Synthesis of functionalized derivatives of the gamma-secretase modulator BMS-932481 and identification of its major metabolite.

In an effort to improve physical properties by introducing polar functionality into the bicyclic pyrimidine gamma-secretase modulator (GSM) clinical candidate BMS-932481, we prepared several oxidative products of BMS-932481. Among the analogs that were prepared, the C-5 alcohol 3 was identified as the predominant metabolite of BMS-932481 found in rat and human liver microsomes. Alcohol 3 was determined to be chemically unstable, leading to the hypothesis that 3 may lead to the production of reactive species both in vitro and in vivo.

The γ-Secretase Modulator, BMS-932481, Modulates Aß Peptides in the Plasma and Cerebrospinal Fluid of Healthy Volunteers.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aß39, Aß40, and Aß42 while increasing Aß37 and Aß38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aß40 and Aß42 were observed with no change in total Aß; in CSF, modest decreases in total Aß were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aß42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aß lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.

Robust Translation of γ-Secretase Modulator Pharmacology across Preclinical Species and Human Subjects.

The amyloid-ß peptide (Aß)-in particular, the 42-amino acid form, Aß1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aß synthesis or increase the clearance of Aß have entered clinical trials, including γ-secretase inhibitors, anti-Aß antibodies, and amyloid-ß precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aß1-42 production, and may also decrease Aß1-40 while simultaneously increasing one or more shorter Aß peptides, such as Aß1-38 and Aß1-37. GSMs are particularly attractive because they do not alter the total amount of Aß peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, ß-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aß1-42 and Aß1-40 levels while increasing Aß1-38 and Aß1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

Synthesis of pyrimido[4,5-c]azepine- and pyrimido[4,5-c]oxepine-based γ-secretase modulators.

This Letter describes an efficient ring-closing metathesis approach to 2-chloro-4-amino-pyrimido[4,5-c]azepines and 2-chloro-4-amino-pyrimido[4,5-c]oxepines. These chlorides were applied to the synthesis of several potent γ-secretase modulators (GSMs).

Design and optimization of tricyclic gamma-secretase modulators.

Beginning with a diaminotriazine screening hit, several series of novel, tricyclic gamma-secretase modulators (GSMs) were designed. The SAR of several related series of GSMs is presented, and the in vivo profile of a lead molecule from the series is described.

Discovery of furo[2,3-d][1,3]thiazinamines as beta amyloid cleaving enzyme-1 (BACE1) inhibitors.

This Letter describes the synthesis and structure-activity relationships of a series of furo[2,3-d][1,3]thiazinamine BACE1 inhibitors. The co-crystal structure of a representative thiazinamine 2e bound with the BACE1 active site displayed a binding mode driven by interactions with the catalytic aspartate dyad and engagement of the biaryl amide toward the S1 and S3 pockets. This work indicates that furo[2,3-d]thiazine can serve as a viable bioisostere of the known furo[3,4-d]thiazine.

Macrocyclic prolinyl acyl guanidines as inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of acyl guanidines which inhibit the BACE-1 enzyme are presented. The prolinyl acyl guanidine chemotype (7c), unlike compounds of the parent isothiazole chemotype (1), yielded compounds with good agreement between their enzymatic and cellular potency as well as a reduced susceptibility to P-gp efflux. Further improvements in potency and P-gp ratio were realized via a macrocyclization strategy. The in vivo profile in wild-type mice and P-gp effects for the macrocyclic analog 21c is presented.

Pharmacodynamics of selective inhibition of γ-secretase by avagacestat.

A hallmark of Alzheimer's disease (AD) pathology is the accumulation of brain amyloid ß-peptide (Aß), generated by γ-secretase-mediated cleavage of the amyloid precursor protein (APP). Therefore, γ-secretase inhibitors (GSIs) may lower brain Aß 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 Aß 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) Aß 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 Aß40 and Aß42 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 Aß40. Consistent with the 137-fold selectivity measured in cell culture, we identified doses of avagacestat that reduce CSF Aß 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.

Monosubstituted γ-lactam and conformationally constrained 1,3-diaminopropan-2-ol transition-state isostere inhibitors of ß-secretase (BACE).

The synthesis, evaluation, and structure-activity relationships of a class of γ-lactam 1,3-diaminopropan-2-ol transition-state isostere inhibitors of BACE are discussed. Two strategies for optimizing lead compound 1a are presented. Reducing the overall size of the inhibitors resulted in the identification of γ-lactam 1i, whereas the introduction of conformational constraint on the prime-side of the inhibitor generated compounds such as the 3-hydroxypyrrolidine inhibitor 28n. The full in vivo profile of 1i in rats and 28n in Tg 2576 mice is presented.

Synthesis and in vivo evaluation of cyclic diaminopropane BACE-1 inhibitors.

The synthesis, evaluation, and structure-activity relationships of a set of related constrained diaminopropane inhibitors of BACE-1 are described. The full in vivo profile of an optimized inhibitor in both normal and P-gp deficient mice is compared with data generated in normal rats.

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 Aß levels, but did not lower rat brain Aß 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.

Viable mouse gene ablations that robustly alter brain Aß levels are rare.

BACKGROUND: Accumulation of amyloid-ß (Aß) peptide in the brain is thought to play a key pathological role in Alzheimer's disease. Many pharmacological targets have therefore been proposed based upon the biochemistry of Aß, but not all are equally tractable for drug discovery. RESULTS: To search for novel targets that affect brain Aß without causing toxicity, we screened mouse brain samples from 1930 novel gene knock-out (KO) strains, representing 1926 genes, using Aß ELISA assays. Although robust Aß lowering was readily apparent in brains from a BACE1 KO strain, none of the novel strains exhibited robust decreases in brain Aß, including a GPR3 KO strain, which had previously been proposed as an Aß target. However, significantly increased Aß was observed in brain samples from two KO strains, corresponding to genes encoding the glycosylphosphatidylinositol mannosyl transferase PIGZ and quinolinate phosphoribosyltransferase (QPRT). CONCLUSIONS: Thus, gene ablations that are permissive for mouse survival and that also have a robust effect on Aß levels in the brain are rare.

Synthesis and SAR of hydroxyethylamine based phenylcarboxyamides as inhibitors of BACE.

A series of N-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-methoxybenzylamino)-butan-2-yl)benzamides has been synthesized as BACE inhibitors. A variety of P2 and P3 substituents has been explored, and these efforts have culminated in the identification of several 1,3,5-trisubstituted phenylcarboxyamides with potent BACE inhibitory activity.

Identification and Preclinical Evaluation of the Bicyclic Pyrimidine γ-Secretase Modulator BMS-932481.

A triazine hit identified from a screen of the BMS compound collection was optimized for potency, in vivo activity, and off-target profile to produce the bicyclic pyrimidine γ-secretase modulator BMS-932481. The compound showed robust reductions of Aß1-42 and Aß1-40 in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aß1-37 and Aß1-38 were observed, with no change in the total amount of Aß1-x produced. No Notch-based toxicity was observed, and the overall preclinical profile of BMS-932481 supported its further evaluation in human clinical trials.

P-glycoprotein efflux and other factors limit brain amyloid beta reduction by beta-site amyloid precursor protein-cleaving enzyme 1 inhibitors in mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Amyloid beta (Abeta) 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, beta-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 Abeta. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Abeta formation in cultured cells with IC(50) values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Abeta peptides by mass spectrometry showed that these inhibitors decreased Abeta by inhibiting BACE1. An assay for Abeta1-40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Abeta1-40, but not brain Abeta1-40, 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 Abeta1-40 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 Abeta1-40 and brain Abeta1-40 dose responses for these three compounds revealed differences in relative ED(50) values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Qualitative and quantitative characterization of the amyloid beta peptide (Abeta) population in biological matrices using an immunoprecipitation-LC/MS assay.

Targeting the metabolism of amyloid beta peptides (Abeta) is currently the leading experimental approach to treatment of Alzheimer's disease (AD). Described here is an immunoprecipitation-liquid chromatography/mass spectrometry (ip-LC/MS) assay to simultaneously characterize and quantitate different forms of Abeta in biological samples. The 4G8 antibody, specific for the 17-24 amino acid epitope of Abeta was employed to selectively isolate Abeta from in vitro samples for subsequent LC-MS analysis. A high resolution accurate mass hybrid linear ion trap-Orbitrap, LTQ-Orbitrap mass spectrometer was used to identify forms of 12 Abeta in H4-APP751 swe cell extracts based on ab initio calculations, accurate mass measurements, isotopic modeling and by de novo peptide sequencing using tandem mass spectrometry. The quantitative LC-MS analysis was performed on a linear ion trap mass spectrometer, LTQ, in full scan mode, this mode of operation enables sensitive detection levels and post-acquisition data mining for different forms of Abeta for quantitative assessment. Dosing studies with three known inhibitors of Abeta production, sulindac sulfide (SSide), BMS-299897 ('897) and compound W (CW) are reported to demonstrate the utility and analytical characteristics of the assay. This assay has the potential to provide insight into the formation of Abeta; increase understanding of drug mechanisms; and to contribute to drug efficacy studies.

[3H]BMS-599240--a novel tritiated ligand for the characterization of BACE1 inhibitors.

In this report we describe a novel radioligand, [(3)H](S)-2-((S)-3-Acetylamino-3-sec-butyl-2-oxo-pyrrolidin-1-yl)-N-[(1S,2R)-1-benzyl-2-hydroxy-3-(3-methoxy-benzylamino)-propyl]-4-phenyl-butyramide ([(3)H]BMS-599240), that exhibits robust specific binding in homogenates from cell cultures overexpressing beta-site amyloid precursor protein cleaving enzyme-1 (BACE1). Radioligand binding exhibited high affinity, K(d)=2 nM, commensurate with its inhibitory potency against BACE1. Inhibition of radioligand binding in the presence of a range of different BACE1 inhibitors exhibited the same rank order of potency as for inhibition of BACE1 enzymatic activity. BACE1-dependent binding of the radioligand was also demonstrated in mouse brain homogenates, where genetic ablation of BACE1 eliminated high affinity binding. Thus, the radioligand [(3)H]BMS-599240 is a novel tool potentially useful for evaluation of BACE1 enzyme in biological samples, and for evaluation of inhibitor binding to BACE1.

Synergistic inhibition of Aß production by combinations of γ-secretase modulators.

Alzheimer's disease is associated with the accumulation of amyloid-ß (Aß) in the brain. In particular, the 42-amino acid form, Aß1-42, is thought to play a key role in the disease. It is therefore of interest that diverse compounds, known as γ-secretase modulators (GSM), can selectively decrease Aß1-42 production without inhibiting the production of other forms of Aß. Here we describe the novel discovery of synergistic inhibition of Aß by certain combinations of GSMs. Cell cultures were treated with pairwise combinations of GSMs to determine how Aß peptide production was affected. Analysis of isobolograms and calculation of the combination index showed that BMS-869780 and GSM-2 were highly synergistic. Additional combinations of GSMs revealed that inhibition of Aß occurred only when one GSM was of the "acid GSM" structural class and the other was of the "non-acid GSM" class. A total of 15 representative acid/non-acid GSM combinations were shown to inhibit Aß production, whereas 10 pairwise combinations containing two acid GSMs or containing two non-acid GSMs did not inhibit Aß. We also discovered that lasalocid, a natural product, is a potent GSM. Lasalocid is unique in that it did not synergize with other GSMs. Synergism did not translate in vivo perhaps because of biochemical differences between the cell culture model and brain. These findings reinforce the pharmacological differences between different structural classes of GSMs, and may help to exploit the potential of γ-secretase as a drug target.

Soluble Abeta and cognitive function in aged F-344 rats and Tg2576 mice.

Recent findings suggest that Alzheimer's dementia may be mediated by soluble beta amyloid (Abeta) more than the deposits of aggregated, insoluble Abeta, and vulnerability to cognitive deficits after scopolamine challenge may help identify AD even in patients that are still pre-symptomatic. The objectives of the present experiments were to determine if vulnerability to cognitive deficits after scopolamine challenge is related to levels of soluble Abeta, and if levels of soluble Abeta are more closely related to cognitive deficits than levels of insoluble Abeta, even in aged, transgenic mice, after they have developed very high levels of insoluble Abeta. Aged F-344 rats and young mice over-expressing the Swedish mutation in the human amyloid precursor protein (APPsw; Tg2576+) had elevated levels of soluble Abeta, and were more vulnerable to scopolamine challenge in the Morris water maze (MWM), relative to young rats and Tg2576- mice; but, among individual animals, higher levels of soluble Abeta were not correlated with vulnerability to scopolamine. On the other hand, in aged Tg2576+ mice, cognitive deficits were related to levels of soluble Abeta, not insoluble Abeta, despite the fact that the levels of insoluble Abeta were thousands of times higher than the levels of soluble Abeta. The results of the present experiments suggest that vulnerability to cognitive deficits after scopolamine challenge is not related to elevated levels of soluble Abeta, but that high levels of soluble Abeta are more closely correlated with cognitive deficits than the amount insoluble Abeta, even after large amounts of aggregated, insoluble Abeta have been deposited.

Discovery of S3-Truncated, C-6 Heteroaryl Substituted Aminothiazine ß-Site APP Cleaving Enzyme-1 (BACE1) Inhibitors.

Truncation of the S3 substituent of the biaryl aminothiazine 2, a potent BACE1 inhibitor, led to a low molecular weight aminothiazine 5 with moderate activity. Despite its moderate activity, compound 5 demonstrated significant brain Aß reduction in rodents. The metabolic instability of 5 was overcome by the replacement of the 6-dimethylisoxazole, a metabolic soft spot, with a pyrimidine ring. Thus, truncation of the S3 substituent represents a viable approach to the discovery of orally bioavailable, brain-penetrant BACE1 inhibitors.