A rational chemical intervention strategy to circumvent bioactivation liabilities associated with a nonpeptidyl thrombopoietin receptor agonist containing a 2-amino-4-arylthiazole motif.

The current study examined the bioactivation potential of a nonpeptidyl thrombopoietin receptor agonist, 1-(3-chloro-5-((4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-yl)carbamoyl)pyridine-2-yl)piperidine-4-carboxylic acid (1), containing a 2-carboxamido-4-arylthiazole moiety in the core structure. Toxicological risks arising from P450-catalyzed C4-C5 thiazole ring opening in 1 via the epoxidation-->diol sequence were alleviated, since mass spectrometric analysis of human liver microsome and/or hepatocyte incubations of 1 did not reveal the formation of reactive acylthiourea and/or glyoxal metabolites, which are prototypic products derived from thiazole ring scission. However, 4-(4-fluoro-3-(trifluoromethyl)phenyl)thiazol-2-amine (2), the product of hydrolysis of 1 in human liver microsomes, hepatocytes, and plasma, underwent oxidative bioactivation in human liver microsomes, since trapping studies with glutathione led to the formation of two conjugates derived from the addition of the thiol nucleophile to 2 and a thiazole- S-oxide metabolite of 2. Mass spectral fragmentation and NMR analysis indicated that the site of attachment of the glutathionyl moiety in both conjugates was the C5 position in the thiazole ring. Based on the structures of the glutathione conjugates, two bioactivation pathways are proposed, one involving beta-elimination of an initially formed hydroxylamine metabolite and the other involving direct two-electron oxidation of the electron-rich 2-aminothiazole system to electrophilic intermediates. This mechanistic insight into the bioactivation process allowed the development of a rational chemical intervention strategy that involved blocking the C5 position with a fluorine atom or replacing the thiazole ring with a 1,2,4-thiadiazole group. These structural changes not only abrogated the bioactivation liability associated with 1 but also resulted in compounds that retained the attractive pharmacological and pharmacokinetic attributes of the prototype agent.

Metabolism of capsaicin by cytochrome P450 produces novel dehydrogenated metabolites and decreases cytotoxicity to lung and liver cells.

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and omega-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).

Bioactivation of benzylamine to reactive intermediates in rodents: formation of glutathione, glutamate, and peptide conjugates.

The in vivo and in vitro disposition of benzylamine was investigated in rats. Benzylamine was metabolized to only a small extent by rat liver subcellular fractions. In contrast, it was extensively metabolized in vivo in rats. In vivo studies performed with stable isotope-labeled benzylamine enabled rapid mass spectrometric identification of metabolites present in rat bile and urine. The major metabolite of benzylamine was the hippuric acid formed by glycine conjugation of benzoic acid. LC/MS analysis of bile and urine obtained from rats dosed with 1:1 equimolar mixture of either d(0):d(7)- or d(0):d(2)-benzylamine showed the presence of several glutathione adducts in addition to the hippuric acid metabolite. The presence of various glutathione adducts indicated that benzylamine was metabolized to a number of reactive intermediates. Various metabolic pathways, including those independent of P450, were found to produce these intermediates. A previously undocumented pathway included the formation of a new carbon-nitrogen bond that led to a potentially reactive intermediate, Ar-CH(2)-NH(CO)-X, capable of interacting with various nucleophiles. The origin of this reactive intermediate is postulated to occur via the formation of either a formamide or carbamic acid metabolites. Metabolites which were produced by the reaction of this intermediate, Ar-CH(2)-NH(CO)-X with nucleophiles included S-[benzylcarbamoyl] glutathione, N-acetyl-S-[benzylcarbamoyl]cysteine, S-[benzylcarbamoyl] cysteinylglycine, S-[benzylcarbamoyl] cysteinylglutamate, N-[benzylcarbamoyl] glutamate, and an oxidized glutathione adduct. Bioactivation of amines via this pathway has not been previously described. The oxidative deamination of benzylamine yielding the benzaldehyde was demonstrated to be a precursor to the hippuric acid metabolite and S-benzyl-L-glutathione. The formation of the S-benzyl-L-glutathione conjugate showed that a net displacement of amine from benzylamine had taken place with a subsequent addition of glutathione at the benzylic position. In addition to these novel pathways, a number of other glutathione-derived adducts formed as a result of epoxide formation was characterized. It was demonstrated that benzylamine was converted by rat P450 2A1 and 2E1 to benzamide that was rapidly metabolized to an epoxide. Mechanisms are proposed for the formation of various GSH adducts of benzylamine.

P450-mediated metabolism of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)- [1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole- 5-carboxamide (DPC 423) and its analogues to aldoximes. Characterization of glutathione conjugates of postulated intermediates derived from aldoximes.

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N-S bond or a nitrogen-oxygen juxtaposed amide with a C-S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon-hydrogen bond in forming this GSH adduct. The lack of isotope effect in forming the aldoxime from [13CD2]DPC 602 suggests its formation does not occur through the imine intermediate. Instead the data supports the postulated mechanism of hydroxylamine and nitroso intermediates as precursors to the aldoxime. However, the formation of the GSH adduct from [13CD2]DPC 602 did show a significant intramolecular kinetic isotope effect (kH/kD = 2.3) since a carbon-deuterium bond had to be broken on the aldoxime prior to the formation of the adduct. A stable nitrile oxide derived from DPC 602 was postulated as the reactive intermediate responsible for forming this unique GSH adduct.