Development of Dipeptidic hGPR54 Agonists

Article abstract of DOI:10.1002/cmdc.201600331

A series of dipeptides were designed as potential agonists of the human KiSS1-derived peptide receptor (hGPR54). While the sequence Arg-Trp-NH₂was the most efficient in terms of affinity, we established a convergent synthetic strategy to optimi

Full text of DOI:10.1002/cmdc.201600331

DOI: 10.1002/cmdc.201600331
Full Papers
Development of Dipeptidic hGPR54 Agonists
Christelle Doebelin,[a] Isabelle Bertin,[b] Sꢀverine Schneider,[a] Martine Schmitt,[a] Jean-
Jacques Bourguignon,[a] Caroline Ancel,[c] Valerie Simonneaux,[c] Frꢀdꢀric Simonin,*[b] and
Frꢀdꢀric Bihel*[a]
A series of dipeptides were designed as potential agonists of
the human KiSS1-derived peptide receptor (hGPR54). While the
sequence Arg-Trp-NH2 was the most efficient in terms of affini-
ty, we established a convergent synthetic strategy to optimize
the N terminus. Using two successive Sonogashira cross-cou-
pling reactions on a solid-supported peptide, we were able to
introduce various alkynes at the N terminus to afford com-
pounds with sub-micromolar affinities for hGPR54. However,
functional assays indicated the benzoylated dipeptide Bz-Arg-
Trp-NH2 as the most promising compound in terms of agonis-
tic properties. Interestingly, this compound appeared much
more stable than the endogenous neuropeptide kisspeptin,
both in serum and in liver microsomes of rats. This compound
was also found to be able to induce a significant in vivo in-
crease in testosterone levels in male rats.
Introduction
GPR54, also known as the KiSS1-derived peptide receptor (or
KiSS1R), belongs to the G-protein-coupled receptor (GPCR) su-
perfamily and is coupled to Gq/11 GTP binding protein.[1]
hGPR54 is a member of the GPCR subfamily called the RF-
amide receptors, which also includes NPFF1R, NPFF2R, GPR10,
and GPR103. This subfamily of GPCR is known to bind endoge-
nous RF-amide neuropeptides that share the C-terminal se-
quence Arg-Phe-NH2. In 2001, three independent research
groups identified kisspeptin,[2] a 54-residue peptide, as the en-
dogenous ligand for GPR54, a known suppressor of metasta-
sis.[3] Kisspeptin was originally named metastin for its ability to
inhibit metastasis, but was later identified as a potent stimula-
tor of gonadotropin-releasing hormone (GnRH) release, and
thus critical for regulation of reproduction.[4] Expressed in sev-
eral parts of the brain (hypothalamus, hippocampus, amygdala,
etc.),[5] GPR54 and its endogenous ligand kisspeptin were also
localized in dorsal root ganglia (DRG) neurons, and are in-
volved in the regulation of pain sensitivity in rodents.[6] Recent-
ly, pharmacological experiments have investigated the role of
various forms of kisspeptin in pubertal development and adult
reproduction. However, kisspeptin-10 (Kp10) shows a poor
metabolic stability, due to the cleavage of the Gly–Leu peptide
bond by matrix metalloproteinases (MMPs).[7] To circumvent
this metabolic instability, several Kp10 analogues were report-
ed with modifications of the peptidic sequence including un-
natural d-amino acids and peptidomimetic bonds.[7,8] We re-
cently published a study about the effects of N-terminal dele-
tions of endogenous RF-amide peptides on their affinity for
NPFF1R, NPFF2R, GPR10, GPR54, and GPR103.[9] We showed
that systematic and sequential N-terminal deletions of Kp10
decrease the affinity for hGPR54, but do not affect the agonis-
tic character. Even the smaller dipeptide (Bz-RF-NH2) was found
to be a full agonist in the micromolar range (pEC50 =6.40,
E
max =100%).[9b] Several acylated RF-NH2 have been previously
reported as antagonists of NPFF receptors, and they were
stable enough to show potent in vivo efficacies at low doses
on several animal models after intravenous administration.[10]
Starting from Bz-RF-NH2, we propose herein a structure–activi-
ty relationship study of this dipeptide, using an original solid-
phase synthesis based on sequential palladium-catalyzed reac-
tions.
Results and Discussion
Previous works have shown that C-terminal phenylalanine may
efficiently be replaced by tryptophan[11] or tyrosine.[8a] Using
a classical Fmoc strategy on solid phase, we performed a scan
of the C-terminal position, replacing the phenylalanine residue
with other amino acids (1–12, Table 1). Only l-tryptophan led
to a significant gain of affinity (3, Ki =974 nm), whereas all
other substitutions resulted in a loss of affinity. Next, we per-
formed an exploration of the vicinity of the benzamide moiety
at the N terminus (Table 2, 13–19), but all the substitutions led
also to a decrease in affinity. Alanine scans and N-terminal de-
letion studies performed on the Kp10 sequence have already
shown the importance of the phenylalanine residue at posi-
tion 5,[10a,12] highlighting the potential presence of a hydropho-
bic pocket in the GPR54 binding site. In an attempt to explore
[a] Dr. C. Doebelin, S. Schneider, Dr. M. Schmitt, Dr. J.-J. Bourguignon,
Dr. F. Bihel
Facultꢀ de pharmacie, UMR7200, CNRS, University of Strasbourg, 74 route
du Rhin, 67400 Illkirch (France)
E-mail: fbihel@unistra.fr
[b] I. Bertin, Dr. F. Simonin
UMR7242, CNRS, University of Strasbourg, ESBS, 67412 Illkirch (France)
E-mail: simonin@unistra.fr
[c] Dr. C. Ancel, Dr. V. Simonneaux
UPR3212, CNRS, University of Strasbourg, ICIN, 67000, Strasbourg (France)
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600331.
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38 with a purity of 95% at 210 nm (HPLC). After purification by
chromatography, 38 was obtained in 65% overall yield. After
this encouraging initial Sonogashira CCR, the trimethylsilyl pro-
tecting group of 36 was selectively removed with TBAF in THF
at room temperature to afford the corresponding supported
terminal alkyne, which was directly engaged in the second So-
nogashira CCR using phenylbromide or phenyliodide under
the same conditions previously used. Surprisingly, 26 was ob-
tained in only 13% yield with phenyliodide, whereas no reac-
tion was observed with phenylbromide (Table 3, entries 1 and
2). Therefore, we decided to undertake the chemical optimiza-
tion of this second Sonogashira CCR (Table 3). Cerezo et al. pre-
viously showed that Suzuki–Miyaura CCR on supported pep-
tides is feasible using microwave (MW) heating for a short time
(t=10 min).[14] Indeed, starting from supported Arg-Phe deriva-
tive 36, and using MW heating at 808C for only 2 min, we
were able to obtain 25 in 13% yield (Table 3, entry 3). This
result was modest, but still remarkable as we used phenylbro-
mide as reagent. Instead of increasing the heating conditions
(temperature or time), we choose to screen a series of palladi-
um catalysts (Table 3, entries 3–8). All of them afforded 25 in
yields ranging from 8 to 18%, similar to Pd(PPh3)2Cl2 (13%
yield), except for Pd(P(tBu)3)2, which led to a promising 42%
yield. However, Pd(P(tBu)3)2 is difficult to handle, as it is known
to be air sensitive, as is the tri-tert-butylphosphine P(tBu)3. Con-
sequently, we used the air-stable tributylphosphonium salt de-
scribed by Netherton and Fu as an in situ precursor of
P(tBu)3.[15] We screened a series of five palladium catalysts in
presence of P(tBu)3·HBF4, and all of them led to yields ranging
from 30 to 39% (Table 3, entries 9–13). Whereas heating is cru-
cial to initiate the reaction (entry 15), an increase in heating
time up to 20 min did not improve yield (entry 14). Phenylio-
dide was a bit more reactive than phenylbromide, and almost
no reaction occurred with phenylchloride. Although the yield
was still modest (~40% overall yield), compound 25 was re-
covered up to 88% purity (HPLC) after a simple trituration in
Et2O. Moreover, we compared the resulting compound 25 with
that previously obtained by directly coupling Ph-ꢀ-C6H4-COOH
to the supported dipeptide Arg-Phe-NH-Rink resin. HPLC and
1H NMR were identical, and no racemization was detected.
Based on this efficient procedure, we synthesized a series of
derivatives of compound 26 (Table 4, 38–46). In this series
based on the Arg-Trp scaffold, overall yields were higher than
that obtained with the sequence Arg-Phe, ranging from 38 to
85%. In terms of affinity, all of them exhibited affinities in the
micromolar range for hGPR54, showing that the phenylacety-
lene moiety does not tolerate any substitution. Based on their
affinities, three compounds (3, 26, and 27) were selected for
further evaluation of their agonistic properties, and also for
their metabolic stability (Table 5). We compared them with the
pentapeptide 47 (Bz-FGLRF-NH2), which has a phenylalanine
residue at the N terminus, crucial for affinity,[8c] but also the se-
quence Phe-Gly-Leu-Arg, known to be hydrolyzed in serum.[7]
Interestingly, the dipeptide 3 appeared to be a potent full
agonist of hGPR54 (EC50 =69 nm). In contrast, the introduction
of phenylacetylene (26) or styryl (27) moieties led to more
rigid molecules, improving affinity, but altering agonistic prop-
Table 1. Exploration of the C terminus of Bz-RF-NH2.
Compd
Sequence
hGPR54 Ki [nm][a]
or Inh. [%][b]
Kp10
1
H-YNWNSFGLRF-NH2
Bz-Arg-Phe-NH2
Bz-Arg-Hph-NH2
Bz-Arg-Trp-NH2
Bz-DArg-Trp-NH2
Bz-Arg-DTrp-NH2
Bz-DArg-DTrp-NH2
Bz-Arg-Trp(Me)-NH2
Bz-Arg-Tyr-NH2
0.5ꢁ0.1
7932ꢁ857
0%
974ꢁ259
0%
3%
2%
4%
23%
0%
17%
2%
0%
2[c]
3
4
5
6
7
8
9
Bz-Arg-His-NH2
Bz-Arg-Leu-NH2
Bz-Arg-Asn-NH2
Bz-Arg-Gln-NH2
10
11
12
[a] [3H]Kp13 was used as radioligand for competition assays; values are
the meanꢁSEM of at least two independent determinations performed
in duplicate. [b] Percent inhibition at 5 mm. [c] Hph=homophenylalanine.
this additional lipophilic pocket, we grafted an aromatic ring
to the benzamide moiety at various positions using spacers
(Table 2, 20–33). The para position appeared more favorable
than the meta position, and the best compounds were those
with a rigid spacer, such as a phenylacetylene group (26, Ki =
326 nm) or a styryl group (27, Ki =322 nm). Interestingly, the
more flexible 29 with a phenethyl group displayed lower affini-
ty (Ki =5.2 mm) than 25 with a rigid spacer (Ki =1.555 mm). As
observed previously (Table 1), tryptophan at the C terminus led
to a significant improvement over phenylalanine.
Following a classical optimization process, we next intro-
duced chemical diversity on the phenylacetylene moiety. All
the compounds listed in Table 2 were prepared by following
a standard solid-phase strategy, in which all the N-terminal
moieties were prepared separately as carboxylic acids (see the
Supporting Information). As the introduction of chemical diver-
sity around the phenylacetylene group would require many
carboxylic acids to prepare as precursors, we developed an in-
novative and convergent chemical strategy aimed at introduc-
ing chemical diversity through palladium-catalyzed cross-cou-
pling reactions performed directly on the dipeptide supported
on resin (Scheme 1). Palladium-catalyzed cross-coupling reac-
tions (CCR) have already been applied to solid-phase synthe-
sis,[13] including Suzuki–Miyaura, Heck, Negishi, Sonogashira,
and Buchwald–Hartwig. However, these reactions were mainly
applied on supported heterocycles, compatible with high tem-
peratures and strong bases. In our case, the challenge was to
perform racemization-free Sonogashira CCR on a supported
peptide. To improve the optimization of compound 26, we
chose to perform two successive Sonogashira CCRs
(Scheme 1). Starting from the supported para-iodobenzamide
34, a first Sonogashira CCR was performed with trimethylsilyl-
acetylene under standard conditions: PdCl2(PPh3)2/CuI in the
presence of Et3N in DMF at room temperature. To characterize
the cross-coupling reaction, the resin, as well as TMS and tBu
moieties, were cleaved with TFA to afford the terminal alkyne
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Table 2. Exploration of the N termini of Bz-RF-NH2 (1) or Bz-RW-NH2 (3).
Compd
R1C(O)-Arg-Xaa-NH2
R1
hGPR54[a]
Xaa
Inh. [%][b]
Ki [nm][c]
1
3
Phe
Trp
66
100
7932ꢁ857
974ꢁ259
Ph
13
14
15
16
17
18
2-C6H4-Cl
3-C6H4-Cl
4-C6H4-Cl
2-C6H4-OMe
3-C6H4-OMe
4-C6H4-OMe
Phe
Phe
Phe
Phe
Phe
Phe
40
39
27
29
44
17
>10 mm
>10 mm
>10 mm
>10 mm
>10 mm
>10 mm
19
Phe
35
>10 mm
20
21
2-C6H4-Ph
3-C6H4-Ph
Phe
Phe
21
24
>10 mm
>10 mm
22
23
Phe
Trp
70
100
4872ꢁ1489
747ꢁ95
4-C6H4-Ph
24
25
26
meta
para
para
Phe
Phe
Trp
43
92
100
>10 mm
1555ꢁ348
326ꢁ28
27
para
Trp
100
322ꢁ91
28
29
meta
para
Phe
Phe
32
62
>10 mm
5208ꢁ608
30
31
meta
para
Phe
Phe
34
89
>10 mm
3135ꢁ345
32
33
meta
para
Phe
Phe
23
60
>10 mm
>10 mm
[a] [3H]Kp13 was used as radioligand for competition assays. [b] Percent inhibition at 5 mm. [c] Values are the meanꢁSEM of at least two independent de-
terminations performed in duplicate.
erties. Still, 26 and 27 are full agonists of hGPR54 in the sub-
micromolar range. Next, we investigated the metabolic stability
of these compounds (Table 5). Whereas the pentapeptide 47
showed poor stability both in serum and in liver microsomes,
dipeptides 3, 26, and 27 appeared much more stable under
both conditions. As peripheral administration of kisspeptin is
known to increase circulating luteinizing hormone (LH) and
testosterone,[16] peptides 3, 26 and 27 were tested for their
ability to increase the production of testosterone in compari-
son with kisspeptin (Figure 1). Compounds were tested at
10 mgkgꢂ1 (i.p.) in male rats, but 26 appeared poorly soluble
in the vehicle (ethanol/Ringer [1:1]), and was not tested. De-
spite acting as full agonist at GPR54 in the sub-micromolar
range, 27 was not potent enough to stimulate testosterone.
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Scheme 1. Supported synthetic strategy leading to compounds 25, 26, and 38–46.
Table 3. Optimization of the second Sonogashira CCR.
Entry
PhX
Pd source
Ligand
MW[a]
Yield [%][b]
T [8C]
t [min]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
PhI
Pd(PPh3)2Cl2
Pd(PPh3)2Cl2
Pd(PPh3)2Cl2
Pd(PPh3)4
Pd(dppf)Cl2
Pd(P(tBu)3)2
Pd(MeCN)2Cl2
Pd(PhCN)2Cl2
Pd(OAc)2
Pd(PPh3)2Cl2
Pd(P(tBu)3)2
Pd(MeCN)2Cl2
Pd(PhCN)2Cl2
Pd(PhCN)2Cl2
Pd(PhCN)2Cl2
Pd(PhCN)2Cl2
Pd(PhCN)2Cl2
–
–
–
–
–
–
–
–
RT
RT
13[c]
0
13
18
8
42
15
16
30
39
28
33
31
34
0
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhBr
PhI
80
80
80
80
80
80
80
80
80
80
80
80
2
2
2
2
2
2
2
2
2
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
P(tBu)3·HBF4
2
2
20
RT
80
80
2
2
43
1
PhCl
[a] Microwave. [b] Yield over nine steps determined by HPLC/UV using caffeine as external standard. [c] Yield after 16 h.
Conversely, the small benzoylated dipeptide 3 led to a signifi-
cant increase in testosterone, similar to that induced by kiss-
peptin. This result is particularly interesting if we consider the
metabolic stability of 3 in comparison with kisspeptin. As the
Arg-Phe-NH2 sequence is known to bind efficiently to both
NPFF1 and 2 receptors, we tested 3 on both receptors, and we
found micromolar affinities (NPFF1R: Ki =2.4 mm, NPFF2R: Ki =
1.0 mm) in the same range of its affinity for hGPR54.
and although exhibiting a lower EC50 value (69 nm) than kiss-
peptin (<1 nm), it clearly induced a significant increase in
levels of circulating testosterone when administered to rats at
a dose of 10 mgkgꢂ1 (i.p.). In conclusion, 3 is a promising com-
pound for optimization, and more synthetic work will be re-
quired to improve its selectivity toward other RFamide recep-
tors such as NPFF1R and NPFF2R.
Experimental Section
Conclusions
Chemistry
Using a rational design along with metal-catalyzed reactions
on supported dipeptides, we were able to develop promising
ligands (26 and 27) of hGPR54. However, these compounds ap-
peared to be less potent agonists than the simple benzoylated
dipeptide Bz-Arg-Trp-NH2 (3). This small dipeptide appeared to
be much more stable than the endogenous peptide kisspeptin,
Materials: Chemicals and solvents were used without further pu-
rification. Compounds were purified using Armen spot flash chro-
matography on silica gel Merck 60 (particle size 0.040–0.063 mm),
or on simply connect C18 from AIT. Yields refer to isolated com-
pounds, with purity >95% as determined by HPLC. 1H and
13C NMR spectra were recorded at 400/500 MHz and 101/128 MHz,
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respectively. All chemical shift values (d) and coupling constants (J)
are quoted in ppm and in Hz, respectively. Microwave irradiations
were performed using Biotage Initiator EXP.
Table 4. Yields obtained for 26, 38–46, and their affinities for hGPR54.
General solid-phase peptide synthesis procedures: All peptides
were synthesized by manual solid-phase synthesis by using an
Fmoc strategy with a Rink-amide resin SS (Advanced ChemTech,
0.75 mmolgꢂ1). The side-chain protective groups included tert-
butyl (tBu) for tyrosine, tert-butoxycarbonyl (Boc) for tryptophane,
trityl (Trt) for histidine, asparagine, and glutamine, and 2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine. Fmoc
deprotection was performed by using a solution of 2 % piperidine
and 2% DBU in DMF. Coupling reactions were performed by the
addition of N,N-diisopropylethylamine (DIEA, 4.5 equiv), Fmoc-pro-
tected amino acid or carboxylic acid (3 equiv), and (benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP,
2.85 equiv) in CH2Cl2, and the mixture was agitated for 1 h. All cou-
pling reactions were monitored by Kaiser test. Resin cleavage was
performed by using a solution of TFA/thioanisole/triisopropylsilane
(92:5:3). The mixture was filtered, and the peptide solution was
concentrated in vacuo, and precipitated by adding cold Et2O. The
mixture was centrifuged, and the resulting crude peptide was puri-
fied by flash chromatography (Simply connect C18 column (50 g,
40–60 mm) from AIT. Phase A: 0.05% TFA in H2O, phase B: MeOH;
eluent: 10!100% phase B in 30 min. Final compounds 1–33 and
47 were recovered with purity >95% as determined by HPLC (see
the Supporting Information).
Compd
R
Yield [%][a]
hGPR54 Ki [nm][b]
26
38
39
40
41
42
43
44
45
46
Ph
H
326ꢁ28
>10 mm
>10 mm
5290ꢁ1398
7122ꢁ2764
2181ꢁ685
>10 mm
65
75
85
39
42
75
38
73
52
2-C6H4-Cl
3-C6H4-Cl
4-C6H4-Cl
3-C6H4-OMe
4-C6H4-OMe
2-pyridinyl
3-pyridinyl
4-pyridinyl
>10 mm
2891ꢁ496
2897ꢁ492
[a] Isolated yield over nine steps. [b] [3H]Kp13 was used as radioligand for
competition assays; values are the meanꢁSEM of at least two independ-
ent determinations performed in duplicate.
N-(benzoyl)-Arg-Trp-NH2, trifluoroacetate salt 3: 1H NMR
(500 MHz, CD3OD): d=7.79–7.72 (m, 2H), 7.61–7.59 (m, 1H), 7.58–
7.52 (m, 1H), 7.50–7.40 (m, 2H), 7.31–7.23 (m, 1H), 7.12 (s, 1H),
7.08–7.02 (m, 1H), 7.0–6.95 (m, 1H), 4,72 (dd, J=5.8, 8.0 Hz, 2H),
4.52 (dd, J=6.3, 8.2 Hz, 1H), 3.23–3.14 (m, 4H), 1.91–1.80 (m, 1H),
1.80–1.73 (m, 1H), 1.56 ppm (s, 2H); 13C NMR (126 MHz, CD3OD):
d=176.9, 174.0, 170.9, 158.9, 138.3, 135.3, 133.3, 129.9, 129.1,
128.9, 125.0, 122.8, 120.2, 119.6, 112.6, 111.1, 55.4, 55.2, 42.2, 30.1,
29.2, 26.5 ppm; MS (MM-ES-APCI) m/z [M+H]+ calcd for
C24H29N7O3: 464.2, found 464.2.
Table 5. Functional activity toward hGPR54 and metabolic stability.
Compd
EC50 [nm][a]
Stability
Rat serum CLint
[b]
t1/2 [min]
47 Bz-Phe-Gly-Leu-Arg-Phe-NH2
0.55ꢁ0.06
69ꢁ1.8
32ꢁ4
36ꢁ5
3
Bz-Arg-Trp-NH2
Ph-ꢀ-Bz-Arg-Trp-NH2
Ph-=-Bz-Arg-Trp-NH2
121ꢁ28 <3
26
27
700ꢁ99
429ꢁ8
<3
3.4ꢁ3
390ꢁ48
196ꢁ10
[a] hGPR54 calcium mobilization assay: values are the meanꢁSEM of at
least two independent determinations performed in duplicate. [b] Clear-
ance [mLminꢂ1 (mg protein)ꢂ1] determined in rat liver microsomes.
General procedure to introduce trimethylsililylacetylene on
solid-phase by Sonogashira reaction (35–36): Supported N-(4-io-
dobenzoyl)-l-Arg-Xaa-NH-resin (Xaa=l-Phe or l-Trp(tBu)) were
synthesized using the general solid-phase peptide synthesis proce-
dures. In a syringe with frit, resin was suspended in anhydrous
DMF (c=0.1 mmolmLꢂ1), followed by the addition of trimethyla-
mine (20 equiv) and trimethylsilylacetylene (5 equiv). Reaction mix-
ture was degassed with nitrogen, before adding PdCl2(PPh3)2
(0.2 equiv) and CuI (0.4 equiv). The reaction was stirred overnight
with an orbital mixer at room temperature. Resin was then washed
successively with DMF, CH2Cl2, MeOH, and Et2O.
General procedure to trimethylsilyl deprotection and second So-
nogashira coupling reaction on solid phase (25–26, 38–46): TBAF
(1m in THF) was introduced in a syringe containing the supported
dipeptide 36 or 37, and stirred for 3 h at room temperature. Solid
phase was then washed successively with THF, DMF, CH2Cl2, MeOH,
and Et2O. Resin was transferred to a microwave reactor, and a mix-
ture of anhydrous DMF/piperidine [v/v] (c=0.28 mmolmLꢂ1) was
added, followed by haloaryl or heteroaryl (5 equiv). Reaction mix-
ture was degassed with nitrogen, and PdCl2(PhCN)2 (0.05 equiv),
PtBu3·HBF4 (0.1 equiv) and CuI (0.1 equiv) were introduced. The mi-
crowave tube was then capped properly and the reaction mixture
was stirred for 2 to 20 min at 808C under microwave irradiation.
Resin was then transferred to a syringe with frit, and was washed
successively with DMF, CH2Cl2, MeOH, Et2O. Cleavage of the pep-
Figure 1. Effect of GPR54 agonists on circulating testosterone in adult male
rats. Kisspeptin and compounds 3 and 27 were dissolved in ethanol/Ringer’s
solution (1:1) and injected intraperitoneally (10 mgkgꢂ1). Testosterone was
measured by using a commercial radioimmunoassay kit from blood sampled
30 min post-injection. Values are the meanꢁSEM of n=6 rats per experi-
mental point; statistical significance: *p<0.05 relative to control.
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tide–resin complexes was performed by treatment with trifluoro-
acetic acid/triisopropylsilane/thioanisole (92:3:5, v/v/v) at room
temperature for 2 h. After filtration of the resin, anhydrous Et2O at
48C was added to the soluble material to cause precipitation of
the crude peptide, which was collected as a pellet by centrifuga-
tion. Purification was performed as described previously for classi-
cal peptides, and final compounds (25–26, 38–46) were recovered
with purity >95% by HPLC.
N-(4-(3-methoxyphenyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroa-
cetate salt 42: 1H NMR (400 MHz, CD3OD): d=7.87–7.73 (m, 2H),
7.68–7.57 (m, 3H), 7.39–7.28 (m, 2H), 7.15 (s, 1H), 7.14–7.05 (m,
3H), 7.05–6.92 (m, 2H), 4.74 (dd, J=5.5, 8.3 Hz, 1H), 4.49 (dd, J=
6.3, 8.0 Hz, 1H), 3.84 (s, 3H), 3.27–3.18 (m, 2H), 3.15 (t, J=7.3 Hz,
2H), 1.92–1.76 (m, 2H), 1.62–1.50 ppm (m, 2H); 13C NMR (101 MHz,
CD3OD): d=176.9, 173.8, 169,9, 161.1, 158.6, 138.0, 134.3, 132.6,
130.8, 128.9, 128.8, 128.2, 125.2, 125.0, 124.8, 122.5, 119.9, 119.3,
117.7, 116.3, 112.4, 110.7, 92.7, 89.1, 55.9, 55.4, 55.2, 41.9, 30.7, 26.2,
24.2 ppm; MS (MM-ES-APCI) m/z [M+H]+ calcd for C33H35N7O4:
594.2, found 594.2.
N-(4-phenylethynylbenzoyl)-Arg-Phe-NH2, trifluoroacetate salt
1
25: H NMR (500 MHz, CD3OD): d=7.83–7.86 (m, 2H), 7.60–7.63 (m,
2H), 7.52–7.56 (m, 2H), 7.38–7.42 (m, 3H), 7.21–7.26 (m, 2H), 7.16–
7.21 (m, 2H), 7.11–7.16 (m, 1H), 4.66 (dd, J=5.36, 9.14 Hz, 1H),
4.53 (dd, J=6.3, 8.2 Hz, 1H), 3.13–3.23 (m, 3H), 2.87–2.99 (m, 1H),
1.82–1.92 (m, 1H), 1.71–1.82 (m, 1H), 1.52–1.64 ppm (m, 2H);
13C NMR (126 MHz, CD3OD): d=176.4, 174.1, 170.1, 159.1, 138.2,
134.4, 133.2, 133.0, 130.8, 130.4, 130.1, 129.9, 129.3, 128.8, 128.3,
124.5, 93.2, 89.7, 56.1, 55.4, 42.4, 39.3, 30.2, 26.7 ppm; MS (MM-ES-
APCI) m/z [M+H]+ calcd for C30H32N6O3: 525.2, found 525.2.
N-(4-(4-methoxyphenyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroa-
cetate salt 43: 1H NMR (400 MHz, CD3OD): d=7.85–7.74 (m, 2H),
7.70 (d, J=7.0 Hz, 1H), 7.65–7.55 (m, 2H), 7.31–7.21 (m, 3H), 7.11
(s, 1H), 7.05–6.88 (m, 4H), 4.69 (dd, J=5.7, 8.7 Hz, 1H), 4.39 (dd,
J=7.2 Hz, 1H), 3.84 (s, 3H), 3.26–3.13 (m, 2H), 3.10 (t, J=7.0 Hz,
2H), 1.87–1.70 (m, 2H), 1.58–1.40 ppm (m, 2H); 13C NMR (101 MHz,
CD3OD): d=178.3, 173.9, 169.8, 164.0, 159.3, 136.5, 135.6, 133.4,
132.2, 131.6, 131.3, 129.1, 124.9, 122.5, 122.5, 119.5, 116.6, 116.5,
113.7, 112.4, 93.4, 87.6, 56.2, 55.7, 55.4, 42.0, 26.3, 24.7, 24.3 ppm;
MS (MM-ES-APCI) m/z [M+H]+ calcd for C33H35N7O4: 594.2, found
594.2.
N-(4-ethynylbenzoyl)-Arg-Trp-NH2, trifluoroacetate salt 38:
1H NMR (400 MHz, CD3OD): d=7.75 (d, J=8.3 Hz, 2H), 7.60 (d, J=
8.0 Hz, 1H), 7.53 (d, J=8.3 Hz, 2H), 7.28 (d, J=8.0 Hz, 1H), 7.12 (s,
1H), 7.08–7.01 (m, 1H), 7.01–6.91 (m, 1H), 4.71 (dd, J=5.5, 8.3 Hz,
1H), 4.45 (dd, J=6.5, 8.0 Hz, 1H), 3.69 (s, 1H), 3.26–3,17 (m, 2H),
3.17–3.06 (m, 2H), 1.87–1.72 (m, 2H), 1.60–1.45 ppm (m, 2H);
13C NMR (101 MHz, CD3OD): d=176.3, 174.2, 169.9, 159.3, 138.3,
135.1, 133.4, 129.1, 127.7, 125.1, 122.8, 120.2, 119.7, 119.6, 116.9,
111.0, 83.9, 81.6, 55,8, 55.6, 42.2, 29.8, 29.0, 26.6 ppm; MS (MM-ES-
APCI) m/z [M+H]+ calcd for C26H29N7O3: 488.2, found 488.2.
N-(4-(2-pyridinyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroacetate
salt 44: 1H NMR (400 MHz, CD3OD): d=8.63–8.61 (m, 1H), 8.03–
7.91 (m, 1H), 7.84 (d, J=8.5 Hz, 2H), 7.75 (d, J=7.8 Hz, 1H), 7.69
(d, J=8.3 Hz, 2H), 7.61 (d, J=7.8 Hz, 1H), 7.58–7.47 (m, 1H), 7.30
(d, J=8.0 Hz, 1H), 7.15 (s, 1H), 7.11–7.01 (m, 1H), 7.01–6.90 (m,
1H), 4.73 (dd, J=5.5, 8.3 Hz, 1H), 4.47 (dd, J=6.9, 7,7 Hz, 1H),
3.24–3.18 (m, 2H), 3.14 (t, J=7.0 Hz, 2H), 1.93–1.74 (m, 2H), 1.62–
1.44 ppm (m, 2H); 13C NMR (101 MHz, CD3OD): d=177.4, 174.4,
170.3, 159.2, 151.0, 143.7, 139.8, 138.6, 135.8, 133.6, 129.7, 129.6,
127.2, 125.8, 125.3, 123.0, 122.8, 119.9, 117.0, 112.9, 111,2, 92.0,
90.5, 56.1, 55.8, 42.5, 30.0, 29.1, 26.8 ppm; MS (MM-ES-APCI) m/z
[M+H]+ calcd for C31H32N8O3: 565.2, found 565.2.
N-(4-(2-chlorophenyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroace-
tate salt 39: 1H NMR (400 MHz, CD3OD): d=7.81 (d, J=8.28 Hz,
2H), 7.66–7.51 (m, 4H), 7.53–7.47 (m, 1H), 7.43–7.31 (m, 2H), 7.29
(d, J=8.03 Hz, 1H), 7.13 (s, 1H), 7.10–7.02 (m, 1H), 7.02–6.92 (m,
1H), 4.72 (dd, J=5.65, 8.41 Hz, 1H), 4.45 (dd, J=6.53, 8.03 Hz, 1H),
3.27–3.15 (m, 2H), 3.13 (t, J=7.15 Hz, 2H), 1.87–1.76 (m, 2H), 1.61–
1.45 ppm (m, 2H); 13C NMR (101 MHz, CD3OD): d=177.0, 174.0,
170.0, 158.7, 138.1, 137.0, 134.7, 134.6, 132.7, 131.4, 130.6, 129.1,
128.2, 127.8, 124.9, 122.6, 120.0, 119.5, 119.4, 116.6, 112.5, 110.8,
94.5, 89.4, 55.7, 55.4, 42.0, 29.6, 28.7, 26.4 ppm; MS (MM-ES-APCI)
m/z [M+H]+ calcd for C32H32ClN7O3: 598.2, found 598.2.
N-(4-(3-pyridinyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroacetate
salt 45: 1H NMR (400 MHz, CD3OD): d=8.86–8.75 (m, 1H), 8.65–
8.53 (m, 1H), 8.24 (d, J=8.0 Hz, 1H), 7.83 (d, J=8,5 Hz, 2H), 7.74–
7.68 (m, 1H), 7.66 (d, J=8.3 Hz, 2H), 7.61 (d, J=8.0 Hz, 1H), 7.29
(d, J=8.0 Hz, 1H), 7.14 (s, 1H), 7.08–7.02 (m, 1H), 7.02–6.93 (m,
1H), 4.76–4.67 (m, 1H), 4.46 (dd, J=7.2 Hz, 1H), 3.37 ꢂ3.32 (m,
1H), 3.18–3.22 (m, 1H), 3.13 (t, J=7.2 Hz, 2H), 1.90–1.74 (m, 2H),
1.61–1.46 ppm (m, 2H); 13C NMR (101 MHz, CD3OD): d=176.7,
173.8, 169.7, 158.5, 150.5, 142.9, 137.9, 135.1, 132.8(2C), 129.0,
128.9, 128.6, 126.7, 124.7, 122.4, 122.1, 119.8, 119.2, 116.3, 112.3,
110.6, 93.9, 91.0, 55.4, 55.2, 41.8, 30.6, 29.4, 26.1 ppm; MS (MM-ES-
APCI) m/z [M+H]+ calcd for C31H32N8O3: 565.2, found 565.2.
N-(4-(3-chlorophenyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroace-
1
tate salt 40: H NMR (400 MHz, CD3OD): d=7.79 (d, J=8.3 Hz, 2H),
7.61–7.59 (m, 3H), 7.55 (s, 1H), 7.49–7.46 (m, 1H), 7.44–7.37 (m,
2H), 7.30 (d, J=8.0 Hz, 1H), 7,14 (s, 1H), 7.08–7.01 (m, 1H), 7.01–
6.92 (m, 1H), 4.71 (dd, J=5.5, 8.3 Hz, 1H), 4.45 (dd, J=7.0, 7.5 Hz,
1H), 3.25–3.16 (m, 2H), 3.13 (t, J=7.2 Hz, 2H), 1.90–1.71 (m, 2H),
1.63–1.47 ppm (m, 2H); 13C NMR (101 MHz, CD3OD): d=176.8,
174.0, 170.0, 158.4, 138.0, 135.5, 134.7, 132.8, 132.3, 131.4, 131.2,
130.2, 129.0, 127.6, 125.9, 124.9, 122.6, 120.1, 119.4, 116.6, 112.5,
110.7, 91.1, 90.6, 55.6, 55.3, 42.0, 40.4, 28.7, 26.8 ppm; MS (MM-ES-
APCI) m/z [M+H]+ calcd for C32H32ClN7O3: 598.2, found 598.2.
N-(4-(4-chlorophenyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroace-
tate salt 41: H NMR (400 MHz, CD3OD): d=7.69 (d, J=8.3 Hz, 2H),
N-(4-(4-pyridinyl)ethynylbenzoyl)-Arg-Trp-NH2, trifluoroacetate
salt 46: 1H NMR (400 MHz, CD3OD): d=8.88–8.71 (m, 2H), 8.01–
7.95 (m, 2H), 7.88–7.81 (d, J=8.5 Hz, 2H), 7.77–7.66 (d, J=8.5 Hz,
2H), 7.61 (d, J=8.0 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.14 (s, 1H),
7.09–7.00 (m, 1H), 7.00–6.93 (m, 1H), 4.72 (dd, J=5.5, 8.3 Hz, 1H),
4.47 (dd, J=6.2, 7.9 Hz, 1H), 3.21–3.15 (m, 2H), 3.14 (t, J=7.0 Hz,
2H), 1.89–1.74 (m, 2H), 1.58–1.56 ppm (m, 2H); 13C NMR (101 MHz,
CD3OD): d=176.8, 173.8, 169.6, 158.7, 146.2, 146.0, 138.0, 136.2,
133.4, 129.2, 128.8, 125.7, 124.8, 122.5, 119.9, 119.4, 116.5, 112.4,
110.7, 88.5, 88.3, 55.5, 55.3, 41.9, 29.5, 26.3, 24.2 ppm; MS (MM-ES-
APCI) m/z [M+H]+ calcd for C31H32N8O3: 565.2, found 565.2
1
7.54–7.48 (m, 3H), 7.44 (d, J=8.5 Hz, 2H), 7.32 (d, J=8.5 Hz, 2H),
7.20 (d, J=8.0 Hz, 1H), 7.03 (s, 1H), 6.98–6.94 (m, 1H), 6.92–6.83
(m, 1H), 4.66–4.57 (m, 1H), 4.38 (dd, J=6.4, 8.4 Hz, 1H), 3.14–3.06
(m, 2H), 3.04 (t, J=7.2 Hz, 2H), 1.79–1.65 (m, 2H), 1.52–1.39 ppm
(m, 2H); 13C NMR (101 MHz, CD3OD): d=176.8, 173.8, 170.0, 168.9,
138.2, 136.0, 134.6, 134.2, 132.6, 130.0, 128.9, 127.8, 124.7, 122.5,
119.9, 119.5, 119.3, 116.6, 112.4, 110.7, 94.6, 90.3, 55.3, 55.2, 41.9,
29.7, 28.7, 26.2 ppm; MS (MM-ES-APCI) m/z [M+H]+ calcd for
C32H32ClN7O3: 598.2, found 598.2.
&
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
6
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
pound remaining based on chromatographic peak area versus
time were plotted, and the slope was calculated by linear fitting of
the curve. The microsomal intrinsic clearance [CLint, expressed in
mLminꢂ1 (mg protein)ꢂ1] was calculated using the equation:[17]
Biological methods
Materials: Probenecid was from Sigma–Aldrich (Saint Quentin Fal-
lavier, France) and Fluo-4 acetoxymethyl (AM) ester from Molecular
Probes (Invitrogen, Cergy Pontoise, France). Kp-10 and NPFF were
from Polypeptide (Strasbourg, France). RFRP-3 was from Tebu-Bio
(Le Perray-en-Yvelines, France). [3H]FFRF-amide (13.6 Cimmolꢂ1
was from the CEA (Saclay, France). [3H]Kp13 (90 Cimmolꢂ1) was
from Hartmann Analytic (Braunschweig, Germany).
0:693 ꢃ ðincubation volumeÞ
CLint
¼
ð1Þ
)
t1=2 ꢃ ðmg microsomal proteinÞ
In vitro metabolic stability in rat serum: All incubations were per-
formed individually for each test compound. Compounds 3, 26,
27, and 47 (50 mm) were incubated at 378C in rat serum (200 mL).
Final concentration of DMSO was 0.5%. At different times (0, 15,
30, 60, and 120 min), aliquots (35 mL) of the reaction mixture were
quenched with 100 mL of cold acetonitrile. Upon centrifugation of
the resulting mixture, supernatants were analyzed by a generic
HPLC method performed on a Dionex Ultimate 3000 (Waters XSe-
lect CSH C18 column, 5 mm, 4.6ꢂ50 mm) with the following param-
eters: Flow rate: 1 mLminꢂ1, column temperature: 308C, solvent
system: A (MeOH) and B (0.05% TFA in H2O), t=0–12 min, 20!
100% A then t=12–15 min, 100% A. The ratio of product was de-
termined by integration of UV spectra recorded at 202 nm. Meta-
bolic stability was determined by the disappearance of the tested
compound over time. The natural logarithm linear plots of the per-
cent of compound remaining based on chromatographic peak
area versus time were plotted, and the slope was calculated by
linear fitting of the curve. The in vitro half-life (t1/2, min) was esti-
mated by using 0.693/k, in which k is the biotransformation rate
constant and corresponds to the slope of the ln-linear curve.
Adult male NMRI mice (Taconic, Ll Skensved) weighing 30–32 g
were housed five animals per cage. The animals were kept under
12 h/12 h light/dark cycle, in a temperature-controlled room, with
free access to food and water and acclimatized in the same cage
for at least seven days before the experiment. All experiments
were conducted in accordance with the Declaration of Helsinki, the
Danish National Guide for Care and Use of Laboratory Animals, and
the European Communities Council Directive (86/609/EEC; license
number 2005/561–962).
Radioligand binding assays: Binding assay conditions were essen-
tially as described previously.[9a] Briefly, membranes from CHO cells
stably expressing hGPR54 were incubated with 0.15 nm [3H]Kp13.
Nonspecific binding levels were determined in the presence of
1 mm Kp10. NPFF1R- or NPFF2R-containing membranes were incu-
bated with 10 or 3 nm [3H]FFRF-NH2, respectively. Nonspecific
[3H]FFRF-NH2 binding levels were determined in the presence of
1 mm RFRP-3 (NPFF1R) or 1 mm NPFF (NPFF2R). Competition-type
experiments were performed at 258C, under binding equilibrium
conditions (1 h, 0.25 mL final volume) in the presence of increasing
concentrations of unlabeled peptides or compounds to be tested
for their binding affinity. Membrane-bound radioactivity was sepa-
rated from free radioligand by rapid filtration through a 96-well
GF/B Unifilter apparatus (PerkinElmer Life and Analytical Sciences,
Courtaboeuf, France) and quantified using a TopCount scintillation
counter (PerkinElmer).
Effect of GPR54 agonists on circulating testosterone in adult
male rats: In vivo assay conditions were essentially as described
previously.[18] Briefly, the mice (n=6) received an intraperitoneal
(i.p.) injection of the test compounds (kisspeptin, 3, and 27 dis-
solved in ethanol/Ringer’s solution [1:1]) at a dose of 10 mgkgꢂ1
(where Ringer’s solution consists of 6.5 mgmLꢂ1 NaCl,
0.42 mgmLꢂ1 KCl, 0.25 mgmLꢂ1 CaCl2, and 0.2 mgmLꢂ1 NaHCO3).
Trunk blood was collected after decapitation 30 min after treat-
ment. The blood was centrifuged for 10 min at 3000 rpm, and
serum was collected and stored at ꢂ208C until hormone levels
were determined. Free serum testosterone was measured using
Calcium mobilization: hGPR54-expressing CHO cells were loaded
with 2.5 mm Fluo-4 AM in the presence of 2.5 mm probenecid, as
described previously.[9a] Compounds were serially diluted in 10 mm
HEPES pH 7.4, 0.4 mm NaH2PO4, 137.5 mm NaCl, 1.25 mm MgCl2,
1.25 mm CaCl2, 6 mm KCl, 10 mm glucose and 1 mgmLꢂ1 BSA). Ag-
onist-evoked increases in intracellular calcium were recorded over
time (5 s intervals over 220 s) at 378C through fluorescence emis-
sion at 520 nm (excitation at 485 nm) by using a Flexstation III (Mo-
lecular Devices, Sunnyvale, CA, USA). Peak response amplitudes
were normalized to basal and maximal (cells permeabilized with
20 mm digitonin) fluorescence levels.
a
direct radioimmunoassay (RIA) kit (DPC coat-a-count RIA
method; Siemens Medical Solutions, Mçlndal, Sweden).
Acknowledgements
This work was supported by the Centre National de la Recherche
Scientifique (CNRS), Universitꢀ de Strasbourg, and by grants from
the Agence National de la Recherche (ANR-08-EBIO 014.02, ANR-
13-BSV1-0001). This work was published within the LABEX ANR-
10-LABX-0034_Medalis framework and received financial support
from the Government of France managed by “Agence National
de la Recherche” under “Programme d’investissement d’avenir”.
In vitro metabolic stability in rat liver microsomes: All incuba-
tions were performed individually for each test compound. Com-
pounds 3, 26, 27, and 47 (50 mm) were incubated at 378C under
standard incubation conditions: phosphate buffer (pH 7.4), NADPH
(1 mm), and rat liver microsomes (0.1 mg protein). The final con-
centration of DMSO was 0.5%. At different times (0, 5, 15, 30, and
45 min), aliquots (35 mL) of the reaction mixture were quenched
with 100 mL of cold acetonitrile. Upon centrifugation of the result-
ing mixture, supernatants were analyzed by a generic HPLC
method performed on a Dionex Ultimate 3000 (Waters XSelect CSH
C18 column, 5 mm, 4.6ꢂ50 mm) with the following parameters:
Flow rate: 1 mLminꢂ1, column temperature: 308C, solvent system:
A (MeOH) and B (0.05% TFA in H2O), t=0–12 min, 20!100% A
then t=12–15 min, 100% A. The ratio of product was determined
by integration of UV spectra recorded at 202 nm. Metabolic stabili-
ty was determined by the disappearance of the tested compound
over time. The natural logarithm linear plots of the percent com-
Keywords: agonists · GPR54 · Sonogashira cross-coupling ·
solid-phase peptide synthesis · testosterone
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Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
8
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
The simpler, the better: By using palla-
dium-catalyzed reactions on supported
peptides, we synthesized a series of di-
peptides that act as agonists of the
human KiSS1-derived peptide receptor
(hGPR54). However, we showed that the
simple N-benzoylated dipeptide Bz-RW-
NH2 is sufficient to induce a significant
increase in levels of circulating testos-
terone when administered to rats at
a dose of 10 mgkgꢂ1 (i.p.).
C. Doebelin, I. Bertin, S. Schneider,
M. Schmitt, J.-J. Bourguignon, C. Ancel,
V. Simonneaux, F. Simonin,* F. Bihel*
&& – &&
Development of Dipeptidic hGPR54
Agonists
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
9
ꢁ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ