Identification and synthesis of a novel selective partial PPARdelta agonist with full efficacy on lipid metabolism in vitro and in vivo.

The aim was to identify a novel selective PPARdelta agonist with full efficacy on free fatty acid (FFA) oxidation in vitro and plasma lipid correction in vivo. Using the triple PPARalpha,gamma,delta agonist 1 as the structural starting point, we wanted to investigate the possibility of obtaining selective PPARdelta agonists by modifying only the acidic part of 1, while holding the lipophilic half of the molecule constant. The structure-activity relationship was guided by in vitro transactivation data using the human PPAR receptors, FFA oxidation efficacy performed in the rat muscle L6 cell line, and in vivo rat pharmacokinetic properties. Compound 7 ([4-[3,3-bis-(4-bromo-phenyl)-allylthio]-2-chloro-phenoxy]-acetic acid) was identified as a selective, partial agonist with good oral pharmacokinetic properties in rat. Chronic treatment of high fat fed ApoB100/CETP-Tgn mice with 7 corrected the plasma lipid parameters and improved insulin sensitivity. These data suggest that selective PPARdelta agonists have the potential to become a novel treatment of dyslipidemia.

Selective PPAR agonists for the treatment of type 2 diabetes.

Type 2 diabetes is a metabolic disease characterized by increased plasma glucose and insulin as well as dyslipidemia. If left untreated, chronic diseases will develop that are associated with neuropathic damage and higher mortality risk. Using a rational drug design, novel compounds have been developed that selectively activate the human PPAR receptors, leading to lessening of hyperglycemia and hyperinsulinemia as well as reduction of lipid levels in conjunction with an increase of the beneficial HDL-cholesterol. These PPAR agonists showed increased potency and efficacy compared to previously marketed insulin sensitizers. Lead compounds with desirable pharmacokinetic properties were chosen for further testing in several animal models. The in vivo activity of some synthetic ligands, capable of activating two or all three members of peroxisome proliferator-activated receptors (PPAR) family of receptors, suggested that they may have improved efficacy in type 2 diabetes therapy. Here, we briefly summarize the development of some novel PPAR agonists identified by our group in recent years.

Large dimeric ligands with favorable pharmacokinetic properties and peroxisome proliferator-activated receptor agonist activity in vitro and in vivo.

Two potent nonselective, but PPARalpha-preferring, PPAR agonists 5 and 6 were designed and synthesized in high yields. The concept of dimeric ligands in transcription factors was investigated by synthesizing and testing the corresponding dimers 7, 8a, and 8b in PPAR transactivation assays. The three dimeric ligands all showed agonist activity on all three PPAR receptor subtypes, but with different profiles compared to the monomers 5 and 6. Despite breaking all the "rule of five" criteria, the dimers had excellent oral bioavailability and pharmacokinetic properties, resulting in good in vivo efficacy in db/db mice. X-ray crystal structure and modeling experiments suggested that the dimers interacted with the AF-2 helix as well as with amino acid residues in the lipophilic pocket close to the receptor surface.

Arylcyanoguanidines as activators of Kir6.2/SUR1K ATP channels and inhibitors of insulin release.

Phenylcyanoguanidines substituted with lipophilic electron-withdrawing functional groups, e.g. N-cyano-N'-[3,5-bis-(trifluoromethyl)phenyl]-N' '-(cyclopentyl)guanidine (10) and N-cyano-N'-(3,5-dichlorophenyl)-N' '-(3-methylbutyl)guanidine (12) were synthesized and investigated for their ability to inhibit insulin release from beta cells, to repolarize beta cell membrane potential, and to relax precontracted rat aorta rings. Structural modifications gave compounds, which selectively inhibit insulin release from betaTC6 cells (e.g. compound 10: IC(50) = 5.45 +/- 1.9 microM) and which repolarize betaTC3 beta cells (10: IC(50) = 4.7 +/- 0.5 microM) without relaxation of precontracted aorta rings (10: IC(50) > 300 microM). Inhibition of insulin release from rat islets was observed in the same concentration level as for betaTC6 cells (10: IC(50) = 1.24 +/- 0.1 microM, 12: IC(50) = 3.8 +/- 0.4 microM). Compound 10 (10 microM) inhibits calcium outflow and insulin release from perifused rat pancreatic islets. The mechanisms of action of 10 and 12 were further investigated. The compounds depolarize mitochondrial membrane from smooth muscle cells and beta cell and stimulate glucose utilization and mitochondrial respiration in isolated liver cells. Furthermore, 10 was studied in a patch clamp experiment and was found to activate Kir6.2/SUR1 and inhibit Kir6.2/SUR2B type of K(ATP) channels. These studies indicate that the observed effects of the compounds on beta cells result from activation of K(ATP) channels of the cell membrane in combination with a depolarization of mitochondrial membranes. It also highlights that small structural changes can dramatically shift the efficacy of the cyanoguanidine type of selective activators of Kir6.2/SUR2 potassium channels.

6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives potently and selectively activate ATP sensitive potassium channels of pancreatic beta-cells.

6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide derivatives were synthesized and characterized as activators of adenosine 5'-triphosphate (ATP) sensitive potassium (K(ATP)) channels in the beta-cells by measuring effects on membrane potential and insulin release in vitro. The effects on vascular tissue in vitro were measured on rat aorta and small mesenteric vessels. Selected compounds were characterized as competitive inhibitors of [(3)H]glibenclamide binding to membranes of HEK293 cells expressing human SUR1/Kir6.2 and as potent inhibitors of insulin release in isolated rat islets. 6-Chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (54) was found to bind and activate the SUR1/Kir6.2 K(ATP) channels in the low nanomolar range and to be at least 1000 times more potent than the reference compound diazoxide with respect to inhibition of insulin release from rat islets. Several compounds, e.g., 3-propylamino- (30), 3-isopropylamino- (34), 3-(S)-sec-butylamino- (37), and 3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (53), which were found to be potent and beta-cell selective activators of K(ATP) channels in vitro, were found to inhibit insulin secretion in rats with minimal effects on blood pressure and to exhibit good oral pharmacokinetic properties.

Novel tricyclic-alpha-alkyloxyphenylpropionic acids: dual PPARalpha/gamma agonists with hypolipidemic and antidiabetic activity.

Synthesis and structure-activity relationships of tricyclic alpha-ethoxy-phenylpropionic acid derivatives guided by in vitro PPARalpha and PPARgamma transactivation data and computer modeling led to the identification of the novel carbazole analogue, 3q, with dual PPARalpha (EC(50) = 0.36 microM) and PPARgamma (EC(50) = 0.17 microM) activity in vitro. Ten days treatment of db/db mice with 3q improved the insulin sensitivity, as measured by OGTT, better than that seen with both pioglitazone and rosiglitazone treatment, suggesting in vivo PPARgamma activity. Likewise, 3q lowered plasma triglycerides and cholesterol in high cholesterol fed rats after 4 days treatment, indicating in vivo PPARalpha activity. Investigations of the pharmacokinetics of selected compounds suggested that extended drug exposure improved the in vivo activity of in vitro active compounds.

Novel selective PPARdelta agonists: optimization of activity by modification of alkynylallylic moiety.

Y-shaped molecules bearing alkynylallylic moieties were found to be potent and selective PPARdelta activators. The alkynylallylic moiety was synthesized from alkyn-1-ols by hydroalumination followed by a cross-coupling reaction. Series of active compounds 6 were obtained by stepwise changing the structure of the known PPARpan agonist 5 into Y-shaped compounds. The most active and selective compound, 6f, had a PPARdelta potency of 0.13 microM, which is 50-fold more potent than compound 5.

Design of a partial PPARdelta agonist.

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.

Design of potent PPARalpha agonists.

Computational analysis of the ligand binding pocket of the three PPAR receptor subtypes was utilized in the design of potent PPARalpha agonists. Optimum PPARalpha potency and selectivity were obtained with substituents having van der Waals volume around 260. Compound 6 had a PPARalpha potency of 0.002 microM and a selectivity ratio to PPARgamma and PPARdelta of 410 and 2000, respectively.

Structure-activity relationships of dimeric PPAR agonists.

A series of dimeric PPAR agonists were designed and tested for PPAR activity in vitro. The SAR showed that dimeric ligands with a common group or full dimeric ligands had retained or even increased PPARgamma potency. The dimeric agonist concept can be used to fine tune the subtype selectivity of PPAR agonists. The PPARgamma potency could, at least partly, be explained using molecular modeling.

Synthesis and biological activity of 3,3-diamino-sulfonylacrylonitriles as novel inhibitors of glucose induced insulin secretion from beta cells.

Pinacidil analogues, for example, N-cyano-N'-(3,5-dichlorophenyl)-N"-(3-methylbutyl)guanidine, 1, have previously been described as potassium channel openers on beta cells and smooth muscle cells. In the present study 3,3-diamino-sulfonylacrylonitrile, a new bioisostere of the cyanoguanidine group, was investigated. 3,3-Diamino-sulfonylacrylonitriles were prepared in a two step synthesis from the corresponding isothiocyanates and sulfonylacetonitriles. Single crystal X-ray crystallography and NMR spectroscopy were used to establish the structure of 2-(4-chlorophenylsulfonyl)-3-cyclobutylamino-3-(3,5-dichlorophenylamino)acrylonitrile 3i. The analysis confirmed that 3i assumes a staggered conformation considered as the energetically most favourable. The compounds synthesised have been identified as potent inhibitors of glucose stimulated insulin secretion from beta cell lines and rat pancreatic islets with minimal effects on vascular smooth muscle.

Design and synthesis of novel PPARalpha/gamma/delta triple activators using a known PPARalpha/gamma dual activator as structural template.

Using a known dual PPARalpha/gamma activator (5) as a structural template, SAR evaluations led to the identification of triple PPARalpha/gamma/delta activators (18-20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARalpha/gamma/delta activation.