Small heavy-atom and secondary hydrogen kinetic isotope effects (KIEs) can provide detailed information about the mechanism of an exceptional range of chemical reactions.1•2 However, there are significant general limitations in methods for the determination of these KIEs. Because absolute rate measurements are rarely sufficiently precise, small KIEs are usually determined in competition reactions of isotopically labeled and unlabeled materials. This is possible only in systems carefully chosen to allow the precise measurement of isotopomer ratios with appropriate analytical techniques, such as scintillation counting for 3H and l4C KIEs. The synthesis of isotopically labeled materials can be arduous, often prohibitively so, and a new synthesis, competition reaction, and analysis are required for each KIE of interest. A broadly useful alternative, particularly for l3C KIEs, is to employ the high precision of isotope ratio mass spectrometry to study KIEs in materials labeled only at natural abundance.3 A major restriction is that each site of interest must be selectively degradable without isotopic fractionation into an analyzable small molecule, most often CO2.The isotope-and position-specific information inherent in NMR techniques seems ideally suited to measuring KIEs at natural abundance. The utility of 2H NMR for determining large 2H KIEs at natural abundance has been established,4 and in theory, all of the individual KIEs in reactions of complex natural abundance materials can be determined simultaneously!5 In practice, however, NMR quantitation has not been sufficiently precise to be useful with small KIEs, the uncertainty in the few cases tried generally rivaling or exceeding the size of the isotope effects.4•5 We report here a simple general method for attaining chemically significant precision while simultaneously measuring all of the KIEs for reactions at natural abundance.As any reaction proceeds, the starting materials are fractionatively enriched in isotopically slower-reacting components. The

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The L-type amino acid transporter 1 (LAT1, SLC7A5) transports essential amino acids across the blood-brain barrier (BBB) and into cancer cells. To utilize LAT1 for drug delivery, potent amino acid promoieties are desired, as prodrugs must compete with millimolar concentrations of endogenous amino acids. To better understand ligand-transporter interactions that could improve potency, we developed structural LAT1 models to guide the design of substituted analogues of phenylalanine and histidine. Furthermore, we evaluated the structure-activity relationship (SAR) for both enantiomers of naturally occurring LAT1 substrates. Analogues were tested in cis-inhibition and trans-stimulation cell assays to determine potency and uptake rate. Surprisingly, LAT1 can transport amino acid-like substrates with wide-ranging polarities including those containing ionizable substituents. Additionally, the rate of LAT1 transport was generally nonstereoselective even though enantiomers likely exhibit different binding modes. Our findings have broad implications to the development of new treatments for brain disorders and cancer.

The transporter protein Large-neutral Amino Acid Transporter 1 (LAT-1, SLC7A5) is responsible for transporting amino acids such as tyrosine and phenylalanine as well as thyroid hormones, and it has been exploited as a drug delivery mechanism. Recently its role in cancer has become increasingly appreciated, as it has been found to be up-regulated in many different tumor types, and its expression levels have been correlated with prognosis. Substitution at the meta position of aromatic amino acids has been reported to increase affinity for LAT-1; however, the SAR for this position has not previously been explored. Guided by newly refined computational models of the binding site, we hypothesized that groups capable of filling a hydrophobic pocket would increase binding to LAT-1, resulting in improved substrates relative to parent amino acid. Tyrosine and phenylalanine analogs substituted at the meta position with halogens, alkyl and aryl groups were synthesized and tested in cis-inhibition and trans-stimulation cell assays to determine activity. Contrary to our initial hypothesis we found that lipophilicity was correlated with diminished substrate activity and increased inhibition of the transporter. The synthesis and SAR of meta-substituted phenylalanine and tyrosine analogs is described.

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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.

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