Design, Synthesis, and Biological Evaluation of Beauveriolide Analogues Bearing Photoreactive Amino Acids

Abstract

Beauveriolides I and III, which are naturally occurring cyclodepsipeptides, have been reported to bind to sterol O-acyltransferase (SOAT), inhibiting its ability to synthesize cholesteryl esters. To facilitate an analysis of the binding site(s) of these compounds, we designed beauveriolide analogues 1a-d wherein the Leu or D-allo-Ile residue was replaced by photoreactive amino acids possessing methyldiazirine or trifluoromethyldiazirine in the side chains. The methyldiazirine moiety was installed by reaction of methyl ketones with liquid ammonia to provide imine intermediates, followed by treatment with hydroxylamine-O-sulfonic acid to provide the diaziridines. Subsequent oxidation gave methyldiazirines. In contrast, trifluoromethyldiazirine derivatives were prepared from trifluoromethyl ketones via the oxime intermediates, which were transformed into diaziridines. Subsequent oxidation afforded trifluoromethyldiazirines. The synthesized photoreactive amino acids 3a-d were coupled with 3-hydroxy-4-methyloctanoic acid 4 and dipeptide 5, followed by macrolactamization to provide beauveriolide analogues 1a-d. The SOAT inhibitory activities of 1a-d were found to be as potent as those of beauveriolides I and III. Moreover, 1a-d inhibited SOAT1 selectively rather than SOAT2, which was also consistent with the behavior of beauveriolides I and III. Key words beauveriolide; sterol O-acyltransferase; photoaffinity labeling; diazirineSterol O-acyltransferase (SOAT, also known as acylCoA : cholesterol acyltransferase (ACAT)) catalyzes conversion of cholesterol and long-chain fatty-acyl-CoA to cholesteryl esters (CE).1) Since CE accumulation in plasma is closely related to artery plaque formation, SOAT inhibitors are expected to be therapeutic agents for atherosclerosis.2,3) Recent molecular biological studies have revealed the existence of two different SOAT isozymes-SOAT1 and SOAT2-in mammals. 4-7)SOAT1 is ubiquitously expressed in most tissues and cells, including macrophages, and is related to foam cell formation. On the other hand, SOAT2, which is expressed predominantly in the liver and intestine, is related to food cholesterol absorption and secretion of low-density lipoprotein (LDL). Since SOAT1 and SOAT2 function differently, 8,9) isozyme selectivity is important for developing SOAT inhibitors. So far, a number of SOAT inhibitors have been reported and their isozyme selectivities have been discussed. 2,3,[10][11][12] Beauveriolides I and III (Fig. 1A), isolated from a culture broth of Beauveria sp. FO-6979, inhibit lipid droplet formation in mouse peritoneal macrophages.13,14) These cyclodepsipeptides were found to inhibit CE synthesis by blocking SOAT activity.15) Ohshiro et al. reported that beauveriolides I and III selectively inhibited SOAT1 in a cell-based assay using two Chinese hamster ovary (CHO) cell lines expressing African green monkey SOAT1 (SOAT1-CHO) and SOAT2 (SOAT2-CHO).10) However, beauveriolides I and III inhibit SOAT2 as well as SOAT1 in an enzyme assay using the microsomal fraction prepared from S...

Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis

“…Reaction to form the diazirine 86 via usual HOSA methodology proceeded in only 30% yield, but the subsequent deprotection/protection chemistry proceeded smoothly to give 90 . Another approach (Scheme , route 4) was pursued from Boc d -aspartic acid α- tert -butyl ester 91 where the acid side chain was converted to the Weinreb amide 92 . , Degradation to the ketone 93 using methyl Grignard reagent did not proceed to completion, whereas the use of methyllithium was much more successful, proceeding in 90% yield. As in the previous synthesis, conversion to the diazirine 94 proceeded in low yield.…”

“…Trifluoromethyl-photo-leucine 60 and methionine 58 have been synthesized (Scheme ) in their protected form ( 100 ), and their photoactivation was compared to their methyl counterparts . The trifluoromethyl­diazirines were found to be more efficient at cross-linking but required a much lower 300–330 nm wavelength light to activate than is typical for aliphatic diazirine probes.…”

Selective inhibition of sterolO-acyltransferase 1 isozyme by beauveriolide III in intact cells

“…More importantly, BeauIII was demonstrated to be orally active in atherosclerogenic mouse models, reducing atherosclerotic lesions in the aortae and hearts of apolipoprotein E knockout mice and in low density lipoprotein receptor knockout mice 1, 4 . Furthermore, we prepared a number of beauveriolide derivatives using combinatorial chemistry 1 to examine structure-activity relationships and identified more potent inhibitors 1, 6–12 . During the derivative study, biotin-labeled beauveriolide (Biotin-Beau, Fig.…”

Fluorinated Aliphatic Diazirines: Preparation, Characterization, and Photolabeling Studies

“…Moreover, these compounds were mostly unknown from the synthetic standpoint. We could find only three single molecules in the literature with a linear aliphatic CF 3 -diazirine motif, [27][28][29] while cyclic molecules were not reported. Aliphatic difluoromethyl-substituted diazirines were completely unknown.…”