Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.
which after mutarotation at C(1) may transfer the acyl group back to the 1R-OH. This has been conclusively demonstrated by NMR spectroscopy. 14,30 Other rearranged forms of AGs are also known. When the Mitsunobu synthesis 32 (see Synthesis) is used, lactones of type 5 are byproducts, sometimes in appreciable yields. The mechanism of formation is not clear but presumably involves cyclization of the first-formed intermediate ester. If there were an in vivo route to such lactones via free carboxyl acyl glucuronides, they would doubtless be potent acylating agents, transferring in this case the sugar along with the acyl residue.Concerning 1β-AGs of benzoic acids, the chemical stability/ reactivity may be predicted with some confidence by a Hammett correlation, 33 as discussed in detail later (NMR section). Some preliminary studies have been carried out on the computational chemistry modeling of the relative stabilities of the isomers of the AGs of 2-, 3-, and 4-trifluoromethylbenzoic acids. 34 For nonarylcarboxylic acids, the structure-reactivity is more complex. Certainly branching at the R-carbon is a factor leading to increased stability; thus the AGs of gemfibrozil 6, 35 clofibric acid 7 36 and valproic acid 8, 37 all doubly R-substituted, display long half-lives (from about 6-70 h in pH 7.4 buffer 37 ).AGs derived from NSAIDs such as naproxen 9 and ibuprofen 10 generally have half-lives from 1 to 4 h under the same conditions. 38 In such compounds, an additional complication is the different rates of rearrangement of the (R)-and (S)-AG diastereoisomers. In the case of 2-phenylpropanoic acid itself 11, 38 this rate was found to be about twice as fast for the (2R)as for the (2S)-AG epimer, and this ratio is typical of the 2-aryl propanoate class. Scheme 2. Possible Fates of AGs Scheme 3. Reactivity of Acyl Glucuronides a a Clearly the rearrangement (pathway 2) requires migration of the acyl group at least as far as O(3).
In humans, metabolism of the commonly used nonsteroidal antiinflammatory drug diclofenac 1 yields principally the 4'-hydroxy 2, 5-hydroxy 3, and acyl glucuronide 4 metabolites. All three metabolites have been implicated in rare idiosyncratic adverse reactions associated with this widely used drug. Therefore, for mechanistic toxicological studies of 1, substantial quantities of 2-4 are required and their syntheses and characterization are described here. Key steps were a convenient two-step preparation of aniline 5 from phenol, efficient and selective 6-iodination of amide 18, and high-yielding Ullmann couplings to generate diarylamines 11 and 21. The acyl glucuronide 4 was obtained by Mitsunobu reaction of 1 (free acid) with allyl glucuronate 23 followed by Pd(0) deprotection, using a modification of a published procedure. We report full characterization of 4 and note that this important metabolite has been made available pure and in quantity for the first time. We report also the metabolic fates of the synthetic metabolites: 2 and 3 were glucuronidated in rats, but only 3 formed glutathione adducts in vivo and by enzymatic synthesis via a quinoneimine intermediate. A previously undescribed glutathione adduct of 3 was obtained by enzymatic synthesis. Compound 4 formed an imine-linked protein conjugate as evinced by sodium cyanoborohydride trapping.
We report the syntheses and activities of a wide range of thiazolides [viz. 2-hydroxyaroyl-N-(thiazol-2-yl)amides] against hepatitis B virus replication, with QSAR analysis of our results. The prototypical thiazolide, nitazoxanide [2-hydroxybenzoyl-N-(5-nitrothiazol-2-yl)amide; NTZ] 1 is a broad spectrum antiinfective agent, effective against anaerobic bacteria, viruses and parasites. By contrast, 2-hydroxybenzoyl-N-(5-chlorothiazol-2-yl)amide 3 is a novel, potent and selective inhibitor of hepatitis B replication (EC50 = 0.33 μm) but is inactive against anaerobes. Several 4′- and 5′-substituted thiazolides show good activity against HBV; by contrast, some related salicyloylanilides show a narrower spectrum of activity. The ADME properties of 3 are similar to 1, viz. the O-acetate is an effective prodrug and the O-aryl glucuronide is a major metabolite. The QSAR study shows a good correlation of observed EC90 s for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.
The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.
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