Synthesis, spectroscopy, theoretical and biological studies of new gramine-steroids salts and conjugates

• New gramine connections with bile acids and sterols were synthesized.
• Their structures were confirmed by spectral analysis as well as PM5 methods.
• Gramine decreases haemolytic activity of cholic acids.
• Gramine-sterols connections show higher cell membrane-perturbing potency than sterols.
• All new compounds affect erythrocyte shape.

New gramine connections with bile acids (lithocholic, deoxycholic, cholic) and sterols (cholesterol, cholestanol) were synthesized. The structures of products were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. Unexpectedly, the products of the reaction of gramine with cholesterol and cholestanol were symmetrical compounds consisting of two molecules of sterols connected by N(CH3)2 group. All new synthesized compounds interact in vitro with the human erythrocyte membrane and alter discoid erythrocyte shape inducing stomatocytosis or echinocytosis. Increase in the incorporation of the fluorescent dye merocyanine 540 (MC540) into the erythrocyte membrane indicates that new compounds at sublytic concentrations are capable of disturbing membrane phospholipids asymmetry and loosening the molecular packing of phospholipids in the bilayer. Gramine significantly decreases the membrane partitioning properties as well as haemolytic activity of lithocholic acid in its new salt. Moreover, both deoxycholic and cholic acids completely lost their membrane perturbing activities in the gramine salts. On the other hand, the capacity of new gramine-sterols connections to alter the erythrocyte membrane structure and its permeability is much higher in comparison with sterols alone. The dual effect of gramine on the bile acid and sterols cell membrane partitioning activity observed in our study should not be neglected in vivo.

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