Synthesis and evaluation of (+) and (−)‐2,2‐difluorocitrate as inhibitors of rat‐liver ATP‐citrate lyase and porcine‐heart aconitase

Abstract

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 pM for (+)-2,2-difluorocitrate and 3.2 pM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed.The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Ki, = 1.5 pM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki > 300 pM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-' . mM-' at 25°C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.ATP-citrate lyase catalyses the production of cytosolic acetyl-CoA and oxaloacetate from citrate, with the concomitant hydrolysis of ATP to ADP and inorganic phosphate. The acetyl-CoA produced, plays a central role in mammalian fatty acid and cholesterol biosynthesis. In vivo dosing of hydroxycitrate, a potent inhibitor of ATP-citrate lyase reduces de m v o synthesis of fatty acids and cholesterol in the liver and adipose tissues (Sullivan et al., 1973). Inhibition of this enzyme would therefore be a useful strategy for treating hyperlipidaemia or hypercholes terolaemia.A mechanism has been proposed for the enzyme (Walsh, 1979; Wells, 1991) which involves enzyme phosphorylation, transfer of the phosphate to activate citrate, formation of a citryl-CoA intermediate and fragmentation of citryl-CoA to produce acetyl-CoA and oxaloacetate. The final step is a basecatalysed retro-Claisen reaction and will generate an incipient anion on the acetyl fragment. This allows several possibilities for the design of mechanism-based inhibitors of ATP-citrate lyase (Dolle et al., 1991). If the hydrogens on the nascent acetyl-CoA were replaced by fluorine, the incipient anion could have novel decay modes. For example, fluoride loss would generate a carbene within the active site. This could then attack groups within the active site, irreversibly inCurrespondenre to T. N.

Fatty Acids and Breast Cancer: Make Them on Site or Have Them Delivered

“…There are three categories of inhibitors of ACLY: those with a structural relationship to citric acid, a class of benzenesulfonamides, and selected natural product macrolides. The citric acid derivatives include (+) and (−)‐2,2‐difluorocitrate (Saxty et al, ) and (−)‐hydroxycitrate (Watson et al, ). These polar compounds are useful for studying the role of ACLY inhibition in vitro and in vivo, but are limited from potential clinical use by poor pharmacokinetic properties (Zu et al, ).…”

A New, Mild, and Efficient Synthesis of 2,2-Difluoro-3-hydroxyacids through a Selective Haloform Reaction

“…These special properties of fluorine have been exploited for the development of new and effective biochemical tools as well as agrochemical, medicinal, and therapeutic agents, particularly in the field of enzymatic inhibition . Particularly attractive are 2,2-difluoro-3-hydroxyacids, which are versatile intermediates for the preparation of a variety of bioactive materials, such as difluorinated gingerol, a potent inhibitor of prostaglandin biosynthesis, chiral 2,2-difluorocitrate as inhibitors of rat liver ATP citrate lyase and porcine heart aconitase or yeast mitochondria, difluorostatine peptides as potent and specific renin inhibitors, proteolysis inhibitors or ferroelectric liquid crystals, among others. Although a number of procedures have been developed to prepare 2,2-difluoro-3-hydroxyacids, , new, efficient methods of preparation from cheap, easily available fluorinated materials are always desirable.…”

Synthesis of Novel Thiol-Containing Citric Acid Analogs. Kinetic Evaluation of These and Other Potential Active-Site-Directed and Mechanism-Based Inhibitors of ATP Citrate Lyase

“…25 Reversible binding Ki and the determination of time-dependent inactivation were measured by inhibition of the carbon-carbon cleavage activity,28 using 1.0 mM citrate (S = Km) as previously described. 24 Under these conditions, the equation for competitive inhibition, V = Vmax[S]/{fím(l + [IVKi) + S}, reduces to V = Vmax/(2 + [I]/Xi). Reversible inhibition data were fitted to this equation using the program Grafit.29 For the timedependent inhibition, enzyme was incubated with 2 mM inhibitor at 25 °C in TrisrHCl, pH 8.0, buffer.…”

“…Thiolactone 22 was separately obtained by analogous TFA deprotection of pure 20b. Saponification of triester 21 was carried out using 10 equiv of LiOH in aqueous THF (1:2 THF-H2O) to furnish the desired 2-mercaptocitrate (-)-12.18 Methyl disulfide (-)-13 was prepared in quantitative yield following exposure of (-)-12 to S-methylsulfenyl O-methyl thiocarbonate in MeOH.19 Thiol (-)-14 was obtained from thiolactone 22 using a two-step saponification protocol which required initial diester hydrolysis followed by in situ thiolactone ring opening (22 - Methyl tezt-butyl malónate (24) was alkylated with methyl bromoacetate (2 equiv each of NaH and BrCH2-COOMe) in THF to give the mixed tetraester 25 (Scheme 3). Monoacid 26 was obtained upon selective removal of the ferf-butyl ester in 25 (1:1 TFA-CH2CI2, 25 °C, 1.5 h) which, following treatment with thionyl chloride, furnished the acid chloride 27.…”