Development of fluorinated Non-Peptidic ghrelin receptor ligands for potential use in molecular imaging

Article abstract of DOI:10.3390/ijms18040768

The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for¹⁸F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.

Full text of DOI:10.3390/ijms18040768

International Journal of
Molecular Sciences
Article
Development of Fluorinated Non-Peptidic Ghrelin
Receptor Ligands for Potential Use in
Molecular Imaging
Rares¸-Petru Moldovan 1, Sylvia Els-Heindl 2, Dennis J. Worm 2, Torsten Kniess 1, Michael Kluge 3,
Annette G. Beck-Sickinger 2, Winnie Deuther-Conrad 1, Ute Krügel 4,† and Peter Brust 1, ,†
*
1
Helmholtz-Zentrum Dresden-Rossendorf e. V., Institute of Radiopharmaceutical Cancer Research,
04318 Leipzig, Germany; r.moldovan@hzdr.de (R.-P.M.); t.kniess@hzdr.de (T.K.);
w.deuther-conrad@hzdr.de (W.D.-C.)
2
3
4
Institute of Biochemistry, Universität Leipzig, 04103 Leipzig, Germany; sylvia.els@uni-leipzig.de (S.E.-H.);
dennis.worm@uni-leipzig.de (D.J.W.); abeck-sickinger@uni-leipzig.de (A.G.B.-S.)
Department of Psychiatry, Universität Leipzig, 04103 Leipzig, Germany;
Michael.Kluge@medizin.uni-leipzig.de
Rudolf Boehm Institute of Pharmacology and Toxicology, Medical Faculty, Universität Leipzig,
04107 Leipzig, Germany; ute.kruegel@medizin.uni-leipzig.de
*
Correspondence: p.brust@hzdr.de; Tel.: +49-341-234-179-4610
†
These authors contributed equally to this work.
Academic Editor: Suzanne L. Dickson
Received: 26 January 2017; Accepted: 31 March 2017; Published: 5 April 2017
Abstract: The ghrelin receptor (GhrR) is a widely investigated target in several diseases.
However, the current knowledge of its role and distribution in the brain is limited. Recently,
the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-
3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand
with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro
evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical
properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules
for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of
GhrR in the brain.
Keywords: brain; ghrelin receptor; fluorine; positron emission tomography
1. Introduction
The ghrelin receptor (GhrR), also known as growth hormone secretagogue receptor, is critically
involved in various peripheral and central actions of the 28-amino acid orexigenic hormone ghrelin.
The peptide, mainly synthesized in the stomach, was identified to stimulate the release of growth
hormone from the anterior pituitary [
occasional findings report otherwise [
that ghrelin stimulates appetite, promotes body weight gain, reduces glucose-stimulated insulin
secretion, and enhances blood glucose [ 14]. The GhrRs are distributed in various peripheral
1–
4
]. Although thought to be non-expressed in the brain [
5,6],
7,8]. A variety of clinical pharmacological studies demonstrated
9–
tissues (e.g., adipose tissue, pancreatic islets, thyroid, and myocardium). They contribute to
cardiovascular functions and energy homeostasis by regulation of glucose metabolism, lipogenesis, and
thermogenesis [
hypothalamic nucleus arcuatus, the GhrR is involved in the regulation of eating behavior [15
and in limbic areas (e.g., hippocampus, ventral tegmentum), it controls the sensitivity of the
dopaminergic reward circuit [17 21]. This receptor is constitutively high active, and thereby mediates
7,
15–
17]. They are also widely expressed in various regions of the brain. In the
,
18 20],
–
,
Int. J. Mol. Sci. 2017, 18, 768; doi:10.3390/ijms18040768
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2017, 18, 768
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functions in brain regions usually not accessible for peripheral ghrelin, forms homodimers, and
activates Gαq/11 proteins [ 22 26]. Alternatively, it builds heteromers with other G protein–coupled
1
,7,
–
receptors (GPCRs) (e.g., dopamine D1, D2, or serotonin 5-HT2C receptors (Rs)). Thereby, it can
bias signal transduction via allosteric modulation, which allows dynamic alterations of biological
functions [27].
Therefore, in brain areas whose activities are strongly dependent on dopamine signaling, GhrRs
have been implicated in the reward circuit [28
,29], mood [30], cognitive functions [31,32], and sleep [33],
as well as in psychiatric disorders like addiction, depression, and anxiety [34].
Up to now, the relevance of the GhrR in healthy and diseased human brain has been nearly
unknown. Apart from the emerging interest to design appetite-stimulating drugs or therapeutics
against obesity, there is a demand for radioligands suitable for molecular imaging with positron
emission tomography (PET) to investigate alterations of GhrR distribution and kinetics in brain
diseases [23,35]. However, due to the peptidic nature of the endogenous ligand, the in vivo imaging of
GhrR in the brain with PET remains a challenging task [36–39].
GhrR selective ligands had already been reported before ghrelin was discovered and identified as
an endogenous ligand [40]. Given the peptidic nature of ghrelin, many of the synthetically developed
ligands for GhrR possess a peptidic or peptidomimetic motif, mainly structurally related to enkephalin,
substance P, and ghrelin itself [5,40]. Though peptidic ligands may enter brain areas next to median
eminence via fenestrated capillaries [41], they have a limited access to other areas (e.g., the limbic
system) [42].
To date, a variety of small molecules acting at the GhrR was discovered (for a review see [43]).
Therefore, we aimed to develop fluorine-bearing highly affine and selective, non-peptidic ligands
with physicochemical properties to cross the blood-brain barrier as lead molecule(s) to establish in
the following step an 18F-labeled radiotracer for PET qualified for GhrR imaging and quantification.
To achieve this, we selected as the lead compound one of the most affine and selective, non-peptidic
GhrR ligands reported so far (Scheme 1, (S)-9, Ki = 0.01 nM [44]) [45,46]. Compound (S)-9 possesses
a fluorine atom at an aromatic position, which offers a challenging late-step incorporation of the
18F-label due to the rather low activation for the nucleophilic substitution (SN2) reaction. Similar
compounds which derived from a common lead molecule as (S)-9 revealed oral bioavailability, brain
uptake, and suppressed the ghrelin-mediated insulin secretion in rats [45,47]. Potter et al. also
showed favorable pharmacodynamics and brain uptake of a similar, 11C-labeled derivative. However,
its affinity was not sufficient for in vivo GhrR imaging with PET [36]. With regard to the aspects
of late fluorination, the binding and physicochemical studies and the structure-activity analysis,
compound (S)-9
and its enantiomer represent suitable surrogate candidates for an 18F-labeled GhrR
PET radiotracer. Additionally, a new small molecule, (S)-16, was revealed with comparable binding
affinity and reduced partial agonism.

Int. J. Mol. Sci. 2017, 18, 768
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Scheme 1. Synthesis of (S)-
temperature (rt), 20 h, 64%; (
corresponding phenol, DMF, 90 C, 18 h, 95%; (
9
to (S)-14 and (R)-
9
. Reagents and conditions: (
a
) BOP, Et3N, DCM, room
) Cs2CO3,
) corresponding
b) 1,1,1-triethoxy ethane, AcOH, reflux, 3 h, >90%; (c
◦
d
) TFA/DCM, 1:2, rt, 2 h, quantitative; (
e
alkylating agent, K2CO3, CH3CN, 70 ◦C. BOP, benzotriazole-1-yl-oxy-tris-(dimethylamino)-
phosphonium hexafluorophosphate; Et3N, triethylamine; DCM, dichloromethane; AcOH, acetic acid;
DMF, N,N-dimethylformamide; TFA, trifluoroacetic acid; DCM, dichloromethane.
2. Results
2.1. Chemistry
The lead compound (S)-
The synthesis started from the commercially available (R)-tert-butyl 3-(aminomethyl)piperidine-1-
carboxylate ((R)- , Scheme 1), which was coupled with the carboxylic acid in presence of the coupling
reagent BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) to
give amide (R)- that was further cyclized with 1,1,1-triethoxy ethane in thermal reaction conditions
to give the 6-methylpirimid-2-one intermediate (R)- . Cs2CO3 promoted diaryl ether formation
followed by trifluoroacetic acid (TFA)-mediated tert-butyloxycarbonyl (Boc) group removal delivered
the key building block (S)- in ~55% yield over five steps. N-alkylation with 2-iodopropane gave
(S)- in 89% yield. From a bulk of (S)- a small series of fluorinated derivatives ((S)-10, (S)-11, (S)-12
and (S)-13; see Scheme 1) was synthesized by alkylating the free amino group at the piperidine
subunit in basic reaction conditions. In parallel, the hitherto unreported (R)- enantiomer was
obtained by using the same synthesis sequence (Scheme 1, reactions (a)–(e)) starting from (S)-tert-butyl
3-(aminomethyl)piperidine-1-carboxylate ((S)- ). Additionally, to avoid possible radiolysis during
9
was synthesized as previously described with some modifications [44].
1
2
3
4
7
9
7
,
9
1
the radiofluorination process, compound (S)-14 lacking the fluorine atom at the phenyl subunit was
synthesized starting from (S)-4 in >80% yield over three steps (Scheme 1, reactions (c)–(e)).
Next, we focused on the possibility of introducing a fluorine atom at the pyrimidine 2-position.
To achieve this, compound (R)-3 was cyclized to the corresponding pyrimidine-3(4H)-one by trimethyl
orthoformate under thermal reaction conditions ((R)-15, Scheme 2). Diaryl ether formation, Boc-group
removal, and piperidine N-alkylation with 2-iodopropane were performed as described above to
give (S)-16 depicted in Scheme 2. Straight forward fluorination at the aminal position of (S)-16 by

Int. J. Mol. Sci. 2017, 18, 768
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tetrabutylammonium fluoride (TBAF) in N,N-dimethylformamide (DMF) at elevated temperature led
to (S)-17 in moderate yield (23%, see Scheme 2).
Boc
N
N
N
N
N
O
O
N
F
O
N
F
N
Cl
a
)
b
c
( )
(
(
)
N
O
N
O
N
3
(R)-
N
Br
F
Br
(R)-15
(S)-16
(S)-17
Scheme 2. Synthesis of compounds (S)-16 and (S)-17
.
Reagents and conditions: (
a
) trimethyl
◦
orthoformate, AcOH, reflux, 1 h, >90%; (
b
) i. Cs2CO3, 2-fluoro-4-bromophenol, DMF, 90 C, 18 h, 95%;
◦
ii. TFA/DCM, 1:2, rt, 2 h, quantitative; iii. corresponding alkylating agent, K2CO3, CH3CN, 70 C, 89%;
◦
(c) TBAF, DMF, 130 C 16 h, 23%. TBAF, tetrabutylammonium fluoride.
Further on, together with the lead compound (S)-9, eight fluorinated compounds (R)-9, (S)-10,
(S)-11, (S)-12, (S)-13, (S)-14, (S)-16, and (S)-17, are proposed for further structure affinity/activity
studies to select candidates for radiolabeling with fluorine-18.
2.2. Biological Investigations
2.2.1. Binding Affinities at the Human Ghrelin Receptor (hGhrR)
All the herein newly developed compounds (R)-
(S)-17 together with the previously described compound (S)-
affinities at the hGhrR using [125I-His9]ghrelin [48
49] as competitive radioligand. Unlabeled ghrelin
served as control with an IC50 of 1.7 nM, comparable to previous experiments [50]. (S)- was the
9
, (S)-10, (S)-11, (S)-12, (S)-13, (S)-14, (S)-16, and
9
have been assessed for their binding
,
9
starting compound for modification with an IC50 of 2.2 nM (Figure 1A). A Ki value of 0.01 nM was
previously published for this derivative [44]. However, no KD and raw IC50 values were given, limiting
the comparability of the compound properties. Substitution at the piperidine nitrogen influenced the
receptor binding. Compounds (S)-10 and (S)-11 had only low affinity to the receptor, indicated by
about 30-fold higher IC50 values. Notably, longer substituents resulted in a further increase of the IC50
((S)-12: 574 nM). Introduction of a fluorine at the isopropyl group ((S)-13) decreased affinity by about
the power of ten. The orientation of the piperidine carbon connected to the azaquinazolinone moiety
is of minor importance, as shown by the synthesized (R)-9, which has only a slightly lower affinity
than (S)-9.
Figure 1. Binding affinity (
(S)- , (R)- , (S)-14, and (S)-16. Compounds were tested at membranes of HEK293 cells stably transfected
with the hGhrR for competitive binding properties against [125I]ghrelin (
); Data are means SEM
(n 4). Agonistic properties of the compounds were analyzed by inositol phosphate accumulation in
COS7 cells stably transfected with the hGhrR ( ). Data were normalized to ghrelin (100% = maximal
A) and agonistic properties (B) of the non-peptidic candidate compounds
9
9
A
±
≥
B
efficacy, 0% = constitutive receptor activity) and given in percent as means
experiments performed in duplicates.
±
SEM of
≥
3 independent

Int. J. Mol. Sci. 2017, 18, 768
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In addition, the significance of the fluorine atom at the aromate and the methyl group at the
azaquinazolinone moiety were investigated. Removal of the fluorine in (S)-14 led to a loss of affinity
by a factor of about five. Interestingly, whereas removal of the methyl group preserved the binding
affinity ((S)-16), the substitution with fluorine ((S)-17) did not and caused a severe loss of binding
affinity with an IC50 of 151 nM (Table 1).
Table 1. Competitive binding assay at HK293 cell membranes expressing hGhrR (human ghrelin
receptor) with [125I-His9]ghrelin.
1
Compound
R1
R2
R3
MW
IC50 [nM]
pIC50
Ghrelin
1.7
8.78 ± 0.04
(S)-9
(R)-9
F
F
Me
Me
489
489
2.2
3.9
8.66 ± 0.05
8.41 ± 0.05
(S)-10
(S)-11
(S)-12
F
F
F
Me
Me
Me
493
507
521
69
66
7.16 ± 0.11
7.18 ± 0.09
6.24 ± 0.12
574
(S)-13
(S)-14
(S)-16
(S)-17
F
H
F
Me
Me
H
507
471
475
493
13
12
7.90 ± 0.13
7.91 ± 0.10
8.57 ± 0.06
6.82 ± 0.09
2.7
151
F
F
1
Mean values ± SEM (n ≥ 4).
2.2.2. Biological Activity at the hGhrR
Molecules with the highest affinity in the binding assay were tested for their biological activity
(Table 2). For that purpose, the inositol phosphate accumulation assay was used [43 44]. The ghrelin
,
receptor was stimulated with different concentrations of the compounds for 2 h in the absence
of ghrelin.
Table 2.
Inositol phosphate accumulation assay to determine agonistic potencies of
candidate molecules.
1
1
Compound
EC50 (nM)
pEC50
Emax (%) 2
n 3
(S)-9
(R)-9
(S)-10
(S)-11
(S)-13
(S)-14
(S)-16
0.7
0.6
1.6
2.9
1.4
2.1
1.0
9.17 ± 0.19
9.26 ± 0.21
8.80 ± 0.14
8.54 ± 0.15
8.87 ± 0.13
8.67 ± 0.20
8.99 ± 0.20
63 ± 6
59 ± 6
64 ± 5
58 ± 4
57 ± 4
42 ± 4
40 ± 4
≥3
≥3
≥3
≥3
≥3
≥3
≥3
1 Mean values
EC50, Half maximal effective concentration.
±
SEM; 2 data refer to ghrelin with Emax as 100%; 3 number of independent experiments in duplicates.

Int. J. Mol. Sci. 2017, 18, 768
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Ghrelin served as control with an EC50 of 1.0 nM (pEC50 = 9.00
±
0.05, Emax = 100%
±
3%),
which was comparable to data from previous experiments [49
partial agonists at the GhrR with similar potencies in the low nanomolar range. (R)-
EC50 values of 0.6 and 1.0 nM, respectively, which are similar to the agonistic potency of the starting
molecule (S)- of 0.7 nM (Table 2). The efficacies of (S)- , (R)- , (S)-10, (S)-11 and (S)-13 were all in the
,
50]. All compounds were found to be
9
and (S)-16 have
9
9
9
same range, as indicated by a maximal receptor activation (Emax) of around 60%.
These data indicate that the stereochemical configuration at the chiral carbon of the piperidine
((S)-
removal of the methyl group at the azaquinazolinone in (S)-16 did not alter the binding affinity
compared to (S)- , but weakened the partial agonism of the molecule, since (S)-16 showed the lowest
9 versus (R)-9) is neither relevant for the binding affinity nor for the agonistic potency. Importantly,
9
agonistic efficacy of all compounds (Figure 1B).
Due to the partial agonism of the compounds in the absence of ghrelin they act as competitive
partial antagonists at the GhrR in the presence of ghrelin. As expected, the agonistic efficacies of the
compounds correlated with their antagonistic properties. Compound (S)-16 exhibited the strongest
antagonistic properties (see supplemental information Table S1 and Figure S1).
2.2.3. Selectivity Studies
The selectivity of the herein reported compounds was assessed by standard screening assays (see
methods). As target receptors, the vesicular acetylcholine transporter, the adenosine A1Rs and A2ARs,
the
investigated. Notably, most of the receptor-specific radioligands used for these assays could not be
displaced by the GhrR ligands reported here. Compounds (S)- and (R)- had a moderate binding
affinity to the 2 receptor (Ki) of 87 23 and 53 7 nM, respectively. Compounds (S)-11 and (S)-12
had a Ki of 27 8 and 64 14 nM, respectively, at 4 nAChRs. Moreover, the most promising
GhrR ligand (S)- did not show significant responses ( 50% inhibition or stimulation in biochemical
assays) at adrenergic 1ARs and 1BRs, chemokine CXCR3Rs, muscarinic M1Rs and M2Rs, serotonin
α7, α4β2, and α3β4 nicotinic acetylcholine receptors (nAChRs), the oxytocin Rs, σ1 and σ2Rs, were
9
9
σ
±
±
±
±
α3β
9
≥
α
α
5-HT2BRs, and the potassium channel hERG in a commercial screening by Eurofins Panlabs (Taiwan;
see Supplementary Materials Table S2).
2.2.4. Distribution Coefficient and Non-Specific Binding
Physicochemical properties of a drug such as lipophilicity and non-specific binding to
membranes and plasma proteins are crucial parameters of a potential radiotracer, in particular if
it is supposed to pass the blood brain barrier (BBB). The lipophilicity (logD7.4) was estimated using
the commercially available ACD/Labs-12.5 software and the non-specific binding was estimated by
the high-performance liquid chromatography (HPLC)-based hydrophobicity index at immobilized
artificial membranes (CHI IAM) [51] for seven fluorine substituted candidate molecules and (S)-13
without fluorine (Table 3).
Table 3. Calculated logD7.4 values and experimentally determined CHI IAM (chromatographic
hydrophobicity index on immobilized artificial membrane) values of GhrR ligands.
1
Compound
cLogD7.4
CHI IAM
(S)-9
(R)-9
2.1
2.1
3.4
3.1
3.0
3.1
2.4
2.9
43.0
42.8
39.6
42.2
43.3
42.8
42.8
42.3
(S)-10
(S)-11
(S)-12
(S)-13
(S)-14
(S)-16
1
calculated with ACD/Labs-12.5.

Int. J. Mol. Sci. 2017, 18, 768
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Since compounds with a logD7.4 between 2 and 3 are expected to enter the brain by lipid-mediated
free diffusion [52], all compounds presented in this study are suggested to be suitable as brain imaging
agents. Further, all investigated ligands meet the requirements of a CHI IAM
non-specific binding of a suitable PET tracer [53].
≤
50 as criterion for the
3. Discussion
A large number of medicinal chemistry studies have focused on the development of small
molecules acting at the GhrR. Their applicability for therapeutic purposes has been investigated
in various preclinical and clinical studies [5,40,43,54]. Apart from that, the present study was
aimed at investigating the synthesis of parent compounds which fulfill the pharmacological and
physicochemical requirements for brain-penetrating receptor ligands and which provide the possibility
to introduce fluorine at a position that enables a late-stage incorporation of the positron emitter 18F.
In particular, compound (S)-9, its enantiomer, and the newly synthesized compound (S)-16 meet
these criteria.
Compound (S)-
optimal pharmacological properties [44
9
attracted attention due to its sub-nanomolar affinity to the GhrR combined with
46]. It contains a fluorine atom at aromatic position (see Table 1,
–
R1). The ortho-ether function and the meta-bromine atom in respect to the fluorine atom only slightly
activate this aromatic position for a putative SN2 radiofluorination. However, aromatic radiolabeling is
generally difficult and occasionally requires the implementation of highly reactive leaving groups (e.g.,
ammonium or iodonium salts) and harsh reaction conditions (e.g., high temperature, long reaction
time, strong base) [35]. On the other hand, the introduction of the 18F radiolabel at aliphatic position is
supposed to be more easily performed (see Table 1, R2).
The modification of synthesis of the lead compound (S)-
led to an improved yield of 49% over five steps [44]. The strategy is based on a late-step derivatization
using compound (S)- as a key building block. The moiety of the piperidine N-substituent was
investigated using both linear ((S)-10, (S)-11, (S)-12) and branched fluoroalkanes ((S)-13, see Scheme 1).
For comparative in vitro investigation, the (R)- enantiomer was synthesized by the same synthesis
9 previously described by Hanrahan et al.
7
9
procedure (Scheme 1). Moreover, the possibility of introducing fluorine at pyrimidine 2-position was
proven. The resulting desmethyl derivative (S)-16 was synthesized using trimethyl orthoformate at the
cyclization step to (R)-15 (reaction (a), Scheme 2), which was subsequently fluorinated at the aminal
position to produce (S)-16 with moderate yield (23%).
In the competitive radioligand binding assay for GhrR, the lead molecule (S)-9 displayed the
most promising binding affinity in contrast to molecules with substitution of the isopropyl group at
the piperidine subunit. Fluoroethyl, fluoropropyl, and fluorobutyl substitution caused a loss in the
GhrR binding affinity (Table 1). The implementation of a 3-fluoro-2-yl group also led to a slightly
lower binding affinity. Whereas the substitution of the methyl group by hydrogen at the pyrimidine
2-position (compound (S)-16) had no effect on binding affinity compared to compound (S)-9, its
substitution with fluorine (compound (S)-17) impaired this property and made (S)-17 useless as the
lead substance for 18F labeling. This is probably due to the impact of the highly electronegative
fluorine atom on the electron density distribution of the corresponding aromatic ring (see Scheme 2
and Table 1).
Surprisingly, in the case of the (R)-9 enantiomer, the binding affinity was nearly unchanged, unlike
the description for similar other compounds [45].
Further characterization of the pharmacological activity of the candidates revealed that (S)-9 and
the newly synthesized molecules are highly potent partial agonists at this receptor. This is a new
finding, since until now (S)-9 was described solely as a GhrR antagonist [41].
In the additionally performed antagonist experiments, compound (S)-9 had lower partial
antagonistic activity than described by Hanrahan et al. [44]. This may be explained by different read-out
parameters and cellular systems used in both studies. While the inositol phosphate accumulation
serves as an indicator for the Gα
q protein-coupled GhrR activity, alterations in the intracellular Ca2+

Int. J. Mol. Sci. 2017, 18, 768
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represent a more downstream event, dependent on the inositol phosphate signaling but in addition
also on other pathways via Gi and Gβγ proteins [55]. Moreover, results after short incubation times
rather reflect kinetic effects and may not allow optimal equilibria conditions between receptor and
ligands. This explanation is supported by a study where GSK1614343-elicited effects that were higher
in the Ca2+ assay compared to the inositol phosphate accumulation assay [56]. It can be assumed that
potential side effects by competitive antagonism of these compounds would affect the GhrR function
only in a few regions easily accessible for ghrelin, such as the arcuate nucleus.
The non-occupied GhrR possesses constitutive activity in various brain regions (including
hippocampus and amygdala) [42]. Hence in the absence of ghrelin, the effects of the potential ligands
on the receptor should be also considered for (S)-
a higher activity GhrR was observed by the addition of compounds (S)-
9
and the newly synthesized compounds. Indeed,
and (R)- at a concentration
9
9
of 10−5 M. Similar agonistic effects were described for GSK1614343, another non-peptidic ligand [57].
With regard to PET imaging, radiolabeled non-peptidic GhrR ligands are expected to have
virtually no effects on the function of the target. The radiolabel and the high sensitivity of its detection
allow the use of concentrations which are much lower than those needed to elicit any functional
effect [35]. Although agonistic or antagonistic properties are very important for therapeutic drugs, this
is not applicable for PET tracers.
Potential side effects of GhrR PET tracers are unlikely related to the receptor function but to
potential high-affinity binding to other targets which would impair the quantitation of the receptor
parameters. The most promising compound (S)-9 did not show significant binding at adrenergic
α
1ARs and 1BRs, chemokine CXCR3Rs, muscarinic M1Rs and M2Rs, serotonin 5-HT2BRs, and the
α
potassium channel hERG. Also, the
any influence on PET imaging [35].
σ
2 receptor binding (Ki = 87
±
23 nM) is obviously too low to be of
All new GhrR ligands are expected to cross the BBB, as indicated by their LogD7.4 values.
The derivatives with a linear fluoroalkyl group ((S)-11 and (S)-12) and in particular the fluoroethyl
substituted ((S)-10) had the highest LogD, and thereby may be less appropriate as potential brain
imaging agent. On the other hand, for (S)-10 the lowest non-specific binding in vivo is predicted.
This suggests that membrane permeability and non-specific binding are not necessarily correlated.
Notably, (S)-9, (R)-9, and (S)-16 have favorable physiochemical properties for brain penetration and
low non-specific binding, and are therefore the three most applicable compounds of this study for
future radiolabeling. The introduction of the 18F-isotope at any of the target molecules is possible by
the nucleophilic (SN2) substitution of a suitable leaving group (e.g., nitro, iodonium, or ammonium
salt), as previously described [35]. The implementation of the leaving group at the aryl position of
(S)-9 or (S)-16 can be performed by the synthetic strategy described in Schemes 1 and 2, respectively
(e.g., Scheme 1 R1 = isopropyl, R2 = NO2).
In summary, a novel small series of fluorinated GhrR ligands was synthesized by modifying
the structure of compound (S)-
9. While fluoro derivatizations performed at the N-piperidine and
pirimidine-2-position led to a decrease in the GhrR affinity, the enantiomer (R)-
9
showed high GhrR
binding in the low nanomolar range. The physicochemical evaluation suggests that all GhrR ligands
are able to cross the BBB and to exhibit low non-specific binding. Furthermore, the compounds are of
high selectivity towards the GhrR. These findings qualify compounds (S)-9, its enantiomer, and (S)-16
as suitable starting molecules for the development of an 18F-labeled GhrR PET tracer for brain imaging.
4. Materials and Methods
4.1. General Methods
Unless otherwise noted, moisture sensitive reactions were conducted under dry nitrogen or
argon. All chemicals and reagents were purchased from commercially available sources and used
without further purification. Thin-layer chromatography (TLC): Silica gel 60 F254 plates (Merck KGaA,
Darmstadt, Germany). Flash chromatography (fc): Silica gel 60, 40–64 µm (Merck). Room temperature

Int. J. Mol. Sci. 2017, 18, 768
9 of 16
◦
(rt) was 21 C. Mass spectrometry (MS): MAT GCQ (Thermo Finnigan MAT GmbH, Bremen, Germany).
1H, 13C spectra were recorded on VARIAN “MERCURY plus” (300 MHz for 1H NMR, 75 MHz for
13C NMR) and VARIAN “MERCURY plus” and BRUKER DRX-400 (400 MHz for 1H NMR, 100 MHz
for 13C NMR, 377 MHz);
δ in ppm related to tetramethylsilane; coupling constants (J) are given with
0.1 Hz resolution. Multiplicities of NMR signals are indicated as follows: s (singlet), d (doublet),
t (triplet), m (multiplet), dd (doublet of doublets), dt (doublet of triplets). ESI/Ion trap mass spectra
(LRMS) were recorded with a Bruker Esquire 3000 plus instrument (Bruker Corporation, Billerica,
MA, USA). High resolution mass spectra were recorded on a FT-ICR APEX II spectrometer (Bruker
Daltonics; Bruker Corporation) using electrospray ionization (ESI) in positive ion mode. Lipophilicity
at pH = 7.4 (cLogD7.4) was calculated using ACD/Labs-12.5.
4.2. Procedures and Compound Characterization
tert-Butyl (R)-3-((3-amino-6-chloropicolinamido)methyl)piperidine-1-carboxylate ((R)-
Procedure 1. BOP (3.1 g, 1.2 equiv, 6.9 mmol) and Et3N (1.2 mL, 3 equiv, 17.4 mmol) were added to
a solution of amine (1.6 g, 1.3 equiv, 7.5 mmol) and acid (1 g, 1 equiv, 5.8 mmol) in 25 mL DCM,
and the reaction mixture was stirred at rt for 20 h. After completion, the NaHCO3 aqueous was added
(20 mL) and the whole was extracted with 2 20 mL DCM. Drying over Na2SO4 followed by solvent
3). General
1
2
×
evaporation under reduced pressure afforded a thick oil which was purified by flash chromatography
◦
(silica, ethyl acetate (EA)/petroleum ether (40–65 C) (IH), 1:4 to 1:1) to give (R)-
3
(1.3 g, 64% yield) as
colorless solid.
tert-Butyl (R)-3-((6-chloro-2-methyl-4-oxopyrido[3,2-d]pyrimidin-3(4H)-yl)methyl)piperidine-
1-carboxylate ((R)- ). General Procedure 2. Compound (1 g, 1 equiv, 2.6 mmol), in 1.1.1-triethoxy
ethane (2 mL, 5 equiv, 13 mmol) and AcOH (170 L, 1 equiv, 2.6 mmol) was warmed for 3 h at
4
3
µ
135 ◦C. Upon completion (TLC), the solvent was removed under reduced pressure and the residue
was purified by flash chromatography (silica, EA) to give (R)-4 (960 mg, 90% yield) as tan solid.
tert-Butyl (R)-3-((6-(4-bromo-2-fluorophenoxy)-2-methyl-4-oxopyrido[3,2-d]pyrimidin-3(4H)-yl)
methyl)piperidine-1-carboxylate ((R)-
3 equiv, 3.8 mmol) was added to a solution of (R)-
3 equiv, 3.8 mmol) in 5 mL DMF, and the reaction was performed at 90 ◦C overnight. An aqueous
solution of NaHCO3 (20 mL) was added and the mixture was extracted with DCM (2 20 mL). Drying
5). General Procedure 3. 4-Bromo-2-fluorophenol (370 mg,
4
(500 mg, 1 equiv, 1.3 mmol) and Cs2CO3 (1.2 g,
×
over Na2SO4 and solvent elimination gave a solid which was purified by flash chromatography (silica,
EA) to give (R)-5 (630 mg, 90% yield) as white solid.
(S)-6-(4-Bromo-2-fluorophenoxy)-2-methyl- 3-(piperidin-3-ylmethyl)pyrido[3,2-d]pyrimidin-4
(3H)-one). General Procedure 4. The Boc-protected (R)-5 (900 mg, 1.6 mmol) was dissolved in 2 mL
TFA/DCM (1:2) and stirred at rt for 2 h. Elimination of the solvent gave a thick oil which was diluted
with 10 mL DCM and washed with 10 mL NaHCO3 aqueous solution. Drying over Na2SO4 followed
by solvent evaporation under reduced pressure gave (S)-7 as white tan solid in quantitative yield.
(S)-6-(4-Bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]
pyrimidin-4(3H)-one ((S)-
to a solution of (S)- (44.7 mg, 1 equiv, 0.1 mmol) and K2CO3 (41.4 mg, 3 equiv, 0.3 mmol) in CH3CN
(2 mL), and the reaction was performed at 70 ◦C overnight. H2O (10 mL) was then added at room
temperature and the mixture was extracted with DCM (2 10 mL). The combined organic phases
9) General Procedure 5. 2-iodopropane (30 µL, 3 equiv, 0.3 mmol) was added
7
×
were dried over Na2SO4, the solvent was evaporated, and the resulting residue was purified by flash
1
chromatography (silica, CHCl3/MeOH, 9:1) to give (S)-
(400 MHz, CDCl3):
3.47–3.28 (m, 2H), 3.21 (d, J = 11.2 Hz, 1H), 3.04 (m, 1H), 2.85–2.52 (m, 2H), 2.72 (s, 3H), 2.34–2.33 (m,
1H), 2.00–1.88 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H), 1.32 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3):
9
(43 mg, 89%yield) as white solid. H NMR
δ
(ppm) = 8.01 (d, J = 8.8 Hz, 1H), 7.45–7.14 (m, 4H), 4.22 (m, 1H), 3.93 (m, 1H),
δ
(ppm) = 60.6, 160.3, 155.5, 154.0, 153.0, 141.6, 139.9 (d, J = 10.1 Hz), 139.7, 133.6, 127.7 (d, J = 3.8 Hz),
124.9, 120.4 (d, J = 21.6 Hz), 118.6, 117.8 (d, J = 8.3 Hz), 57.7, 52.8, 47.3, 34.3, 32.2, 27.4, 23.1, 22.1, 17.4,
16.3. HRMS (ESI+): m/z (%) = 489.1293, calcd. 489.1296 [M + H]+.

Int. J. Mol. Sci. 2017, 18, 768
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(S)-6-(4-Bromo-2-fluorophenoxy)-3-((1-(2-fluoroethyl) piperidin-3-yl)methyl)-2-methylpyrido
[3,2-d]pyrimidin-4(3H)-one ((S)-10) was obtained according to General Procedure 5. 1H NMR
(400 MHz, CDCl3):
J = 7.8 Hz, 1H), 3.96 (s, 1H), 2.90 (dd, J = 57.0, 36.6 Hz, 4H), 2.69 (s, 3H), 2.47 (d, J = 47.8 Hz, 3H), 1.98–1.63
(m, 3H), 1.31 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, CDCl3):
(ppm) = 160.3 (d, J = 7.4 Hz), 155.6,
δ (ppm) = 7.99 (d, J = 8.8 Hz, 1H), 7.48–7.12 (m, 4H), 4.89–4.41 (m, 2H), 4.14 (d,
δ
154.1, 153.1, 141.5, 139.9 (d, J = 12.0 Hz), 139.5, 133.8, 127.8 (d, J = 3.7 Hz), 125.0, 120.5 (d, J = 21.5 Hz),
118.6, 117.9 (d, J = 8.2 Hz), 58.2 (d, J = 20.2 Hz), 57.4, 53.7, 47.6, 35.3, 27.7, 27.6, 23.9, 23.2. HRMS (ESI+):
m/z (%) = 493.1049, calcd. 493.1045 [M + H]+.
(S)-6-(4-Bromo-2-fluorophenoxy)-3-((1-(3-fluoropropyl)piperidin-3-yl)methyl)-2-methylpyrido
[3,2-d]pyrimidin-4(3H)-one ((S)-11) was obtained according to General Procedure 5. 1H NMR
(400 MHz, CDCl3):
(m, 1H), 4.18 (m, 1H), 3.93 (m, 1H), 3.39–2.88 (m, 2H), 2.76 (m, 2H), 2.67 (s, 3H), 2.70–2.25 (m, 2H),
2.22–1.82 (m, 3H) 1.83 (m, 2H), 1.50–1.12 (m, 2H). 13C NMR (100 MHz, CDCl3):
(ppm) = 160.4, 160.3,
δ (ppm) = 7.98 (d, J = 8.8 Hz, 1H), 7.51–7.16 (m, 4H), 4.64–4.48 (m, 1H), 4.48–4.34
δ
155.6, 154.1, 153.1, 141.5, 139.9 (d, J = 12.0 Hz), 139.5, 133.7, 127.8 (d, J = 3.7 Hz), 125.1, 120.6, 120.4,
118.6, 117.9 (d, J = 8.2 Hz), 64.8, 57.1, 54.7, 53.1, 47.6, 40.2, 34.8, 27.7, 26.4, 26.1, 23.2. HRMS (ESI+): m/z
(%) = 507.1205, calcd. 507.1202 [M + H]+.
(S)-6-(4-Bromo-2-fluorophenoxy)-3-((1-(4-fluorobutyl) piperidin-3-yl)methyl)-2-methylpyrido
[3,2-d]pyrimidin-4(3H)-one ((S)-12) was obtained according to General Procedure 5. 1H NMR
(400 MHz, CDCl3):
4.38 (t, J = 5.3 Hz, 1H), 4.25–3.85 (m, 2H), 2.79 (m, 2H), 2.68 (s, 3H), 2.46 (m, 2H), 2.29 (s, 1H), 2.09 (m,
2H), 1.84–1.52 (m, 7H), 1.26 (m, 1H). 13C NMR (100 MHz, CDCl3):
(ppm) = 160.2, 154.3 (t, J = 126.9
δ (ppm) = 7.99 (d, J = 8.8 Hz, 1H), 7.56–6.98 (m, 4H), 4.49 (t, J = 5.8 Hz, 1H),
δ
Hz), 141.4, 139.9 (d, J = 11.9 Hz), 139.4, 133.9, 127.7 (d, J = 3.8 Hz), 124.9 (d, J = 1.5 Hz), 120.5, 120.3,
118.4, 117.8, 83.7 (d, J = 164.7 Hz), 58.2, 57.5, 53.2, 48.0, 35.6, 28.3, 28.3, 28.1, 24.6, 24.0, 22.2. HRMS
(ESI+): m/z (%) = 521.1355, calcd. 521.1358 [M + H]+.
6-(4-Bromo-2-fluorophenoxy)-3-(((3S)-1-
methylpyrido [3,2-d]pyrimidin-4(3H)-one ((S)-13) was obtained according to General Procedure
5. 1H NMR (400 MHz, CDCl3):
(ppm) = 7.98 (d, J = 8.8 Hz, 1H), 7.46–7.11 (m, 4H), 4.29–3.86
(1-fluoropropan-2-yl) piperidin-3-yl)methyl)-2-
δ
(m, 2H), 3.86–3.28 (m, 1H), 3.20–2.88 (m, 2H), 2.75–2.55 (m, 1H), 2.68 (s, 3H), 2.50 (m, 1H), 2.28 (m,
2H), 1.93–1.67 (m, 3H), 1.38–1.22 (m, 2H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
δ
(ppm) = 160.4 (d, J = 16.1 Hz), 154.4 (t, J = 127.0 Hz), 141.6, 140.0 (d, J = 12.1 Hz), 139.6, 133.8, 128.9,
127.9 (d, J = 3.7 Hz), 125.8, 125.1, 120.6 (d, J = 21.5 Hz), 118.7, 118.0 (d, J = 8.2 Hz), 56.5, 52.6, 52.5, 47.7,
35.3, 29.8, 27.9, 23.4, 23.2, 10.8. HRMS (ESI+): m/z (%) = 507.1203, calcd. 507.1202 [M + H]+.
(R)-6-(4-Bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]
1
pyrimidin-4(3H)-one ((R)-
CDCl3):
9
) was obtained according to General Procedures 1–5. H NMR (400 MHz,
δ
(ppm) = 8.01 (d, J = 8.8 Hz, 1H), 7.45–7.14 (m, 4H), 4.21 (s, 1H), 3.93 (m, 1H), 3.45–3.25
(m, 2H), 3.25–2.95 (m, 2H), 2.86–2.49 (m, 2H), 2.73 (s, 3H), 2.34–2.33 (m, 1H), 2.00–1.88 (m, 3H), 1.40
(d, J = 6.6 Hz, 3H), 1.32 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ (ppm) = 160.7, 160.3, 155.6,
154.0, 141.6, 140.0 (d, J = 12.0 Hz), 139.7, 133.6, 127.8 (d, J = 3.7 Hz), 124.9, 120.5 (d, J = 21.6 Hz),
118.7, 117.9 (d, J = 8.3 Hz), 58.0, 52.8, 47.4, 47.0, 34.2, 27.3, 23.1, 22.0, 17.3, 16.3. HRMS (ESI+):
m/z (%) = 489.1292, calcd. 489.1296 [M + H]+.
(S)-6-(4-Bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)pyrido[3,2-d]pyrimidin-
1
4(3H)-one ((S)-16) was obtained according to General Procedures 1–5. H NMR (400 MHz, CDCl3):
δ
(ppm) = 8.18–7.89 (m, 2H), 7.62–7.13 (m, 4H), 4.00 (d, J = 6.9 Hz, 2H), 2.79–2.74 (m, 3H), 2.38–2.70
(m, 3H), 1.92–1.53 (m, 3H), 1.26 (m, 1H), 1.08 (t, J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3):
(ppm) = 160.6, 159.1, 155.6, 153.0, 146.4, 142.3, 140.0, 135.7, 127.7 (d, J = 3.5 Hz), 124.9, 120.4 (d,
δ
J = 21.6 Hz), 118.3, 118.0 (d, J = 8.3 Hz), 55.4, 52.0, 49.7, 48.6, 35.1, 27.9, 23.5, 17.9, 17.4. HRMS (ESI+):
m/z (%) = 475.1139, calcd. 475.1139 [M + H]+.
(S)-6-(4-Bromo-2-fluorophenoxy)-2-fluoro-3- ((1-isopropylpiperidin-3-yl)methyl)pyrido[3,2-d]
pyrimidin-4(3H)-one ((S)-17). TBAF (87 mg, 2 equiv, 0.33 mmol) was added to a solution of (S)-16
(80 mg, 1 equiv, 0.16 mmol) in 2 mL DMF and the reaction mixture was heated to 130 C for 16 h.
)
◦

Int. J. Mol. Sci. 2017, 18, 768
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Upon cooling, water (5 mL) was added and the solution was extracted with DCM (2
over Na2SO4 followed by solvent elimination gave an oil which was purified by flash chromatography
(silica, CH2Cl2/MeOH, 18:1 to 9:1) to give (S)-17) (19 mg, 23% yield) as light brown solid. H NMR
×
5 mL). Drying
1
(400 MHz, CDCl3):
δ (ppm) = 7.71 (s, 1H), 7.54–7.34 (m, 2H), 7.18 (dd, J = 14.9, 8.7 Hz, 2H), 3.26 (m,
5H), 2.35 (m, 4H), 1.86 (m, 2H), 1.36 (t, J = 6.9 Hz, 6H), 1.03 (m, 1H). 13C NMR (100 MHz, CDCl3):
δ
(ppm) = 166.5, 159.6, 155.8, 155.6 (d, J = 25.7 Hz), 139.92 (d, J = 12.1 Hz), 134.5, 133.6, 129.1, 128.1 (d,
J = 3.6 Hz), 125.2, 121.0, 120.4 (d, J = 21.4 Hz), 118.1 (d, J = 8.2 Hz), 115.8, 57.6, 51.8, 48.4, 41.8, 29.7, 27.0,
24.0, 19.7, 15.5. HRMS (ESI+): m/z (%) = 493.1041, calcd. 493.1045 [M + H]+.
4.3. Biology In Vitro Characterization of Compounds
4.3.1. Radioligand Binding Assay
Two micrograms of human GhrR membranes (prepared from HEK293 cells stably expressing
the fusion protein hGhrR-eYFP), 60 pM [125I-His9]ghrelin (PerkinElmer, Waltham, MA, USA), and
different concentrations of the test compound were incubated in HEPES buffer (20 mM HEPES, 20 mM
CaCl2, 0.8 mM MgCl2, 1% Pefabloc, 1% bovine serum albumin, pH 7.4) in a 96-well plate on a shaker
(200 rpm) for 2.5 h at rt. Each concentration was tested in duplicate. The membrane-bound radioligand
was trapped on a Filtermat B (PerkinElmer) pre-soaked with 0.1% polyethylenimine in H2O by using
a filtermat harvester (PerkinElmer). After drying for 20 min at 55 ◦C, a Meltilex scintillation sheet
(PerkinElmer) was melted onto the filtermat and the samples were counted in a Microbeta scintillation
counter (PerkinElmer). For each compound, all data points (n = 4–6) from at least two independent
experiments were combined and IC50 values were obtained by a sigmoidal dose-response fit. Data
were normalized to no competition (100%, minimal ghrelin effect) and full competition (0%, maximal
ghrelin effect).
4.3.2. Inositol Phosphate Accumulation Assay
Briefly, 60,000 to 80,000 cells/well of COS7 cells, stably expressing the fusion protein hGhrR-eYFP,
were seeded in 48-well plates in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal
calf serum (FCS) and 0.4 mg/mL hygromycin B. The next day, the cells were labeled with
[2-3H]-myo-inositol (2
µCi/mL, PerkinElmer). Stimulation with different concentrations of the
compounds alone or with 10−8 M ghrelin together with different concentrations of the compounds
(antagonist experiments) was performed in duplicates for 2 h [58]. Cell debris was removed and the
supernatant purified on an anion-exchange resin (BioRad, Hercules, CA, USA). The obtained data
were analyzed with GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Dpm (decay
per minute) values were normalized to ghrelin effects (100% = maximal efficacy, 0% = constitutive
receptor activity). EC50/IC50 values were calculated from at least three independent experiments. Emax
is defined as the difference between maximal and minimal effect in presence of the compound.
4.3.3. Determination of CHI IAM Values
The chromatographic hydrophobicity index on immobilized artificial membranes (CHI IAM)
was experimentally determined by a HPLC method described by Valko and co-workers [51] using a
IAM PC DD2 column (100
and 50 mM ammonium acetate as eluent and acetanilide, acetophenone, 1,4-dinitrobenzene, anisole
propiophenone, valerophenone, and octanophenone as references.
×
4.6 mm, 10 µM, Regis Technologies, IL, USA), a gradient of acetonitrile,
4.3.4. Selectivity Studies
All compounds were investigated regarding their potential to bind to rat
human 2, 4, and 7 nAChRs, human A1 and A2A receptors, human oxytocin receptor, and rat
VAChT at 100 nM and 1 µM by radioligand displacement studies using the following conditions:
σ1 and σ2 receptors,
α
4
β
α
3
β
α

Int. J. Mol. Sci. 2017, 18, 768
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(a) rat
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 120 min incubation at rt;
(b) rat M dextrallorphan, 50 mM TRIS-HCl, pH 7.4,
2 receptor = rat liver homogenate, [3H]DTG + 1
σ
1 receptor = rat brain homogenate, [3H]pentazocine, 50 mM TRIS-HCl, pH 7.4, 120 mM
σ
µ
120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 120 min incubation at rt;
(c) rat VAChT = homogenates of transfected PC12 cells (by courtesy of Ali Roghani, Texas Tech
University, Lubbock, TX, USA), [3H]vesamicol, 50 mM TRIS-HCl, pH 7.4, 120 mM NaCl, 5 mM
KCl, 2 mM CaCl2, 1 mM MgCl2, 60 min incubation at rt;
(d) human oxytocin receptor = homogenates of transfected CHO cells (by courtesy of Bice Chini,
Isituto di Neuroscienze, Milano, Italy), [3H]oxytocin, 50 mM TRIS-HCl, pH 7.4, 5 mM MgCl2,
0.1% BSA, 60 min incubation at rt;
(e) human nAChR = homogenates of transfected HEK (
courtesy of Dominik Feuerbach, Novartis, Basel, Switzerland), [3H]epibatidine (
[3H]MLA (
α
4
β
2,
α
3
β
4) and SH-SY5Y (
α
7) cells (by
2, 4) or
α
4
β
α3β
α
7), 50 mM TRIS-HCl, pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
60 min incubation at rt;
(f) human adenosine receptors = homogenates of transfected CHO cells (by courtesy of Karl-Norbert
Klotz, Julius-Maximilians-Universität, Würzburg, Germany), [3H]DPCPX (A1) or [3H]ZM241385
(A2A), 50 mM TRIS-HCl, pH 7.4, 1 mM MgCl2 (A1) or 50 mM TRIS-HCl, pH 7.4, 100 mM NaCl,
5 mM MgCl2, 1 M EDTA (A2A), 120 min (A1) or 60 min (A2A) incubation at rt.
The incubations were terminated by rapid filtration via glass-fiber filters presoaked in PEI
(0.3%, 60 min at rt), and the filter-bound radioactivity was counted by liquid scintillation counting
(Tricarb, PerkinElmer).
For compounds displacing >50% of the radioligand at 1 µM or 100 nM, Ki values were determined
by investigating seven concentrations of the test compound (0.01 nM to 10 µM) as described.
Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/4/768/s1.
Acknowledgments: We thank the staff of the Institute of Analytical Chemistry, Department of Chemistry and
Mineralogy of the Universität Leipzig, for the NMR spectral analysis. We thank Tina Spalholz for performing
the radioligand displacement experiments for the selectivity studies and Andrea Suhr for HPLC investigations
regarding CHI IAM determination. The work was supported by Deutsche Forschungsgemeinschaft (DFG)
AOBJ:624810 (to U.K.) and the Sino-German-Center for Research Promotion (SGC) GZ1236 (to U.K.)
Author Contributions: Michael Kluge, Peter Brust, and Ute Krügel initiated the study. Rares¸-Petru Moldovan
conceived and performed the chemistry, Sylvia Els-Heindl, Dennis J. Worm, and Annette G. Beck-Sickinger
planned and performed the GhrR binding affinity assay, and the agonist and the antagonist studies, Torsten Kniess
performed and interpreted the CHI IAM experiments, Winnie Deuther-Conrad, Peter Brust, and Ute Krügel
planned and performed the selectivity studies. All co-authors analyzed and discussed the data and wrote
the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AcOH
GhrR
BOP
Acetic acid
Ghrelin receptor
Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate
Blood-brain barrier
BBB
Boc
tert-Butyloxycarbonyl
CHI IAM
DCM
DMF
EA
Chromatographic hydrophobicity index on immobilized artificial membranes
Dichloromethane
N,N-Dimethylformamide
Ethyl acetate
HPES
HPLC
HRMS
4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
High-performance liquid chromatography
High resolution mass spectrometry

Int. J. Mol. Sci. 2017, 18, 768
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IC50
IH
The half maximal inhibitory concentration
Petroleum ether (40–65 C)
◦
M
Molarity
MW
PET
SPECT
TBAF
Et3N
TFA
Molecular weight
Positron emission tomography
Single-photon emission computed tomography
Tetrabutylammonium fluoride
Triethylamine
Trifluoroacetic acid
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