A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aβ in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aβ EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10 h.

Cleavage of the amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) is the first proteolytic step in the production of amyloid-β (Aβ), the principal component of senile plaques in Alzheimer's disease (AD). While inhibition of BACE1 represents one of the promising therapeutic strategies for AD, development of centrally active, small molecule inhibitors have been challenging. Here we describe in vitro and in vivo characterization of a novel, brain-penetrant BACE1 inhibitor. Potency of the compound was evaluated in various cell-free and whole-cell assays. In vivo Aβ-lowering was assessed in wild-type (WT), Tg2576 transgenic and P-glycoprotein knockout (Pgp KO) mice at 3 hours following subcutaneous injection of the compound (1, 3, 10, 30, 100, 300 mg/kg doses) or vehicle. Brain, plasma and CSF samples were harvested for measurement of Aβx-40 and Aβx-42 by sandwich ELISAs. Drug exposures were determined in brain and plasma samples by LC-MS. The compound potently inhibited total Aβ and sAPPβ production in whole-cell assays (H4 cells) overexpressing human WT APP with IC50 values of 30 and 56 nM, respectively. In WT mice, subcutaneous administration of the BACE inhibitor resulted in dose-dependent brain Aβ lowering at 3h post-drug, with significant effects observed at 100 and 300 mg/kg doses (Aβx-40 reduction of 39% and 59%, and Aβx-42 reduction of 44% and 57%, respectively). These doses also significantly lowered CSF Aβx-40 in the same animals (43% and 54%, respectively). In line with other reports, we found that equivalent doses of the compound were less efficacious at reducing Aβ in the Tg2576 mice compared to WT mice. Aβ-lowering was most robust, although still not complete, in the Pgp KO mice, with maximum effect of 65% Aβx-40 reduction at 300 mg/kg dose. At this dose, free drug brain exposures were greatly in excess of in vitro IC50 values. Present studies demonstrate robust central Aβ reduction in wild-type mice by acute administration of a novel brain penetrant BACE1 inhibitor. Analysis of pharmacokinetic-pharmacodynamic relationships suggest that, unlike the effect of γ-secretase, in vivo BACE1 inhibition may results in an incomplete reduction of Aβ.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Aβ 1-42 and 1-40 peptides have been used extensively as biomarkers in Alzheimer's disease (AD) and may have utility as markers of therapeutic response. A series of natural history studies were completed to examine Aβ variability and further explore the use of this endpoint in Phase I trials. Aβ endpoints were also evaluated as pharmacodynamic (PD) endpoints in a Phase I first-in-human study to test the effects of a novel gamma secretase inhibitor, PF-3084014. All studies included young, healthy volunteers aged 18-55 years. For the first natural history study (N=20), plasma samples were collected daily over a 2-week period and on Days 1 and 13 at baseline, 3, 6, 9, 12, 16 and 24 hours. In the second study (N=14), matched plasma and cerebrospinal fluid (CSF) samples were collected by direct lumbar puncture (LP) every 6 hours over a 24-hour period. In a third study (N=20), serial matched plasma and lumbar CSF samples were collected from catheterized volunteers on two separate days over a 14-hour period. A Phase I randomized crossover single-dose study (N=10) was conducted to test central PD activity of PF-3084014 on Aβ peptides using serial CSF assessments. Generally, intra-subject variability of Aβ in plasma and CSF was lower than inter-subject variability, particularly when Aβ over time was parameterized as an area-under-the-curve assessment. No 24-hour diurnal variability was observed in the direct LP study. However, a systematic upward drift in CSF Aβ was noted in studies using serial lumbar catheterization. Preliminary analysis showed poor correlation between plasma and CSF Aβ levels. Subsequent power analyses suggested that CSF Aβ endpoints can be used in small Phase I studies with the objective of detecting relatively small changes as proof of pharmacological activity. In the Phase I CSF study, PF-3084014 showed good peripheral activity as evidenced by dose-dependent decreases in plasma Aβ levels, although central exposure and central PD activity were poor. Aβ endpoints in plasma and CSF can be used in Phase I studies to test PD of amyloid precursor protein processing modulators. PF-3084014 is a peripheral, functionally active gamma secretase inhibitor.

The thiazole-diamide series (1) has been identified as highly potent gamma-secretase inhibitors. Several representative compounds showed IC(50) values of <0.3 nM. The synthesis and SAR, as well as a radiolabeled synthesis of [(3)H]-2a, are described.

The synthesis and structure-activity relationships of a series of 6-phenyl-2-aminopyridines that potently and selectively inhibit the neuronal isoform of nitric oxide synthase (nNOS) are described. Compound 14bi from this series exhibits potent in vivo activity in harmaline-induced cGMP formation in rat cerebellum, a functional model of nNOS inhibition, and in the PCP-induced hypermotility model in the rat. These results suggest that 14bi may be a useful reagent for evaluating potential therapeutic applications of nNOS inhibitors in the central nervous system.

The synthesis and SAR of a series of 6-(4-(substituted)phenyl)-2-aminopyridines as inhibitors of nitric oxide synthase are described. Compound 3a from this series shows potent and selective inhibition of the human nNOS isoform, with pharmacokinetics sufficient to provide in vivo inhibition of nNOS activity.

The synthesis of a series of alkylcarbamates of 1,5-methano-2,3,4,5-tetrahydro-1H-2-benzazepin-7-ol is reported. Many of these compounds are potent acetylcholinesterase (AChE) inhibitors. The in vitro AChE inhibition, cholinergic effects, acute toxicity, and elevation of brain acetylcholine levels in vivo of this series of compounds are described. A representative compound, 1d (5.6 mg/kg, po), was able to reverse hemicolinium-3-induced amnesia in the mouse passive avoidance assay.

The synthesis of a series of 1,2,3,3a,8,8a-hexahydroindeno[2,1-b]pyrrole 5-alkylcarbamates and their resolution are reported. These compounds are structurally related to physostigmine with substitution of a methylene group in place of the NMe group at position 8 of physostigmine. Many of these 8-carbaphysostigmine analogues are more potent acetylcholinesterase inhibitors in vitro and less toxic in vivo than physostigmine. The (-)-enantiomer (e.g., 1d and 1g) possessing the same absolute configuration at C3a and C8a as that of physostigmine, is about 6 to 12-fold more potent at inhibiting acetylcholinesterase than the corresponding (+)-enantiomer (e.g., 1e and 1h).

9-Amino-1,2,3,4-tetrahydroacridine (THA) has been reported to cause improvement in patients with senile dementia of the Alzheimer's type. We have examined some effects of THA in vitro and in vivo to define its mechanism of action. In vitro, THA inhibits acetylcholinesterase (AChE) (IC50 = 223 nM) and blocks [3H]AFDX-116 (M2) and [3H]telenzepine (M1) binding (IC50 s of 1.5 and 9.1 microM respectively). In vivo levels of THA were 10-fold higher in brain than plasma following 3.2 mg/kg i.p., a dose which was found to be active in reversing amnesia induced by scopolamine assessed in T-maze tests in rats and passive avoidance tests in mice. Additionally, these brain concentrations were above the IC50 of THA for AChE inhibition. THA (5.6-17.8 mg/kg i.p.) also elevated acetylcholine levels in the rat CNS. THA-induced side effects were blocked by the central muscarinic antagonist, scopolamine, but not by the peripheral antagonists methscopolamine and glycopyrrolate, nor by nicotinic antagonists. We conclude that brain AChE inhibition by THA is sufficient to explain its purported therapeutic activity in Alzheimer's disease and that its favorable brain/plasma distribution in vivo may account for its central cholinergic action without inducing the severe peripheral cholinergic effects typically seen with other AChE inhibitors.