Hybrid Pharmacophores as Carbonic Anhydrase Inhibitors

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes encoded in most organisms of the tree of life and classified in eight evolutionarily unrelated families: α-, β-, γ-, δ-, ζ-, η-, θ- and ι-CAs. The CAs catalyse a simple, but physiologically crucial reaction for all living beings, that is the reversible hydration of carbon dioxide to bicarbonate and proton. The CAs are involved in respiration, pH and CO2 homeostasis, transport of CO2/HCO3- and a multitude of biosynthetic reactions in many organisms. Fifteen α-class CA isoforms were identified in human, which are further implicated in electrolytes secretion in many tissues/organs, metabolic reactions such as gluconeogenesis, lipogenesis, ureagenesis, bone resorption, calcification, and carcinogenesis. As a result, a multitude of human physio/pathological processes which show abnormal expression or activity of specific human CAs could be target of pharmacological intervention based on CA modulation. In addition, a number of α-, β-, γ- and η-CAs was identified in many bacteria, fungi and protozoa that are human pathogens. In this context, CAs were shown to be crucial for the virulence, growth or acclimatization of the parasites in the hosts with their inhibition, that produces growth impairment and defects in the pathogen, being a promising strategy for chemotherapy. The research activity included in this Ph.D. thesis fits in the context of the spreading interest of the scientific community on CAs as drug targets for the treatment of a multitude of disorders. Thus, a set of projects involving drug-design, synthesis and biological evaluation of new CAs modulators, among which most based on the multi-target approach, were the focus of the three-years Ph.D. cycle. In recent years, the choice of multi-potent hybrid agents is spreading worldwide and overcoming the co-administration of multiple drugs because of improved pharmacokinetics, better patient compliance, reduced drug-drug interactions as well as a synergistic effect in the treatment of the pathology. Thus, in the present Ph.D. thesis I exploited the validated efficacy of CA inhibition in the treatment of many disorders for designing several multi-target strategies including CAIs against cancer, inflammation, glaucoma and infections. My activity included both design, synthesis and in vitro kinetic evaluation by a Stopped-flow assay of the new CAI derivatives. As first and second projects of the Ph.D. period, two series of multi-target nonsteroidal anti-inflammatory drug (NSAID) - CAI derivatives for the management of rheumatoid arthritis were reported, which differ for the type of linker, amide or ester, connecting the active portions. A coumarin scaffold was chosen as CAI to induce a selective inhibition of CA IX and XII, that are overexpressed in inflamed tissues. In spite of a different plasma stability, and thus predicted pharmacokinetics, a subset of multi-potent derivatives from both series showed more efficient pain-relieving action than clinically used NSAIDs in a rat model of rheumatoid arthritis. Of note, the derivatives were shown in vitro and in silico to produce cyclooxygenase 1/2 inhibition even as integral hybrids. In contrast, for pharmacologically hitting glaucoma, CAIs of the sulfonamide type were assembled with portions able to block the β1/2-adrenergic receptors that, as hCA II, IV and XII, are involved in the pathogenesis of the ocular disorder. Multi-potent derivatives identified within the series were evaluated in a rabbit model of glaucoma and showed to possess more effective internal ocular pressure lowering properties as eye drops than the leads, clinically used dorzolamide and timolol, and even the combination of them which is a globally marketed antiglaucoma medication. Both sulfonamide and coumarin scaffolds were incorporated as CA IX and XII inhibitors in nitric oxide (NO)-donor derivatives based on a benzofuroxan scaffold, that was shown to induce a potent cytotoxic action against several types of cancer cells as well as an antimetastatic activity. Further, benzofuroxans were recently shown to possess activity against Trypanosoma cruzi, Mycobacterium Tuberculosis and Leishmania, which was assumed to be enhanced by inhibiting the α- and β-CAs encoded in these pathogens. To date, the outcomes of this study are limited to design, synthesis and in vitro kinetic assessment. As it was recently found that two of the world’s most marketed sweeteners, saccharin (SAC) and acesulfame K (ACE), selectively inhibit the tumor-associated CAs IX and XII over ubiquitous CAs, we planned a drug design strategy that considered SAC and ACE as leads and produced a new CAI chemotype, namely the 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one 1,1-dioxide (BTD). Many synthesized derivatives showed enhanced potency and in some cases selectivity, when compared to the leads against the target CA IX and XII over off-target isoforms. A subset of compounds displayed effective cytotoxic action against A549, PC-3 and HCT-116 cell lines and anticancer effects on apoptotic markers. During my Ph.D. period, I performed a multitude of kinetic studies with inhibitors and activators of several isoforms of CA belonging to the classes α, β, γ, δ, ζ, η and θ. These studies allowed to draw up the inhibition or activation profiles of hundreds of derivatives from collaborators of us. Additionally, I performed the ex novo complete kinetic characterization of the CA isoforms identified in the protozoan human parasite Entamoeba histolytica and in the coral Stylophora pistillata.