Síntesis de nuevos compuestos inhibidores de glicolato oxidasa. Aplicación en hiperoxaluria primaria y evaluación biológica

Abstract

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism caused by a deficiency in the hepatic enzyme alanine-glyoxylate aminotransferase (AGT), which is in charge of glyoxylate detoxification. The consequent accumulation of the substrate, glyoxylate, leads to its oxidation into oxalate mainly by the enzyme lactate dehydrogenase (LDH) in the cytosol and, in a minor extent, by glycolate oxidase (GO) in the peroxisome. GO also catalyzes glyoxylate production from glycolate. Since oxalate constitutes and end-product of the metabolism, once the renal excretion is exceeded, it starts depositing as insoluble calcium oxalate crystals that initially damage kidneys and liver, and ends causing systemic oxalosis, being the combined kidney-liver transplantation the unique healing option for these patients. One of the current approaches aiming at the search of a non-surgical treatment for the disease is the substrate reduction therapy (SRT). In this sense, GO has been validated as a safe and efficient target for SRT in a PH1 mouse model. On that basis, and considering the structural requirements previously established for the development of GO inhibitors, two new families of GO inhibitors have been prepared: oxamic acid derivatives and salicylic acid derivatives. All of them share a common structure with a polar head, either salicylic acid or oxamic acid-based, and a hydrophobic side chain, for which different substitution options have been explored. Thus, a total of 70 compounds have been synthesized, purified and characterized, as well as evaluated against isolated GO and in hyperoxaluric mouse hepatocytes, so as to determine their efficacy on oxalate diminution. Within the oxamic acid derivatives, N-octiloxamic acid, an established GO inhibitor, was used as reference for the development of three new sub-families: 2-oxoacetic, 3-oxopropanoic, and 4-oxobutanoic acid derivatives. Regarding the salicylic acid derivatives, the furylsalicylates initially synthesized resulted to be moderate GO inhibitors and efficient agents decreasing oxalate in cultures, with EC50 values at the low micromolar range. These furylsalicylates also served as a base for structural modulation, so that the introduction of both flexible and rigid spacers between the polar head and the side chain was explored, as well as different side chain options. As a result, potency against GO was considerably improved. Docking studies allowed the binding mode prediction of the salicylic acid derivatives with GO, and this, along with the biological data gathered for these compounds, led to the establishment of preliminary structure-activity relationships for salicylates with activity as GO inhibitors and capacity to decrease oxalate production in cell culture. For some compounds, discrepancies between the activity against GO and the phenotypic effect in cells were observed. Thus, so as to determine the existence of alternative biological targets whereby these compounds might be exerting their biological effect, representative compounds were tested against LDH, which is also involved in oxalate production. As a result, salicylic acid derivatives, specifically aminomethylfuryl derivatives, have been identified as potent dual GO/LDHA inhibitors. This, along with their drug-like structure and easy two-step synthesis, makes them promising candidates for the development of a multi-target approach for the treatment of PH1.