Tirofiban hydrochloride monohydrate receptor Our last area o

Our last area of exploration was the piperidine core as shown in . We rationalised that the basicity of the nitrogen (measured p=7.0) may be responsible for the affinity for the hERG receptor and we therefore sought to moderate this by incorporation of electronegative groups at the 3-position. To this end, the enantiomeric pairs of the -F ( & ; predicted p=5.5) and -OMe ( & predicted p=6.4) were synthesized and compared to the unsubstituted analogue . For the fluoro substitution, we observed modest reduction in the absolute affinities for hERG despite an increase in lipophilicity. In the case of the (,) isomer , we achieved a dramatic improvement in GPR119 potency (EC=7nM) with high efficacy (123%) leading to improved LLE (4.9) and hERG selectivity (>250-fold). The methoxy analogues had significant increases in metabolic instability (Cl=71 & 65μL/min/mg for compounds and , respectively). It was of interest to note that for the F and OMe, different enantiomers of the pairs were preferred in each case and that the potency and efficacy of the (,) OMe Tirofiban hydrochloride monohydrate receptor was significantly reduced. With this knowledge of the structure–activity relationships within this series, we elected to fix the core as the (,)-F isomer and synthesise examples of what we felt to be optimal combinations as shown in . Incorporation of a pyridyl group into the CF aryl ring delivered compounds & which were potent against GPR119 with hERG affinities >10μM and that were stable in rat and human microsomes (Cl=4 & 3μL/min/mg, respectively). The benzothiazole was found to have excellent potency (EC=12nM) with improved selectivity against hERG (>390-fold). Switching to a sulphone R substituent, gave which was the most potent compound to date (EC=4nM) and with high LLE (5.7) and robust efficacy (85%). The affinity for hERG was 4.2μM leading to a selectivity in excess of 1000-fold, and the compound was metabolically stable (Cl=6μL/min/mg). The benzothiazole combinations & were potent and compound was notable for having good potency (EC=24nM) in combination with low affinity for the hERG channel (IC=16μM). In parallel with this optimisation work, further profiling of compound had revealed that this compound had many attractive features. The compound displayed no inhibition towards five major isoforms of cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) in a high throughput fluorescence assay, with IC values >25μM. Plasma protein binding showed reasonable free drug levels consistently across species (mouse=9.9% free; rat=8.1% free; dog=6.8% free; human=7.6% free). The solubility as measured on crystalline material was modest at 18μM but when coupled with good cellular permeability as measured in an in vitro CACO-2 assay (apical to basolateral =57×10cm/s, efflux ratio=0.4 at a compound concentration of 10μM) or MDCK assay (apical to basolateral =17×10cm/s, efflux ratio=0.8 at a compound concentration of 10μM) it was predicted that this would lead to good absorption in vivo. The pharmacokinetic profiles of were determined in vivo in three pre-clinical species; mouse, rat and dog (). The compound showed good pharmacokinetic properties across species, with low clearance (rat and dog) and good bioavailability (mouse, rat). A bile duct cannulation study in rat indicated no biliary or renal elimination. This was consistent with the excellent correlation of in vitro/in vivo scaling of clearance from hepatocytes. Compound was tested against the murine form of GPR119 and was found to retain potency (EC=147nM in an in vitro cAMP assay) with an efficacy of 74% relative to that of OEA. Based on the favourable mouse pharmacokinetics with this compound, it was investigated in vivo for its potential to control the glucose excursion in a mouse (C57BL6/JAX) oral glucose tolerance test (OGTT). In order to establish that this glucose control was mediated by GPR119, compound was tested in an OGTT with both wild-type and GPR119 knock-out mice at a dose of 20mg/Kg. A significant glucose lowering effect was observed in the wild-type mice however, to our surprise, an effect of similar magnitude was also observed in the knock-out mice group indicating that the effects observed were not exclusively mediated through GPR119 (). Reducing the dose of compound led to lower glucose lowering effects (data not shown) but crucially no separation of wild-type and knock-out activities.