Engineering a Potent, Long-Acting, and Periphery-Restricted Oxytocin Receptor Agonist with Anorexigenic and Body Weight Reducing Effects.

The effects of oxytocin on food intake and body weight reduction have been demonstrated in both animal models and human clinical studies. Despite being efficacious, oxytocin is enzymatically unstable and thus considered to be unsuitable for long-term use in patients with obesity. Herein, a series of oxytocin derivatives were engineered through conjugation with fatty acid moieties that are known to exhibit high binding affinities to serum albumin. One analog (OT-12) in particular was shown to be a potent full agonist at the oxytocin receptor (OTR) in vitro with good selectivity and long half-life (24 h) in mice. Furthermore, OT-12 is peripherally restricted, with very limited brain exposure (1/190 of the plasma level). In a diet-induced obesity mouse model, daily subcutaneous administration of OT-12 exhibited more potent anorexigenic and body weight reducing effects than carbetocin. Thus, our results suggest that the long-acting, peripherally restricted OTR agonist may offer potential therapeutic benefits for obesity.

Novel Oxazolidinone-Based Peroxisome Proliferator Activated Receptor Agonists: Molecular Modeling, Synthesis, and Biological Evaluation.

A series of new peroxisome proliferator activated receptors (PPARs) chiral ligands have been designed following the accepted three-module structure comprising a polar head, linker, and hydrophobic tail. The majority of the ligands incorporate the oxazolidinone moiety as a novel polar head, and the nature of the hydrophobic tail has also been varied. Docking studies using the crystal structure of an agonist bound to the ligand binding domain of the PPARα receptor have been performed as a tool for their design. Suitable synthetic procedures have been developed, and compounds with different stereochemistries have been prepared. Evaluation of basal and ligand-induced activity proved that several compounds showed agonist activity at the PPARα receptor, thus validating the oxazolidinone template for PPAR activity. In addition, two compounds, 2 and 4, showed dual PPARα/PPARγ agonism and interesting food intake reduction in rats.

A PEGylated analog of the gut hormone oxyntomodulin with long-lasting antihyperglycemic, insulinotropic and anorexigenic activity.

Peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor (GLP1R) are rapidly gaining favor as antidiabetic agents, since in addition to increasing glucose-dependent insulin secretion, they also cause weight loss. Oxyntomodulin (OXM), a natural peptide with sequence homology to both glucagon and GLP-1, has glucose-lowering activity in rodents and anorectic activity in rodents and humans, but its clinical utility is limited by a short circulatory half-life due to rapid renal clearance and degradation by dipeptidyl peptidase IV (DPP-IV). Here, we describe the development of a novel DPP-IV-resistant, long-acting GLP1R agonist, based on derivatization of a suitably chosen OXM analog with high molecular weight polyethylene glycol (PEG) ('PEGylation'). PEG-OXM exerts an anti-hyperglycemic effect in diet-induced obese (DIO) mice in a glucose-dependent manner, with a maximally efficacious dose of 0.1mg/kg, and reduces food intake and body weight with a minimally efficacious dose of 1mg/kg. If this pharmacology is recapitulated in patients with type 2 diabetes, these results indicate PEG-OXM as a potential novel once-weekly GLP-1 mimetic with both glucose-lowering activity and weight loss efficacy.

The synthesis and characterization N-methyl-3-phenyl-norbornan-2-amine (Camfetamine™).

N-Methyl-3-phenyl-norbornan-2-amine (N-methyl-3-phenylbicyclo[2.2.1]heptan-2-amine, Camfetamine(™) ) is available from a number of online legal highs/research chemicals' vendors. Although it was developed as an analeptic by Merck in the early 1960s, it was never commercialized. However, the Association of Independent Research Chemical Retailers (AIRCR), an umbrella organization for a number of online vendors, has redeveloped it for use as a recreational drug. N-Methyl-3-phenyl-norbornan-2-amine is closely related to fencamfamine which has been widely used as a central nervous system (CNS) stimulant and appetite suppressant. In this paper we describe the synthesis of N-methyl-3-phenyl-norbornan-2-amine, its characterization and interpretations of its electron impact, and electrospray ionization mass spectra.

Chemical polysialylation: design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo.

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid-oxyntomodulin (PSA-OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA-OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA-OXM at 15 µmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.

Synthesis and pharmacological evaluation of novel 4-alkyl-5-thien-2'-yl pyrazole carboxamides.

The synthesis of three series of novel 4-alkyl-5-(5'-chlorothiophen-2'-yl)-pyrazole-3-carbamoyl analogues of rimonabant with affinity for the CB1 cannabinoid receptor subtype is reported. Amongst the novel derivatives, compounds 21j, 22a, 22c, and 22f showed affinity values expressed as Ki ranging from 5.5 to 9.0 nM. Derivative 23e revealed a good CB1 affinity (K(i) = 11.7 nM) and the highest CB1 selectivity of the whole series (K(i)CB2/K(i)CB1 = 384.6). These new compounds appeared to be able to pass the blood brain barrier and to counteract the activity of cannabinoid agonist. According to the results of mice vas deferens assays, as in the case of rimonabant, derivatives 21a, 22a, and 22b showed inverse agonist activity. In contrast, as a preliminary result to be confirmed, compound 23a exhibited neutral antagonist profile. According to the data obtained through an acute animal model, selected compounds 21a, 22a, and 23a evidenced the capability to significantly reduce food intake. At specific conditions, the effect of the novel compounds were higher than that induced by rimonabant. Amongst the novel CB1 antagonist compounds, 23a may represent a useful candidate agent for the treatment of obesity and its metabolic complications, with reduced side effects relative to those instead observed with rimonabant.

Expeditious synthesis of saponin P57, an appetite suppressant from Hoodia plants.

Pregnane glycoside P57, the appetite suppressant component from Hoodia, was synthesized expeditiously, featuring preparation of the aglycone Hoodigogenin A from digoxin and assembly of the deoxytrisaccharide with glycosyl o-alkynylbenzoates as donors.

Influence of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) on appetite and thrombosis.

Preclinical pharmacological characterization of a novel inhibitor (UM8190) of prolylcarboxypeptidase (PRCP) was investigated. We synthesized and evaluated a library of proline-based analogs as prospective recombinant PRCP (rPRCP) inhibitors and inhibitors of PRCP-dependent prekallikrein (PK) activation on human pulmonary artery endothelial cells (HPAEC). Among the newly synthesized compounds, UM8190 was further characterized in vivo using methods that encompassed a mouse carotid artery thrombosis model and animal model of food consumption. (S)-N-dodecyl-1-((S)-pyrrolidine-2-carbonyl) pyrrolidine-2-carboxamide [Compound 3 (UM8190)] was selected for further evaluation from the initial assessment of its PRCP inhibitory action (K(i)= 43 µM) coupled with its ability to block PRCP-dependent PK activation on HPAEC (K(i)= 34 µM). UM8190 demonstrated excellent selectivity against a panel of carboxypeptidases and serine proteases and blocked bradykinin (BK) generation and BK-induced permeability by 100%, suggesting that it may be useful in preventing the local production of large amounts of BK. Furthermore, UM8190 showed an anorexigenic effect when systemically administered to fasted mice, reducing food intake in a dose- and time-dependent manner. In a mouse carotid artery thrombosis model, it also demonstrated an antithrombotic effect. UM8190 is a selective PRCP inhibitor and it may represent a new anorexigenic, and antithrombotic drug, that works by inhibiting PRCP-mediated mechanisms.

Cultivation practices and manufacturing processes to produce Hoodia gordonii extract for weight management products.

Hoodia gordonii (Masson) Sweet ex Decne., is a succulent shrub, indigenous to the arid regions of southern Africa. Indigenous people have historically utilised certain species of Hoodia, including H. gordonii, as a source of food and water. Studies by the Council for Scientific and Industrial Research (CSIR, South Africa) identified that extracts of H. gordonii had appetite suppressant activity associated with specific steroid glycosides. A programme to develop weight management products based around this discovery was implemented in 1998. An agronomy programme was established which demonstrated that it was possible to cultivate this novel crop on a commercial scale (in excess of 70 ha). In parallel, a food grade manufacturing process was developed consisting of four main steps: harvesting of H. gordonii plant stems, comminution, drying under controlled conditions and extraction using food grade solvents. Appropriate Quality Control (QC) procedures were developed. The extraction process is capable of delivering a consistent composition despite natural variations in the composition of the dried H. gordonii. Specifications were developed for the resulting extract. The intended use of the standardised H. gordonii extract was as a functional food ingredient for weight management products. Other development studies on characterisation, toxicology and pharmacology are reported separately.

Chemical characterisation of Hoodia gordonii extract.

The chemical composition of a solvent extract of Hoodia gordonii termed 'H.gordonii extract' has been characterised by hyphenated chromatographic methods and traditional analytical techniques. The extract consists of a mixture of steroid glycosides, fatty acids, plant sterols and polar organic material. High performance liquid chromatography (HPLC) with ultra violet (UV) and mass spectrometric (MS) detection was used to quantify and confirm the identity of a number of steroid glycosides (73.7% w/w) present in the extract. Gas chromatography (GC) with MS and flame ionisation detection (FID) was applied to determine the fatty acid (3.12% w/w) sterol (0.39% w/w) and alcohol (0.03% w/w) content of a saponified sample of the extract. Polar organic material was quantified by gravimetric methodology using C(18) SPE separation and was determined to be a minimum of 3% w/w. Moisture content was measured by Karl Fischer coulometric titration (0.81% w/w). The protein content was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with SYPRO Ruby staining and a negative result was determined with a limit of detection of <0.001%w/w of protein per band. The chemical composition of the extract remained stable for 19 months when stored in re-sealable plastic bags at ambient (21-24°C) temperature and <60% relative humidity.

Synthesis and SAR study of 4-arylpiperidines and 4-aryl-1,2,3,6-tetrahydropyridines as 5-HT2C agonists.

A series of substituted 4-arylpiperidines and a smaller family of 4-aryl-1,2,3,6-tetrahydropyridines were synthesized and their biological activity at the 5-HT(2C) receptor studied to determine whether either series showed noteworthy agonist activity. Structure-activity relationships were developed from the performed receptor binding assays and functional studies, and the results of the analysis are presented herein.

Biostable and PEG polymer-conjugated insect pyrokinin analogs demonstrate antifeedant activity and induce high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae).

The pyrokinins (PK) are multifunctional neuropeptides found in a variety of arthropod species, including the pea aphid Acyrthosiphon pisum (Hemiptera: Aphidae). A series of biostable pyrokinin analogs based on the shared C-terminal pentapeptide core region were fed in solutions of artificial diet to the pea aphid over a period of three days and evaluated for antifeedant and aphicidal activity. The analogs contained either modified Pro residues Oic or Hyp and or a d-amino acid in key positions to enhance resistance to tissue-bound peptidases and retain activity in a number of PK bioassays. A series of PK analogs conjugated with two lengths of polyethyleneglycol (PEG) polymers were also evaluated in the aphid feeding assay. Three of the biostable PK analogs demonstrated potent antifeedant activity, with a marked reduction in honeydew formation and very high mortality after 1 day. In contrast, a number of unmodified, natural pyrokinins and several other analogs containing some of the same structural components that promote biostability were inactive. Two of the most active analogs, Oic analog PK-Oic-1 (FT[Oic]RL-NH(2)) and PEGylated analog PK-dF-PEG(8) [(P(8))-YF[dF]PRL-NH(2)], featured aphicidal activity calculated at LC(50)'s of 0.042nmol/µl [0.029µg/µl] (LT(50) of 1.0 day) and 0.126nmol/µl (LT(50) of 1.3 days), respectively, matching the potency of some commercially available aphicides. Notably, a PEGylated analog of a PK antagonist can block over 55% of the aphicidal effects of the potent PK agonist PK-Oic-1, suggesting that the aphicidal effects are mediated by a PK receptor. The mechanism of this activity has yet to be established, though the aphicidal activity of the biostable analogs may result from disruption of digestive processes by interfering with gut motility patterns, a process shown to be regulated by the PKs in other insects. The active PK analogs represent potential leads in the development of selective, environmentally friendly aphid pest control agents.

Synthesis of Hoodigogenin A, aglycone of natural appetite suppressant glycosteroids extracted from Hoodia gordonii.

14ß-hydroxy pregnane glycosides extracted from Hoodia gordonii, a succulent plant isolated from Apocynaceae are suggested to have appetite suppressant properties in animals and humans. However, limited reports on biological studies concerning the appetite suppressant properties are available in the open literature. One reason for that is the poor availability of these glycosteroids because H. gordonii is a protected plant and the yield of extraction lies between 0.003% and 0.02%. Starting from 3α,12α-diacetoxy-pregnanone 1, we disclose in this report the synthesis of Hoodigogenin A, the aglycone of the natural 14ß-hydroxy pregnane glycosides extracted from H. Gordonii.

Highly enantioselective catalytic asymmetric synthesis of a (R)-sibutramin precursor.

The first highly enantioselective, catalytic asymmetric synthesis of di-des-methylsibutramine 3 is described. Dienamide 10, prepared by acetic acid anhydride quenching of the condensation product of nitrile 4 with a methallyl magnesium chloride, proved to be an excellent substrate for ruthenium-catalyzed asymmetric hydrogenation with atropisomeric diphosphine ligands. Hydrogenation with a ruthenium/(R)- MeOBiPheP catalyst at S/C = 500, gave the chiral amide (R)-9 in 98.5% ee in almost quantitative yield. After acidic amide hydrolysis the desired amine (R)-3 was obtained without erosion of enantioselectivity. It is anticipated that the overall process will be amenable to large-scale production.

Synthesis and evaluation of dibenzothiazepines: a novel class of selective cannabinoid-1 receptor inverse agonists.

A novel class of CB1 inverse agonists was discovered. To efficiently establish structure-activity relationships (SARs), new synthetic methodologies amenable for parallel synthesis were developed. The compounds were evaluated in a mammalian cell-based functional assay and in radioligand binding assays expressing recombinant human cannabinoid receptors (CB1 and CB2). In general, all of the compounds exhibited high binding selectivity at CB1 vs CB2 and the general SAR revealed a lead compound 11-(4-chlorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12e) which showed excellent in vivo activity in pharmacodynamic models related to CB1 receptor activity. The low solubility that hampered the development of 12e was solved leading to a potential preclinical candidate 11-(3-chloro-4-fluorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12h).

5-HT2C receptor agonists with potential anorectic activity.

Substituted 2,3,4,4a-tetrahydropyrazino[2,1-c][1,4]benzoxazin-5-(1H)-ones were synthesized and evaluated as 5-HT(2C) receptor agonists for the possible treatment of obesity. A number of compounds exhibited 5-HT(2C) agonist binding activity with compound 19 showing the most potent in vitro activity.

Diaryl dihydropyrazole-3-carboxamides with significant in vivo antiobesity activity related to CB1 receptor antagonism: synthesis, biological evaluation, and molecular modeling in the homology model.

A number of analogues of diaryl dihydropyrazole-3-carboxamides have been synthesized. Their activities were evaluated for appetite suppression and body weight reduction in animal models. Depending on the chemical modification of the selected dihydropyrazole scaffold, the lead compounds--the bisulfate salt of (+/-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 26 and the bisulfate salt of (-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 30--showed significant body weight reduction in vivo, which is attributed to their CB1 antagonistic activity and exhibited a favorable pharmacokinetic profile. The molecular modeling studies also showed interactions of two isomers of (+/-)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid morpholin-4-ylamide 9 with CB1 receptor in the homology model similar to those of N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (rimonabant) 1 and 4S-(-)-3-(4-chlorophenyl)-N-methyl-N'-[(4-chlorophenyl)-sulfonyl]-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamidine (SLV-319) 2.

Novel sulfamide analogs of oleoylethanolamide showing in vivo satiety inducing actions and PPARalpha activation.

Long chain saturated and unsaturated alkyl sulfamide and propyl sulfamide derivatives, analogs of oleoylethanolamide, have been synthesized and evaluated in vivo and in vitro as peroxisome proliferator activated receptor alpha (PPARalpha) activators. Additionally, the anorexic effects of the new compounds have been studied in vivo in food-deprived rats. Among the active compounds N-octadecyl-N'-propylsulfamide (7) has been identified as a potent hypolipidemic compound, a potent feeding suppressant, and a concentration-dependent activator of PPARalpha.

Discovery and pharmacological evaluation of growth hormone secretagogue receptor antagonists.

The discovery and pharmacological evaluation of potent, selective, and orally bioavailable growth hormone secretagogue receptor (GHS-R) antagonists are reported. Previously, 2,4-diaminopyrimidine-based GHS-R antagonists reported from our laboratories have been shown to be dihydrofolate reductase (DHFR) inhibitors. By comparing the X-ray crystal structure of DHFR docked with our GHS-R antagonists and GHS-R modeling, we designed and synthesized a series of potent and DHFR selective GHS-R antagonists with good pharmacokinetic (PK) profiles. An amide derivative 13d (Ca2+ flux IC50 = 188 nM, [brain]/[plasma] = 0.97 @ 8 h in rat) showed a 10% decrease in 24 h food intake in rats, and over 5% body weight reduction after 14-day oral treatment in diet-induced obese (DIO) mice. In comparison, a urea derivative 14c (Ca2+ flux IC50 = 7 nM, [brain]/[plasma] = 0.0 in DIO) failed to show significant effect on food intake in the acute feeding DIO model. These observations demonstrated for the first time that peripheral GHS-R blockage with small molecule GHS-R antagonists might not be sufficient for suppressing appetite and inducing body weight reduction.

A thienopyridazinone-based melanin-concentrating hormone receptor 1 antagonist with potent in vivo anorectic properties.

Melanin-concentrating hormone receptor antagonists containing thieno- and a benzopyridazinone cores were designed and tested as potential anorectic agents. These ligands showed high affinity for the receptor, potent functional activity in vitro, and good oral bioavailabilty in rats. The thiophene analogue exhibited low iv clearance, long half-life, and high brain penetration. In obese rats, the thienopyridazinone demonstrated a dose-dependent reduction in feeding and body weight with doses between 1 and 10 mg kg-1.