Warfarin Analogues Research Articles

Warfarin Analog Inhibition of Human CYP2C9-Catalyzed S-Warfarin 7-Hydroxylation

Human metabolism of the S-warfarin enantiomer is catalyzed primarily by cytochrome P4502C9 (CYP2C9), which, because of the enzyme's broad drug substrate specificity, leads to drug-S-warfarin interactions. Several warfarin analogs have been synthesized and used to determine whether they exhibit diminished interactions with CYP2C9. The kinetics of the warfarin analogs' inhibition of human liver microsomal CYP2C9 catalyzed metabolism of S-warfarin to S-7-hydroxywarfarin have been investigated. R- and S-7-fluorowarfarin were both predominantly competitive inhibitors, whereas racemic 6-fluorowarfarin and racemic 6,7,8-trifluorowarfarin were predominantly mixed inhibitors with some competitive inhibition. For the alcohols produced by reductive methylation of the side chain of R- and S-warfarin, the R-enantiomer did not inhibit S-warfarin metabolism, whereas the S-enantiomer was primarily a competitive inhibitor. The fluorine substituted warfarins and the S-warfarin alcohol apparently bind with high affinity to CYP2C9. Thus their use clinically (if efficacious) would not prevent CYP2C9 associated warfarin-drug interactions. The R-warfarin alcohol did not inhibit CYP2C9 catalyzed metabolism of S-warfarin and is less likely than warfarin to participate in CYP2C9 associated warfarin-drug interactions.

THE CHARACTERIZATION OF POTENT NOVEL WARFARIN ANALOGS

Racemic sodium warfarin, Coumadin,® is widely used in the prevention of thromboembolic disease. The present study was undertaken to characterize three novel classes of warfarin analogs, and to compare them with the warfarin enantiomers. All three classes of compounds inhibit vitamin K epoxide reductase, the enzyme inhibited by racemic warfarin. The alcohol and the ester analogs have reduced protein binding compared with R-(+)-warfarin 1 1 R-(+)-warfarin will be designated as R-warfarin; S-(−)-warfarin will be designated as S-warfarin . The ester and the fluoro-derivatives have similar in vivo anticoagulant activity in the rat to that of S-(−)-warfarin 1 . Thus, it is possible to synthesize novel warfarin analogs that differ from racemic warfarin or its enantiomers in certain selected properties. © 1997 Elsevier Science Ltd

Competitive Inhibition of HIV-1 Protease by Warfarin Derivatives

The oral anticoagulant warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl)-benzopyran-2-one) is a structurally novel low micromolar competitive inhibitor of HIV-1 protease in vitro. It was recently reported that warfarin inhibits HIV-1 infection in U-1 monocytes and viral production in ACH-2 lymphocytes (Bourinbaiar, A. S. et al., (1993) AIDS 7, 129-130.). Our results demonstrate that warfarin and a series of structurally related analogs inhibit the viral protease, the most potent analog having an IC 50 = 1.9 μM. Kinetic analysis reveals inhibition by warfarin occurs in a competitive manner, with K i = 3.3 μM. While it is unclear whether the cellular inhibition previously reported is due to inhibition of HIV-1 protease, the warfarin analogs are a novel class of nonpeptide HIV-1 protease inhibitors.

Azidowarfarin photoaffinity probes of purified rat liver cytochrome P4501A1

Substrate specificity differences between various forms of cytochrome P450 (P450) are governed by substrate binding site amino acid residue differences. To determine the identities of these residues, four analogs of warfarin, a thoroughly investigated anticoagulant drug which is regio- and stereoselectively metabolized by many P450s, have been synthesized as photoaffinity probes. The probes 4′-, 6-, 7-, and 8-azidowarfarin were readily photolyzed in neutral solution by 254-nm light, with half-lives of less than 15 s. When the azidowarfarins were photolyzed in the presence of β-naphthoflavone-inducible P4501A1 (2.5 μ m) at −196 °C and the P450 was subsequently reconstituted for warfarin metabolism, 50% inactivation was achieved with 160 μ m 4′-azidowarfarin, 64 μ m 6-azidowarfarin, 127 μ m 7-azidowarfarin, and 29 μ m 8-azidowarfarin. This inactivation is irreversible. When these concentrations of the azidowarfarins were photolyzed prior to addition to P4501A1, less inhibition of P450 activity was detected and the inhibition was reversible. The CO-ferrous P450 spectrum of P4501A1 at 448 nm was diminished when photoactivated azidowarfarins bound to and inactivated the enzyme, with essentially no formation of P420 except in the case of 4′-azidowarfarin. The inactivation of P4501A1 by photoactivated 4′-azidowarfarin was prevented by 50% by 1.2 m m R-warfarin or 0.3 m m 4′-nitrowarfarin, consistent with the latter being a better P4501A1 substrate than R-warfarin. The photoinactivation of P4501A1 by each of the azidowarfarins was prevented to variable extents by R-warfarin or by 4′-, 6-, 7-, or 8-nitrowarfarin. Taken together these results demonstrate that all four azidowarfarins are potentially useful photoaffinity probes of the substrate binding site amino acid residues of P450s.

The in vitro ketone reduction of warfarin and analogues: Substrate stereoselectivity, product stereoselectivity and species differences

The in vitro metabolic ketone reduction of warfarin and its 4'-analogues acenocoumarol (4'-nitrowarfarin) and 4'-chlorowarfarin has been investigated using microsomal and cytosolic fractions of several species. Both subcellular fractions showed ketone reductase activity. The cytosolic fractions, in most species, exhibited strong substrate stereoselectivity as well as product stereoselectivity, i.e. the R(+)enantiomer was preferred as a substrate to be reduced mainly to the RS alcohol. Phenobarbital and methylcholanthrene induced cytosolic ketone reductase activity in the rat 5- to 11-fold and 3- to 7.4-fold, respectively. The microsomal fractions also showed substrate and product stereoselectivity. Contrary to the cytosolic fractions a general pattern for substrate and product stereoselectivity could not be seen. Stereoselectivity seemed species dependent (e.g. sheep vs bovine and pig). Rat liver microsomes showed practically no ketone reductase activity. Induction by phenobarbital or methylcholanthrene resulted in only a slight rise, if any, in rat microsomal ketone reductase activity. Both cytosolic and microsomal ketone reductases proved to be NADPH dependent. Substitution of the 4'-hydrogen of warfarin resulted in a change in reduction rates and in some cases, even in a change in substrate or product stereoselectivity. The data indicate that microsomal ketone reductases are different from cytosolic ketone reductases. Prelog's rule for product stereoselectivity of metabolic ketone reduction, when applied to the ketone reduction of the warfarin analogues did not agree with all data presented here.

Stereoselective metabolism of conformational analogues of warfarin by beta-naphthoflavone-inducible cytochrome P-450.

Previous studies have shown that the structurally related oral anticoagulants warfarin and phenprocoumon are regioselectively hydroxylated in the 6- and 8-positions by hepatic microsomes obtained from 3-methylcholanthrene (3-MC) or beta-naphthoflavone (BNF) pretreated rats. Stereoselectivity for hydroxylation is also observed and favors (R)-warfarin but (S)-phenprocoumon. The possibility that the stereoselectivity of warfarin hydroxylation is a function of the solution conformation of the drug was tested with conformationally restricted analogues. In these experiments the analogues were incubated with microsomes obtained from BNF-pretreated rats and any stereoselectivity associated with 6- and 8-hydroxylation was determined. The R enantiomer of cyclocoumarol, the cyclic ketal analogue of warfarin, was found to be selectively hydroxylated, in contrast to the S enantiomer of warfarin 4-methyl ether, the ring-opened analogue. The latter compound is known to have a preferred solution conformation similar to that of phenprocoumon. The results suggest that at the active site of BNF-induced cytochrome P-450 (R)-warfarin is metabolized in its cyclic hemiketal tautomer, a form which spatially mimics the preferred solution conformation of (S)-phenprocoumon.