ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinium.

Sir: Clinical and nutritional studies have indicated that high cholesterol levels in the blood may be one of the major causes of atherosclerosis and coronary heart disease. In this context the metabolism of cholesterol in liver is of paramount importance because liver seems to be the sole organ supplying serum cholesterol and because the conversion of cholesterol into bile acids in liver is quantitatively the most important pathway for its elimination from the body. These facts suggest that the inhibition of endogenous cholesterol synthesis in liver could lead to a lowering of its level in the blood and several studies have been made for this purpose.'-'" In a previous paper from this laboratory'), citrinin, known as an antibiotic, was isolated from cultures of the fungus Pythium ultimum as an inhibitor of cholesterol synthesis in a rat liver enzyme system. Hypocholesterolemic activity of this antibiotic was shown in both liver and serum when administered orally to rats. Further work in this laboratory to detect specific inhibitors of cholesterol synthesis produced by micro-organisms has led to the isolation of three metabolites (1), ML-236A, ML-236B, and ML-236C, produced by Penicillium citrinum. The present communication describes the production, isolation and some biochemical and biological activities of these compounds.* Preliminary abstracts of these studies have been published.' '71 More recently BROWN et al.s> have isolated ML-236B from cultures of Penicillium brevicornpactum as an antifungal metabolite (designated compactin). P. citrinum SANK 18767 was grown aerobically in a medium containing 3 % malt extract, 2 glucose and 0.1 % peptone in a 6,000-liter fermentor for 96 hours. The culture filtrate (2,900 liters from 3,000-liter culture broth) was concentrated in vacuo to 450 liters and the active compounds were extracted with ethyl acetate at pH 4. The extract was concentrated in vacuo to dryness and the resultant pellet (327 g) was applied to a column of silica gel in n-hexane. ML-236C was first eluted from the column with n-hexane acetone (95: 5) and then ML-236B with n-hexane acetone (85:15), following which ML-236A was eluted with acetone. The active fractions containing the individual metabolites were separately concentrated in vacuo to dryness. The dried product containing ML-236A (194 g) was dissolved in ethyl acetate and washed successively with saturated Na2CO3 and NaCI solutions. The dried product from the ethyl acetate layer (70 g) was adsorbed on a silica gel column in benzene. After washing the column with benzene ethyl acetate (8: 2), ML-236A was eluted with benzene methanol (95: 5), and the active fractions were concentrated to dryness, giving 9 g of ML236A as a white powder. The dried fraction containing ML-236B (38 g) was dissolved in benzene (500 ml) and the insoluble materials were removed by filtration. The filtrate was concentrated and allowed to stand overnight, and the resultant white crystals were collected by filtration. The compound was recrystallized from benzene and then from ethanol, giving 10.5 g of ML-236B as white crystals. The ML-236C-containing product (3.2 g) was dissolved in dichloromethane and then applied to a column of silica gel. The column was washed with dichloromethane and then developed with dichloromethane ethyl acetate (95: 5). The active eluate was concentrated to dryness, giving 2.1 g of ML-236C as an oily substance. Fig. I shows the inhibitory effects of the three metabolites on cholesterol synthesis; 11C-acetate incorporation into digitonin-precipitable sterols in a rat liver enzyme system was measured by the method of KNAUSS et al.9 Of the three compounds, the major metabolite ML-236B was ML 236A R=-OH ML-236B R=-000CH(CH,)CH,CH,