Design and synthesis of potent and selective 1,3,4-trisubstituted-2-oxopiperazine based melanocortin-4 receptor agonists

The design and synthesis of a series of potent 1,3,4-trisubstituted-2-oxopiperazine based MC4 agonists are described. The tripeptidomimetic analogs (12a,b and 23) and the dipeptidomimetic 27 displayed single-nanomolar binding affinity and agonist potency for MC4R and excellent selectivity for MC4R relative to MC1R.     

Melanocortin subtype-4 receptor agonists containing a piperazine core with substituted aryl sulfonamides

The biological activity for a set of melanocortin-4 receptor (MC4R) agonists containing a piperazine core with an ortho-substituted aryl sulfonamide is described. Compounds from this set had binding and functional activities at MC4R less than 30 nM. The most selective compound in this series was >25,000-fold more potent at MC4R than MC3R, and 490-fold more potent at MC4R than MC5R. This compound also reduced food intake after oral dosing at 25, 50, and 100 mg kg(-1) in fasted mice.     

Synthesis and biological evaluation of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R)

A novel series of imidazole-based small molecule antagonists of the melanocortin 4 receptor (MC4-R) is reported. Members of this series have been identified, which exhibit sub-micromolar binding affinity for the MC4-R, functional potency <100nM, and good oral exposure in rat. Antagonists of the MC4-R are potentially useful in the therapeutic treatment of involuntary weight loss due to advanced age or disease (e.g. cancer or AIDS), an area of large, unmet medical need.     

Synthesis and biological activity of novel peptide mimetics as melanocortin receptor agonists

A series of novel peptidomimetic analogs was prepared containing cyclohexyl, phenyl, or heterocyclic groups to ostensibly orient the guanidine or mimic of an arginine in a putative melanocortin receptor ligand pharmacophore. Some binding affinity at the melanocortin receptors MC(3) and MC(4) was noted. In silico docking also indicated that the relative positions of the hydrogen-bonding sites and hydrophobic regions of the compounds are reasonably well matched to the receptor-binding site. This may present a lead entry into a selective series of MC(4)R agonists.     

Synthesis of novel melanocortin 4 receptor agonists and antagonists containing a succinamide core

A novel series of piperazines appended to a succinamide backbone were synthesized and found to have a high affinity for the melanocortin-4 receptor (IC(50)s ranging from <0.1 to 200 nM). Both agonists and antagonists of MC4R were prepared by modifying the groups attached to the right-hand side of the succinamide. This series also exhibits a high level of selectivity (up to 7000-fold) over mouse MC1R and human MC3R.     

A potent and selective nonpeptide antagonist of the melanocortin-4 receptor induces food intake in satiated mice

Optimization on a series of piperazinebenzylamines resulted in analogues with low nanomolar binding at the human MC4 receptor but weak affinity (Ki > 500 nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist (Ki = 3.2 nM) with a selectivity of 240-fold over MC3R. It proved to be an insurmountable antagonist in a cAMP assay. Compound 14c potently stimulated food intake in satiated mice when given by intracerebroventricular administration.     

Spiroindane based amides as potent and selective MC4R agonists for the treatment of obesity

We report a series of potent and selective MC4R agonists based on spiroindane amide privileged structures for potential treatments of obesity. Among the synthetic methods used, Method C allows rapid synthesis of the analogs. The series of compounds can afford high potency on MC4R as well as good rodent pharmacokinetic profiles. Compound 1r (MK-0489) demonstrates MC4R mediated reduction of food intake and body weight in mouse models. Compound 1r is efficacious in 14-day diet-induced obese (DIO) rat models.     

Structure-activity relationship of a series of cyclohexylpiperidines bearing an amide side chain as antagonists of the human melanocortin-4 receptor

A series of cyclohexylpiperazines was synthesized as potent and selective antagonists of the human MC4 receptor. Compound 14t displayed binding affinity (Ki) of 4.2 and 1100 nM at MC4R and MC3R, respectively.     

Potent and orally active non-peptide antagonists of the human melanocortin-4 receptor based on a series of trans-2-disubstituted cyclohexylpiperazines

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of energy homeostasis. Recent studies have shown that blockade of the MC4R reverses tumor-induced weight loss in mice. Herein, we describe the synthesis and identification of potent and selective non-peptide antagonists of the human MC4R from a series of 2-ethoxycarbonylcyclohexyl-piperazines. Compound 12i was found to possess low nanomolar affinity for the MC4R, and exhibit oral bioavailability in rats. More importantly, when administered orally to mice (10 mg/kg), it led to statistically significant increases in food intake over a 24-h period.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Inverse agonist activity of agouti and agouti-related protein

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.     

Novel piperazines: potent melanocortin-4 receptor antagonists with anxiolytic-like activity

In the present study, we found that a novel piperazine compound, 11a, showed a moderate affinity (IC(50)=333nM) for the MC4 receptor. We developed the new type of piperazine compounds and found that mono-piperazine 11b exhibited a high-affinity (IC(50)=40.3nM) for the MC4 receptor. We also found that a series of biphenyl analogues exhibited a high-affinity for the receptor, and in particular, compound 11j exhibited the highest affinity for the MC4 receptor with an IC(50) value of 14.5nM. Furthermore, some of these compounds, when administered orally, significantly reversed the stress-induced anxiety-like behavior in rats. In this paper, we report the synthesis, structure-activity relationships, and oral activity of the novel mono-piperazines as MC4 receptor antagonists.     

Synthesis and SAR of potent and orally bioavailable tert-butylpyrrolidine archetype derived melanocortin subtype-4 receptor modulators

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.     

Chimeras of the agouti-related protein: insights into agonist and antagonist selectivity of melanocortin receptors

The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone alpha-MSH and antagonized by the agouti-related protein (AGRP). Whereas alpha-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.     

Novel selective melanocortin 4 receptor antagonist induces food intake after peripheral administration

We synthesized a new series of small cyclic melanocyte-stimulating hormone (MSH) analogues and screened them for binding affinity at the four MSH binding melanocortin (MC) receptors. We identified a novel substance HS131, with about 20-fold higher affinity for the MC4 receptor than the MC3 receptor. This substance proved to be antagonist for all the four MC receptors in a cAMP assay. HS131 is a six amino acid long peptide, has a molecular weight below 1000, and has only two amino acids in common with the natural MSH peptides. HS131 potently and dose dependently increased food intake after i.c.v. administration. Moreover, s.c. administration of HS131 (1.0 mg/kg) increased food intake, suggesting that HS131 may be able to pass the blood brain barrier. This cyclic low molecular weight peptidomimetic will enable studies of the functional role of the MC4 receptors by peripheral administration and it may be used as a template for further development of low molecular weight substances for the MC receptors.     

Sub-nanomolar hMC1R agonists by end-capping of the melanocortin tetrapeptide His-D-Phe-Arg-Trp-NH(2)

Twenty three derivatives of the core fragment His(6)-D-Phe(7)-Arg(8)-Trp(9)-NH(2) end-capped with carboxylic and sulfonic acids were synthesized and evaluated at human melanocortin receptors (hMC1, hMC3, and hMC4Rs). The SAR within this series allowed us to map the hMCRs near the His(6) binding site and design a superpotent MC1R agonist, LK-184, Ph(CH(2))(3)CO-His-D-Phe-Arg-Trp-NH(2) (19) with EC(50) 0.01 nM (5 nM at MC3 and MC4Rs).     

Synthesis and characterization of trans-4-(4-chlorophenyl)pyrrolidine-3-carboxamides of piperazinecyclohexanes as ligands for the melanocortin-4 receptor

A series of trans-N-alkyl-4-(4-chlorophenyl)pyrrolidine-3-carboxamides of piperazinecyclohexanemethylamines was synthesized and characterized for binding and function at the melanocortin-4 receptor (MC4R), and several potent benzylamine derivatives were identified. Compound 18v was found to bind MC4R with potent affinity (K_i = 0.5 nM) and high selectivity over the other melanocortin subtypes and behaved as a functional antagonist (IC₅₀ = 48 nM).     

Synthesis and structure-activity relationships of novel dipeptides and reduced dipeptides as ligands for melanocortin subtype-4 receptor

A series of benzylic piperazines (e.g., 4 and 5) attached to an 'address element', the dipeptide H-D-Tic-D-p-Cl-Phe-OH, 3 has been identified as ligands for the melanocortin subtype-4 receptor (MC4R). We describe herein the structure-activity relationship (SAR) studies on the N-terminal residue of the 'address element'. Several novel dipeptides and reduced dipeptides with high MC4R binding affinities and selectivity emerged from this SAR study.     

Design, synthesis, and evaluation of proline based melanocortin receptor ligands

A series of proline based melanocortin ligands has been developed on the basis of initial piperazine leads by using a more conformationally rigid scaffold. A number of these novel ligands showed significant binding affinity for MC3 and MC4 receptors.