Lucile Anthore-Dalion

Emmanuel Nicolas

Thibault Cantat

Catalytic Metal-free Deoxygenation of Nitrous Oxide with Disilanes

Keywords: nitrous oxide, organocatalysis, reduction, fluoride, disilanes

Due to its high kinetic stability, conditions to reduce the greenhouse gas N2O are limited, hence requiring a better understanding of its chemistry and of the N-O bond cleavage. In this work, N2O was deoxygenated under metal-free conditions. Using disilanes as reducing agents and a catalytic amount of fluoride anions or alkoxides allowed a mild reduction at ambient pressure and temperature. DFT calculations unveiled the mechanism, which shows a nucleophilic addition of a silyl anion to the central N atom of N2O and release of N2 from a pseudo-Brook rearrangement.

In the natural nitrogen cycle, nitrous oxide, also called "laughing gas", is the last intermediate of the denitrification process, which transforms nitrates into atmospheric dinitrogen. [START_REF] Lehnert | Reversing Nitrogen Fixation[END_REF] Because of human activities, such as agriculture, waste management, and industrial productions of adipic and nitric acid, its atmospheric concentration has significantly increased in the last centuries (20% since 1750), 2 raising environmental issues. N2O is indeed the most potent ozonedepleting substance and a greenhouse gas, [START_REF] Dameris | Depletion of the Ozone Layer in the 21st Century. Angew[END_REF] estimated to be 298 times more potent than CO2. [START_REF] Epa | Gas Inventory Report[END_REF] This has led to a growing interest in its chemical reactivity and to the development of end-of-the-pipe technologies to reduce industrial emissions. [START_REF] Nirisen | The Development of a N2O Abatement Catalyst: from Laboratory Scale to Plant Testing[END_REF] From a synthetic point of view, nitrous oxide is kinetically highly stable [START_REF] Johnston | Interpretation of the Data on the Thermal Decomposition of Nitrous Oxide[END_REF] and a poor ligand for transition metals. [START_REF] Tolman | Binding and Activation of N2O at Transition-Metal Centers: Recent Mechanistic Insights[END_REF] Hence, reactions with this gas often require harsh conditions in presence of heterogeneous catalysts (typically >200 °C and >25 bar for alkenes oxidation). [START_REF] Leont'ev | Modern Chemistry of Nitrous Oxide[END_REF] Only few examples of reactions with N2O occur under homogeneous and milder reaction conditions. [START_REF] Severin | Synthetic Chemistry with Nitrous Oxide[END_REF] In most cases, N2O has been used as an oxidant (E°(N2O/N2) = 1.77 V vs SHE) 10 and usually reacts with highly reactive organometallic species via oxygen atom transfer. 11 This reactivity has enabled the development of transition metal-catalyzed transformations where N2O is deoxygenated in the presence of reductants such as Grignard reagents, 12 phosphines, 13 aldehydes, 14 alcohols 15 or CO. [START_REF] Zeng | CO Oxidation by N2O Homogeneously Catalyzed by Ruthenium Hydride Pincer Complexes Indicating a New Mechanism[END_REF] Recently, Milstein and coworkers showed that the reduction of nitrous oxide can even be performed using H2, with a PNP pincer ruthenium complex as catalyst (Scheme 1). [START_REF] Zeng | Hydrogenation and Hydrosilylation of Nitrous Oxide Homogeneously Catalyzed by a Metal Complex[END_REF] So far, there is however no example of metalfree deoxygenation of N2O under mild conditions.

To better apprehend the reactivity of the N-O bond and overcome the kinetic stability of N2O, we chose to use disilane derivatives as reducing agents. They are indeed efficient and mild deoxygenating agents due to the high oxophilicity of silicon, and easier to activate than hydrogen thanks to a lower bond dissociation energy (79.7 kcal.mol -1 in Me3SiSiMe3 vs 104 kcal.mol -1 in H2). [START_REF] Dávalos | Thermochemistry and Dissociative Photoionization of Si(CH3)4, BrSi(CH3)3, ISi(CH3)3, and Si2(CH3)6 Studied by Threshold Photoelectron-Photoion Coincidence Spectroscopy[END_REF] Moreover, N2O is known to react with tetramethylsilane in absence of catalysts but only under harsh conditions (>2 000 bar, >360 °C); [START_REF] Hagen | High-Pressure Reactions of Small Covalent Molecules. 9. Reaction of Methylsilanes with Covalent Oxides[END_REF] a transformation used for the passivation of electronical devices by chemical vapor deposition ("Semi Insulating Polycrystalline Silicon" (SIPOS)). [START_REF] Fayolle | Analysis and Modeling of SIPOS Deposition in a Hot Wall Tubular Reactor. Part II: Modeling of SIPOS Deposition[END_REF] Milstein et al. have also used their ruthenium catalyst to perform N2O reduction with hydrosilanes under milder reaction conditions (3.4 bar, 65 °C, Scheme 1). [START_REF] Zeng | Hydrogenation and Hydrosilylation of Nitrous Oxide Homogeneously Catalyzed by a Metal Complex[END_REF] We show herein that the deoxygenation of N2O can even be carried out under ambient conditions (1 bar, 20 °C) without any transition metal by using disilanes in the presence of a catalytic amount of fluoride anions or alkoxides (Scheme 1). Mechanistic investigations unveiled the mechanism at play in the N-O bond cleavage. Scheme 1. Catalytic reduction of nitrous oxide with H2 or organosilanes.

To start our investigations, we selected hexamethyldisilane (1) as a readily available and moisture stable reductant. Fluoride anions were screened as catalysts since they are known to efficiently activate disilanes. 21,[START_REF] Hiyama | Generation of Metal-Free Silyl Anions from Disilanes and Fluoride Catalyst. Synthetic Reactions with Aldehydes and 1,3-Dienes[END_REF] Exposing a DMSO solution of disilane 1 to an atmosphere of N2O (1 bar), in the presence of a catalytic amount of CsF (10 mol%), resulted in the formation of 77 % hexamethyldisiloxane (2) within 4 h at 20 °C. Analysis of the gas phase by gas chromatography (GC) indicated the quantitative conversion of N2O into N2 (N2/N2O 99:1) (Table 1, entry 1). No reaction took place without catalyst, even after 3 days (Table 1, entry 2). [START_REF] Spialter | The Cleavage of Silanes by Oxides of Nitrogen[END_REF] Strictly anhydrous conditions were necessary to avoid the competitive deoxygenation of H2O leading to H2 production (Figure S8). Remarkably, the reaction could be scaled-up to 1.78 mmol of disilane 1 without any modification of the protocol, and furnished disiloxane 2 in 75% yield. Other alkali metal fluoride salts (LiF, NaF, KF) did not catalyze the reaction, certainly due to their lower solubilities (Table 1, entries 3-5). In contrast, with tetramethyl ammonium fluoride (TMAF), an anhydrous and solid surrogate of tetrabutyl ammonium fluoride, an increased activity was noted (Table 1,entry 6). The reaction proved to be however much slower with the moisture stable KHF2 and tetrabutylammonium difluorotriphenyl-silicate (TBAT) as catalysts (Table 1, entries 7 and8). In these latter cases, the fluoride anion is indeed not naked, and hence less reactive.

Disilanes can also be activated by non-fluorinated bases such as alkoxides. 24 In fact, both KOMe and KOtBu catalyzed the reaction, leading to full conversion of N2O within 1 h and 2 h, respectively (Table 1, entries 9 and10). At this stage, KOMe and KOtBu could act as precatalysts for the generation of a silanolate species. 21a,c This hypothesis was validated by the successful use of KOSiMe3 as a catalyst (Table 1, entry 11).

Interestingly, no reaction was detected in either THF or MeCN, even when 18-crown-6 was added to help solubilize CsF. In contrast, when a DMSO/THF 1:1 mixture was used as solvent, the reaction proceeded but required 48 h to reach full conversion, showing a detrimental effect of THF (Table S2). This observation was ascribed to a lower solubility of both the fluorinated base and N2O in less polar solvents.

Table 1. Catalyst screening for the reduction of N2O with disilane 1. [a] Entry Cat. Time Conversion of 1 (%) [b] Yield in 2 (%) [b] N2/N2O 8 [d] TBAT [a] Conditions: N2O (1 bar, ≈2 mL), (Me3Si)2 (0.12 mmol), catalyst (12 µmol, 10 mol%), DMSO-d6 (0.5 mL). [b] Conversions and yields measured by GC-MS analysis. Internal standard: 1,3,5-trimethoxybenzene. The difference between conversion and yield is ascribed to the volatility of the silylated compounds and to the production of Me3SiF, Me3SiOMe or Me3SiOtBu in the course of the reaction. [c] Corrected GC-ratios. Residual amount of N2 from the atmosphere <10% [d] Scrambling of R3Si residues: Ph3SiF and Ph3SiOSiMe3 are detectable in GC-MS.

[e] H2 is also detected in GC (<2%). See SI for more details (p. S7).

To test the influence of the stereoelectronic properties of the silane reductant, the deoxygenation of N2O was carried out with (PhMe2Si)2 ( 3) and (Ph2MeSi)2 (4) (Table 2, entries 2 and 3). Interestingly, the more Lewis acidic the disilane was, the faster the reaction proceeded, as reflected in the yields in the corresponding siloxanes, after 1 h, of 55%, 63% and 68%, for disilanes 1, 3 and 4, respectively (Table 2). In contrast, neither (Bpin)2 nor PhMe2Si-Bpin proved successful under these conditions.

Unfortunately, no intermediate could be either isolated or observed by NMR in the course of the reaction or through stoichiometric reactions. Hence DFT calculations were used (for computational details see SI, part V), to track how the N-O bond is cleaved under mild and metal-free conditions to promote the reduction of the kinetically stable N2O molecule. According to the experimental results, CsF and KOSiMe3 show a similar activity (Table 1, entries 1 and 11), and the latter was hence selected as a catalyst in the mechanism depicted in Scheme 2. The role of the fluoride anion (or alkoxides) as a precatalyst (mechanism A) or as a catalyst (mechanism B) is also explored, thereafter.

Table 2. Screening of disilanes for the reduction of N2O [a] Entry R3SiSiR3 Yield [b] after 1 h

Final yield [b] N2/N2O [c] 1 (Me3Si)2 (1) 55% 77% (4 h) 99:1 2 (PhMe2Si)2 (3) 63% 93% (3 h) 100:0 3 [d] (Ph2MeSi)2 (4) 68% 93% (2 h) 100:0

[a] Conditions: N2O (1 bar, ca. 2 mL), R3SiSiR3 (0.12 mmol), catalyst (12 µmol, 10 mol%), DMSO-d6 (0.5 mL). [b] Yields measured by GCanalysis (disilane 1) or by NMR-analysis (compounds 3 and 4). Internal standard: 1,3,5-trimethoxybenzene. [c] Corrected GC-ratios. Residual amount of N2 from the atmosphere <10%. [d] To overcome the poor solubility of the starting material in DMSO, the reaction mixture was stirred 2 h at 20 °C under argon prior to adding N2O. See SI for more details (p. S11).

The silanolate Me3SiO -(I) can react either with N2O or with (Me3Si)2 (1). Whereas the activation barrier of the reaction with N2O was high in energy (ΔG ‡ = +44.4 kcal.mol -1 , Figure S10), the nucleophilic addition of silanolate I to disilane 1 appeared more favorable (ΔG ‡ = +18.5 kcal.mol -1 ). After release of the siloxane (Me3Si)2O ( 2), it generates a highly nucleophilic Me3Si -anion (III), in an overall exergonic sequence (ΔG= -4.7 kcal.mol -1 ) (Figure 1c). This result is supported by the observation in NMR of the instantaneous formation of Me3SiOSiMe3 ( 2) when KOSiMe3 and disilane 1 are mixed (Figure S7).

Scheme 2. Two plausible mechanisms: with the fluoride anion or the alkoxides as precatalysts (A) or as catalysts (B).

In contrast to silanolate I, silyl anion III efficiently reacts with N2O via a nucleophilic attack. Considering that the two degenerate LUMOs of N2O are mostly developed on the two nitrogen atoms (Figure 1a), the nucleophilic attack can occur either on the central or the terminal nitrogen centers. Computational results show a significant difference in the activation barrier energy of both pathways: the nucleophilic attack of Me3Si -(III) on the central nitrogen is kinetically favored by 10.8 kcal.mol -1 (TS3 and TS'3) and, under the applied conditions (20 °C), the attack on the terminal N atom is hardly accessible (ΔG ‡ =+26.2 kcal.mol -1 ). As such, the less stable Me3Si(N2O) -intermediate IV is the major product. Little is known about the ambivalent reactivity of N2O as an electrophile. According to DFT calculations, metal-hydride complexes attack preferentially at the terminal nitrogen. 25 Likewise, non-metallic systems, such as N-heterocyclic carbenes reported by Severin et al. 26 or frustrated Lewis pairs, 27 have been experimentally shown to react via the terminal nitrogen of N2O. The selectivity observed in our system is reversed and follows the principle of least nuclear motion, [START_REF] Mayr | Farewell to the HSAB Treatment of Ambident Reactivity[END_REF] with an addition of the silyl anion to the nitrogen atom bearing the highest positive charge and a corresponding transition state (TS3) that minimizes the bond elongations in the N2O fragment (Figure 1a and SI). Such an intramolecular rearrangement has also been proposed for CO2 reduction with disilanes by Skrydstrup et al. 21a,c and in the reaction of KHMDS with N2O to synthesize potassium azide, where a [1,4]-silicon shift has been suggested. [START_REF] Liu | Trimethylsilyl-Induced N-O Bond Cleavage in Nitrous Oxide-Derived Aminodiazotates[END_REF] The overall reaction between N2O and hexamethyldisilane ( 1) is thus highly exergonic by 120.1 kcal.mol -1 , and the first nucleophilic substitution at the silicon atom is the rate determining step (RDS), with an energetic barrier of 18.5 kcal.mol -1 (TS1, Figure 1c). With this mechanism in hands, we then investigated the role of the catalytic species, using the fluoride system as a model. Addition of F -to the hexamethyldisilane reductant (1) generates the Me3Si - anion (III), in agreement with previous findings, [START_REF] Hiyama | Generation of Metal-Free Silyl Anions from Disilanes and Fluoride Catalyst. Synthetic Reactions with Aldehydes and 1,3-Dienes[END_REF] and this nucleophile is competent to deoxygenate N2O as discussed there above. Yet, its regeneration can proceed via two different pathways: either through the route described in Figure 1c, where the fluoride anion is playing the role of a precatalyst (Scheme 2, A), or through a route involving an actual catalytic activity of F -(Scheme 2, B). The latter implies the displacement of a fluoride anion from Me3SiF, by the Me3SiO -silanolate (I). This reaction proceeds via an associative pathway, with the formation of a stable pentacoordinated intermediate V (Figure 2). The energy barrier involved in the regeneration of the free fluoride anion is computed at 20.8 kcal.mol -1 , 2.3 kcal.mol -1 greater than the energy span computed for the transformation catalyzed by the Me3SiO -silanolate. This small difference in energy suggests that F -is a precatalyst in the reduction of N2O by hexamethyldisilane.

In the reduction of N2O with hexamethyldisilane ( 1), the formation and regeneration of the silyl anion Me3Si -(III) controls the rate of the reaction. The latter attacks N2O preferentially at the central nitrogen and a subsequent barrier-less pseudo-Brook rearrangement releases N2. This mechanism also explains the trend observed with phenyl substituted disilanes 2 and 3 (Table 2). Indeed, with the more acidic disilanes 2 and 3, the rate of the reaction is no longer controlled by the regeneration of the silyl anion but by the addition of the silyl anion to N2O, which involves low energy barriers of 15.0 kcal.mol -1 and 15.2 kcal.mol -1 , respectively (see SI, Figures S12,S13). Thanks to this lower energy span, phenyl substituted disilanes 2 and 3 react faster than disilane 1 (Table 2).

In summary, we have developed the first transition metal-free catalytic deoxygenation of N2O. The kinetic stability of this gas has been overcome by using disilanes as mild reducing agents, under ambient conditions (1 bar, 20 °C), and in presence of catalytic amounts of fluoride anion or alkoxides. According to DFT calculations, the reaction proceeds through nucleophilic addition of a silyl anion to the central nitrogen of N2O. The cleavage of the N-O bond then results from a pseudo-Brook rearrangement to release N2 gas, triggered by the formation of a strong Si-O bond.

Figure 1 .

 1 Figure 1. N2O structure and computed mechanism. [a] NBO charges and frontier molecular orbitals of N2O [b] Main resonance structures of N2O [c] Reaction mechanism starting from silanolate I. Gibbs free energies in kcal.mol -1 computed at the B3LYP-D3/6-31G* (C,H,F,N,O) and B3LYP-D3/6-311+G** (Si) level of theory, using PCM=DMSO to account for solvation From the Me3Si(N2O) -intermediate (IV), an essentially barrierless and highly exergonic [1,2]-pseudo-Brook rearrangement occurs through the 3-center transition state TS4 (ΔG= -102 kcal.mol - 1 ), in which the cleavage of the N-O bond is compensated by the synchronized formation of the stable Si-O bond, to release N2.Such an intramolecular rearrangement has also been proposed for CO2 reduction with disilanes by Skrydstrup et al.21a,c and in the reaction of KHMDS with N2O to synthesize potassium azide, where a[1,4]-silicon shift has been suggested.[START_REF] Liu | Trimethylsilyl-Induced N-O Bond Cleavage in Nitrous Oxide-Derived Aminodiazotates[END_REF] The overall reaction between N2O and hexamethyldisilane (1) is thus highly exergonic by 120.1 kcal.mol -1 , and the first nucleophilic substitution at the silicon atom is the rate determining step (RDS), with an energetic barrier of 18.5 kcal.mol -1 (TS1, Figure1c).

Figure 2 .

 2 Figure 2. Possible pathway for the regeneration of silyl anion Me3Si -(III) in presence of a catalytic amount of fluoride anions. Gibbs free energies in kcal.mol-1 computed at the B3LYP-D3/6-31G* (C,H,F) and B3LYP-D3/6-311+G** (Si) level of theory, using PCM=DMSO to account for solvation.

ACKNOWLEDGMENT

For financial support of this work, we acknowledge the CEA, CNRS, ANR (project RecyclingSiB) and the CHARMMMAT LABEX. We thank CINES for the allowance of computer time (Project No. c2019086494).

AUTHOR INFORMATION

Corresponding Author * lucile.anthore@cea.fr and thibault.cantat@cea.fr

Notes

The authors declare no competing financial interests.

ASSOCIATED CONTENT Supporting Information

Detailed descriptions of experimental and computational methods are given in the Supporting Information, available free of charge on the ACS Publications website.


Khoubaib Ben

Haj Salah

Mathieu Maingot

Anne-Laure Blayo

Céline M'kadmi

Marjorie Damian

Sophie Mary

Sonia Cantel

Jérémie Neasta

Catherine Oiry

Sylvie Péraldi-Roux

Gimena Fernandez

Guadalupe García Romero

Mario Perello

Jacky Marie

Jean-Louis Banères

Jean-Alain Fehrentz

Séverine Denoyelle

Development of non-peptidic inverse agonists of the ghrelin receptor

INTRODUCTION

The ghrelin receptor, a G protein-coupled receptor identified as the growth hormone secretagogue receptor (GHSR), 1 is known for regulating numerous central and peripheral processes including, among others, growth hormone (GH) secretion, adiposity, feeding and glucose metabolism. Indeed, administration of its endogenous agonist, a stomach-derived 28amino acid peptide named ghrelin, 2 induces blood glucose elevation and the reduction of circulating insulin in humans and rodents. 3 Moreover, ghrelin is the most potent orexigenic peptide hormone known and its administration to humans or rodents potently increases food intake. 4 Knowing that GHSR exhibits a high constitutive activity, an inverse agonist compound capable of selectively targeting the receptor and reducing its constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In an effort to find efficient GHSR ligands, we previously developed a heterocyclic series that presented high affinity and selectivity for this receptor. These new ligands, based on the 3,4,5-trisubstituted 1,2,4-triazole scaffold (Figure 1), included four points of diversity and their synthesis, starting from an optically pure amino acid, comprised a few steps with moderate to good yields. We demonstrated that the configuration of the chiral center was retained during the synthetic process. 5-7 An intensive structure activity relationship study, carried out over several hundred triazole compounds, allowed us to evaluate the ability of these compounds to activate or inactivate GHSR signaling. Moreover, very interestingly, some of our antagonist compounds were able to antagonize food intake elicited by an agonist such as hexarelin, while they were unable to antagonize GH secretion elicited by the same agonist. This was the case for JMV2959 (Figure 1, compound 1), a GHSR antagonist which has since been broadly described in the literature 8 to hold potent anti-obesity but also anti-addictive properties. The first described inverse agonist of GHSR was an analog of substance P (SPA). 9 Even though [D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]-substance P was able to selectively reduce the constitutive activity of the receptor, it also acted as a biased ligand toward neuropeptide and chemokine receptors. 10-11 However, after several truncations of the original sequence, Holst et al. identified the C-terminal pentapeptide wFwLL-NH2 (in its carboxamide version) as the peptide core responsible for the inverse agonist activity. 12 Moreover, the presence of a lysine residue at the N-terminus of the peptide core K-(D-1-Nal)-FwLL-NH2 led to a specific inverse agonist almost as potent as the substance P analog. 13 Most recently, our team found that LEAP2 (liver-expressed antimicrobial peptide 2), an endogenous ligand described as antagonist of GHSR, 14 behaved as inverse agonist of the receptor and as competitive antagonist of ghrelin-induced inositol phosphate production and calcium mobilization. 15 We also demonstrated that the entire sequence of LEAP2 was not necessary for its biological activity. Indeed, the N-terminal part alone, namely the LEAP2 (1-14-NH2) analog, maintained receptor binding and biological activity, and was able to inhibit ghrelininduced food intake in mice. In addition to these substance P and LEAP2 peptide analogs, only a few non peptidic inverse agonists of GHSR has been reported in the literature so far (for a review see 16 ). In order to overcome the lack of potent small molecules capable of selectively reducing the constitutive activity of GHSR, we decided to search for inverse agonists based on our 1,2,4-triazole scaffold. The first structural modulations with minor changes in the R 1' , R 2' , R 3' and R 4' groups did not lead to inverse agonists. However, the introduction of a secondary chiral center at position 3 of the triazole ring and the elongation with the Leu-Leu dipeptide sequence allowed us to obtain a partial inverse agonist with moderate affinity toward the receptor. 17 In light of these encouraging results, we decided to carry on our investigation by making structural changes on our initial antagonist, JMV2959. First, we chose to decorate its primary amine with alkyl or carbonyl groups.

We were able to introduce various substituents including alkanes, alkenes, substituted aromatic rings and heterocycles. Then, we modulated the R 3' substituent of the 1,2,4-triazole scaffold.

Herein we report the design, the synthesis and the biological evaluation of this new series of ligands. Having characterized all the compounds by NMR, LC-MS and HRMS, an in vitro SAR study was performed in order to determine their pharmacological properties and their ability to reduce the basal activity of GHSR. In order to determine whether this specific 1,2,4-triazole scaffold was active on endogenously expressed GHSR, we tested one of the most effective ligands for its ability to modulate the insulinostatic effect of ghrelin in rat isolated pancreatic islets. At last, we investigated the ability of this lead compound to affect food intake under different experimental settings in mice.

RESULTS AND DISCUSSION

Design and syntheses. We first designed a set of ligands (compounds 2-8) on which the primary amine of JMV2959 (compound 1) was substituted by various alkyl groups. Compounds 2-8 were synthesized following the same procedure previously described to provide compound 1. 18 Briefly, the triazole derivative (iv) was obtained in four steps starting from Boc-(D)-Trp-OH as shown in Scheme 1. First, Boc-(D)-Trp-OH (i) was reacted with 4-methoxybenzylamine (ii) in the presence of BOP reagent and DIEA in DCM for 2 hours at room temperature. The resulted amide was transformed into the corresponding thioamide (iii) using the Lawesson's reagent in DME at 80 °C for 3 hours. The cyclization into the triazole heterocycle was performed by reacting the thioamide (iii) with 3-phenylpropanehydrazide in the presence of silver benzoate (PhCO2 ˉAg + ) and acetic acid (AcOH) in DCM at room temperature for 12 hours. 19 After removal of the Boc protecting group using a solution of 4N HCl in dioxane at 50 °C for 30 minutes, the structural diversity was introduced on the triazole derivative (iv) by coupling various N-substituted glycines in the presence of BOP reagent and DIEA in DCM for 2 hours at room temperature. Depending on the structure of the N-substituent on the glycine, a last deprotection step was required to afford the final compounds: a solution of 4N HCl in dioxane at 50 °C for the removal of the Boc and tBu protecting groups and a solution of diethylamine (Et2NH) in DMF at room temperature for the removal of the Fmoc protecting group. The second set of ligands (compounds 9-35) was also designed starting from compound 1 (Scheme 2). A classical peptide coupling reaction between the primary amine of 1 and a large panel of carboxylic acids allowed us to introduce the structural diversity on substituent R 2 . The coupling reaction was conducted under basic conditions (DIEA) in the presence of HATU reagent in DCM for 2 hours at room temperature. In the case of ligands decorated with either an amino function (compounds 11 and 23) or a hydroxyl group (compounds 21 and 22), the protecting group, required to carry on the coupling reaction, was removed in a final step using a solution of 4N HCl in dioxane at 50 °C for 30 min. The last set of ligands (compounds 36-46) included several structural modulations but all were comprised of a -cyano--phenylethylene moiety (potentially substituted in position para of the phenyl group) in place of substituent R 2 (Scheme 3). First, we varied the nature of substituent R 3 .

We decided to introduce either a phenyl group (compounds 36-39), a morpholine group (compounds 40-42), an indole group (compounds 43 and 44), a piperidine group (compound 45) or a diethylamine group (compound 46). We used appropriate substituted hydrazides (intermediates v) that we reacted with thioamide (iii) in the presence of PhCO2 ˉAg + and AcOH in DCM at room temperature for 12 hours. Removal of the Boc protecting group led to triazole intermediates (vi). At this step of the syntheses, we had the possibility to modulate the spacer between these triazole intermediates and the 2-cyano-3-phenylacrylyl moiety bearing substituent R 4 . While we kept a glycine residue for most of the ligands, we modified the linker of compounds 36-39. For instance, compound 36, which had no spacer, was obtained by directly coupling alphacyano-4-methoxycinnamic acid with intermediate (vi) under the same conditions described above.

The spacer of compound 37 was introduced by using 2-hydroxyacetic acid, which allowed us to make an ester bond with alpha-cyano-4-methoxycinnamic acid in contrast to the usual amide bond.

For compounds 38 and 39, we simply replaced the methylene bridge by an ethylene bridge (compound 38) or an ethylidene bridge (compound 39). At last, diversity of substituent R 4 resulted from the use of various para substituted alpha-cyanocinnamic acids that were reacted with deprotected beforehand intermediate (vii) via the same classical peptide coupling conditions previously described. (f) HATU, DIEA, DCM, 2 h; (g) para substituted alpha-cyanocinnamic acids, HATU, DIEA, DCM, 2h. For R 3 and R 4 , see Table 3.

All final compounds were purified by reversed-phase preparative HPLC. Depending on the intricacy of the purification, the purity assessed by analytical reversed phase C18 HPLC was found around 95% for most compounds and greater than 98% for the rest, and the structures of the compounds were confirmed by HRMS, 1 H NMR, and 13 C NMR (see supporting information for more details).

Evaluation of the ligands in GHSR expressing HEK293T cells. Binding and Pharmacological Activities of the Ligands. We first analyzed in vitro whether the different compounds directly bound to GHSR expressed in HEK293T cells (Tables 123). Ki values were determined from binding competition experiments with fluorescent ghrelin using the homogenous time-resolved fluorescence (HTRF) assay as previously described. 20 We also measured the Inositol Phosphate 1 (IP1) response of each compound (10 -5 M) and calculated it as a percentage of the maximal stimulation promoted by ghrelin (10 -7 M). We compared this value with the percentage of the basal response of the receptor (value in square brackets) in order to determine the pharmacological activity of each compound toward the receptor. A ligand was considered as agonist when it increased the basal activity of the receptor, as inverse agonist when it reduced it and as antagonist when it did not induce any modulation. The high basal level of IP1 production in GHSR-expressing cells, representing 65-75% of the maximal IP1 production promoted by ghrelin, resulted from the high constitutive activity of GHSR as previously shown. 9, 21 Parent compound JMV2959 (ligand 1) was taken as reference. First, we tested the compounds with alkylated amine (ligands 2-8). As shown in Table 1, all ligands lost some affinity toward the receptor compared to JMV2959. Steric hindrance did not seem to play a role since ligands 3, 4, 5, 7 and 8 presented comparable Ki values while bearing more or less bulky substituents. However, the presence of a carboxyl group (ligand 6) should be prohibited as it drastically reduced the affinity of the compound (Ki = 617 nM). Concerning the activity of the ligands, all behaved as agonists or partial agonists of GHSR. In light of these results, alkylation of the amine function of compound JMV2959 did not appear as a valid approach to obtain inverse agonists of the receptor.

Therefore, we decided to acylate the amine function with various carboxylic acids offering a wide range of substituents R 2 (Table 2). In terms of affinity, this approach seemed promising as it lowered the Ki value for most of the ligands. Some ligands even showed Ki values below the nanomolar range (ligands 14, 30 and 32). Only a cyano group directly linked to the amide bond prevented the compound to bind to the receptor (ligand 12, Ki = 1.6 μM). It is noteworthy that all the ligands bearing a -cyanoethylene group substituted in position  by an unsaturated ring such as phenyle (ligands 27-32), thiophene (ligand 33), pyrrole (ligand 34) or indole (ligand 35), behaved as inverse agonists of the receptor. The substituent in position para of the phenyl group (ligands 28-32) did not seem to affect the affinity nor the activity of the ligands. As shown in Table 3, modification of the spacer (Y) between the triazole core and the 2-cyano-3-phenylacrylyl moiety bearing substituent R 4 had an impact on the properties of the ligands. Indeed, shortening the spacer (ligand 36) or replacing the amide bond by an ester bond (ligand 37) cannot be envisaged since it leads to partial agonists. At last, replacement of a phenyl group by various R 3 substituents (ligands [40][41][42][44][45][46] was well tolerated as it led to potent inverse agonists of the receptor with good binding affinities, except for ligand 43, which behaved as a partial agonist. a Values reported in the table were obtained from experiments performed in HEK293T cells stably expressing GHSR. b Ki values were determined from HTRF competition binding assays. Values are expressed as means  SEM of two independent experiments, each experiment being performed in triplicate. c Activities were determined by HTRF inositol phosphate (IP) assay. IP response of each ligand (10 -5 M) is given as percentage of the maximal stimulation promoted by ghrelin (10 -7 M). The percentage in square brackets refers to the basal response of the receptor also given as percentage of the maximal ghrelin response. a Values reported in the table were obtained from experiments performed in HEK293T cells stably expressing GHSR. b Ki values were determined from HTRF competition binding assays. Values are expressed as means  SEM of two independent experiments, each experiment being performed in triplicate. c Activities were determined by HTRF inositol phosphate (IP) assay. IP response of each ligand (10 -5 M) is given as percentage of the maximal stimulation promoted by ghrelin (10 -7 M). The percentage in square brackets refers to the basal response of the receptor also given as percentage of the maximal ghrelin response. Pharmacological Activities of the Inverse Agonist Ligands. We selected the ligands that behaved as inverse agonists and further tested their ability to affect the basal level of IP1 production in HEK293T cells expressing GHSR (Table 4, Figure 2). We determined Emax (maximal inhibition effect on the basal response promoted by the receptor) and EC50 values, and compared them to reference inverse agonists SPA and K-(D-1-Nal)-FwLL-NH2. As shown in Table 4, most of our ligands were at least as potent as the reference compounds. Some were even more potent than SPA (ligands [START_REF] Damian | Ghrelin Receptor Conformational Dynamics Regulate the Transition from a Preassembled to an Active Receptor:Gq Complex[END_REF][START_REF] Kozasa | Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged Alpha and Beta Gamma Subunits[END_REF][START_REF] Mcewen | Fluorescent BODIPY-GTP Analogs: Real-Time Measurement of Nucleotide Binding to G Proteins Anal[END_REF]44). Moreover, ligands described as partial inverse agonists (ligands [START_REF] M'kadmi | Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling[END_REF][START_REF] Mary | Ligands and Signaling Proteins Govern the Conformational Landscape Explored by a G Protein Coupled Receptor[END_REF][START_REF] Yanagi | The Homeostatic Force of Ghrelin[END_REF] showed less effect on the basal response (Emax values of between 18 and 43% inhibition of the basal response) than full inverse agonists which were able to reduce the basal response up to more than 80% (ligands [START_REF] Damian | Ghrelin Receptor Conformational Dynamics Regulate the Transition from a Preassembled to an Active Receptor:Gq Complex[END_REF][START_REF] Kozasa | Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged Alpha and Beta Gamma Subunits[END_REF][START_REF] Mcewen | Fluorescent BODIPY-GTP Analogs: Real-Time Measurement of Nucleotide Binding to G Proteins Anal[END_REF]44). As shown in Figure 1, ligands 28, 29, 30 and 44 presented better pharmacological profiles in term of Emax and/or EC50 values compared to SPA and K-(D-1-Nal)-FwLL-NH2. Table 4. Pharmacological activity of inverse agonist ligands 18, 21-23, 27-35, 38-42, 44-46 a Values reported in the table were obtained from experiments performed in HEK293T cells stably expressing GHSR. b EC50 values were obtained from concentration-response curves of inositol phosphate (IP) production (HTRF assay) and are expressed as means  SEM of three independent experiments, each experiment being performed in triplicate. c Emax values were obtained from these plots and are given as percentage inhibition of basal receptor response at a concentration of 10 -5 M for each compound. Activity of Inverse Agonists on the Purified GHSR. GHSR-mediated G protein activation.

a Ligand IP EC50 b (nM) IP Emax c (%) Ligand IP EC50 b (nM) IP Emax c (%) SPA 66  10 78 33 8.6  4 65 K-(D-1-Nal)-FwLL-NH2 12
We used the purified receptor assembled into lipid nanodiscs in order to evaluate whether the impact of selected ligands 27, 29, 30 and 44 on GHSR signaling observed in HEK293T cells resulted from a direct effect on the receptor independently of the cellular context. Note that ligand 28 was excluded due to solubility issues, and replaced by ligand 27 (same structure without the methyl substituent R 4 ). The ability of the ligands to affect GHSR activation was assessed by monitoring GHSR-mediated Gq protein activation in a GTPγS binding assay (Figure 3). We fixed the maximal GHSR-catalyzed G protein activation with ghrelin, and we compared our ligands to reference inverse agonist SPA. As expected, the purified apo GHSR assembled into lipid nanodiscs displayed a significant constitutive activity (Figure 3). Concerning ligands 27, 29, 30 and 44, they were able to significantly reduce Gq protein activation by the receptor. Ligand 44 has proven to be even more potent than SPA as it almost fully inhibited the basal activity of the purified receptor. This indicated that our ligands exerted an inverse agonist activity on GHSR-dependent basal activation of Gq. Thus, these data suggest that the inverse agonist activity of our ligands observed in HEK293T cells is directly related to their interaction with GHSR independently of the cellular context. Impact of Inverse Agonists on GHSR Conformation. We previously reported that the pharmacological properties of GHSR ligands were intricately related to their ability to stabilize specific receptor conformations. 22 To assess whether the inverse agonist activity of ligands 27, 29, 30 and 44 was indeed associated with their ability to stabilize an inactive conformation similar to that observed with SPA, we analyzed the conformational features of GHSR using a fluorescence resonance energy transfer (FRET)-based approach (Figure 4). 22 To this end, the purified monomeric receptor stabilized into nanodiscs was labeled with a fluorescence acceptor (AF488) and donor (AF350) in the cytoplasmic ends of TM1 and TM6, respectively. As shown in Figure 4, addition of ghrelin or SPA at saturating concentrations induced a significant change in the FRET-monitored proximity ratio, indicative of a change in the distance between the two fluorescent probes. These variations were not due to changes in the mobility or orientation of the fluorophores, as the anisotropy of fluorophores attached to the receptor was not altered by treatment with ligands. Specifically, the full agonist ghrelin triggered a decrease in the proximity ratio, whereas the inverse agonist SPA had the opposite effect. All our ligands triggered an increase in the proximity ratio similar to that observed with SPA, suggesting that these compounds stabilize similar GHSR conformations. Correlated with GTPγS binding assay results, these data indicate that our ligands exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific, inactive receptor conformation. Evaluation of ligand 29 on endogenously expressed GHSR. Effect of ligand 29 in rat isolated pancreatic islets. To determine whether the substituted 1,2,4-triazole scaffold was active on native GHSR, we tested the effect of ligand 29 on the modulation of insulin secretion in rat isolated pancreatic islets (Figure 5). Knowing that ghrelin-evoked GHSR activation inhibits glucosestimulated insulin secretion in isolated islets of Langerhans, 23 we used this biological system to test ligand 29. This ligand was selected because (i) it appeared as one of the most promising compounds in term of pharmacological profile (Ki value of 3  1 nM, Emax value of 83% inhibition of the basal level, and EC50 value of 27  12 nM), and (ii) it has a hydroxyl substituent on the aryl group that would favor its solubility in biological fluids and, consequently, would increase its chances to be used as a drug. Isolated pancreatic islets were therefore incubated with a stimulating glucose concentration (8.3 mM) in the presence or absence of ghrelin (10 nM) and ligand 29 (1 μM) and then insulin release was quantified (Figure 5). As previously shown, ghrelin decreased glucose-stimulated insulin secretion. Importantly, simultaneous treatment with both ghrelin and ligand 29 blocked the inhibitory action of ghrelin on insulin secretion. These findings indicate that ligand 29 binds to pancreatic GHSR and prevents its ghrelin-evoked activation. Effect of Ligand 29 on Food Intake in Mice. First, we tested the effect of K-(D-1-Nal)-FwLL-NH2 and ligand 29 on spontaneous food intake during the dark cycle, when mice consume the majority of their food. K-(D-1-Nal)-FwLL-NH2 was used here as the reference inverse agonist because of its higher specificity compared to SPA. ICV administration of K-(D-1-Nal)-FwLL-NH2 and ligand 29 significantly reduced ad libitum food intake in the early dark phase period, from 18:00 h to 23:00 h (Figure 6A) but did not affect overnight food intake, as compared to vehicle treatment (2623  240, 2179  333 and 2591  197 mg, respectively). Importantly, K-(D-1-Nal)-FwLL-NH2 and ligand 29 treatments did not affect body weight nor induce any sickness-like behavior such as a spiky coat, hunched posture, altered breathing rate, labored movements, reduced activity and/or subdued behavior. Then, we tested the ability of K-(D-1-Nal)-FwLL-NH2 and ligand 29 to affect ghrelin-induced food intake (Figure 6B). Both GHSR ligands reduced ghrelininduced food intake; however, K-(D-1-Nal)-FwLL-NH2 treatment reduced ghrelin-induced food intake by 67.6  5.6% while ligand 29 treatment reduced ghrelin-induced food intake by 30.3  4.8%. Thus, these observations suggest that ligand 29 behaves as a GHSR antagonist in vivo. Indeed, since the physiological impact of the constitutive GHSR activity is uncertain, demonstrating the ability of a given GHSR ligand to display inverse agonist activity in vivo has proved to be challenging. We have shown that the compensatory hyperphagia that follows a fasting event requires GHSR but occurs in a ghrelin-independent manner. 24 Thus, we investigated the ability of K-(D-1-Nal)-FwLL-NH2 and ligand 29 to affect the compensatory hyperphagia in a fasting-refeeding protocol that follows a fasting event (Figure 6C-D). Notably, K-(D-1-Nal)-FwLL-NH2 and ligand 29 treatments significantly decreased the cumulative food intake between days 1 and 5 of refeeding (Figure 6C). In particular, K-(D-1-Nal)-FwLL-NH2 and ligand 29 reduced the compensatory hyperphagia by 20.1  3.5 and 12.4  3.9%, respectively, as compared to the vehicle treatment (Figure 6D). This in vivo evidence supports the notion that ligand 29 modulates ghrelin-independent aspects of GHSR activity.

CONCLUSION

We synthesized a new series of 3,4,5-trisubstituted 1,2,4-triazole derivatives based on our antagonist JMV2959 (compound 1). We modulated the structure by introducing various substituents at different positions on the scaffold and generated 45 new ligands. We carried out an in vitro SAR study in order to determine the pharmacological properties of the ligands and their ability to reduce the basal activity of GHSR. We showed that 21 ligands behaved as inverse agonists of GHSR since they were able to significantly inhibit its basal activity. Moreover, most of these ligands were at least as potent as the reference compounds SPA and K-(D-1-Nal)-FwLL-NH2. Four ligands (28-30 and 44) proved to be even more potent than SPA by inhibiting the basal response up to 80%. In order to show a direct interaction between the ligands and GHSR, we tested the activity of some selected inverse agonists on purified GHSR assembled into lipid nanodiscs.

We proved that these ligands exerted an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. At last, we investigated the effect of one of the most promising inverse agonist (ligand 29) on both the modulation of insulin secretion in rat isolated pancreatic islets and food intake in mice. By directly binding to GHSR, ligand 29 revealed to block the inhibitory action of ghrelin on insulin secretion.

Moreover, ICV administration of ligand 29 reduced ghrelin-induced food intake in mice, as well as the compensatory hyperphagia that follows a fasting event. In conclusion, this 3,4,5trisubstituted 1,2,4-triazole ring has proven to be a promising scaffold for the development of efficient inverse agonists of GHSR in order to treat obesity-related metabolic diseases.

EXPERIMENTAL SECTION

General Chemistry Methods. All chemicals for synthesis were purchased from Sigma-Aldrich Chemical Co., Thermo Fisher GmbH, Fluka TM and Iris Biotech GmbH, and used without further purification. All solvents were purchased from Sigma Aldrich and Honeywell in gradient grade or reagent quality. Classical purifications were performed with flash column chromatography using silica gel (VWR, 40-63 μm). HPLC purifications were run on a Waters 4000 preparative apparatus using a C18 Deltapak column (100 mm x 40 mm, 15μm, 100 Å), with UV detection at 214 nm and 254 nm, at a flow rate of 50 mL/min of a mixture of A, water with 0.1% TFA, and B, acetonitrile with 0.1% TFA, in gradient mode. Samples for LC-MS analyses were prepared in methanol (gradient grade). The LC-MS system consisted of a Waters Alliance 2695 HPLC coupled to a Water Micromass ZQ spectrometer (electrospray ionization mode, ESI+). All analyses were carried out using a Phenomenex Onyx reversed-phase column (25 x 4.6 mm). A flow rate of 3 mL/min and a gradient from 0 to 100% of B over 2.5 min were used. Eluent A: water/0.1% FA ; eluent B: acetonitrile/0.1% FA. UV detection was performed at 214 nm. 1 H-NMR and 13 C-NMR spectra were recorded on Bruker Advance spectrometers at 300, 400 or 500 MHz and 75, 100 or 125 MHz, respectively. Chemical shifts (δ) are reported in parts per million (ppm) relative to the residual peak of the solvent (2.50 and 39.52 ppm for DMSO, 3.31 and 49 ppm for MeOD) according to the literature. 25 Data are reported as follows: chemical shift (δ in ppm), multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, br s=broad singlet, m=multiplet), coupling constants (J = Hz), integration and assignment. HRMS analyses were performed using a microTOF-Q spectrometer (electrospray ionization mode, ESI+). The lyophilizer from Cryonext (Saint Gely du Fesc, France) was used with a freezing trap at -90 °C to lyophilize compounds purified by reverse phase preparative chromatography.

General Procedure: Compound 1 (compound 4 in reference 18), triazoles (iv, vi and vi) and not commercially available hydrazides (v) were synthesized following the same procedures previously described. 18, 26 General Procedure: Method A (for ligands 2-8). Triazole iv (100 mg, 0.2 mmol, 1eq) was coupled with carboxylic acids (1.1 equiv), in the presence of BOP (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. The mixture was then concentrated in vacuo, and the residue was dissolved in EtOAc. The organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure to yield the desired compound. The compounds that did not require a final deprotection were purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method B (for ligands . Compound 1 (100 mg, 0.18 mmol, 1eq) was coupled with carboxylic acids (1.1 equiv), in the presence of HATU (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. The mixture was condensed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure to yield the desired compound. The compounds that did not require a final deprotection were purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method C (for ligand 36). Appropriate triazole vi (100 mg, 1eq) was coupled with alpha-cyano-4-methoxycinnamic acid (1.1 equiv), in the presence of HATU (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. The mixture was condensed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure to yield the desired compound that was purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method D (for ligand 37). Appropriate triazole vi (100 mg, 1eq) was coupled with 2-hydroxyacetic acid (1.1 equiv), in the presence of HATU (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. The mixture was condensed under reduced pressure and the residue was dissolved in EtOAc. The organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure. The resulting compound was purified by flash chromatography on silica gel with a gradient of EtOAc to EtOAc/MeOH 9:1 as eluent and then reacted with alphacyano-4-methoxycinnamic acid (1.1 equiv), in the presence of EDC (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. After removal of solvents under reduced pressure, the residue was dissolved in EtOAc and the organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure to yield the desired compound that was purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method E (for ligand [38][39][40][41][42][43][44][45][46]. Appropriate Boc-protected triazole vii (100 mg, 1eq) was treated with 4N HCl in dioxane (5 mL) for 30 min at 50 °C. When removal of the Boc-protecting group was confirmed by HPLC analysis, MeOH was added and the reaction mixture was condensed under reduced pressure. Additions of MeOH followed by evaporations were repeated several times in order to remove excess of HCl. The resulting deprotected amine was coupled with appropriate para-substituted alpha-cyano-cinnamic acid (1.1 equiv), in the presence of HATU (1.1 equiv) and DIEA (pH=9) for 2 h, in DCM. The mixture was condensed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was successively washed with aqueous solutions of 1M KHSO4, saturated NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and condensed under reduced pressure to yield the desired compound that was purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method F (for ligands 2,4,6,[START_REF] Munoz | The Antiproliferative Action of [D-ARG 1 , D-PHE 5[END_REF][START_REF] M'kadmi | Antagonism, and Inverse Agonism Bias at the Ghrelin Receptor Signaling[END_REF][START_REF] Mary | Ligands and Signaling Proteins Govern the Conformational Landscape Explored by a G Protein Coupled Receptor[END_REF][START_REF] Yanagi | The Homeostatic Force of Ghrelin[END_REF]. Removal of the protecting group was performed at 50 °C with a solution of 4N HCl in dioxane (2 mL) for 30 min. The mixture was then condensed under reduced pressure, diluted with MeOH, and condensed again under reduced pressure. Additions of MeOH followed by evaporations were repeated several times in order to remove excess of HCl. The resulting deprotected compounds were then purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient.

General Procedure: Method G (for ligands 6 and 8). Removal of the Fmoc protecting group was performed at room temperature with Et2NH (10 eq, 1 mmol/ml) in DMF for 2 h. The mixture was then condensed under reduced pressure and the compounds were purified by preparative HPLC on a C18 column using a water/acetonitrile/TFA 0.1% gradient. (2). By methods A and F: 89 mg (0.17 mmol, 85%), orange wax (purity HPLC > 99%). LC-MS (ES): tR = 1.30 min, m/z 523.3 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.90 (d,J = 1.8 Hz,1H,NH),9.35 (d,J = 8.1 Hz,1H,NH),2H,NH2 TFA salt),7.35 (d,J = 8.1 Hz,1H,CH aryl), 6H,CH aryl), 7.09-7.02 (m, 2H, CH aryl), 6.91 (t, J = 7.3 Hz, 1H, CH aryl), 6.71 (br s, 4H, CH aryl), 1H,CH), 5.05 (d, J = 16.9 Hz, 1H, CH2), 4.99 (d, J = 16.9 Hz, 1H, CH2), 3.69 (s,3H,OCH3),2H,CH2),3.44 (d,J = 14.2 Hz,7.3 Hz,1H,CH2),3.32 (d,J = 14.2 Hz,7.3 Hz,1H,CH2),4H,2 CH2),2.41 (br s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 164. 8, 158.7, 154.4, 154.1, 140.3, 136.1, 128.3 (4C), 127.5 (2C), 127.0 (2C), 126.2, 124.3, 121.0, 118.5, 117.9, 114.1 (2C), 111.4, 109.2, 55.1, 48.7, 45.2, 44.9, 32.5, 32.2, 29.1, 26. 3.72 (d, J = 13.4 Hz, 1H, CH2), 3.70 (s, 3H, OCH3), 3.44 (dd, J = 14.3 Hz, 6.9 Hz, 1H, CH2), 3.33 (d,J = 14.3 Hz,7.8 Hz,1H,CH2), 2.92-2.79 (m, 4H, 2 CH2), 2.60 (br s, 6H, 2 CH3). 13 C NMR ( 75MHz, DMSO-d6): δ 163. 8, 158.7, 154.2, 154.1, 104.4, 136.1, 128.3 (4C), 127.4 (2C), 127.2, 127.0, 126.1, 124.2, 120.9, 118.4, 118.0, 114.1 (2C), 111.4, 109.3, 57.1, 55.1, 45.0, 44.8, 42.9 (2C) 6.71 (br s, 4H, CH aryl), 1H,CH),2H,CH2),3.69 (s,3H,OCH3),2H,CH2),3.43 (dd,J = 14.2 Hz,7.4 Hz,1H,CH2),3.31 (dd,J = 14.2 Hz,7.4 Hz,1H,CH2),4H,2 CH2), 2.77-2.67(m, 2H, CH2), 1.08 (t, J = 7.2 Hz, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6): δ 164. 7, 158.7, 154.3, 154.0, 140.4, 136.0, 128.3 (4C), 127.3 (2C), 127.2, 126.9, 126.1, 124.2, 120.9, 118.4, 117.9, 114.1 (2C), 111.4, 109.3, 55.1, 46.8, 45.0, 44.9, 41.8, 32.2, 29.2, 26.0, 10.7. HRMS (ESI): m/z calculated for C32H37N6O2 [M+H] + : 537.2978. Found: 537.2977. (5). By method A: 70 mg (0.13 mmol, 65%), orange wax (HPLC, purity 140.4, 136.1, 128.3 (4C), 127.3 (2C), 127.0, 126.1, 124.3, 120.9, 118.4-118.0 (3C), 114.1 (2C), 111.4, 109.3, 55.1, 52.1, 48.2, 45.0, 44.8, 39.8, 32.2, 28.9, 26.0, 8.7, 8.4 (dd, J = 14.1 Hz, 7.1 Hz, 1H, CH2), 2.89-2.73 (m, 4H, 2 CH2). 13 C NMR (75 MHz, DMSO-d6): δ 167. 8,164.5,158.4,154.2,153.8,140.5,136.0,128.3 (4C),127.3 (2C),126.9,126.1,124.4,120.9,118.4,117.8,114.0 (2C),111.4,109.3,55.0,47.0,46.6,45.3,44.2,40.5,32.3,29.3,26.1. HRMS (ESI): m/z calculated for C32H35N6O4 [M+H] + : 567.2720. Found: 567.2722. [START_REF]m/z calculated for C41H40N7O3[END_REF]. By method A: 84 mg (0.14 mmol, 70%), orange amorphous powder (HPLC, purity > 98%). LC-MS (ES): tR = 1.43 min, m/z 599.4 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.86 (d, J = 0.9 Hz, 1H), 9.30 (d,J = 8.2 Hz,1H),1H,NH),5H,CH aryl), 7H,CH aryl), 7.08-7.01 (m, 2H, CH aryl), 6.90 (t, J = 7.4 Hz, 1H, CH aryl), 6.71 (s, 4H, CH aryl), 1H,CH), 5.02 (s, 2H, CH2), 4.01-3.90 (s, 2H, CH2), 3.68 (s,3H,OCH3),2H,CH2),3.41 (dd,J = 14.2 Hz,7.2 Hz,1H,CH2),3.30 (dd,J = 14.2 Hz,7.5 Hz,1H,CH2),4H,2 CH2). 13 C NMR (75 MHz, DMSO-d6): δ 164. 6, 158.7, 154.3, 154.0, 140.3, 136.0, 131.3, 130.0 (2C), 129.1, 128.7 (2C), 128.3 (4C), 127.4 (2C), 127.1, 126.9, 126.2, 124.2, 121.0, 118.4, 117.9, 114.1 (2C), 111.4, 109.2, 55.0, 49.7, 46.5, 45.0, 44.9, 32.2, 29.2, 26. 1H, CH aryl), 6.72 (s,4H,CH aryl), 1H,CH),5.00 (s,2H,CH2),5H,OCH3 and CH2),3.43 (dd,J = 14.2 Hz,7.0 Hz,1H,CH2),3.34 (dd,J = 14.2 Hz,7.9 Hz,1H,CH2),2H,CH2),1H,CH),6H,3 CH2),2H,CH2), (m,2H,CH2). 13 C NMR (75 MHz, DMSO-d6): δ 163. 5,158.7,154.1 (2C),140.4,136.1,128.3 (4C),127.3 (2C),127.2,127.0,126.1,124.3,120.9,118.5,118.0,114.1 (2C),111.4,109.3,56.1,55.1,45.0,44.8,32.3,29.1,26.7 (2C),26.1. HRMS (ESI): m/z calculated for C35H42N7O2 [M+H] + : 592.3400. Found: 592.3401.

(R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2- (methylamino)acetamide
(R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2- (diethylamino)acetamide
(R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2- (benzylamino)acetamide
(R)-N- [2-(1H-indol-3-yl) -1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2-formamidoacetamide [START_REF] Holst | High Constitutive Signaling of the Ghrelin Receptor-Identification of a Potent Inverse Agonist[END_REF]. By method B: 56 mg (0.10 mmol, 58%), beige amorphous powder (HPLC, purity = 95%). LC-MS (ES): tR = 1.44 min, m/z 537.2 [M+H] + . 1 H-NMR (500 MHz, methanol-d4): δ 8.12 (s, 1H, CHO), 7.35 (d,J = 8.0 Hz,1H,CH aryl), 7.32 (d,J = 7.5 Hz,1H,CH aryl), 3H,CH aryl), 7.08 (t, J = 8.0 Hz, 1H, CH aryl), 7.00-6.97 (m, 3H, CH aryl), 6.93 (t, J = 8.0 Hz, 1H, CH aryl), 3H,CH aryl), 6.54 (d,J = 8.5 Hz,1H,CH aryl), 5.42-5.39 (dd, J = 9.5 Hz, 6.0 Hz, 1H, CH), 4.87 (d,J = 16.0 Hz,1H,CH2),4.75 (d,J = 16.0 Hz, 1H, CH2), 3.82 (d,J = 4.5 Hz,2H,CH2),3.70 (s,3H,OCH3),J = 14.0 Hz,9.5 Hz,1H,CH2), (dd, J = 14.0 Hz, 6.0 Hz, 1H, CH2), 2.89-2.84 (m, 2H, CH2), 2.78 (t, J = 7.0 Hz, 2H, CH2). 13 C-NMR (125 MHz, methanol-d4): δ 170. 5, 164.1, 160.9, 157.0, 156.4, 140.8, 137.9, 129.6 (2C), 129.3 (2C), 128.7 (2C), 128.4, 127.6, 127.1, 125.2, 122.6, 120.1, 118.9, 115.3 (2C), 112.4, 110.2, 55.7, 47.3, 47.0, 41.7, 33.9, 30.3, 27.6. HRMS (ESI): m/z calculated for C31H33N6O3 [M+H] + : 537.2614. Found: 537.2614. [START_REF] Jarpe | D-Arg 1 ,D-Phe 5 ,D-Trp 7,9 ,Leu 11 ]Substance P Acts as a Biased Agonist Toward Neuropeptide and Chemokine Receptors[END_REF]. By method B: 54 mg (0.10 mmol, 54%), orange amorphous powder (HPLC, purity = 95%). LC-MS (ES): tR = 1.53 min, m/z 551.1 [M+H] + . 1 H-NMR (500 MHz, methanol-d4): 7.35 (d,J = 8.0 Hz,1H,CH aryl), 7.31 (d,J = 8.0 Hz,1H,CH aryl), 3H,CH aryl), 7.08 (t, J = 8.0 Hz, 1H, CH aryl), 3H,CH aryl), 6.93 (t, J = 8.0 Hz, 1H, CH aryl), 6.60 (d,J = 8.5 Hz,2H,CH aryl), 6.54 (d,J = 8.5 Hz,2H,CH aryl), J = 9.5 Hz,6.0 Hz,1H,CH),4.86 (d,J = 16.5 Hz,1H,CH2),4.75 (d,J = 16.5 Hz,1H,CH2),3.77 (s,2H,NMR (400 MHz,): δ 7.35 (d,J = 8.4 Hz,1H,CH aryl), 7.30 (d,J = 8.0 Hz,1H,CH aryl),3H,CH aryl), 7.09 (t, J = 8.0 Hz, 1H, CH aryl), 4H,CH aryl), 6.61 (d,J = 8.8 Hz,2H,CH aryl), 6.55 (d,J = 8.8 Hz,2H,CH aryl), 5.41 (dd,J = 9.2 Hz,6.0 Hz,1H,CH),4.95 (d,J = 16.4 Hz,1H,CH2),4.83 (d,J = 16.4 Hz,1H,CH2),3.83 (s,2H,CH2),3.70 (s,3H,OCH3),J = 14.0 Hz,9.2 Hz,1H,CH2),J = 14.0 Hz,6.0 Hz, 1H, CH2), 2.99-2.85 (m, 2H, CH2), 2.78 (t, J = 7.6 Hz, 2H, CH2). 13 C-NMR (100 MHz, methanol-d4): δ 170. 4, 161.0, 157.3, 156.4, 155.1, 140.4, 137.9, 129.7 (2C), 129.3 (2C), 128.9 (2C), 127.8, 126.6, 125.2, 122.7, 120.2, 118.8, 115.4 (2C), 112.5, 109.9, 77.8, 76.6, 55.7, 47.8, 47.1, 43.2, 33.5, 30.1, 27.4. HRMS (ESI): m/z calculated for C33H33N6O3 [M+H] + : 561.2614. Found, 561.2614. (13). By method B: 34 mg (0.06 mmol, 34%), beige amorphous powder (HPLC, purity > 94%). LC-MS (ES): tR = 1.61 min, m/z 563.2 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 7.36 (d,J = 8.4 Hz,1H,CH aryl), 7.30 (d,J = 8.0 Hz,1H,CH aryl), 3H,CH aryl), [START_REF]m/z calculated for C41H40N7O3[END_REF].09 (t, J = 7.2 Hz, 1H, CH aryl), 4H,CH aryl), 4H,CH aryl), 2H,CH vinyl), J = 9.2 Hz,2.8 Hz,1H,CH vinyl), 5.42 (dd,J = 9.2 Hz,6.0 Hz,1H,CH),4.93 (d,J = 16.8 Hz,1H,CH2),4.80 (d,J = 16.8 Hz,1H,CH2), 3.87 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 3.57-3.51 (dd, J = 14.0 Hz, 9.2 Hz, 1H, CH2), 3.47-3.40 (dd, J = 14.0 Hz, 6.0 Hz, 1H, CH2), 2.95-2.85 (m, 2H, CH2), 2.78 (t, J = 7.6 Hz, 2H, CH2). C-NMR (100 MHz, methanol-d4): δ 171. 1, 168.5, 161.0, 157.2, 156.5, 140.6, 137.9, 131.6, 129.7 (2C), 129.3 (2C), 128.8 (2C), 128.4, 127.7, 127.3, 126.9, 125.2, 122.7, 120.2, 118.9, 115.4 (2C), 112.5, 110.1, 55.7, 47.6, 47.1, 43.2, 33.7, 30.3, 27.5. HRMS (ESI): m/z calculated for C33H35N6O3 2.76 (t, J = 8.0 Hz, 2H, CH2). 13 C-NMR (125 MHz, methanol-d4): δ 171. 5, 170.5, 161.0, 157.3, 156.4, 140.4, 137.9, 134.9, 133.0, 129.7 (2C), 129.6 (2C), 129.3 (2C), 128.9 (2C), 128.5 (2C), 128.3, 127.8, 126.7, 125.2, 122.7, 120.2, 118.9, 115.4 (2C), 112.5, 110.0, 55.6, 47.8, 47.1, 43.7, 33.5, 30.1, 27. 166.8, 161.1, 157.5, 156.5, 151.0, 140.0, 137.9, 137.8, 136.7, 133.7, 129.8 (2C), 129.6, 129.3 (2C), 129.0 (2C), 128.3, 127.9, 126.2, 125.3, 122.7, 120.2, 118.8, 117.8, 115.5 (2C), 112.5, 109.7, 55.7, 48.2, 47.2, 43.7, 33.2, 30.0, 27.3 Hz, 1H, CH2), 2.97-2.86 (m, 2H, CH2), 2.77 (t, J = 8.0 Hz, 2H, CH2). 13 C-NMR (100 MHz, methanol-d4): δ 175. 2, 171.3, 161.1, 157.6, 157.3, 156.5, 140.2, 137.9, 131.3 (2C), 129.7 (2C), 129.3 (2C), 128.9 (2C), 128.3, 127.8, 127.2, 126.4, 125.2, 122.7, 120.2, 118.8, 116.4 (2C), 115.4 (2C), 112.5, 109.9, 55.7, 48.0, 47.1, 43.4, 42.7, 33.3, 30.3, 27.4. HRMS (ESI): m/z calculated for C38H39N6O4 [M+H] + : 643.3027. Found: 643.3037. [START_REF] Mary | Ligands and Signaling Proteins Govern the Conformational Landscape Explored by a G Protein Coupled Receptor[END_REF]. By methods B and F: 66 mg (0.10 mmol, 56%), white amorphous powder (HPLC, purity > 99%). LC-MS (ES): tR = 1.66 min, m/z 657.2 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 7.36 (d,J = 8.4 Hz,1H,CH aryl), 7.30 (d,J = 8.0 Hz,1H,CH aryl),3H,CH aryl), 7.09 (t, J = 7.6 Hz, 1H, CH aryl), 7.02-6.92 (m, 6H, CH aryl), 6.69 (d,J = 8.4 Hz,2H,CH aryl),4H,CH aryl) 6H,CH aryl,CH vinyl), 3H,CH aryl), 7.08 (t, J = 7.2 Hz, 1H, CH aryl), 7.00-6.92 (m, 4H, CH aryl), 4H,CH aryl,CH vinyl), 5.45 (dd,J = 9.2 Hz,6.4 Hz,1H,CH),4.95 (d,J = 16.4 Hz,1H,CH2),4.85 (d,J = 16.4 Hz, 1H, CH2), 3.93 (s, 2H, CH2), 3.69 (s, 3H, OCH3), 3.57-3.52 (dd, J = 14.0 Hz, 9.2 Hz, 1H, CH2), J = 14.0 Hz,6.4 Hz,1H,CH2),2H,CH2), 2.78 (t, J = 7.6 Hz, 2H, CH2). 13 C-NMR (100 MHz, methanol-d4): δ 171. 3, 169.0, 161.0, 157.3, 156.5, 152.1, 146.3, 142.4, 140.4, 137.9, 136.2, 131.0, 130.0 (2C), 129.7 (2C), 129.3 (2C), 128.9 (2C), 128.4, 127.8, 126.7, 125.2, 122.7, 121.3, 120.2, 118.9, 115.4 (2C), 112.5, 110.0, 55.7, 47.8, 47.1, 43.5, 33.6, 30.2, 27.5. HRMS (ESI): m/z calculated for C39H39N6O3 [M+H] + : 639.3078. Found: [START_REF] Gottlieb | NMR Chemical Shifts of Common Laboratory Solvents as Trace Impurities[END_REF]. By methods B: 68 mg (0.10 mmol, 58%), white amorphous powder (HPLC, purity > 97%). LC-MS (ES): tR = 1.85 min, m/z 657.3 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 7.62-7.51 (m, 3H, CH aryl), 7.35 (d,J = 8.4 Hz,1H,CH aryl), 7.33 (d,J = 8.0 Hz,1H,CH aryl), 6H,CH aryl,CH vinyl), 7.00-6.91 (m, 4H, CH aryl), 5H,CH aryl,CH vinyl), J = 9.2 Hz,6.0 Hz,1H,CH),4.95 (d,J = 16.8 Hz,1H,CH2),4.84 (d,J = 16.8 Hz,1H,CH2), 3.92 (s, 2H, CH2), 3.69 (s, 3H, OCH3), 3.57-3.51 (dd, J = 14.0 Hz, 9.2 Hz, 1H, CH2), 3.47-3.42 (dd, J = 14.0 Hz, 6.0 Hz, 1H, CH2), 2.96-2.84 (m, 2H, CH2), 2.78 (t, J = 7.6 Hz, 2H, CH2). 13 C-NMR (100 MHz, methanol-d4): δ 171. 3, 168.9, 166.3, 163.8, 161.0, 157.3, 156.5, 141.0, 140.5, 137.9, 132.6, 131.0, 130.9, 129.7 (2C), 129.3 (2C), 128.9 (2C), 127.8, 126.7, 125.2, 122.7, 121.2, 120.2, 118.9, 117.0, 116.7, 115.4 (2C), Inositol Phosphate Assay. The inositol phosphate accumulation assay was carried out on the adherent HEK293T cell line expressing the SNAP-GHSR in a 96-well plate format at a density of 50 000 cells/well. IP1 production was measured using the IP-One HTRF kit (Cisbio Bioassays ref 621PAPEC) as described previously. 20 Briefly, cells were stimulated for 30 min at 37 °C with the ligand to be tested in 70 μL of the IP1 stimulation buffer. An anti-IP1 antibody labeled with Lumi4-Tb (15 μL) and an IP1-d2 derivative (15 μL) were added to the cells. The medium was incubated for 1 h at room temperature. Signals at 665 and 620 nm were detected using a PHERAstar (BMG LABTECH) fluorescence reader. EC50 values from dose-response curves were obtained using GraphPad Prism 5.0 software.

(R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2- acetamidoacetamide
(R)-N-[2-(2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3- yl)ethylamino)-2-oxoethyl]acrylamide
(R)-N-[2-(2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3- yl)ethylamino)-2-oxoethyl]-3-(4-hydroxyphenyl)propanamide
Receptor Preparation and Labeling. For GTPγS binding assays, the monomeric wild-type human GHSR in lipid nanodiscs was prepared as previously described. 27 For FRET measurements, the ghrelin receptor with a TAG amber codon at the position encoding F71 and a single reactive cysteine at position 255 was produced in lipid nanodiscs and subsequently labeled with Click-IT Alexa Fluor 488 DIBO Alkyne (LifeTechnologies) and Alexa Fluor 350 maleimide, as described. 28 G Protein Preparation. His6-tagged Gαq was expressed in Sf9 cells and purified using nickelnitrilotriacetic acid (Ni-NTA) affinity chromatography. 29 Gβ1 was expressed with His6-tagged Gγ in Sf9 cells and the Gβγ dimer purified using Ni-NTA chromatography combined with ion exchange chromatography. 29 GTPγS Binding. GTPγS binding experiments were carried out using the fluorescent BODIPY-FL GTPγS analog. 30 Association of BODIPY-FL GTPγS to the G protein was monitored using a fluorescence spectrophotometer (Cary Eclipse, Varian) with the excitation wavelength set at 500 nm and the emission wavelength at 511 nm. Reaction conditions were 100 nM Gαq, 500 nM Gβ1γ2,

Figure 1 .

 1 Figure 1. Structure of the 3,4,5-trisubstituted 1,2,4-triazole scaffold and compound 1 (JMV2959)

Scheme 1 .

 1 Scheme 1. Synthesis of Compounds 1-8 a

Scheme 2 .

 2 Scheme 2. Synthesis of Compounds 9-35 a

  .

Scheme 3 .

 3 Scheme 3. Synthesis of Compounds 36-46 a

a

  Values reported in the table were obtained from experiments performed in HEK293T cells stably expressing GHSR. b Ki values were determined from HTRF competition binding assays. Values are expressed as means  SEM of two independent experiments, each experiment being performed in triplicate. c Activities were determined by HTRF inositol phosphate (IP) assay. IP response of each ligand (10 -5 M) is given as percentage of the maximal stimulation promoted by ghrelin(10 -7 M). The percentage in square brackets refers to the basal response of the receptor also given as percentage of the maximal ghrelin response.

Figure 1 .

 1 Figure 1. Concentration-response curves of inverse agonist ligands at promoting IP1 production.

Figure 3 .

 3 Figure 3. Effect of inverse agonist ligands on GHSR-catalyzed Gq protein activation. BODIPY-

Figure 4 .

 4 Figure 4. Effect of inverse agonist ligands on GHSR conformation. Proximity ratio changes

Figure 5 .

 5 Figure 5. Effect of ligand 29 on ghrelin-induced inhibition of insulin secretion in rat pancreatic

Figure 6 .

 6 Figure 6. (A) 5-hour early dark cycle food intake in mice ICV-treated with vehicle, K-(D-1-Nal)-

  0. HRMS (ESI): m/z calculated for C31H35N6O2 [M+H] + : 523.2821. Found: 523.2819. (R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2-(dimethylamino)acetamide (3). By method A: 71 mg (0.13 mmol, 66%), beige wax (purity HPLC > 99%). LC-MS (ES): tR = 1.30 min, m/z 537.2 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.89 (d, J = 1.6 Hz, 1H, NH), 9.79-9.63 (m, 1H, NH TFA salt), 9.43 (d, J = 8.3 Hz, 1H, NH), 7.38-7.30 (m, 2H, CH aryl), 5H, CH aryl), 2H, CH aryl), 1H, CH aryl),6.73 (br s, 4H, CH aryl), 1H, CH), 5.00 (s, 2H, CH2),3.78 (d, J = 13.4 Hz, 1H, CH2),

  , 32.2, 29.1, 26.1. HRMS (ESI): m/z calculated for C32H37N6O2 [M+H] + : 537.2978. Found: 537.2979. (R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2-(ethylamino)acetamide (4). By methods A and F: 62 mg (0.12 mmol, 58%), white amorphous powder (purity HPLC > 98%). LC-MS (ES): tR = 1.33 min, m/z 537.3 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.87 (d, J = 1.2 Hz, 1H, NH), 9.31 (d, J = 8.2 Hz, 1H, NH), 8.73-8.68 (m, 2H, NH2 TFA salt), 7.34 (d, J = 8.1 Hz, 1H, CH aryl), 7.31-7.11 (m, 6H, CH aryl), 7.08-7.01 (m, 2H, CH aryl), 6.90 (t, J = 7.4 Hz, 1H, CH aryl),

>

  98%). LC-MS (ES): tR = 1.35 min, m/z 445.3[M+H -4-methoxybenzyl] + , 565.3 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.88 (s, 1H, NH), 9.40 (d, J = 8.5 Hz, 1H, NH), 9.32-9.21 (m, 1H, NH TFA salt), 7.37-7.29 (m, 2H, CH aryl), 7.28-7.11 (m, 5H, CH aryl), 7.09-7.01 (m, 2H, CH aryl), 6.91 (t, J = 7.4 Hz, 1H, CH aryl), 4H, CH aryl), 1H, CH), 5.02 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 2H, CH2), 2H, CH2), 8H, 4 CH2), 6H, 2 CH3).13 C NMR (75 MHz, DMSO-d6): δ 164.0, 158.7, 154.2 (2C),

  . HRMS (ESI): m/z calculated for C34H41N6O2 [M+H] + : 565.3291. Found: 565.3290. (R)-2-[2-(2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3yl)ethylamino)-2-oxoethylamino]acetic acid (6). By methods A, F and G: 16 mg (0.03 mmol, 14%), white amorphous powder (HPLC, purity > 95%). LC-MS (ES): tR = 1.36 min, m/z 567.3 [M+H] + . 1 H NMR (300 MHz, DMSO-d6): δ 10.84 (d, J = 1.6 Hz, 1H, NH), 9.32 (d, J = 8.1 Hz, 1H, NH), 7.34 (d, J = 7.6 Hz, 1H, CH aryl), 7.28-7.12 (m, 7H, NH, CH aryl), 7.08-7.00 (m, 2H, CH aryl), 6.89 (t, J = 7.6 Hz, 1H, CH aryl),6.68 (s, 4H, CH aryl), 1H, CH), 2H, CH2), 3.78 (s, 4H, 2 CH2), 3.69 (s, 3H, OCH3), 3.42 (dd, J = 14.1 Hz, 7.6 Hz, 1H, CH2), 3.29

  0. HRMS (ESI): m/z calculated for C37H39N6O2[M+H] + : 599.3134. Found: 599.3140.(R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2-(4-aminopiperidin-1-yl)acetamide(8). By methods A and G: 90 mg (0.15 mmol, 76%), orange wax (HPLC, purity > 95%). LC-MS (ES): tR = 1.14 min, m/z 592.4[M+H] + .1 H NMR (300 MHz, DMSO-d6): δ 10.90 (s, 1H, NH), 9.44 (d, J = 8.4 Hz, 1H, NH), 8.26 (br s, 3H, NH3 TFA salt), 7.38-7.31 (m, 2H, CH aryl), 7.29-7.10 (m, 5H, CH aryl), 7.09-7.01 (m, 2H, CH aryl), 6.92 (t, J = 7.4 Hz,

13

 13

[

  M+H] + : 563.2771. Found: 563.2772. amorphous powder (HPLC, purity > 99%). LC-MS (ES): tR = 1.71 min, m/z 613.3 [M+H] + . 1 H-NMR (500 MHz, methanol-d4): δ 7.85 (d, J = 8.0 Hz, 2H, CH aryl), 7.55 (t, J = 7.0 Hz, 1H, CH aryl), 7.46 (t, J = 8.0 Hz, 2H, CH aryl), 7.35 (d, J = 8.0 Hz, 1H, CH aryl), 7.30 (d, J = 8.0 Hz, 1H, CH aryl), 7.22-7.15 (m, 3H, CH aryl), 7.09 (t, J = 7.0 Hz, 1H, CH aryl), 7.00-6.91 (m, 4H, CH aryl), 6.60-6.55 (m, 4H, CH aryl), 5.47-5.44 (dd, J = 9.5 Hz, 6.0 Hz, 1H, CH), 4.98 (d, J = 16.5 Hz, 1H, CH2), 4.86 (d, J = 16.5 Hz, 1H, CH2), 4.04-3.96 (m, 2H, CH2), 3.67 (s, 3H, OCH3), 3.57-3.53 (dd, J = 14.0 Hz, 9.5 Hz, 1H, CH2), 3.47-3.43 (dd, J = 14.0 Hz, 6.0 Hz, 1H, CH2), 2.95-2.85 (m, 2H, CH2),

  5. HRMS (ESI): m/z calculated for C37H37N6O3 [M+H] + : 613.2922. Found: 613.2917. (R)-N-[2-(2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3yl)ethylamino)-2-oxoethyl]-6-cyanonicotinamide (20). By method B: 59 mg (0.09 mmol, 51%), red amorphous powder (HPLC, purity > 95%). LC-MS (ES): tR = 1.70 min, m/z 639.2 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 9.09 (s, 1H, CH 8.35 (dd, J = 8.4 Hz, 2.4 Hz, 1H, CH aryl), 7.96 (dd, J = 8.4 Hz, 1.2 Hz, 1H, CH aryl), 7.35 (d, J = 8.0 Hz, 1H, CH aryl), 7.29 (d, J = 8.0 Hz, 1H, CH aryl), 7.24-7.16 (m, 3H, CH aryl), 7.09 (t, J = 7.4 Hz, 1H, CH aryl), 7.01 (s, 1H, CH aryl), 6.97-6.92 (m, 3H, CH aryl), 6.62 (s, 4H, CH aryl), 5.50-5.46 (dd, J = 9.2 Hz, 6.4 Hz, 1H, CH), 5.02 (d, J = 16.4 Hz, 1H, CH2), 4.93 (d, J = 16.4 Hz, 1H, CH2), 4.03 (s, 2H, CH2), 3.69 (s, 3H, OCH3), 3.58-3.52 (dd, J = 14.0 Hz, 9.2 Hz, 1H, CH2), 3.49-3.44 (dd, J = 14.0 Hz, 6.4 Hz, 1H, CH2), 3.02-2.90 (m, 2H, CH2), 2.78 (t, J = 8.0 Hz, 2H, CH2). 13 C-NMR (100 MHz, methanol-d4): δ 171.1,

  . HRMS (ESI): m/z calculated for C37H35N8O3 [M+H] + : 639.2827. Found: 639.2831. (R)-N-[2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3-yl)ethyl]-2-(2-(4-hydroxyphenyl)acetamido)acetamide (21). By methods B and F: 62 mg (0.10 mmol, 54%), white amorphous powder (HPLC, purity > 99%). LC-MS (ES): tR = 1.64min, m/z 643.2 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 7.35 (d, J = 8.0 Hz, 1H, CH aryl), 7.29 (d, J = 8.0 Hz, 1H, CH aryl), 7.23-7.15 (m, 3H, CH aryl), 3H, CH aryl), 4H, CH aryl),6.73 (d, J = 8.8 Hz, 2H, CH aryl), 4H, CH aryl), 5.42 (dd, J = 9.2 Hz, 6.0 Hz, 1H, CH), 4.94(d, J = 16.8 Hz, 1H, CH2), 4.82 (d, J = 16.8 Hz, 1H, CH2), 3.76 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 3.54-3.48 (dd, J = 14.0 Hz, 9.2 Hz, 1H, CH2),3.45 (s, 2H, CH2), J = 14.0 Hz, 6.0

  , 5.43 (dd, J = 9.2 Hz, 6.0 Hz, 1H, CH), 4.97 (d, J = 16.4 Hz, 1H, CH2), 4.87 (d, J = 16.4 Hz, 1H, CH2), 3.76 (s, 2H, amorphous powder (HPLC, purity > 94%). LC-MS (ES): tR = 1.82 min, m/z 639.3 [M+H] + . 1 H-NMR (400 MHz, methanol-d4): δ 7.58-7.54 (m, 3H, CH aryl),

  E)-N-[2-(2-(1H-indol-3-yl)-1-(4-(4-methoxybenzyl)-5-phenethyl-4H-1,2,4-triazol-3yl)ethylamino)-2-oxoethyl]-3-(4-fluorophenyl)acrylamide

  ABBREVIATIONS

Table 1 .

 1 Binding and Pharmacological Activity of Ligands 1-8 a

	Ligand	R 1	Ki b (nM)	IP-[basal] c (%)	Pharmacological profile
	1	-NH2.HCl	43  10	65-[65]	antagonist
	2	-NH-CH3	96  22	100-[76]	agonist
	3	-N(CH3)2	200  28	96-[76]	agonist
	4	-NH-CH2-CH3	300  32	82-[61]	partial agonist
	5	-N(CH2-CH3)2	230  31	88-[72]	partial agonist
	6		617  41	80-[72]	partial agonist
	7		136  19	88-[72]	partial agonist
	8		220  21	85-[72]	partial agonist

Table 2 .

 2 Binding and Pharmacological Activity of Ligands 9-35 a

	Ligand	R 1	Ki Pharmacological profile
	9	-H	12  2	79-[62]	partial agonist
	10	-CH3	10  3	93-[62]	partial agonist
	11	-CH2-NH2	130  22	85-[64]	partial agonist
	12	-CN	1600  112	76-[67]	partial agonist
	13		4  1	90-[69]	partial agonist
	14		0.45  0.2	83-[79]	partial agonist
	15		3.4  1	70-[70]	antagonist
	16		28  8	77-[74]	partial agonist
	17		166  23	72-[70]	antagonist

b (nM) IP-[basal] c (%) inverse agonist 28 1.0  0.3 19-[74] inverse agonist 29 3  1 22-[77] inverse agonist 30 0.5  0.2 12-[77] inverse agonist 31 2.4  1 22-[70] inverse agonist 32 0.6  0.2 23-[74] inverse agonist 33 9.7  2 31-[70] inverse agonist 34 8.5  2 23-[70] inverse agonist 35 6.3 2 19-[74] inverse agonist

Table 3 .

 3 Binding and Pharmacological Activity of Ligands 35-46 a

ACKNOWLEDGMENTS

100 nM BODIPY-FL GTPγS, and 20 nM receptor in lipid disks. The receptor, the ligands (10 μM final concentration), and the G protein were first incubated for 30 min at room temperature, and fluorescence was measured after addition of BODIPY-FL GTPγS and 10 min incubation at 15 °C. FRET Measurements. For FRET measurements, the receptor, the ligand, and the Gαqβ1γ2 protein were incubated for 30 min at room temperature. GHSR and ligand concentrations were in the 0.5 and 10 μM range, respectively, with a 1:5 receptor-to-G protein molar ratio. Fluorescence emission spectra were recorded between 360 and 700 nm on a Cary Eclipse spectrofluorimeter (Varian) with an excitation at 346 nm. Buffer contributions were systematically subtracted. The proximity ratio was calculated from the emission spectra as described. 31 Insulin Secretion Experiments in Isolated Rat Pancreatic Islets. All animal care and experiments complied with the directive 2010/63/EU and were approved by the local animal care and use committee Languedoc-Roussillon under reference CEEA-LR-19002. Male Wistar rats (Charles River, France) were housed in a temperature and humidity-controlled room under a 12 h light/dark cycle and had food and water ad libitum. Rats weighed 280-320 g at the time of the experiments. Rats were anesthetized with isoflurane and killed by decapitation. The common bile duct was cannulated, and the pancreas was filled by injection of the collagenase V solution (10 mL, 1 mg/mL), excised, and digested at 37 °C for 10 min. After three successive sedimentations using PBS and 5 mM glucose, islets of Langerhans were hand-picked under a microscope. Then, islets were stabilized for 1 h (5% CO2, 37 °C) in buffer A (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 24 mM NaHCO3, and 0.1% (w/v) BSA, pH 7.4) containing 2.8 mM glucose. Batches of five islets were incubated for 1 h at 37 °C, in 1 mL of buffer A with 0.1% (w/v) BSA containing 2.8 or 8.3 mM glucose in the absence or presence of the tested ligands. At the end of the incubation period, supernatants were collected and stored at -20 °C until the insulin assay. Experiments were performed in quintuplicate. Insulin was quantified using the Cisbio insulin ultrasensitive kit according to the manufacturer's instructions.

Food intake experiments in mice. Experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the US National Research Council (2011, Guide for the care and use of laboratory animals) and all efforts were made to minimize suffering. All protocols received approval from the Institutional Animal Care and Use Committee of the IMBICE (ID 10-0112). Studies were performed using 3-to 5-monthold C57BL6/J wild type male mice generated in the animal facility of the IMBICE. Mice were housed under controlled room temperature (22  1°C) and photoperiod (12 h light/dark cycle from 6:00 h to 18:00 h) with regular chow and water available ad libitum, except when indicated. Thirtythree mice were implanted with intracerebroventricular (ICV) cannulas and then used to test the effect of K-(D-1-Nal)-FwLL-NH2 or ligand 29 on food intake under three experimental conditions: spontaneous ad libitum food intake, ghrelin-induced food intake and fast-refeeding. Briefly, mice were first stereotaxically implanted with an ICV single indwelling guide cannula in the lateral brain ventricle (placement coordinates: antero-posterior: -0.34 mm, medio-lateral: +1.0 mm and dorso-ventral: -2.3 mm) as previously described. 24 After surgery, mice were individually housed and allowed to recover for at least 5 days. In these days, mice were made accustomed to handling by removal of the dummy cannula and connection to an empty cannula connector. For the experiments, mice were ICV-injected with 2 μl of vehicle alone (artificial cerebrospinal fluid) or containing 1 nmol/mouse of K-(D-1-Nal)-FwLL-NH2 or ligand 29. The dose of K-(D-1-Nal)-FwLL-NH2 was chosen based on previous studies showing that it reduces both ad libitum food intake in the early dark phase period and ghrelin-induced food intake in wild-type mice while it does not affect food intake in GHSR-deficient mice. 13, 24 All ICV injections were made over 2 min through a 30-gauge needle that extend 0.5 mm below the guide cannula, which was connected by polyethylene tubing to a Hamilton syringe. The needle was left in place for 2 min following the injection, to prevent back flow of the injected solution. To test the effect of the GHSR ligands on spontaneous food intake, mice were ICV-injected with vehicle (n=11), K-(D-1-Nal)-FwLL-NH2 (n=11) or ligand 29 (n=11) right before lights turn off (at 18:00 h), and food intake was assessed 5 and 12 h later. After 3 days of recovery, mice were randomly used to test the effect of the GHSR ligands on ghrelin-induced food intake. Specifically, mice were ICV-injected with vehicle (n=11), K-(D-1-Nal)-FwLL-NH2 (n=11) or ligand 29 (n=11) around 9:00 h, and then ICV-injected with 0.02 nmol/mouse of ghrelin 15 min later. Food intake was assessed 2 h after ghrelin treatment.

After 3 days of recovery, mice were randomly used to test the effect of the GHSR ligands on a fast-refeeding condition. In particular, food was removed from the home cages at 10:00 h, and mice ICV-treated with vehicle (n=13), K-(d-1-Nal)-FwLL-NH2 (n=13) or ligand 29 (n=7) every 8 h, starting around 16:00 h of the first day of fasting and finishing around 08:00 h of the second day of fasting. Thus, each mouse received 6 ICV injections during the 48 h fasting period. Mice were refed at 10:00 h, and food intake was monitored every 1 h, until 14:00 h of the first day of refeeding.

Then, body weight and food intake were daily assessed at 10:00 h during 7 days after refeeding.

In all the experiments, food intake was assessed by subtracting the remaining weight of the pellets, at any given time, to the initial weight. At the end of the experiment, mice were euthanized and perfused. Brains were extracted, frozen, coronally cut and visualized in a microscope to verify the correct location of ICV cannulas.

Statistical analysis. Data were expressed as mean  standard error of the mean (SEM).

Depending on the design of the experiment, data were analyzed with one-way or two-way ANOVA followed by the Tukey's post hoc test to assign significance to the comparisons between groups.

Equality of variance was analyzed using Bartlett's test. Statistical analysis was performed using GraphPad Prism 6.0 (GraphPad Software). Differences were considered statistically significant when p < 0.05.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:

Copies of 1 H and 13 C NMR spectra of all final ligands 1-46 (PDF)

AUTHOR INFORMATION

Corresponding Author *E-mail: severine.denoyelle@umontpellier.fr.

ORCID

Notes

The authors declare no competing financial interest.