Quantitative projection of human brain penetration of the H₃ antagonist PF-03654746 by integrating rat-derived brain partitioning and PET receptor occupancy

<p>1. Unbound brain drug concentration (<i>C</i>_b,u), a valid surrogate of interstitial fluid drug concentration (<i>C</i>_ISF), cannot be directly determined in humans, which limits accurately defining the human <i>C</i>_b,u:<i>C</i>_p,u of investigational molecules.</p> <p>2. For the H₃R antagonist (1<i>R</i>,3<i>R</i>)-<i>N</i>-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-lmethyl)phenyl]cyclobutane-1-carboxamide (<b>PF-03654746</b>), we interrogated <i>C</i>_b,u:<i>C</i>_p,u in humans and nonhuman primate (NHP).</p> <p>3. In rat, <b>PF-03654746</b> achieved net blood–brain barrier (BBB) equilibrium (<i>C</i>_b,u:<i>C</i>_p,u of 2.11).</p> <p>4. In NHP and humans, the PET receptor occupancy-based <i>C</i>_p,u IC₅₀ of <b>PF-03654746</b> was 0.99 nM and 0.31 nM, respectively, which were 2.1- and 7.4-fold lower than its <i>in vitro</i> human H₃ <i>K</i>_i (2.3 nM).</p> <p>5. In an attempt to understand this higher-than-expected potency in humans and NHP, rat-derived <i>C</i>_b,u:<i>C</i>_p,u of <b>PF-03654746</b> was integrated with <i>C</i>_p,u IC₅₀ to identify unbound (neuro) potency of <b>PF-03654746</b>, <i>n</i>IC₅₀.</p> <p>6. The <i>n</i>IC₅₀ of <b>PF-03654746</b> was 2.1 nM in NHP and 0.66 nM in human which better correlated (1.1- and 3.49-fold lower) with <i>in vitro</i> human H₃ <i>K</i>_i (2.3 nM).</p> <p>7. This correlation of the <i>n</i>IC₅₀ and <i>in vitro h</i>H₃ <i>K</i>_i suggested the translation of net BBB equilibrium of <b>PF-03654746</b> from rat to NHP and humans, and confirmed the use of <i>C</i>_p,u as a reliable surrogate of <i>C</i>_b,u.</p> <p>8. Thus, <i>n</i>IC₅₀ quantitatively informed the human <i>C</i>_b,u:<i>C</i>_p,u of <b>PF-03654746</b>.</p

Discovery and Characterization of a Novel Dihydroisoxazole Class of α‑Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Potentiators

Positive allosteric modulators (“potentiators”) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) enhance excitatory neurotransmission and may improve the cognitive deficits associated with various neurological disorders. The dihydroisoxazole (DHI) series of AMPAR potentiators described herein originated from the identification of <b>7</b> by a high-throughput functional activity screen using mouse embryonic stem (mES) cell-derived neuronal precursors. Subsequent structure-based drug design using X-ray crystal structures of the ligand-binding domain of human GluA2 led to the discovery of both PF-04725379 (<b>11</b>), which in tritiated form became a novel ligand for characterizing the binding affinities of subsequent AMPAR potentiators in rat brain homogenate, and PF-04701475 (<b>8a</b>), a prototype used to explore AMPAR-mediated pharmacology in vivo. Lead series optimization provided <b>16a</b>, a functionally potent compound lacking the potentially bioactivatable aniline within <b>8a</b>, but retaining desirable in vitro ADME properties

The Discovery and Characterization of the α‑Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Potentiator <i>N</i>‑{(3<i>S</i>,4<i>S</i>)‑4-[4-(5-Cyano-2-thienyl)phenoxy]tetrahydrofuran-3-yl}propane-2-sulfonamide (PF-04958242)

A unique tetrahydrofuran ether class of highly potent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiators has been identified using rational and structure-based drug design. An acyclic lead compound, containing an ether-linked isopropylsulfonamide and biphenyl group, was pharmacologically augmented by converting it to a conformationally constrained tetrahydrofuran to improve key interactions with the human GluA2 ligand-binding domain. Subsequent replacement of the distal phenyl motif with 2-cyanothiophene to enhance its potency, selectivity, and metabolic stability afforded <i>N</i>-{(3<i>S</i>,4<i>S</i>)-4-[4-(5-cyano-2-thienyl)­phenoxy]­tetrahydrofuran-3-yl}­propane-2-sulfonamide (PF-04958242, <b>3</b>), whose preclinical characterization suggests an adequate therapeutic index, aided by low projected human oral pharmacokinetic variability, for clinical studies exploring its ability to attenuate cognitive deficits in patients with schizophrenia

Discovery and Characterization of (<i>R</i>)‑6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1‑<i>c</i>][1,4]oxazin-4(9<i>H</i>)‑one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu₅ negative allosteric modulator (NAM) <b>7</b>. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu₅ NAMs. Increasing the sp³ character of high-throughput screening hit <b>40</b> afforded a novel morpholinopyrimidone mGlu₅ NAM series. Its prototype, (<i>R</i>)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido­[2,1-<i>c</i>]­[1,4]­oxazin-4­(9<i>H</i>)-one (PF-06462894, <b>8</b>), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound <b>8</b> did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that <b>8</b> did not form any reactive metabolites. However, <b>8</b> caused the identical microscopic skin lesions in NHPs found with <b>7</b>, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance

Discovery and Preclinical Characterization of 1‑Methyl‑3-(4‑methylpyridin‑3‑yl)‑6‑(pyridin‑2‑ylmethoxy)‑1<i>H</i>‑pyrazolo-[3,4‑<i>b</i>]­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 (<b>1</b>), 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)-1<i>H</i>-pyrazolo­[3,4-<i>b</i>]­pyrazine (PF470, <b>14</b>) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound <b>14</b> 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 <b>14</b> 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

Discovery and Characterization of (<i>R</i>)‑6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1‑<i>c</i>][1,4]oxazin-4(9<i>H</i>)‑one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu₅ negative allosteric modulator (NAM) <b>7</b>. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu₅ NAMs. Increasing the sp³ character of high-throughput screening hit <b>40</b> afforded a novel morpholinopyrimidone mGlu₅ NAM series. Its prototype, (<i>R</i>)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido­[2,1-<i>c</i>]­[1,4]­oxazin-4­(9<i>H</i>)-one (PF-06462894, <b>8</b>), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound <b>8</b> did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that <b>8</b> did not form any reactive metabolites. However, <b>8</b> caused the identical microscopic skin lesions in NHPs found with <b>7</b>, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance