Synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs.

Thiazolides are polypharmacology agents with at least three mechanisms of action against a broad spectrum of parasites, bacteria and viruses. In respiratory viruses they inhibit the replication of orthomyxoviridae and paramyxoviridae at a post-translational level. Nitazoxanide 1a, the prototype thiazolide, was originally developed as an antiparasitic agent and later repurposed for the treatment of viral respiratory infections. The second generation thiazolides following nitazoxanide, such as the 5-chloro analogue RM-5038 2a, are also broad-spectrum antiviral agents as we have reported. Both 1a and its effective circulating metabolite, tizoxanide 1b, are 5-nitrothiazole derivatives, while RM-5038 2a and its de-acetyl derivative RM-4848 2b are the corresponding 5-chloro derivatives. Recently 1a has completed phase II-III clinical trials in the United States, Canada, Australia and New Zealand in a total of 2865 adults and adolescents of at least 12 months of age with viral acute respiratory illness. Since its biodisposition is primarily seen in the gastro-intestinal tract, its efficacy in systemic viral diseases requires relatively high oral doses. The chemical synthesis of new derivatives with a better systemic absorption was therefore urgently needed. In order to improve their systemic absorption, new amino-ester prodrug derivatives of 1b and RM4848 2b were prepared and tested for their animal pharmacology, pharmacokinetics and toxicology. RM-5061 8a in rats showed 7-fold higher blood concentration compared to 1a: absolute bioavailability increased from 3 to 20%, with a good safety profile in animal safety pharmacology and toxicology.

Glucuronides from metabolites to medicines: a survey of the in vivo generation, chemical synthesis and properties of glucuronides.

Covering: 1998 to 2011. Previous review: Nat. Prod. Rep., 1998, 15, 173-186. The fourteen years that have passed since the previous review on this topic have seen a significant increase of interest in many aspects of glucuronide chemistry and biology. Glucuronides are the most important class of phase 2 xenobiotic metabolites and typically act in a detoxifying role. While this is generally true for O-alkyl and O-aryl glucuronides, a number of glucuronides are known to be pharmacologically active per se. Additionally the use of glucuronide prodrugs, notably to ameliorate the cytotoxicity of anticancer agents, has markedly increased. Whereas the previous review covered only the synthesis of O-glucuronides, we now include N-, S- and C-glucuronides also and discuss both synthetic and biological aspects. Synthetic methods for all classes of glucuronides are reviewed and updated, together with advances in the enzymatic synthesis of glucuronides and methods for their detection. Finally we discuss the biological reactivity of glucuronides where known, including the important morphine-6-glucuronide. A lively debate has continued for several years on whether O-acyl glucuronide metabolites of carboxylic acids are toxic, affecting both the safety assessment of well-used drugs and new drug development programmes. We summarise the current understanding, together with other known examples of interaction between glucuronides and macromolecules.

Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives.

The anticonvulsant carbamazepine 1 is associated with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metabolism of 1 has been implicated in the pathogenesis of the ADRs. We report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogues of 1 that were intended to minimize reactive metabolite formation via arene oxidation and 10,11-epoxidation. Halo analogues were obtained either by rearrangement of halogenated N-arylindoles or from specifically halogenated iminodibenzyl derivatives. In rat hepatocytes, none of the analogues underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but aromatic hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of aromatic hydroxylation required at least monochlorination or difluorination of 1. In human liver microsomes, difluoro analogue 5b underwent 10,11-epoxidation but gave no arene oxidation.

Thiazolides as novel antiviral agents. 1. Inhibition of hepatitis B virus replication.

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 (EC(50) = 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 EC(90) for intracellular virions with thiazolide structural parameters. Finally we discuss the mechanism of action of thiazolides in relation to the present results.

Cyclization of the acyl glucuronide metabolite of a neutral endopeptidase inhibitor to an electrophilic glutarimide: synthesis, reactivity, and mechanistic analysis.

The neutral endopeptidase inhibitor (2R)-2-[(1-{[(5-ethyl-1,3,4-thiadiazol-2-yl)amino]carbonyl}cyclopentyl)methyl]pentanoic acid 2 is metabolized to acyl glucuronide 3. Unprecedentedly, at pH 7.4, 3 does not undergo the O-acyl migration characteristic of acyl glucuronides but rapid, eliminative cyclization (t1/2 at 37 degrees C, 10.2 min) to glutarimide 4. Glucuronide 3 was synthesized efficiently via acylation of benzylglucuronate with N-benzyloxymethyl-protected 2. Glucuronide and imide reacted rapidly in aqueous solution, pH 7.4, with amino acids and glutathione to form stable amides and unstable thioesters. Imide 4 acylated eight lysine Nepsilon-amino groups of human serum albumin. Rapid cyclization of 3 was attributed to attack on the ester linkage by an unusually nucleophilic glutaramide NH (pKa in 2 = 9.76). N-propyl 3 was refractory to acyl migration and cyclization. This suggested a synthetic strategy for preparing analogues of 2 that form chemically stable acyl glucuronides.

Investigation of the immunogenicity of diclofenac and diclofenac metabolites.

Oral administration of the non-steroidal anti-inflammatory drug diclofenac (DCF) is associated with a high incidence of adverse drug reactions, some of which are thought to be mediated by the immune system. It has been proposed that metabolic activation of DCF and covalent binding to protein generates an antigenic determinant that stimulates immune cells; however, the nature of the metabolite remains ill-defined. The aim of this study was to synthesize and evaluate the antigenic potential of DCF metabolites in the mouse. DCF and DCF metabolites were administered via subcutaneous injection over a 5-day period to BALB/C strain mice to induce immune activation. Proliferation was measured by the addition of [(3)H] thymidine to ex vivo isolated draining auricular lymph node cells. Results were compared with those provoked by exposure to 2,4-dinitrochlorobenzene. Lymph node activation was observed following treatment with 2,4-dinitrochlorobenzene, 5-hydroxy DCF quinoneimine and 4'-hydroxy DCF quinoneimine, but not DCF acyl glucuronide or DCF itself. Interestingly, lymph node cells from 5-hydroxy DCF treated mice were also found to proliferate, when compared with cells from vehicle-treated mice, while 4'-hydroxy DCF did not stimulate lymph node cell activation. The reactivity of 5-hydroxy DCF quinoneimine was confirmed by synthesis and characterization of an N-acetyl cysteine adduct. These data show that formation of 5-hydroxy DCF and subsequent autoxidation provides an antigenic determinant for immune cell activation in the mouse.

Identification of isoflavone derivatives as effective anticryptosporidial agents in vitro and in vivo.

We report the preparation and antiparasitic activity in vitro and in vivo of a series of isoflavone derivatives related to genistein. These analogues retain the 5,7-dihydroxyisoflavone core of genistein: direct genistein analogues (2-H isoflavones), 2-carboethoxy isoflavones, and the precursor deoxybenzoins were all evaluated. Excellent in vitro activity against Cryptosporidium parvum was observed for both classes of isoflavones in cell cultures, and the lead compound 19, RM6427, shows high in vivo efficacy against an experimental infection.

Syntheses and characterization of the acyl glucuronide and hydroxy metabolites of diclofenac.

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.