Potent CYP19 (aromatase) 1-[(benzofuran-2-yl)(phenylmethyl)pyridine, -imidazole, and -triazole inhibitors: synthesis and biological evaluation.

The synthesis of a series of novel 1-[(benzofuran-2-yl)phenylmethyl]-pyridine, -imidazole, and -triazole derivatives is described. All the compounds were evaluated in vitro for inhibitory activity against aromatase (P450(AROM), CYP19), using human placental microsomes. The 6-methoxy- and 6-hydroxy-substituted benzofuran derivatives were shown to be potent CYP19 inhibitors (IC(50) = 0.01-1.46 microM) with activity greater than that observed for the unsubstituted parent compounds and inhibitory activity comparable with or greater than the reference compound arimidex (IC(50) = 0.6 microM). Six of the benzofuran derivatives were subjected to in vitro cytotoxicity assays, using rat liver hepatocytes with cytotoxicity determined from alteration in cell morphology and lactate dehydrogenase enzyme retention over a period of 24 h, and selectivity (CYP17, 17beta-HSD types 1 and 3, CYP24, and CYP26) determination; negligible inhibitory activity was observed, suggesting a good selectivity for CYP19. The pyridine benzofuran 4a containing the 4-fluorophenyl group was the most promising (IC(50) = 44 nM; LC(50) >100 microM) compared with arimidex (IC(50) = 600 nM; LC(50) > 200 microM).

Membrane dips over nuclei correlate with DNA synthesis in spreading hepatocytes.

Spreading of hepatocytes on different supports was examined using scanning electron microscopy. Positively charged Primaria plates gave a uniform morphology in 2 h. The spreading was rapid and the surface of the cells showed early prominent dips. The hepatocytes had one or two of these structures corresponding with nuclearity of the cells. The nuclear origin of the dips was confirmed after 6 h. The indentations contained solid structures the number, size, and shape of which were identical to the nucleoli seen by light microscopy. The spreading on the other supports was less uniform. Nuclear dips appeared more slowly and were less marked initially in their depths. The nuclear dipping was independent of cell density and took place under conditions under which the cells undergo phenotypic changes during culture. Individual phenotypic changes occur at different times and rates so that the initial signal for their onset cannot be determined with any certainty. However, the appearance of the dips was accompanied by DNA synthesis in the normally quiescent cells. The process stopped when the dipping was completed. The unavoidable change in nuclear morphology in spread cells may explain why maintenance of a spherical shape circumvents inappropriate DNA synthesis and maintains hepatocyte differentiation in vitro.

Early membrane depressions in hepatocytes cultured on Primaria supports.

The topography of spreading hepatocytes on positively charged Primaria plates was examined using scanning electron microscopy. The cells acquired a uniform morphology within 2 h. The spreading was rapid and the surface of the cells showed early prominent depressions or dips. The hepatocytes had either one or two of these structures which corresponded to the frequency of mononuclear and binuclear cells, respectively. The nuclear origin of the dips was strengthened after 6 h. They contained solid structures, whose number, size and shape were the same as nucleoli. The membrane dipping was independent of cell density and took place under conditions where phenotypic changes can occur. Kidney proximal tubule cells had no dips. Co-cultures of hepatocytes and kidney proximal tubule cells showed that the cell types behave differently.

Ultrastructural changes in hypothermically preserved hepatocytes.

Hypothermic preservation of hepatocytes on gelatin gels (10 degrees C) provided a stable system for investigating cold-induced changes culminating in cell death. Hepatocyte morphology remained unchanged during 9 days of preservation. Thereafter there was a progressive movement of organelles toward the center of the cell. During this process the mitochondria appeared to have a normal morphology suggesting that they are not the primary cause of the transition. When the movement was completed the mitochondria appeared aggregated and microvilli were no longer apparent on the cell surface.