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(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of β-secretase 1 (β-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aβ1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aβ1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

A series of unique bicyclic aminooxazolines were synthesized and found to exhibit micromolar inhibition of β-secretase-1 (BACE-1). The aminooxazolines were procured by an intramolecular diethylaminosulfur trifluoride (DAST)-mediated ring closure of a benzylic urea onto a secondary alcohol.

A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and applied for the determination of human Aβ1-40 and Aβ1-42 peptides in transgenic mouse plasma to support preclinical pharmacodynamics studies. The method consisted of micro-elution solid phase extraction for sample preparation and LC–MS/MS analysis in the negative ion mode using electrospray ionization for analysis. 15N53-Aβ1-40 and 15N55-Aβ1-42 peptides were used as internal standards. A quadratic regression (weighted 1/concentrations), with an equation y = ax 2 + bx + c, was used to fit calibration curves over the concentration range of 0.500–100 ng/mL for both Aβ1-40 and Aβ1-42 peptides. For quality control samples at 6.00, 40.0 and 80.0 ng/mL from the qualification experiment, the within-run accuracy ranged from −2.69 to 0.583 % with precision values ≤8.23 % for Aβ1-40. Within-run accuracy ranged from −4.83 to 10.1 % with precision values ≤8.87 % for Aβ1-42. Samples from a pharmacodynamics study using Tg2576 transgenic mice were analyzed by this qualified LC–MS/MS method and concentrations were compared to those generated by ELISA. The two methods were shown to be comparable for Aβ1-40 quantification of samples from the Tg2576 amyloid precursor protein transgenic mouse model, but varied slightly for Aβ1-42.

This study was conducted to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of two novel inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1), GNE-629 [(4S,4a'S,10a'S)-2-amino-8'-(2-fluoropyridin-3-yl)-1-methyl-3',4',4a',10a'-tetrahydro-1'H-spiro[imidazole-4,10'-pyrano[4,3-b]chromen]-5(1H)-one] and GNE-892 [(R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one], and to develop a PK-PD model to predict in vivo effects based solely on in vitro activity and PK. GNE-629 and GNE-892 concentrations and PD biomarkers including amyloid β (Aβ) in the plasma and cerebrospinal fluid (CSF), and secreted APPβ (sAPPβ) and secreted APPα (sAPPα) in the CSF were measured after a single oral administration of GNE-629 (100 mg/kg) or GNE-892 (30 or 100 mg/kg) in cynomolgus monkeys. A mechanistic PK-PD model was developed to simultaneously characterize the plasma Aβ and CSF Aβ, sAPPα, and sAPPβ using GNE-629 in vivo data. This model was used to predict the in vivo effects of GNE-892 after adjustments based on differences in in vitro cellular activity and PK. The PK-PD model estimated GNE-629 CSF and free plasma IC₅₀ of 0.0033 μM and 0.065 μM, respectively. These differences in CSF and free plasma IC₅₀ suggest that different mechanisms are involved in Aβ formation in these two compartments. The predicted in vivo effects for GNE-892 using the PK-PD model were consistent with the observed data. In conclusion, a PK-PD model was developed to mechanistically describe the effects of BACE1 inhibition on Aβ, sAPPβ, and sAPPα in the CSF, and Aβ in the plasma. This model can be used to prospectively predict in vivo effects of new BACE1 inhibitors using just their in vitro activity and PK data.

An efficient method for the selective "N1" alkylation of indazoles is described. Use of alpha-halo esters, lactones, ketones, amides, and bromoacetonitrile provides good to excellent yield of the desired N1 products.

The discovery and development of the o-iodoxybenzoic acid (IBX) reaction with certain unsaturated N-aryl amides (anilides) to form heterocycles are described. The application of the method to the synthesis of delta-lactams, cyclic urethanes, hydroxy amines, and amino sugars among other important building blocks and intermediates is detailed. In addition to the generality and scope of this cyclization reaction, this article describes a number of mechanistic investigations suggesting a single electron transfer from the anilide functionality to IBX and implicating a radical-based mechanism for the reaction.

A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2′ and P3 moieties were explored. A conformationally restricted P2′ methyl group provided inhibitors with excellent cell potency (37–137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aβ1–40 at 60 mg/kg (PO).

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a β-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50 = 110 nM) demonstrated a reduction in CSF Aβ in wild type rats after a single dose.

[figure: see text] The direct opening at the bridgehead of oxabicyclo[3.2.1]octenes employing silyl ketene acetals in 4.0-5.0 M lithium perchlorate in diethyl ether has been realized, which gives rise to highly functionalized cycloheptadienes that can be further manipulated for use in natural product synthesis. The bridgehead opening reaction has been employed in the construction of the C(19)-C(27) fragment of Rifamycin S.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aβ), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aβ is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aβ in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aβ lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aβ in rodents and in monkey.

The discovery and development of the o-iodoxybenzoic acid (IBX) reaction with certain unsaturated N-aryl amides (anilides) to form heterocycles are described. The application of the method to the synthesis of δ-lactams, cyclic urethanes, hydroxy amines, and amino sugars among other important building blocks and intermediates is detailed. In addition to the generality and scope of this cyclization reaction, this article describes a number of mechanistic investigations suggesting a single electron transfer from the anilide functionality to IBX and implicating a radical-based mechanism for the reaction.

Despitetheseefforts,how-ever,themoleculeofazadirachtinstillremainselusivetototalsynthesis, with its crowded C8 C14 bond bridging its twodomainsbeingthemainobstacle.Inthiscommunicationwewish to report our model studies which culminated in apotential solution to the C8 C14 problem based on atetheringstrategyfollowedbyaradical-basedintramolecularcouplinganddismantlingofthetemporarybridge.Scheme1 depicts the targeted model compound 2, whichcontainsringsystemsrepresentingthetwostructuraldomainsof azadirachtin, and, in retrosynthetic format, the radical-basedproposedstrategyfortheconstructionof2.Thus,itwasreasoned that tethering the two domains through a mixedbromoketal would facilitate the intended C8 C14 couplingreaction,upongenerationoftheradicalspecies4,bya5-exo-trigcyclizationtofurnish3.Subsequentcollapseofthemixedketalbridgewouldthenallowthegenerationofthetargetedring system 2 with the desired C8 C14 bond in place. Thesuccessful execution of this strategywas preceded byanumberofrelatedstudieswhosebriefdescriptionshouldbeinstructive.In search for a suitable trans-decalin system as a startingmaterial with an angular substituent and oxygen function-alities on both rings, we found ester 5 (Scheme2) to be an

An efficient method for the selective “N1” alkylation of indazoles is described. Use of α-halo esters, lactones, ketones, amides, and bromoacetonitrile provides good to excellent yield of the desired N1 products.

[reaction: see text] An efficient strategy for transforming meso-oxabicyclo[3.2.1]octenone 1 into optically active intermediates for macrolide synthesis has been developed. The direct bridgehead opening of optically active oxabicyclo[3.2.1]octene derivative 2 with hydride or a silyl ketene acetal utilizing the highly polar medium lithium perchlorate in diethyl ether resulted in highly functionalized cycloheptenones, which were transformed into the C(19)-C(26) and C(27)-C(32) fragments of Scytophycin C.

Reactive cyclic oxocarbenium ions, generated in situ from internal hemiketals, undergo Baeyer−Villiger oxidation upon exposure to m-chloroperbenzoic acid leading, after hydrolysis of the resultant lactones, to acyclic fragments for use in natural product synthesis.

[reaction: see text] Reactive cyclic oxocarbenium ions, generated in situ from internal hemiketals, undergo Baeyer-Villiger oxidation upon exposure to m-chloroperbenzoic acid leading, after hydrolysis of the resultant lactones, to acyclic fragments for use in natural product synthesis.

Despitetheseefforts,how-ever,themoleculeofazadirachtinstillremainselusivetototalsynthesis, with its crowded C8 C14 bond bridging its twodomainsbeingthemainobstacle.Inthiscommunicationwewish to report our model studies which culminated in apotential solution to the C8 C14 problem based on atetheringstrategyfollowedbyaradical-basedintramolecularcouplinganddismantlingofthetemporarybridge.Scheme1 depicts the targeted model compound 2, whichcontainsringsystemsrepresentingthetwostructuraldomainsof azadirachtin, and, in retrosynthetic format, the radical-basedproposedstrategyfortheconstructionof2.Thus,itwasreasoned that tethering the two domains through a mixedbromoketal would facilitate the intended C8 C14 couplingreaction,upongenerationoftheradicalspecies4,bya5-exo-trigcyclizationtofurnish3.Subsequentcollapseofthemixedketalbridgewouldthenallowthegenerationofthetargetedring system 2 with the desired C8 C14 bond in place. Thesuccessful execution of this strategywas preceded byanumberofrelatedstudieswhosebriefdescriptionshouldbeinstructive.In search for a suitable trans-decalin system as a startingmaterial with an angular substituent and oxygen function-alities on both rings, we found ester 5 (Scheme2) to be an