Transacylation and hydrolysis of the acyl glucuronides of ibuprofen and its α-methyl-substituted analogues investigated by 1H NMR spectroscopy and computational chemistry: Implications for drug design.

Drugs and drug metabolites containing a carboxylic-acid moiety can undergo in vivo conjugation to form 1-ß-O-acyl-glucuronides (1-ß-O-AGs). In addition to hydrolysis, these conjugates can undergo spontaneous acyl migration, and anomerisation reactions, resulting in a range of positional isomers. Facile transacylation has been suggested as a mechanism contributing to the toxicity of acyl glucuronides, with the kinetics of these processes thought to be a factor. Previous 1H NMR spectroscopic and HPLC-MS studies have been conducted to measure the degradation rates of the 1-ß-O-AGs of three nonsteroidal anti-inflammatory drugs (ibufenac, R-ibuprofen, S-ibuprofen) and a dimethyl-analogue (termed here as "bibuprofen"). These studies have also determined the relative contributions of hydrolysis and acyl migration in both buffered aqueous solution, and human plasma. Here, a detailed kinetic analysis is reported, providing the individual rate constants for the acyl migration and hydrolysis reactions observed in buffer for each of the 4 AGs, together with the overall degradation rate constants of the parent 1-ß-O-AGs. Computational modelling of the reactants and transition states of the transacylation reaction using density functional theory indicated differences in the activation energies that reflected the influence of both substitution and stereochemistry on the rate of transacylation/hydrolysis.

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity.

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides.

A host-gut microbial amino acid co-metabolite, p-cresol glucuronide, promotes blood-brain barrier integrity in vivo.

The sequential activity of gut microbial and host processes can exert a powerful modulatory influence on dietary components, as exemplified by the metabolism of the amino acids tyrosine and phenylalanine to p-cresol by gut microbes, and then to p-cresol glucuronide (pCG) by host enzymes. Although such glucuronide conjugates are classically thought to be biologically inert, there is accumulating evidence that this may not always be the case. We investigated the activity of pCG, studying its interactions with the cerebral vasculature and the brain in vitro and in vivo. Male C57Bl/6 J mice were used to assess blood-brain barrier (BBB) permeability and whole-brain transcriptomic changes in response to pCG treatment. Effects were then further explored using the human cerebromicrovascular endothelial cell line hCMEC/D3, assessing paracellular permeability, transendothelial electrical resistance and barrier protein expression. Mice exposed to pCG showed reduced BBB permeability and significant changes in whole-brain transcriptome expression. Surprisingly, treatment of hCMEC/D3 cells with pCG had no notable effects until co-administered with bacterial lipopolysaccharide, at which point it was able to prevent the permeabilizing effects of endotoxin. Further analysis suggested that pCG acts as an antagonist at the principal lipopolysaccharide receptor TLR4. The amino acid phase II metabolic product pCG is biologically active at the BBB, antagonizing the effects of constitutively circulating lipopolysaccharide. These data add to the growing literature showing glucuronide conjugates to be more than merely metabolic waste products and highlight the complexity of gut microbe to host communication pathways underlying the gut-brain axis.

Synthesis, antiviral activity, preliminary pharmacokinetics and structural parameters of thiazolide amine salts.

Background: The thiazolides, typified by nitazoxanide, are an important class of anti-infective agents. A significant problem with nitazoxanide and its active circulating metabolite tizoxanide is their poor solubility. Results: We report the preparation and evaluation of a series of amine salts of tizoxanide and the corresponding 5-Cl thiazolide. These salts demonstrated improved aqueous solubility and absorption, as shown by physicochemical and in vivo measurements. They combine antiviral activity against influenza A virus with excellent cell safety indices. We also report the x-ray crystal structural data of the ethanolamine salt. Conclusion: The ethanol salt of thiazolide retains the activity of the parent together with an improved cell safety index, making it a good candidate for further evaluation.

Discovery and Optimization of a 4-Aminopiperidine Scaffold for Inhibition of Hepatitis C Virus Assembly.

The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced in vitro toxicity, as well as improved in vitro and in vivo ADME properties.

Systemic efficacy on Cryptosporidium parvum infection of aminoxanide (RM-5061), a new amino-acid ester thiazolide prodrug of tizoxanide.

Cryptosporidiosis is a gastrointestinal illness with profuse diarrhoea. Although there are no other Food and Drug Administration (FDA)-approved alternatives for the treatment of cryptosporidiosis, nitazoxanide (NTZ) can be qualified as partially effective. In immunosuppressed conditions, severe and/or disseminated cryptosporidiosis may occur and patients should be treated parenterally. To achieve the goal of developing parenteral treatment for cryptosporidiosis, the current study was undertaken to investigate the in vitro and in vivo anticryptosporidial activity of aminoxanide. This new l-tert-leucyl thiazolide is a soluble prodrug of tizoxanide (TIZ), the main metabolite of NTZ. Confirming the good efficacy of aminoxanide in Cryptosporidium parvum-infected HCT-8 cells with a 50% inhibitory concentration of 1.55 µm (±0.21), in immunosuppressed C. parvum-infected Mongolian gerbils (Meriones unguiculatus), a 5-day treatment with a daily intramuscular dose of 100 mg kg−1 aminoxanide resulted in a 72.5% oocyst excretion inhibition, statistically equivalent to 75.5% in gerbils treated with a 4-fold lower oral dose of NTZ. Cryptosporidium parvum-induced intestinal pathology and inflammation were improved. Aminoxanide provides an injectable form of TIZ that NTZ was unable to do and is a promising drug for which optimization of the formulation should be further explored. These results represent a first promising step towards the goal of developing a parenteral treatment for cryptosporidiosis.

Therapeutic Potential of Nitazoxanide: An Appropriate Choice for Repurposing versus SARS-CoV-2?

The rapidly growing COVID-19 pandemic is the most serious global health crisis since the "Spanish flu" of 1918. There is currently no proven effective drug treatment or prophylaxis for this coronavirus infection. While developing safe and effective vaccines is one of the key focuses, a number of existing antiviral drugs are being evaluated for their potency and efficiency against SARS-CoV-2 in vitro and in the clinic. Here, we review the significant potential of nitazoxanide (NTZ) as an antiviral agent that can be repurposed as a treatment for COVID-19. Originally, NTZ was developed as an antiparasitic agent especially against Cryptosporidium spp.; it was later shown to possess potent activity against a broad range of both RNA and DNA viruses, including influenza A, hepatitis B and C, and coronaviruses. Recent in vitro assessment of NTZ has confirmed its promising activity against SARS-CoV-2 with an EC50 of 2.12 µM. Here we examine its drug properties, antiviral activity against different viruses, clinical trials outcomes, and mechanisms of antiviral action from the literature in order to highlight the therapeutic potential for the treatment of COVID-19. Furthermore, in preliminary PK/PD analyses using clinical data reported in the literature, comparison of simulated TIZ (active metabolite of NTZ) exposures at two doses with the in vitro potency of NTZ against SARS-CoV-2 gives further support for drug repurposing with potential in combination chemotherapy approaches. The review concludes with details of second generation thiazolides under development that could lead to improved antiviral therapies for future indications.

Synthesis and toxicity profile in 293 human embryonic kidney cells of the ß D-glucuronide derivatives of ortho-, meta- and para-cresol.

The formation of ß-glucuronides is a major route by which mammals detoxify and remove breakdown products, such as l-tyrosine, as well as many xenobiotics, from their systems. In humans, dietary l-tyrosine is broken down largely by the action of the anaerobic gut bacterium C. difficile to p-cresol, providing a competitive advantage in the gut microbiota. Ortho- (o-) and meta- (m-), cresols, also present in the environment, may share a common degradative pathway. Relatively little work has been done on cresyl glucuronides. Here, a direct synthesis of o-, m-, and p-cresyl ß-D-glucuronides from methyl 1,2,3,4 tetra-O-acetyl-ß-d-glucuronate and the respective cresol employing trimethylsilyltriflate as promoter is presented. The protected intermediates were hydrolysed using aqueous sodium carbonate to yield the cresyl ß-glucuronides. The toxicities of the o-, m- and p-cresyl ß-D-glucuronides were compared. All three were less toxic to HEK293 cells than their respective cresol precursors: toxicity followed the order o < m < p for Na+ salts and o < p < m for Ca2+ salts. The m-cresyl-glucuronide Ca2+ salt and p-cresyl-glucuronide Na+ salt reduced colony formation by 11% and 9% (v. 30% reduction from the aglycone) respectively, whereas o-cresyl-glucuronide (both Na+ and Ca2+ salts), mildly stimulated HEK293 cell growth.

Acyl glucuronide reactivity in perspective.

Acyl glucuronidation is a common metabolic fate for acidic drugs and their metabolites and, because these metabolites are reactive, they have been linked to adverse drug reactions (ADRs) and drug withdrawals. However, alternative routes of metabolism leading to reactive metabolites (e.g., oxidations and acyl-CoA thioesters) mean that unambiguous proof that acyl glucuronides are toxic is lacking. Here, we review the synthesis and reactivity of these metabolites, and describe the use of molecular modelling and in vitro and in vivo reactivity assessment of acyl glucuronide reactivity. Based on the emerging structure-dependent differences in reactivity and protein adduction methods for risk assessment for acyl glucuronide-forming acid drugs or drug candidates in drug discovery/development are suggested.

Kinetic modelling of acyl glucuronide and glucoside reactivity and development of structure-property relationships.

Acyl glucuronide metabolites have been implicated in the toxicity of several carboxylic acid-containing drugs, and the rate of their degradation via intramolecular transacylation and hydrolysis has been associated with the degree of protein adduct formation. Although not yet proven, the formation of protein adducts in vivo - and subsequent downstream effects - has been proposed as a mechanism of toxicity for carboxylic acid-containing xenobiotics capable of forming acyl glucuronides. A structurally-related series of metabolites, the acyl glucosides, have also been shown to undergo similar degradation reactions and consequently the potential to display a similar mode of toxicity. Here we report detailed kinetic models of each transacylation and hydrolysis reaction for a series of phenylacetic acid acyl glucuronides and their analogous acyl glucosides. Differences in reactivity were observed for the individual transacylation steps between the compound series; our findings suggest that the charged carboxylate ion and neutral hydroxyl group in the glucuronide and glucoside conjugates, respectively, are responsible for these differences. The transacylation reaction was modelled using density functional theory and the calculated activation energy for this reaction showed a close correlation with the degradation rate of the 1-ß anomer. Comparison of optimised geometries between the two series of conjugates revealed differences in hydrogen bonding which may further explain the differences in reactivity observed. Together, these models may find application in drug discovery for prediction of acyl glucuronide and glucoside metabolite behaviour.

Synthesis of MeBmt and related derivatives via syn-selective ATH-DKR.

The unusual α-amino, ß-hydroxy acid MeBmt is a key structural feature of cyclosporin A, an important naturally occurring immunosuppressant and antiviral agent. We present a convergent synthesis of MeBmt which relies on new aspects of dynamic kinetic resolution (DKR) to establish simultaneously the chirality at C(2) and C(3). We also show that this route is applicable to the synthesis of other derivatives.

Second-generation nitazoxanide derivatives: thiazolides are effective inhibitors of the influenza A virus.

AIM: The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza. RESULTS: We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC50s in the range of 0.14-5.0 µM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. CONCLUSION: A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified.

Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis.

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ∼100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ∼90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC50 = 525 nM, Mtb Wayne IC50 = 76 nM, and MDR Mtb patient isolates IC50 = 140 nM) and favorable pharmacokinetic and toxicological profiles.

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.

Mass spectrometric characterization of circulating covalent protein adducts derived from a drug acyl glucuronide metabolite: multiple albumin adductions in diclofenac patients.

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-: derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-: related hypersensitivities had either a single drug-: derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.

Convenient syntheses of halo-dibenz[b,f]azepines and carbamazepine analogues via N-arylindoles.

The dibenz[b,f]azepine heterocyclic system and related molecules with a single 10,11-bond are important templates for well-prescribed drug molecules, notably carbamazepine (anticonvulsant), clomipramine and imipramine (antidepressants). We synthesised a range of halogenated carbamazepine analogues, in connection with metabolic and immunological studies, as probes for structure-metabolism and hypersensitive effects and have published on their metabolic behaviour. While a number of synthetic routes to such analogues are possible, we naturally sought short and efficient methods for our target compounds. In the following report we present an effective two-step synthesis of a range of dibenz[b,f]azepines from appropriate indoles via N-arylation, then acid-catalysed rearrangement, with a critical analysis of other approaches. We showed earlier that this route was effective for fluoro analogues and here present a broader review of its scope. The 5-(carboxamido) side chain of carbamazepine may be added in various ways, affording overall a convenient access to drug molecules.

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.

The generation, detection, and effects of reactive drug metabolites.

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.

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.