Characterization of Novel Glutathione Adducts of a Non-Nucleoside Reverse Transcriptase Inhibitor, (S)-6-Chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in Rats. Possible Formation of an Oxirene Metabolic Intermediate from a Disubstituted Alkyne

Abstract

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Metabolism of (S)-5,6-Difluoro-4-cyclopropylethynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)-quinazolinone, a Non-Nucleoside Reverse Transcriptase Inhibitor, in Human Liver Microsomes. Metabolic Activation and Enzyme Kinetics

“…The various metabolic pathways leading to diconjugates (M1, M2), glutathione conjugates (M3-M6), defluorinated metabolite (M7), a ring-hydroxylated metabolite (M8), glucuronide conjugates (M9 and M10), and a sulfate conjugate (M11) are shown in Scheme 1. The major metabolite was found to be the glucuronide conjugate (M9) of 8-OH DPC 963 (Scheme 1) consistent with previous findings from close structural analogs, efavirenz and DPC 961 (Mutlib et al, , 2000. The structures of these metabolites suggested the existence of multiple metabolic pathways, some of which led to potentially reactive metabolic intermediates.…”

“…It appears that the oxidation of the triple bond in DPC 963 took place to form an unstable oxirene metabolic intermediate, which appeared to be responsible for the production of two isomeric M3 and M4. Such a formation of an oxirene intermediate has been postulated and reported for the metabolism of DPC 963 in the rat and with a close analog, DPC 961 (Mutlib et al, 2000). As shown in Scheme 2, it is postulated that an initial oxidation of the triple bond, catalyzed by cytochrome P450 3A4, results in the formation a putative oxirene intermediate, which undergoes rapid rearrangement with the adjacent cyclopropyl ring to form a reactive cyclobutenyl ketone intermediate.…”

Synthesis of some new fluorine substituted thiobarbituric acid derivatives as anti HIV1 and cyclin-dependent kinase 2 (CDK2) for cell tumor division: Part I

“…The introduction of fluorine atoms to bioactive molecules enhance and improve their pharmacological properties [16][17][18]. Also, presence of hetero-atoms increased membrane, permeability, which enhances the electrostatic force and hydrophobic binding stability against metabolic transformations [18][19][20][21][22]. As part of the interested research program in the fluorine substituted heterocyclic nitrogen systems as biocidal agents [23][24][25][26][27], the present work tends to synthesize, some new fluorinated-thiobarbituric acids full fused in view of their anti-HIV and cyclin-dependent kinase 2.…”

Design, synthesis, antimicrobial activity and anti-HIV activity evaluation of novel hybrid quinazoline–triazine derivatives

“…In context to the above rationale and in continuation of our ongoing program focused on finding new leads with anti-HIV activities [37][38][39][40][41][42] , a novel hybrid series of DATA molecules with a combination of different pharmacophores such as triazine, quinazoline and piperazine that are structurally related to anti-HIV lead compounds, i.e. piperidine linked with DATA-NNRTI derivatives 43 , abacavir having cyclopropyl amine group 44 and DPC 961 45 with quinazoline moiety, are reported ( Figure 2). Herein, we synthesized a new series of ethylene diamine-linked quinazolinetriazine derivatives starting from the triazine ring of DATA.…”