Intramolecular approach to some new D-ring-fused steroidal isoxazolidines by 1,3-dipolar cycloaddition: synthesis, theoretical and in vitro pharmacological studies

Abstract

Intramolecular 1,3-dipolar cycloaddition of alkenyl oxime 5, obtained from trans-3β-acetoxy-16,17-secopregna-5,17(20)-dien-16-al 2 with hydroxylamine hydrochloride, was carried out under reflux in toluene to furnish a ca. 3 : 2 mixture of D-ring-fused isoxazolidine diastereomers 8 and 11a both with cis D/E ring junction stereochemistry. The corresponding reaction of 5 in the presence of a catalytic amount of BF₃·OEt₂ gave a single isomer 8 under milder conditions with an improved yield. The experimental findings on the thermally induced and BF₃-catalysed transformations were supported by calculations of the proposed mechanism at the BLYP/6-31G(d) level of theory. Analogously, cyclization of D-secopregnene aldehyde 2 with N-substituted hydroxylamine derivatives (10b–e) under thermal conditions furnished N-functionalized isoxazolidines 11b–e diastereoselectively, via the corresponding alkenyl nitrones 7b–e. The activities of the 3-deacetylated compounds (9, 12b–e) were tested in vitro on rat testicular C_17,20-lyase: the radioligand incubation assay revealed that 9 exerted a moderate enzyme-inhibitory effect (IC₅₀ = 26 μM), while the other derivatives were found to decrease the enzyme activity by only 68–83%. The antiproliferative activities of the structurally related isoxazolidine derivatives were also determined in vitro on three malignant human cell lines (HeLa, MCF7, and A431) by the microculture tetrazolium assay. The highest cytotoxic activities were displayed by the N-benzyl-substituted derivative 12e (IC₅₀: 14.00, 23.18 and 8.76 μM on HeLa, MCF7 and A341 cells, respectively).