John M. Humphrey

Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

Conformationally restricted inhibitors of the high affinity L-glutamate transporter

Abstract A series of acidic amino acids has been prepared and evaluated in an effort to identify the structural features required for binding to and inhibiting the high affinity uptake system that clears L-glutamate from the synaptic cleft during excitatory amino acid-mediated neurotransmission in the mammalian CNS.

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: rational modifications imparting PP1 selectivity

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.

Use of structure-based design to discover a potent, selective, in vivo active phosphodiesterase 10A inhibitor lead series for the treatment of schizophrenia.

Utilizing structure-based virtual library design and scoring, a novel chimeric series of phosphodiesterase 10A (PDE10A) inhibitors was discovered by synergizing binding site interactions and ADME properties of two chemotypes. Virtual libraries were docked and scored for potential binding ability, followed by visual inspection to prioritize analogs for parallel and directed synthesis. The process yielded highly potent and selective compounds such as 16. New X-ray cocrystal structures enabled rational design of substituents that resulted in the successful optimization of physical properties to produce in vivo activity and to modulate microsomal clearance and permeability.

Discovery of Novel, Potent, and Selective Inhibitors of 3-Phosphoinositide-Dependent Kinase (PDK1)

Analogues substituted with various amines at the 6-position of the pyrazine ring on (4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)pyrazin-2-ylmethanone were discovered as potent and selective inhibitors of PDK1 with potential as anticancer agents. An early lead with 2-pyridine-3-ylethylamine as the pyrazine substituent showed moderate potency and selectivity. Structure-based drug design led to improved potency and selectivity against PI3Kα through a combination of cyclizing the ethylene spacer into a saturated, five-membered ring and substituting on the 4-position of the aryl ring with a fluorine. ADME properties were improved by lowering the lipophilicity with heteroatom replacements in the saturated, five-membered ring. The optimized analogues have a PDK1 Ki of 1 nM and >100-fold selectivity against PI3K/AKT-pathway kinases. The cellular potency of these analogues was assessed by the inhibition of AKT phosphorylation (T308) and by their antiproliferation activity against a number of tumor cell lines.

L-trans-2,3-Pyrrolidine Dicarboxylate: Characterization of a Novel Excitotoxin

This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-glutamate, L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 h) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 microM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 microM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 microM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transported substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes.

Discovery of indole-derived pyridopyrazine-1,6-dione γ-secretase modulators that target presenilin.

Herein we describe design strategies that led to the discovery of novel pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) incorporating an indole motif as a heterocyclic replacement for a naphthyl moiety that was present in the original lead 9. Tactics involving parallel medicinal chemistry and in situ monomer synthesis to prepare focused libraries are discussed. Optimized indole GSM 29 exhibited good alignment of in vitro potency and physicochemical properties, and moderate reduction of brain Aβ42 was achieved in a rat efficacy model when dosed orally at 30mg/kg. Labeling experiments using a clickable, indole-derived GSM photoaffinity probe demonstrated that this series binds to the presenilin N-terminal fragment (PS1-NTF) of the γ-secretase complex.

Design of Pyridopyrazine-1,6-dione γ-Secretase Modulators that Align Potency, MDR Efflux Ratio, and Metabolic Stability

Herein we describe the design and synthesis of a series of pyridopyrazine-1,6-dione γ-secretase modulators (GSMs) for Alzheimers disease (AD) that achieve good alignment of potency, metabolic stability, and low MDR efflux ratios, while also maintaining favorable physicochemical properties. Specifically, incorporation of fluorine enabled design of metabolically less liable lipophilic alkyl substituents to increase potency without compromising the sp(3)-character. The lead compound 21 (PF-06442609) displayed a favorable rodent pharmacokinetic profile, and robust reductions of brain Aβ42 and Aβ40 were observed in a guinea pig time-course experiment.

Diastereoselective Synthesis of 2,3,6-Trisubstituted Piperidines

We report the diastereoselective and chromatography-free syntheses of four 2-phenyl-6-alkyl-3-aminopiperidines. Ring construction was accomplished through a nitro-Mannich reaction linking a nitroketone and phenylmethanimine, followed by a ring-closure condensation. Relative stereocontrol was achieved between C-2 and C-3 by kinetic protonation of a nitronate or by equilibration of the nitro group under thermodynamic control. Stereocontrol at C-6 was accomplished by utilizing a variety of imine reduction methods. The C-2/C-6-cis stereochemistry was established via triacetoxyborohydride iminium ion reduction, whereas the trans relationship was set either by triethylsilane/TFA acyliminium ion reduction or by Lewis acid catalyzed imine reduction with lithium aluminum hydride.

Ligand-protein interactions of selective casein kinase 1δ inhibitors.

Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.