Nucleobase-Derived Nitrones: Synthesis and Antioxidant and Neuroprotective Activities in an In Vitro Model of Ischemia-Reperfusion.

Herein, we report the synthesis, antioxidant, and neuroprotective properties of some nucleobase-derived nitrones named 9a-i. The neuroprotective properties of nitrones, 9a-i, were measured against an oxygen-glucose-deprivation in vitro ischemia model using human neuroblastoma SH-SY5Y cells. Our results indicate that nitrones, 9a-i, have better neuroprotective and antioxidant properties than α-phenyl-N-tert-butylnitrone (PBN) and are similar to N-acetyl-L-cysteine (NAC), a well-known antioxidant and neuroprotective agent. The nitrones with the highest neuroprotective capacity were those containing purine nucleobases (nitrones 9f, g, B = adenine, theophylline), followed by nitrones with pyrimidine nucleobases with H or F substituents at the C5 position (nitrones 9a, c). All of these possess EC50 values in the range of 1-6 µM and maximal activities higher than 100%. However, the introduction of a methyl substituent (nitrone 9b, B = thymine) or hard halogen substituents such as Br and Cl (nitrones 9d, e, B = 5-Br and 5-Cl uracil, respectively) worsens the neuroprotective activity of the nitrone with uracil as the nucleobase (9a). The effects on overall metabolic cell capacity were confirmed by results on the high anti-necrotic (EC50's ≈ 2-4 µM) and antioxidant (EC50's ≈ 0.4-3.5 µM) activities of these compounds on superoxide radical production. In general, all tested nitrones were excellent inhibitors of superoxide radical production in cultured neuroblastoma cells, as well as potent hydroxyl radical scavengers that inhibit in vitro lipid peroxidation, particularly, 9c, f, g, presenting the highest lipoxygenase inhibitory activity among the tested nitrones. Finally, the introduction of two nitrone groups at 9a and 9d (bis-nitronas 9g, i) did not show better neuroprotective effects than their precursor mono-nitrones. These results led us to propose nitrones containing purine (9f, g) and pyrimidine (9a, c) nucleobases as potential therapeutic agents for the treatment of cerebral ischemia and/or neurodegenerative diseases, leading us to further investigate their effects using in vivo models of these pathologies.

Novel Isoxazolidine and γ-Lactam Analogues of Homonucleosides.

Homonucleoside analogues cis-16 and trans-17 having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. Hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective γ-lactam analogues. γ-Lactam analogues having 5-bromouracil and 5-chlorouracil fragments were synthesized by treatment of uracil-containing γ-lactams with NBS and NCS. Isoxazolidine and γ-lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to 100 µM. The cytostatic activities of all compounds toward nine cancerous cell lines was tested. γ-Lactams trans-15e (Cl-Ura) and cis-15h (Theo) appeared the most active toward pancreatic adenocarcinoma cells (Capan-1), showing IC50 values 21.5 and 18.2 µM, respectively. Isoxazolidine cis-15e (Cl-Ura) inhibited the proliferation of colorectal carcinoma (HCT-116).

Novel isoxazolidine analogues of homonucleosides and homonucleotides.

Isoxazolidine analogues of homonucleos(t)ides were synthesized from nucleobase-derived nitrones 20a-20e (uracil, 5-fluorouracil, 5-bromouracil, thymine, adenine) employing 1,3-dipolar cycloadditions with allyl alcohol as well as with alkenylphosphonates (allyl-, allyloxymethyl- and vinyloxymethyl- and vinylphosphonate). Besides reactions with vinylphosphonate the additions proceeded regioselectively to produce mixtures of major cis and minor trans 3,5-disubstituted isoxazolidines (d.e. 28-82%). From vinylphosphonate up to 10% of 3,4-disubstituted isoxazolidines was additionally produced. Vicinal couplings, shielding effects and 2D NOE correlations were employed in configurational assignments as well as in conformational analysis to find out preferred conformations for several isoxazolidines and to observe anomeric effects (pseudoaxial orientation of phosphonylmethoxy groups) for those obtained from vinyloxymethylphosphonate. None of the tested compounds were endowed in vitro with antiviral activity against a variety of DNA and RNA viruses at subtoxic concentrations (up to 250 µM) nor exhibited antiproliferative activity towards L1210, CEM, and HeLa cells (IC50 = ≥100 µM).