John E. Macor

Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable γ-Secretase Inhibitor

During the course of our research efforts to develop a potent and selective γ-secretase inhibitor for the treatment of Alzheimers disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-β precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of γ-secretase (Aβ40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced Aβ40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

Pharmacodynamics of Selective Inhibition of γ -Secretase by Avagacestat

A hallmark of Alzheimer’s disease (AD) pathology is the accumulation of brain amyloid β-peptide (Aβ), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aβ and offer a potential new approach to treat AD. As γ-secretase also cleaves Notch proteins, GSIs can have undesirable effects due to interference with Notch signaling. Avagacestat (BMS-708163) is a GSI developed for selective inhibition of APP over Notch cleavage. Avagacestat inhibition of APP and Notch cleavage was evaluated in cell culture by measuring levels of Aβ and human Notch proteins. In rats, dogs, and humans, selectivity was evaluated by measuring plasma blood concentrations in relation to effects on cerebrospinal fluid (CSF) Aβ levels and Notch-related toxicities. Measurements of Notch-related toxicity included goblet cell metaplasia in the gut, marginal-zone depletion in the spleen, reductions in B cells, and changes in expression of the Notch-regulated hairy and enhancer of split homolog-1 from blood cells. In rats and dogs, acute administration of avagacestat robustly reduced CSF Aβ40 and Aβ42 levels similarly. Chronic administration in rats and dogs, and 28-day, single- and multiple-ascending–dose administration in healthy human subjects caused similar exposure-dependent reductions in CSF Aβ40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aβ levels without causing Notch-related toxicities. Our results demonstrate the selectivity of avagacestat for APP over Notch cleavage, supporting further evaluation of avagacestat for AD therapy.

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

A strategy to minimize reactive metabolite formation: discovery of (S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile as a potent, orally bioavailable corticotropin-releasing factor-1 receptor antagonist.

Detailed metabolic characterization of 8, an earlier lead pyrazinone-based corticotropin-releasing factor-1 (CRF(1)) receptor antagonist, revealed that this compound formed significant levels of reactive metabolites, as measured by in vivo and in vitro biotransformation studies. This was of particular concern due to the body of evidence suggesting that reactive metabolites may be involved in idiosyncratic drug reactions. Further optimization of the structure-activity relationships and in vivo properties of pyrazinone-based CRF(1) receptor antagonists and studies to assess the formation of reactive metabolites led to the discovery of 19e, a high affinity CRF(1) receptor antagonist (IC(50) = 0.86 nM) wherein GSH adducts were estimated to be only 0.1% of the total amount of drug-related material excreted through bile and urine, indicating low levels of reactive metabolite formation in vivo. A novel 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine group in 19e contributed to the potency and improved in vivo properties of this compound and related analogues. 19e had excellent pharmacokinetic properties in rats and dogs and showed efficacy in the defensive withdrawal model of anxiety in rats. The lowest efficacious dose was 1.8 mg/kg. The results of a two-week rat safety study with 19e indicated that this compound was well-tolerated.

Discovery of (R)-4-(8-fluoro-2-oxo-1,2-dihydroquinazolin-3(4H)-yl)-N-(3-(7-methyl-1H-indazol-5-yl)-1-oxo-1-(4-(piperidin-1-yl)piperidin-1-yl)propan-2-yl)piperidine-1-carboxamide (BMS-694153): a potent antagonist of the human calcitonin gene-related peptide receptor for migraine with rapid and efficient intranasal exposure.

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Early chemistry leads suffered from modest potency, significant CYP3A4 inhibition, and poor aqueous solubility. Herein, we describe the optimization of these leads to give 4 (BMS-694153), a molecule with outstanding potency, a favorable predictive toxicology profile, and remarkable aqueous solubility. Compound 4 has good intranasal bioavailability in rabbits and shows dose-dependent activity in validated in vivo and ex vivo migraine models.

The synthesis of a conformationally restricted analog of the anti-migraine drug sumatriptan

The synthesis of 5-N-Methylaminosulfonylmethyl-3-(N-methylpyrrolidin-2-ylmethyl)indole (1), a conformationally restricted analog of the anti-migraine drug, sumatriptan, is described. To incorporate our novel stereogenic replacement for the aminoethyl sidechain in sumatriptan, a convergent synthesis of the 3,5-disubstituted indole (1) was employed which utilized an intramolecular Heck reaction as the cornerstone reaction.

Synthesis and SAR of indole-and 7-azaindole-1,3-dicarboxamide hydroxyethylamine inhibitors of BACE-1.

Heterocyclic replacement of the isophthalamide phenyl ring in hydroxyethylamine (HEA) BACE-1 inhibitors was explored. A variety of indole-1,3-dicarboxamide HEAs exhibited potent BACE-1 enzyme inhibition, but displayed poor cellular activity. Improvements in cellular activity and aspartic protease selectivity were observed for 7-azaindole-1,3-dicarboxamide HEAs. A methylprolinol-bearing derivative (10n) demonstrated robust reductions in rat plasma Aβ levels, but did not lower rat brain Aβ due to poor central exposure. The same analog exhibited a high efflux ratio in a bidirectional Caco-2 assay and was likely a substrate of the efflux transporter P-glycoprotein. X-ray crystal structures are reported for two indole HEAs in complex with BACE-1.

AN IMPROVED SYNTHESIS OF THE UNIQUE ANTI-MIGRAINE AGENT CP-122,288 : A BROMINE ATOM PASSENGER IN AN INTRAMOLECULAR HECK REACTION

Abstract Utilizing a 2,6-dibromoaniline ( 6b ) in place of the original monobromo aniline in the indole forming intramolecular Heck reaction allows for a greater than two-fold improvement in the overall yield of CP-122,288. The second bromine is carried through unmolested, forming a 7-bromoindole analog of CP-122,288 ( 1b ). This result might represent a novel approach to 7-substituted indole derivatives.

Acyl guanidine inhibitors of β-secretase (BACE-1): optimization of a micromolar hit to a nanomolar lead via iterative solid- and solution-phase library synthesis.

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.