Inhibitors and inhibitor conjugates designed for selective inhibition of carbonic anhydrase isoforms

Carbonic anhydrase is a ubiquitous zinc-metalloenzyme that catalyzes the reversible hydration of carbon dioxide, forming bicarbonate and a proton. In humans, fifteen different isoforms of carbonic anhydrase have been identified so far, twelve of which are catalytically active. Because of their various functions, carbonic anhydrases are targeted for numerous different diseases such as edema, glaucoma, epilepsy, and hypoxic tumors. Classic, non-selective carbonic anhydrase inhibitors (CAIs) have been employed to treat various clinical conditions, yet their unspecific inhibition causes several side effects. Because the three-dimensional structure of carbonic anhydrase is highly conserved across all isozymes, previous efforts in designing subtype-selective CAIs have often been met with limited success. One of the most targeted CA isoforms is CA IX, a dimeric transmembrane glycoprotein, due to its involvement in the progression of solid tumors under hypoxic conditions. The inhibition of CA IX has demonstrated the ability to slow tumor progression and induce tumor cell death. In Chapter 2, the most relevant recently developed CA IX targeting strategies are discussed to identify areas which can be improved and expanded upon to advance successful inhibitor technologies. The most effective class of CA inhibitors have proven to be aromatic or heterocyclic sulfonamides, which act by coordinating to the catalytic zinc ion. The general approach to designing new CAIs is expanding the molecule and creating selectivity by establishing additional interactions with the rim of the active site. In Chapter 3, X-ray crystallography data, molecular docking and synthetic efforts were combined to identify how different structural elements of the small molecule inhibitor contribute to potency and selectivity against CA IX. This allows us to utilize all existing knowledge of CA inhibition for the generation of new selective CAIs. In Chapter 4, we describe a new series of halogenated ureido-sulfonamides with improved physicochemical properties designed to overcome the pharmacokinetic challenges that have limited the success of previous inhibitor technologies. Molecular docking was used to select phenyl substituents and their position on the benzene ring. The series was then synthetically accessed, and the synthetic pathway optimized to improve yields and reproducibility. Lastly, the estimated lipophilicities of the series were compared to assess their physicochemical properties and to validate the working hypothesis. Another strategy to induce selectivity for CA IX was utilizing polyethylene glycol (PEG) to conjugate small molecule inhibitors to improve physicochemical properties and reduce cell penetrability. We thereby expanded on previous works by our group by introducing two new targeting groups that were both mono- and bis-conjugated to PEG of different linker lengths, exploring multivalency and allowing for further functionalization to conjugate to delivery platforms such as gold nanoparticles. In Chapter 6, mitochondrial carbonic anhydrases VA and VB were explored as new targets of CA inhibition. Compared to other isoforms, CA VA and VB were not thoroughly investigated. Their association with diseases such as obesity, metabolic conditions, and recently discovered link between CA VB and Alzheimer’s, makes them interesting targets for inhibition. We introduce a chemically diverse series of potential inhibitors against CA VA and VB, utilizing molecular docking in 3D structures modeled by AlphaFold to design and assess the series in silico. We optimized the synthetic pathway to access alpha-alkylated and heterocyclic amide-linked thiadiazole sulfonamide inhibitors.