Design and Synthesis of Minimally Modified Dihydroethidium Derivatives for PET Imaging of Reactive Oxygen Species

1099 Background: Although the reactive oxygen species (ROS) are recognized with an important role in the initiation and progression of pathologies, such as inflammation, cancer, cardiotoxicity, and neurodegenerative diseases,1-3 the quick in vivo kinetics of reactions involving ROS and their low concentrations still stand as main obstacles for their noninvasive imaging assessment. To date, imaging quantification of ROS in tumor microenvironment is at its infancy. One of the few examples reported in the literature involves the radiolabeling of dihydroethidium (DHE) derivatives with significant modifications in the isolated benzene ring of the molecule for 18F radiolabeling.4,5 The rational of our work is to minimally alter the structure of dihydroethidium for the ROS probe design as compared to the significant structural difference between the reported agents5 and DHE so as to maintain the desired chemical and biological properties of DHE. Methods: The precursor di-tert-butyl (2-bromo-5-methyl-6-phenyl-5,6-dihydrophenanthridine-3,8-diyl)dicarbamate for the radiolabeled compound 1 (di-tert-butyl (2-(cyano-11C)-5-methyl-6-phenyl-5,6-dihydrophenanthridine-3,8-diyl)dicarbamate) was successfully synthesized using an eight step synthetic route. Starting from the reaction with benzoyl chloride and 4,49-dinitrophenyl-2-amine, followed by the catalytic amination of the nitro group, to further protect these amines with methyl chloroformate. The cyclization of the middle fused ring was achieved with the reaction with POCl3, continuing with the deprotection of the amines using concentrated HBr for two days. The amines were protected again with di-tert-butyl dicarbamate (to minimize deprotection time when radiolabeling the molecule) and methylated in the nitrogen from the fused ring using MeI. Finally, bromination of the fused ring was achieved through the reaction with Cu(I)Br and liquid Br2. For the synthesis of compound 2 the same methodology was applied starting with the bromo derivative of the benzoyl chloride in the first step. Results: The synthesis for the bromo precursor for the 11C radiolabeled dihydroethidium derivative was achieved. The precursor molecule was fully characterized using NMR spectroscopy and confirmed by MS (ESI) m/z calcd: 580.5230; found: 580.1665, [Mox]+. Conclusions: The design and the synthesis of precursors for minimally modified dihydroethidium derivatives was successfully achieved. Radiolabeling strategies are currently under development for biological and imaging studies. Acknowledgements: This work was partially supported by Cancer Prevention and Research Institute of Texas (CPRIT RP170638) and the Dr. Jack Krohmer Professorship Funds. References: (1) Sabharwal, S. S.; Schumacker, P. T. Mitochondrial ROS in Cancer: Initiators, Amplifiers or an Achilles’ Heel? Nat. Publ. Gr.2014. (2) Taniyama, Y.; Griendling, K. K. Reactive Oxygen Species in the Vasculature Molecular and Cellular Mechanisms. 2003. (3) D’Autreaux, B.; Toledano, M. B. ROS as Signalling Molecules: Mechanisms That Generate Specificity in ROS Homeostasis. Nat. Rev. Mol. Cell Biol.2007, 8 (10), 813-824. (4) Hou, C.; Hsieh, C. J.; Li, S.; Lee, H.; Graham, T. J.; Xu, K.; Weng, C. C.; Doot, R. K.; Chu, W.; Chakraborty, S. K.; et al. Development of a Positron Emission Tomography Radiotracer for Imaging Elevated Levels of Superoxide in Neuroinflammation. ACS Chemical Neuroscience. 2018, pp 578-586. (5) Chu, W.; Chepetan, A.; Zhou, D.; Shoghi, K. I.; Xu, J.; Dugan, L. L.; Gropler, R. J.; Mintun, M. A.; Mach, R. H. Development of a PET Radiotracer for Non-Invasive Imaging of the Reactive Oxygen Species, Superoxide, in Vivo. Org. Biomol. Chem.2014, 12 (25), 4421-4431.