Two routes based on a reductive debromination strategy start-
ing from commercially available bromo-compounds 2 and 3
were considered for the synthesis of a radioligand (Scheme 1).
We set out to evaluate both reductive debromination routes
initially using deuterium gas in the reductive debromination
step, to identify the route best suited to proceed to radio-
labelling (Scheme 1). The two substrates 4 and 5 for reductive
debromination and the non-labelled agonist 1 were synthesised
as outlined in Schemes 2 and 3. Carboxylic acid 2 was converted
to methyl ester 7 using sulfuric acid in methanol followed
by reaction with hydrazine hydrate in ethanol to provide
hydrazide 8. Subsequent coupling of hydrazide 8 with 1-iso-
cyanatonaphthalene gave substrate 4 in good overall yield,
ready for reductive debromination (Scheme 2). Analogously,
substrate 5 and agonist 1 were synthesised in good overall yield
(Scheme 3). It should be noted that due to the poor solubility of
the hydrazides in THF it was necessary to add methanol to
facilitate the reaction with the isocyanates.
Scheme 2 Reagents and conditions: (a) H2SO4, MeOH, reflux, 16 h,
84%; (b) hydrazine hydrate, EtOH, 50 ꢂC, 8 h, 57%; (c) 1-iso-
cyanatonaphthalene, THF, MeOH, 1 h, 78%; (d) D2, Pd/C, DMF, rt, 30.5
With substrates 4 and 5 in hand, we set out to evaluate these
in the nal reductive debromination step. Two critical param-
eters were considered for the reductive debromination, namely h, 36% (ratio [2H]-9 : 1 – 4 : 1).
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Fig. 2 (A) HPLC chromatogram and (B) radio trace of the purified
radioligand [3H]-1.
Scheme 3 Reagents and conditions: (a) hydrazine hydrate, EtOH, 55
ꢂC, 8 h, 32%; (b) 1: 1-isocyanatonaphthalene, THF, MeOH, 1.5 h, 70%; 5:
1-bromo-4-isocyanatonaphthalene, THF, MeOH, 1 h, 81%; (c) D2, Pd/
C, Et3N, DMF, 1 h, >80% (ratio [2H]-1 : 1 – 4 : 1); (d) T2, Pd/C, Et3N, DMF,
1 h, (ratio [3H]-1 : 1 – 5 : 2).
(WT) and GPR139 knockout (KO) mice (see ESI† for details). As
illustrated in Fig. 4A and B, it is evident that [3H]-1 labels both
the brain sections from the WT and GPR139 KO mice with the
same intensity. Furthermore, the [3H]-1 binding was not
signicantly displaced by 10 mM unlabelled 1. The autoradiog-
raphy was repeated under slightly modied conditions using
a HEPES-buffer (20 mM HEPES, 1 mM MgCl2 and 1 mM CaCl2,
0.01% Pluronic, pH 7.4) with a similar result indicating that
[3H]-1 has a high degree of non-specic binding.
To verify the presence of GPR139 in the striatum and
hypothalamus,4–6 quantitative PCR (qPCR) was performed for
both regions on both GPR139 WT and KO mice (see ESI† for
details). Indeed, high levels of GPR139 mRNA could be detec-
ted in both brain regions of the WT mice, but not in KO mice
(Fig. 4C).
reductive debromination of substrate 5 was reproduced on
a 100 mg scale to give >93% conversion in approx. 1 hour ([2H]-
1 : 1 ratio – 2 : 3, see ESI† for details). To further optimise the
route for radiolabelling, triethylamine (3 eq.) was added to
sequester HBr formed during the course of the reaction.
Moreover, because the reaction would be run at higher dilution
during radiosynthesis, the catalyst loading was increased
substantially. This resulted in a signicant increase in deute-
rium incorporation to produce a ꢁ4 : 1 mixture of [2H]-1 vs. 1,
while maintaining a reaction time of approx. 1 hour, and was
judged suitable for radiolabelling (see ESI† for details). Thus,
substrate 5 was subjected to reductive debromination using
tritium gas, triethylamine and palladium on carbon at room
temperature for 1 hour, followed by purication by preparative
HPLC (Fig. 2).
Recombinant GPR139 CHO-k1 cell line
Next, we investigated the binding of [3H]-1 to the CHO-k1
GPR139 cell line (expressing the human GPR139) in a stan-
dard membrane lter-binding assay. Membrane binding to
either CHO-k1 GPR139 or background CHO-k1 cells was per-
formed with 5 nM or 50 nM [3H]-1 in a standard 50 mM Tris
buffer, pH 7.4 (Fig. 5A, see ESI† for details). No difference in the
binding of [3H]-1 to CHO-k1 GPR139 and CHO-k1 membranes
was observed. Furthermore [3H]-1 binding was not displaced by
4 mM unlabelled 1. The lter membrane binding was repeated
with slight modications using two alternative buffers; (A) 50
mM Tris, 2.5 mM CaCl2, pH 7.4; and (B) 20 mM HEPES, 100 mM
NaCl, 10 mM MgCl2, pH 7.4; with similar results. Additionally,
lter binding was assessed by pre-incubation of the GF/C uni-
lters with 0.2% bovine serum albumin or polyethylenimine,
however all conditions resulted in high [3H]-1 binding to the
lters (see ESI Fig. S1†).
In Fig. 3A, the 1H NMR spectra for the aromatic region of
compounds 1, [2H]-1 and [3H]-1 are depicted. Tritium incorpo-
ration for [3H]-1 vs. 1 was estimated to be ꢁ5 : 2 based on 1H NMR
(Fig. 3A and B) with a purity and radiochemical purity of $95%
and a specic activity of 19.3 Ci mmolꢀ1 (see ESI† for details).
[3H]-1 was formulated in absolute ethanol at a radioactive
concentration of 1 mCi mlꢀ1 and the stability during storage
was evaluated by HPLC. [3H]-1 proved to be very stable at ꢀ196
ꢂC (2 and 10 months aer formulation the radiopurity was
determined to $94% and $90%, respectively). In contrast,
when stored at ꢀ21 ꢂC, decomposition was observed (99%
radiopurity at day 0, 92% at day 26, 81% at 74 and 50% at day
321, see ESI† for details).
Native expression of GPR139
To avoid the high level of binding to the lters and potential
To determine the spatial expression of GPR139 in mouse brain, low specic binding due to a fast off-rate of the radioligand;
the distribution of [3H]-1 binding was assessed qualitatively by SPA-bead binding, centrifugation-binding and whole cell
autoradiography in brain sections from 4.5 months old wildtype binding was attempted, but none of these methods gave any
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Fig. 3 (A) 1H NMR of compound 1, [2H]-1 and [3H]-1 (only the aromatic region is shown, see ESI† for the full spectrum). Both the deuterated and
tritiated product contain unlabelled compound 1. The ratio between labelled and unlabelled compound was determined by 1H NMR by using the
integrals for the resonances at 7.67 ppm (corresponding to Hb in compound 1 only) and at 7.47 ppm (corresponding to Ha both in the labelled and
unlabelled compounds). (B) Proton decoupled 3H NMR of [3H]-1.
signicant difference in [3H]-1 binding between the total and protocols are performed in equilibrium and thus allow for
non-specic binding (data not shown).
detection of radioligands with relatively low affinity and higher
GPR139 is expressed at the protein level in the CHO-k1 off-rates.12 It is also possible that the GPR139 receptor protein
GPR139 cell line, as we have previously demonstrated a func- level in the CHO-k1 cells are too low to allow for a sufficient level
tional agonist response to 1 in the same cell line,9 which was of specic binding, as it is not possible to determine GPR139
conrmed in the present study. Using qPCR analysis we receptor levels quantitatively from the measured functional
furthermore veried the presence of GPR139 transcripts in the responses.
CHO-k1 GPR139 cell line but not in the CHO-k1 background cell
line (Fig. 5B, see ESI† for details).
Compound 1 has previously been shown to be metabolically
stable in human and rat liver microsomes.7 10 mM compound 1
Thus, using a variety of radioligand binding assays, we show shows no activity on native CHO-k1 cells and does not change
that [3H]-1 is not a suitable radioligand for detecting GPR139 in the morphology of living CHO-k1 cells during ligand incubation
brain or in CHO-k1 GPR139 cell membranes. This might be due (data not shown). Moreover, no cross-reactivity of compound 1
to the lipophilic character of 1 causing signicant non-specic was observed against a broad Cerep panel of 90 receptors, ion
binding, low affinity or a high off-rate. The former is the most channels and enzymes.7 Collectively, these data show that the
likely explanation as in particular the SPA and centrifugation lack of specic radioligand binding was neither due to
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metabolic instability of compound 1 or a result of low specic
binding to the GPR139 receptor.
During the preparation of this manuscript, scientists at
Janssen R&D reported synthesis and initial characterization of
JNJ-63533054 as a tritium-labelled radioligand.4,10 This radio-
ligand shows specic binding with a Kd of 10 nM in a GPR139-
inducible Trex-CHO cell line in a membrane lter-binding
assay. A comparison of clog P and log S for JNJ-63533054 vs.
[3H]-1 resulted in similar values (clogP: 3.21 vs. 3.40, and log S:
ꢀ4.53 vs. ꢀ5.46. Calculated with MOE 2014.0901)13 and thus
does not provide an explanation for the negative results ob-
tained with [3H]-1. Alternatively, the induced Trex-CHO cell line
has higher expression of GPR139 than the stable CHO-k1 cell
line used in this study, given the potential receptor down-
regulation mediated by the L-Trp and L-Phe present in the cell
culture medium during the cell culturing.4,9 However, we were
not able to detect specic 1 binding to native receptors in brain
slices with autoradiography. This could potentially also be
explained by low receptor expression levels in the discrete brain
regions showing mRNA expression as this has so far not been
determined.
Conclusion
Radioligand [3H]-1 was successfully synthesised using a reduc-
tive debromination route. A variety of binding experiments with
mouse brain tissue and cells recombinantly expressing GPR139
failed to show specic receptor binding, due to a high degree of
non-specic binding of [3H]-1. Collectively, our data demon-
strates that [3H]-1 is not suitable as
a GPR139-specic
radioligand.
Fig. 4 (A) Autoradiograms showing [3H]-1 binding in WT and GPR139
KO mice brains. The coronal brain sections were incubated with 1 nM
or 10 nM [3H]-1 either in the absence (total) or presence (non-specific
binding, NSB) of 10 mM 1. To the right [3H]-microscale in nCi mgꢀ1
tissue equivalent is depicted. (B) Mean ꢃ SD of mean nCi mgꢀ1 tissue
equivalent of the whole brain area. (C) qPCR analysis of GPR139 mRNA
expression in the striatum and hypothalamus of WT and GPR139 KO
mice. Levels are shown relative to the housekeeping gene B2M.
Acknowledgements
H. Lundbeck A/S is thanked for providing compound 1, the
CHO-k1 GPR139 and CHO-k1 cell line. The Lundbeck Founda-
tion and Hørslev Foundation are gratefully acknowledged for
nancial support. The authors thank the Academy of Sciences
of the Czech Republic for nancial support on this project
(RVO: 61388963). S. K. was supported by the European
Fig. 5 (A) [3H]-1 binding to membranes prepared from CHO-k1 GPR139 and CHO-k1 cell lines. Membranes were incubated with 5 nM or 50 nM
[3H]-1 in either the absence (total) or presence (non-specific binding, NSB) of 4 mM unlabelled 1 in a 50 mM Tris buffer, pH 7.4. (B) qPCR analysis
showing GPR139 mRNA levels relative to the reference gene B2M (beta-2 microglobulin) in the CHO-k1 GPR139 stable cell line and CHO-k1 cell
line.
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Cooperation in Science and Technology (COST) Action CM1207:
GPCR-Ligand Interactions, Structures, and Transmembrane
Signalling: a European Research Network (GLISTEN), as well as
the European University Consortium for Pharmaceutical
Sciences (ULLA).
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