Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Target Engagement of a Phosphodiesterase 2A Inhibitor Affecting Long-Term Memory in the Rat.

Inhibition of phosphodiesterase 2A (PDE2A) has been proposed as a potential approach to enhance cognitive functioning and memory through boosting intracellular cGMP/cAMP and enhancing neuroplasticity in memory-related neural circuitry. Previous preclinical studies demonstrated that PDE2A inhibitors could reverse N-methyl-D-aspartate receptor antagonist (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine or ketamine-induced memory deficit. Here, we report that the potent and selective PDE2A inhibitor 4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine (PF-05180999) enhances long-term memory in a contextual fear conditioning model in the rat at the oral dose of 0.3 mg/kg. Target engagement at this efficacious dose was explored using in vivo autoradiography. Converse to the results of a decrease of PDE2A binding (target occupancy) by the PDE2A inhibitor, a paradoxical increase (up to 40%) in PDE2A binding was detected. However, a typical target occupancy curve could be generated by PF-05180999 at much higher doses. In vitro experiments using recombinant PDE2A protein or rat brain homogenate that contains native PDE2A protein demonstrated that increased cGMP after initial PDE2A inhibition could be responsible for the activation of PDE2A enzyme via allosteric binding to the GAF-B domain, leading to positive cooperativity of the dormant PDE2A enzymes. Our results suggest that when evaluating target engagement of PDE2A inhibitors for memory disorder in clinical setting with occupancy assays, the efficacious dose may not fall on the typical receptor/target curve. On the contrary, an increase in PDE2A tracer binding is likely seen. Our results also suggest that when evaluating target occupancy of enzymes, potential regulation of enzyme activities should be considered.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain.

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3',5'-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3',5'-cyclic guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure endogenous 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000ng/g and showed excellent linearity (r2>0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies.

Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).