Bruce Fletcher Johnson

Asymmetric synthesis of tetrabenazine and dihydrotetrabenazine.

The enantioselective synthesis of (+)-tetrabenazine (TBZ) and (+)-dihydrotetrabenazine (DTBZ), agents of significant interest for therapeutic and molecular imaging applications, has been completed in 21% (TBZ) and 16% (DTBZ) overall yield and in >97% ee from the starting dihydroisoquinoline. The synthesis utilizes Sodeokas palladium-catalyzed asymmetric malonate addition to set the initial stereocenter followed by a number of diastereoselective transformations to incorporate the remaining asymmetric centers.

Combinatorial discovery of metal co-catalysts for the carbonylation of phenol

Abstract The palladium-catalyzed carbonylation of phenol to form diphenyl carbonate (DPC) requires the presence of a metal co-catalyst to catalyze the reoxidation of palladium from Pd 0 to Pd 2+ in the presence of oxygen. In this study, we utilize a high throughput screening (HTS) methodology to rapidly study the nature of the co-catalyst with an emphasis on combinations of metal co-catalysts that appear to work in a synergistic manner to increase palladium usage. Critical new developments were made in using a small-scale reactor in diffusion controlled systems. The HTS system is described along with the optimized catalyst packages that were determined. Additionally, the results from HTS were used to better elucidate the mechanism of this potentially important commercial reaction.

Functional Imaging of Oxidative Stress with a Novel PET Imaging Agent, 18F-5-Fluoro-l-Aminosuberic Acid

Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc-) represents a potentially attractive PET biomarker to enable in vivo quantification of xc- activity in response to oxidative stress associated with disease. We have developed a system xc- substrate that incorporates characteristics of both natural substrates, l-cystine and l-glutamate (l-Glu). l-aminosuberic acid (l-ASu) has been identified as a more efficient system xc- substrate than l-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, 18F-5-fluoro-aminosuberic acid (18F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. Methods: 18F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to l-cystine and l-Glu. Affinity toward system xc- was determined via in vitro uptake and inhibition studies using oxidative stress–induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). Results: In vitro assay results determined that l-ASu inhibited system xc- as well as or better than l-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc- uptake of l-ASu than l-Glu. Radiosynthesis of 18F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. 18F-FASu uptake was approximately 5 times greater than 18F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. Conclusion: 18F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. 18F-FASu may provide more sensitive detection than 18F-FDG in certain tumors.

18F-5-fluoro-aminosuberic acid (FASu) as a potential tracer to gauge oxidative stress in breast cancer models.

The cystine transporter (system xC−) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element promoter. We have developed 18F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system xC−. The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC− activity in vivo and its potential for breast cancer imaging. Methods: 18F-FASu specificity toward system xC− was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system xC−–transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species–inducing diethyl maleate increased glutathione levels and 18F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC− inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion: 18F-FASu as a system xC− substrate is a specific PET tracer for functional monitoring of system xC− and OS imaging. By enabling noninvasive analysis of xC− responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the xC− transporter

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).