Karen J. Coffman

Discovery and preclinical profiling of 3-[4-(morpholin-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl]benzonitrile (PF-06447475), a highly potent, selective, brain penetrant, and in vivo active LRRK2 kinase inhibitor.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinsons disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Design and Discovery of 6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943), a Selective Brain Penetrant PDE9A Inhibitor for the Treatment of Cognitive Disorders

6-[(3S,4S)-4-Methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl]-1-(tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (PF-04447943) is a novel PDE9A inhibitor identified using parallel synthetic chemistry and structure-based drug design (SBDD) and has advanced into clinical trials. Selectivity for PDE9A over other PDE family members was achieved by targeting key residue differences between the PDE9A and PDE1C catalytic site. The physicochemical properties of the series were optimized to provide excellent in vitro and in vivo pharmacokinetics properties in multiple species including humans. It has been reported to elevate central cGMP levels in the brain and CSF of rodents. In addition, it exhibits procognitive activity in several rodent models and synaptic stabilization in an amyloid precursor protein (APP) transgenic mouse model. Recent disclosures from clinical trials confirm that it is well tolerated in humans and elevates cGMP in cerebral spinal fluid of healthy volunteers, confirming that it is a quality pharmacological tool for testing clinical hypotheses in disease states associated with impairment of cGMP signaling or cognition.

Synthesis and structure-activity relationships of CP-122,721, a second-generation NK-1 receptor antagonist.

The synthesis and SAR of benzylamine side chain analogs of the NK-1 receptor antagonist CP-99,994 are described. The 5-trifluoromethoxy analog, CP-122,721, shows superior in vivo blockade of NK-1 receptor mediated responses.

Pharmacodynamics and Pharmacokinetics of the γ-Secretase Inhibitor PF-3084014

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 γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 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 EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ 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 Aβ. To further characterize Aβ 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 Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ 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 Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Identification of Multiple 5-HT4 Partial Agonist Clinical Candidates for the Treatment of Alzheimer’s Disease

The cognitive impairments observed in Alzheimers disease (AD) are in part a consequence of reduced acetylcholine (ACh) levels resulting from a loss of cholinergic neurons. Preclinically, serotonin 4 receptor (5-HT(4)) agonists are reported to modulate cholinergic function and therefore may provide a new mechanistic approach for treating cognitive deficits associated with AD. Herein we communicate the design and synthesis of potent, selective, and brain penetrant 5-HT(4) agonists. The overall goal of the medicinal chemistry strategy was identification of structurally diverse clinical candidates with varying intrinsic activities. The exposure-response relationships between binding affinity, intrinsic activity, receptor occupancy, drug exposure, and pharmacodynamic activity in relevant preclinical models of AD were utilized as key selection criteria for advancing compounds. On the basis of their excellent balance of pharmacokinetic attributes and safety, two lead 5-HT(4) partial agonist candidates 2d and 3 were chosen for clinical development.

Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.

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 γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 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 EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ 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 Aβ. To further characterize Aβ 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 Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ 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 Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014

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.

Pharmacodynamics and Pharmacokinetics of the -Secretase Inhibitor PF-3084014 □ S

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 γ-secretase inhibitor that reduces amyloid-β (Aβ) production with an in vitro IC50 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 EC50 of 2.1 μM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Aβ 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 Aβ. To further characterize Aβ 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 Aβ, and the magnitude and duration of Aβ lowering exceeded those of the reductions in B-cell endpoints. Other γ-secretase inhibitors have shown high potency at elevating Aβ 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 Aβ11-40 and Aβ1-43 at doses that potently inhibited Aβ1-40 and Aβ1-42. PF-3084014, like previously described γ-secretase inhibitors, preferentially reduced Aβ1-40 relative to Aβ1-42. Potency at Aβ relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.

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.