Elucidating the structure and cytochrome P450-mediated mechanism for novel metabolites of GDC-0575 in rats.

1. GDC-0575 is an ATP-competitive small-molecule inhibitor of ChK1 that is being developed by Genentech for the treatment of various human malignancies.2. In a radiolabeled mass balance study of GDC-0575 in rats, two novel metabolites, named M12 (-71 Da,) and M17 (+288 Da), were detected as abundant circulating metabolites.3. Subsequent mass spectrometry and nuclear magnetic resonance analysis showed that M12 was a cyclized metabolite of GDC-0575, whereas M17 was its heterodimer to the parent. We further determined that M12 was mainly generated by cytochrome P450 (Cyp) 2d2.4. We proposed the potential mechanism was initiated by the oxidation on the pyrrole ring and subsequent cyclisation of the free primary amine onto C-3 of the pyrrole ring. This was followed by expulsion of cyclopropylcarboxamide and a loss of water to form intermediate I, which can be further oxidised to form M12, or dimerise with another molecule of GDC-0575 as nucleophile to form M17.5. To verify this hypothesis, we attempted to trap the intermediate I with glutathione (GSH) trapping assay and the GSH conjugate on the pyrrole ring was identified. This suggests the oxidation on the pyrrole led to reactive metabolite formation and supported this proposed mechanism.

Characterizing the in vitro species differences in N-glucuronidation of a potent pan-PIM inhibitor GNE-924 containing a 3,5-substituted 6-azaindazole.

1. Glucuronidation of amines has been shown to exhibit large species differences, where the activity is typically more pronounced in human than in many preclinical species such as rat, mouse, dog and monkey. The purpose of this work was to characterize the in vitro glucuronidation of GNE-924, a potent pan-PIM inhibitor, to form M1 using liver microsomes (LM) and intestinal microsomes (IM). 2. M1 formation kinetics varied highly across species and between liver and intestinal microsomes. In LM incubations, rat exhibited the highest rate of M1 formation (CLint,app) at 140 ± 10 µL/min/mg protein, which was approximately 30-fold higher than human. In IM incubations, mouse exhibited the highest CLint,app at 484 ± 40 µL/min/mg protein, which was >1000-fold higher than human. In addition, CLint,app in LM was markedly higher than IM in human and monkey. In contrast, CLint,app in IM was markedly higher than LM in dog and mouse. 3. Reaction phenotyping indicated that UGT1A1, UGT1A3, UGT1A9, UGT2B4 and the intestine-specific UGT1A10 contributed to the formation of M1. 4. This is one of the first reports showing that N-glucuronidation activity is significantly greater in multiple preclinical species than in humans, and suggests that extensive intestinal N-glucuronidation may limit the oral exposure of GNE-924.

Chemoselective sp2-sp3 cross-couplings: iron-catalyzed alkyl transfer to dihaloaromatics.

The chemoselective functionalization of a range of dihaloaromatics with methyl, cyclopropyl, and higher alkyl Grignard reagents via iron-catalyzed cross-coupling is described. The site selectivity of C-X (X = halogen) activation is determined by factors such as the position of the halogen on the ring, the solvent, and the nucleophile. A one-pot protocol for the chemoselective synthesis of mixed dialkyl heterocycles is achieved solely employing iron catalysis.

Degradation of a pharmaceutical in HPLC grade methanol containing trace level formaldehyde.

An anomalous peak was observed in the HPLC/UV analysis of a developmental drug product. High resolution LC/MS revealed that the mass of this degradant was 12 Da greater than the drug substance, corresponding to a net gain of a single carbon atom. The degradant was reproduced by incubating the drug substance with formaldehyde, followed by isolation using normal phase chromatography and structure elucidation by NMR. It was determined to be an analytical artifact caused by the nucleophilic reaction of the drug substance with trace levels of formaldehyde in the methanol diluent. Typical formaldehyde levels in various grades of methanol were determined, leading to the adoption of spectrophotometric purity solvent to mitigate the recurrence of this artifact. This work demonstrates that even ppm levels of impurities in solvents can cause significant degradation of drug product and the HPLC grade solvents are not always suitable for HPLC analysis in drug product development.

Novel mechanism for dehalogenation and glutathione conjugation of dihalogenated anilines in human liver microsomes: evidence for ipso glutathione addition.

The objective of the present study was to investigate the influence of halogen position on the formation of reactive metabolites from dihalogenated anilines. Herein we report on a proposed mechanism for dehalogenation and glutathione (GSH) conjugation of a series of ortho-, meta-, and para-dihalogenated anilines observed in human liver microsomes. Of particular interest were conjugates formed in which one of the halogens on the aniline was replaced by GSH. We present evidence that a (4-iminocyclohexa-2,5-dienylidene)halogenium reactive intermediate (QX) was formed after oxidation, followed by ipso addition of GSH at the imine moiety. The ipso GSH thiol attacks at the ortho-carbon and eventually leads to a loss of a halogen and GSH replacement. The initial step of GSH addition at the ipso position is also supported by density functional theory, which suggests that the ipso carbon of the chloro, bromo, and iodo (but not fluoro) containing 2-fluoro-4-haloanilines is the most positive carbon and that these molecules have the favorable highest occupied molecular orbital of the aniline and the lowest unoccupied orbital from GSH. The para-substituted halogen (chloro, bromo, or iodo but not fluoro) played a pivotal role in the formation of the QX, which required a delocalization of the positive charge on the para-halogen after oxidation. This mechanism was supported by structure-metabolism relationship analysis of a series of dihalogenated and monohalogenated aniline analogues.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

Bioactivation of flutamide metabolites by human liver microsomes.

Flutamide, a widely used nonsteroidal antiandrogen drug for the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. A novel N-S glutathione adduct has been identified in a previous bioactivation study of flutamide (Kang et al., 2007). Due to the extensive first pass metabolism, flutamide metabolites such as 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)phenylamine (Flu-1) have achieved plasma concentrations higher than the parent in prostate cancer patients. In vitro studies in human liver microsomes were conducted to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide metabolites and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. Several GSH adducts (G1, Flu-1-G1, Flu-1-G2, Flu-6-Gs) derived from the metabolites of flutamide were identified and characterized. A comprehensive bioactivation mechanism was proposed to account for the formation of the observed GSH adducts. Of interest were the formation of a reactive intermediate by the desaturation of the isopropyl group of M5 and the unusual bioactivation of Flu-1. Studies using recombinant P450s suggested that the major P450 isozymes involved in the bioactivation of flutamide and its metabolites were CYP1A2, CYP3A4, and CYP2C19. These findings suggested that, in addition to the direct bioactivation of flutamide, the metabolites of flutamide could also be bioactivated and contribute to flutamide-induced hepatotoxicity.

Identification of a novel glutathione conjugate of flutamide in incubations with human liver microsomes.

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

In vitro metabolic activation of thiabendazole via 5-hydroxythiabendazole: identification of a glutathione conjugate of 5-hydroxythiabendazole.

Thiabendazole (TBZ) is a broad-spectrum antihelmintic used for treatment of parasitic infections in animals and humans and as an agricultural fungicide for postharvest treatment of fruits and vegetables. It is teratogenic and nephrotoxic in mice, and cases of hepatotoxicity have been observed in humans. Recent reports have demonstrated a correlation between 5-hydroxythiabendazole (5-OHTBZ) formation, a major metabolite of TBZ, and covalent binding of [(14)C]TBZ to hepatocytes, suggesting another pathway of activation of TBZ. Current in vitro studies were undertaken to probe the bioactivation of TBZ via 5-OHTBZ by cytochrome P450 (P450) and peroxidases and identify the reactive species by trapping with reduced glutathione (GSH). Microsomal incubation of TBZ or 5-OHTBZ supplemented with NADPH and GSH afforded a GSH adduct of 5-OHTBZ and was consistent with a bioactivation pathway that involved a P450-catalyzed two-electron oxidation of 5-OHTBZ to a quinone imine. The same adduct was detected in GSH-fortified incubations of 5-OHTBZ with peroxidases. The identity of the GSH conjugate suggested that the same reactive intermediate was formed by both these enzyme systems. Characterization of the conjugate by mass spectrometry and NMR revealed the addition of GSH at the 4-position of 5-OHTBZ. In addition, the formation of a dimer of 5-OHTBZ was discernible in peroxidase-mediated incubations. These results were consistent with a one-electron oxidation of 5-OHTBZ to a radical species that could undergo disproportionation or an additional one-electron oxidation to form a quinone imine. Overall, these studies suggest that 5-OHTBZ can also play a role in TBZ-induced toxicity via its bioactivation by P450 and peroxidases.

Human in vitro glutathionyl and protein adducts of carbamazepine-10,11-epoxide, a stable and pharmacologically active metabolite of carbamazepine.

The clinical use of carbamazepine (CBZ), an anticonvulsant, is associated with a variety of idiosyncratic adverse reactions that are likely related to the formation of chemically reactive metabolites. CBZ-10,11-epoxide (CBZE), a pharmacologically active metabolite of CBZ, is so stable in vitro and in vivo that the potential for the epoxide to covalently interact with macromolecules has not been fully explored. In this study, two glutathione (GSH) adducts were observed when CBZE was incubated with GSH in the absence of biological matrices and cofactors (e.g., liver microsomes and NADPH). The chemical reactivity of CBZE was further confirmed by the in vitro finding that [14C]CBZE formed covalent protein adducts in human plasma as well as in human liver microsomes (HLMs) without NADPH. The two GSH adducts formed in the chemical reaction of CBZE were identical to the two major GSH adducts observed in the HLM incubation of CBZ, indicating that the 10,11-epoxidation represents a bioactivation pathway of CBZ. The two GSH adducts were isolated and identified as two diastereomers of 10-hydroxy-11-glutathionyl-CBZ by NMR. In addition, the covalent binding of [14C]CBZE was significantly increased in the HLM incubation upon addition of NADPH, indicating that CBZE can be further bioactivated by HLMs. To our knowledge, this is the first time the metabolite CBZE has been confirmed for its ability to form covalent protein adducts and the identity of the two CBZE-glutathionyl adducts has been confirmed by NMR. These represent important findings in the bioactivation mechanism of CBZ.