Synthesis and evaluation of novel aldose reductase inhibitors: Effects on lens protein kinase Cγ

Purpose: To synthesize novel aldose reductase inhibitors (ARI) that will normalize losses in protein kinase Cγ (PKCγ) observed during diabetes and galactosemia. Methods: ARI were synthesized as tricyclic pyrones 1-6 (HAR-1 through HAR-6) from 3-methyl-1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran and [1]benzopyran and were tested by inhibition of aldose reductase enzyme activity in vitro and by inhibition of polyol formation in lens epithelial cells in culture. Identified compounds were further tested in galactosemic rat lens in vivo for (a) normalized PKCγ levels by Western blot, (b) reduction of phosphorylation of the gap junction protein Cx46 by analyses of co-immunoprecipitated proteins, and (c) by normalization of gap junction activity as measured by dye transfer. Results: HAR-1 (1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano[4,3-b][1]benzopyran-3-acetic acid) was identified as an ARI with IC₅₀ for aldose reductase inhibition at 2 nM. Polyol accumulation in lens epithelial cells was reduced by 80% at 10 μM. Rats fed 40% galactose for 9 days had an 80% reduction in PKCγ levels which were normalized by HAR-1 at 100 mg/kg/day, fed orally. Phosphorylation of Cx46 was increased by 50% and this was normalized in HAR-1 treated rats (6 day treatment). Gap junction activity of galactosemic rats was reduced by 55% and this was normalized by HAR-1 in six day-treated rats. Conclusions: HAR-1 is a novel ARI which normalized losses of PKCγ, changes in Cx46 phosphorylation, and gap junction activity.