Discovery of 4-(dimethylamino)quinazolines as potent and selective antagonists for the melanin-concentrating hormone receptor 1

A series of 4-(dimethylamino)quinazoline based antagonists of the melanin-concentrating hormone receptor 1 (MCH-R1) is described. This series was derived from a lead compound, AR129330, identified by HTS of a GPCR-directed library using a functional assay with a constitutively activated (CART) form of the receptor. The preliminary optimization resulted in the identification of compounds 20, 21, and 23.     

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.     

4-arylphthalazin-1(2H)-one derivatives as potent antagonists of the melanin concentrating hormone receptor 1 (MCH-R1)

A novel series of 4-arylphthalazin-1(2H)-one linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of an extensive SAR study probing the effects of substituents on the 4-arylphthalazin-1(2H)-one C-4 aryl group led to the identification of the 4-(3,4-difluorophenyl) derivative as a highly potent MCH-R1 inhibitor with an IC(50)=1nM. However, further investigations showed that this substance has unacceptable pharmacokinetic properties including a high clearance and volume of distribution.     

Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists

The optimization of the distance between two key pharmacophore features within our first hit compounds 1a and 2a led to the identification of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylaminoquinazolines. In particular, ATC0065 (2c), N2-[cis-4-([2-[4-Bromo-2-(trifluoromethoxy)phenyl]ethyl]amino)cyclohexyl]-N4,N4-dimethylquinazoline-2,4-diamine dihydrochloride, bound with high affinity to the MCH-R1 (IC50 value of 16 nM) and showed good metabolic stability in liver microsomes from human and rat.     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Cloning and characterization of rhesus monkey MCH-R1 and MCH-R2

Rhesus monkey MCH-R1 and MCH-R2 receptors were cloned. Amino acid homology is 98.8% between monkey and human MCH-R1, while monkey and human MCH-R2 are 98% homologous. Binding and intracellular signaling characteristics of the monkey receptors were compared with the human homologues. The results demonstrate that MCH binds to the monkey MCH-R1 receptor with a K(d) of 6.5 nM and monkey MCH-R2 with a K(d) of 2.2 nM similar to K(d) values for human MCH-R1 and MCH-R2. Additionally, monkey MCH-R1 couples through G(i)/G(o) and G(q)-type G proteins similar to human MCH-R1 whereas monkey and human MCH-R2 utilize the G(q) signaling pathway.     

Expression of receptors for melanin-concentrating hormone (MCH) in different tissues and cell lines

Melanin-concentrating hormone (MCH) is a potent orexigenic neuropeptide and a physiological antagonist of alpha-melanocyte-stimulating hormone (alpha-MSH) in the brain as well as at peripheral sites, including the pigmentary systems of specific vertebrates. Two receptor subtypes for MCH, MCH-R1 and MCH-R2, have been cloned, but other receptor subtypes are likely to exist. Based on our own data and the current literature, we have compared the expression of different receptors for MCH in various mammalian cell lines and tissues. Summarizing all data currently available, we conclude that the two cloned MCH receptors, MCH-R1 and MCH-R2, exhibit differences in their expression pattern, although MCH-R1 is generally colocalized in all tissues where MCH-R2 expression is found. It appears that MCH-R1 is more abundant and has a wider distribution pattern than MCH-R2. Other hypothetical MCH-R subtypes may be expressed in specific tissues, e.g., in the pigment cell system.     

Identification and pharmacological characterization of a novel human melanin-concentrating hormone receptor, mch-r2.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.     

Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.     

Synthesis and SAR study of pyrrolo[3,4-b]pyridin-7(6H)-one derivatives as melanin concentrating hormone receptor 1 (MCH-R1) antagonists

The discovery and optimization of novel pyrrolo[3,4-b]pyridin-7(6H)-one MCH-R1 antagonists are described. A systematic SAR study probing the effects of aryl-, benzyl- and arylthio-substituents at the 2-position of the pyrrolo[3,4-b]pyridin-7(6H)-ones led to identification of the 2-[(4-fluorophenyl)thio] derivative 7b as a highly potent MCH-R1 antagonist. This compound also has favorable pharmacokinetic properties along with a high metabolic stability and a minimal impact on CYP isoforms and hERG.     

Optimization of piperidin-4-yl-urea-containing melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Reducing hERG-associated liabilities

The discovery and optimization of piperidin-4-yl-urea derivatives as MCH-R1 antagonists is herein described. Previous work around the piperidin-4-yl-amides led to the discovery of potent MCH-R1 antagonists. However, high affinity towards the hERG potassium channel proved to be an issue. Different strategies to increase hERG selectivity were implemented and resulted in the identification of piperidin-4-yl-urea compounds as potent MCH-R1 antagonists with minimized hERG inhibition.     

1-(4-Amino-phenyl)-pyrrolidin-3-yl-amine and 6-(3-amino-pyrrolidin-1-yl)-pyridin-3-yl-amine derivatives as melanin-concentrating hormone receptor-1 antagonists

Derivatives of 1-(4-amino-phenyl)-pyrrolidin-3-yl-amine and 6-(3-amino-pyrrolidin-1-yl)-pyridin-3-yl-amine were identified as potent and functionally active MCH-R1 antagonists. One compound with Ki = 2.3 nM demonstrated good oral bioavailability (32%) and in vivo efficacy in rats.     

Endogenous receptor for melanin-concentrating hormone in human neuroblastoma Kelly cells

Melanin-concentrating hormone (MCH), a cyclic nonadecapeptide, is predominantly expressed in mammalian neurons located in the zona incerta and lateral hypothalamus. Current interest in MCH relates to its role in the control of feeding behaviour. Two receptors for MCH were recently found: MCH-R(1) and MCH-R(2). We show here by RT-PCR analysis and immunofluorescence studies that the human neuroblastoma cell line Kelly expresses MCH and MCH-R(1) but not MCH-R(2). In competition assays using 125I-labelled MCH an inhibitory concentration 50% (IC(50)) of 76nM was determined for MCH, indicating a high affinity of Kelly cells for MCH. MCH induces mitogen-activated protein kinase (MAPK) phosphorylation in Kelly cells but no increase in the intracellular free Ca(2+) concentration. This suggests that MCH signals via Galpha(i)/Galpha(0) in these cells. The presence and functionality of MCH-R(1) renders this neuronal cell a very useful model for future structure-activity studies in a physiological environment mimicking the human brain for the evaluation of potential appetite-regulating drugs.     

Discovery of cyclopentane- and cyclohexane-trans-1,3-diamines as potent melanin-concentrating hormone receptor 1 antagonists

We herein report the optimization of cyclopentane- and cyclohexane-1,3-diamine derivatives as novel and potent MCH-R1 antagonists. Structural modifications of the 2-amino-quinoline and thiophene moieties found in the initial lead compound served to improve its metabolic stability profile and MCH-R1 affinity, and revealed unprecedented SAR when compared to other 2-amino-quinoline-containing MCH-R1 antagonists.     

Identification of a new small molecule chemotype of Melanin Concentrating Hormone Receptor-1 antagonists using pharmacophore-based virtual screening

MCH receptor is a G protein-coupled receptor with two subtypes R1 and R2. Many studies have demonstrated the role of MCH-R1 in feeding and energy homeostasis. It has been proven that oral administration of small molecule MCH-R1 antagonists significantly reduces food intake and causes a dose-dependent weight loss. In this study, two ligand-based pharmacophores were developed and validated based on recently published MCH-R1 antagonists with diverse structures. Successful pharmacophores had one hydrogen bond acceptor, one positive ionizable, one ring aromatic and two or three hydrophobic groups. These 3D-QSAR models were used for virtual screening of the ZINC chemical database resulting in the identification of nine compounds with more than 50% displacement of radiolabeled MCH at a 20 μM concentration. Moreover, four of these compounds showed antagonistic activities in Aequorin functional assay, including MH-3 which is the first MCH-R1 antagonist based on a diazaspiro[4.5]decane scaffold. The most active compounds were also docked into our previously published MCH-R1 homology model to gain insights into their binding determinants. These compounds could represent a viable starting scaffold for the design of potent MCH-R1 antagonists with improved pharmacokinetic properties as an effective treatment for obesity.     

Synthesis and SAR investigations of novel 2-arylbenzimidazole derivatives as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists

Compounds containing 2-arybenzimidazole ring systems linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of structure-activity relationship studies led to the identification of compound 4c as a potent MCH-R1 antagonist (IC₅₀ = 1 nM). This compound also has good metabolic stability, and favorable pharmacokinetic and brain penetration properties. However 4c was found to be potent inhibitor of the hERG potassium channel.     

Melanin-concentrating hormone activates signaling pathways in 3T3-L1 adipocytes

Energy homeostasis is regulated by peripheral signals, such as leptin, and by several orexigenic and anorectic neuropeptides. Recently, we reported that the orexigenic neuropeptide melanin-concentrating hormone (MCH) stimulates leptin production by rat adipocytes and that the MCH receptor (MCH-R1) is present on these cells. Here, we show that MCH-R1 is present on murine 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with 1 micromolar MCH for up to 2 h acutely downregulated MCH-R1, indicating a mechanism of ligand-induced receptor downregulation. Potential signaling pathways mediating MCH-R1 action in adipocytes were investigated. Treatment of 3T3-L1 adipocytes with 1 micromolar MCH rapidly induced a threefold and a fivefold increase in p44/42 MAPK and pp70 S6 kinase activities, respectively. In addition, 3T3-L1 adipocytes transiently transfected with a murine leptin-luciferase promoter construct showed a fourfold and a sixfold increase in leptin promoter-reporter gene expression at 1 h and 4 h, respectively, in response to MCH. Activity decreased to basal levels at 8 h. Furthermore, MCH-stimulated leptin promoter-driven luciferase activity was diminished in the presence of the MAP/ERK kinase inhibitor PD-98059 and in the presence of rapamycin, an inhibitor of pp70 S6 kinase activation. These results provide further evidence for a functional MCH signaling pathway in adipocytes.     

Synthesis of new N-phenylpyrazole derivatives with potent antimicrobial activity

The versatile synthons 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2) were used as precursors for the synthesis of a series of phenylpyrazoles with different aromatic ring systems at position 4. The antimicrobiological evaluation of the newly synthesized compounds was carried out in vitro assays for antifungal and antibacterial activities. Amongst the tested compounds, 4-acetyl-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (1), 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2), 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-(2-aminothiazol-4-yl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (17) showed interesting antimicrobial properties. In particular, all tested compounds produced inhibitory effects against pathogenic yeast (Candida albicans) similar or superior to those of reference drug. In addition, compound 3 showed excellent activity against pathogenic mould (Aspergillus). From structure-activity relationship (SAR) point of view, the attachment of bromoacetyl moiety to pyrazole ring can be considered as a breakthrough in developing a new therapeutic antifungal agent related to phenylpyrazole system.     

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.