Inhibitors of Ergosterol Biosynthesis and Growth of the Trypanosomatid Protozoan Crithidia fascicuZuta*

Six nitrogen-, sulfurand cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 pM for 24-thia-5a,20tcholestan-38-01 (2), 0.009 FM for 24-methyl-24-azaSa,2O.+holestan-38-01 (3), 0.95 MM for (20,21),(24,25)~bis-(methylene)-5a-cholestan-3&ol (4), 0.13 pM for 22-aza-Sa,20&cholestan-3fi-o1 (5), and 0.3 pM for 23-azacholestan-3-01 (7). 23-Thia-5a-cholestan-38-01 (6) had no effect on protozoan growth at concentrations as high as 20 PM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-38-01, ergosta-7,24(28)-dien-3/l-01, ergosta5,7,22,24(28)-tetraen-38-01, cholesta-8,24-dien-3@01, and, in an unusual finding, 14m-methyl-cholesta8,24-dien-38-01 were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-38-01, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost7-en-3/3-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3&ol and ergosta-7,24(28)-dien-38-01, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the Az4(“) side chain double bond. When C. famiculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.