Erika Del Grosso

In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes.

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6′ of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.

Are 1,4- and 1,5-disubstituted 1,2,3-triazoles good pharmacophoric groups?

Over the last decade, 1,2,3‐triazoles have received increasing attention in medicinal chemistry thanks to the discovery of the highly useful and widely applicable 1,3‐dipolar cycloaddition reaction between azides and alkynes (click chemistry) catalyzed by copper salts and ruthenium complexes. After a decade of medicinal chemistry research on 1,2,3‐triazoles, we feel that the time is ripe to demonstrate the real ability of this heterocycle to participate in important and pivotal binding interactions with biological targets while maintaining a good pharmacokinetic profile. In this study, we retrieved and analyzed X‐ray crystal structures of complexes between 1,2,3‐triazoles and either proteins or DNA to understand the pharmacophoric role of the triazole. Furthermore, the metabolic stability, the capacity to inhibit cytochromes, and the contribution of 1,2,3‐triazoles to the overall aqueous solubility of compounds containing them have been analyzed. This information should furnish fresh insight for medicinal chemists in the design of novel bioactive molecules that contain the triazole nucleus.

Solution-Phase Parallel Synthesis and Biological Evaluation of Combretatriazoles

Combretastatin A-4 is an antitumoral and antitubulin agent that is active only in its cis configuration. In the present manuscript, we have synthesized cis-locked combretastatins containing a triazole ring (combretatriazoles). To achieve this, we have developed a column chromatography-free parallel solution-phase synthesis that exploits the reaction between azides and alpha-keto phosphorus ylids, which is known to regioselectively generate the 1,5-disubstituted triazoles. The prepared compounds were screened as antitubulinic agents, allowing us to identify three new compounds with high potency, two of which show a new mechanism of action that induces cells to appear multinucleated and display a high number of mitotic spindles.

IDENTIFICATION OF THE HUMAN UDP-GLUCURONOSYLTRANSFERASES INVOLVED IN THE GLUCURONIDATION OF COMBRETASTATIN A-4.

The stilbenic compound (Z)-combretastatin A-4 (CA-4) has been described as a potent tubulin polymerization inhibitor. In vivo, CA-4 binds to tubulin and inhibits microtubule depolymerization, which results in morphological changes in proliferating endothelial cells. Combretastatin A-4 prodrug phosphate is a leading vascular disrupting agent and is currently being evaluated in multiple clinical trials as a treatment for solid tumors. The aim of this study was to identify and characterize the UDP-glucuronosyltransferase (UGT) isoforms involved in CA-4 glucuronidation by incubation with human liver microsomes and a panel of nine liver-expressed recombinant UGT Supersomes (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17). As we observed, the high rate of formation of CA-4 glucuronide (Vmax = 12.78 ± 0.29 nmol/min/mg protein) and the low Km (6.98 ± 0.65 μM) denoted that UGT1A9 was primarily responsible for the in vitro glucuronidation of CA-4. UGT1A6 was also a significant contributor to CA-4 glucuronidation (Vmax = 3.95 ± 0.13 nmol/min/mg protein and S50 = 44.80 ± 3.54 μM). Furthermore, we demonstrated that the kinetics of CA-4 glucuronidation with liver microsomes but also with a panel of recombinant UGTs is atypical as it fits two different models: the substrate inhibition and also the sigmoidal kinetic model. Finally, experiments conducted to inhibit the glucuronosyltransferase activity in the human liver microsomes assay showed that phenylbutazone, trifluoperazine, propofol, and 1-naphthol effectively inhibited CA-4 glucuronidation.

The metabolic fate of isocombretastatin A-4 in human liver microsomes: identification, synthesis and biological evaluation of metabolites.

Combretastatins, naturally occurring stilbenes, were isolated from Combretum caffrum by Pettit in 1989. Among them, combretastatin A-4 (CA-4) was shown to strongly inhibit the polymerization of tubulin by binding to the colchicine binding site and was the most cytotoxic agent. CA-4 showed toxicity at a nanomolar level against a variety of human cancer cell lines, including multiple drug-resistant cancers. CA-4 also exerts highly selective toxicity in proliferating endothelial cells and, as a consequence, demonstrates strong suppressive activity on tumor blood flow leading to cell death. Moreover, in contrast to colchicine, the antivascular effects of CA-4 are apparent well below the maximal tolerated dose, offering a wide therapeutic window. However, there are two major disadvantages with CA4 as a drug candidate: its low aqueous solubility limits its efficacy in vivo, and its low chemical stability due to the cisdouble bond isomerization to the more thermally stable but nonactive trans form during storage and administration. The first problem was solved by the synthesis of water-soluble prodrugs, including the disodium phosphate prodrug CA-4P (1) 5] and its serinamido derivative AVE-8062 (2). Currently, CA-4P, either as a single agent or in combination therapy, is undergoing several advanced clinical trials worldwide for the treatment of age-related macular degeneration (AMD) or anaplastic thyroid cancer. To overcome the second problem related to the double bond isomerization, several approaches have been undertaken to rigidify the olefin using a heterocyclic compound. In an ongoing project aimed at developing novel tubulin assembly inhibitors, we recently discovered isocombretastatin A-4 (isoCA-4), a structural isomer of the natural product that displays biological activities comparable to those of CA-4. isoCA-4 contains a 1,1-diarylethylene scaffold and is easy to synthesize on a multigram scale without the need to control the olefin geometry, and then definitively solves the problem of the double bond isomerization. Recently, we studied the metabolic fate of CA-4 in rat and human microsomal preparations, and several phase I metabolites (resulting from O-demethylation, aromatic hydroxylation and quinones formation), as well as phase II conjugates, in both cis and trans configuration, were characterized. The role of these metabolites in the pharmacodynamic activity of CA-4 is still unknown. These considerations together with the structural differences between CA-4 and isoCA-4, due to the presence of a 1,1-diarylethylene scaffold, prompted us to examine the metabolic profile of isoCA-4, identifying and synthesizing the metabolites, and then evaluating their biological properties. Herein, we report the results of this study. Possible metabolites of isoCA-4, demethylated/hydroxylated on the Aand B-rings, hydroxylated on the B-ring, or transformed into p-quinones, were prepared according to Scheme 1. Hypothetical metabolites 3–6 arising from O-demethylation or hydroxylation of the Aand B-rings were prepared by the coupling of N-tosylhydrazones with aryl iodides under palladium catalysis. Then, the crude coupling products were O-desilylated using a tetrahydrofuran (THF) solution of tetra-nbutylammonium fluoride (TBAF) to afford the desired phenols 3 and 4, as well as catechols 5 and 6, in good yields. The use of Fremy’s salt as a biomimetic oxidant with isoCA-4 in a mixture of water and dichloromethane furnished p-quinone 7 in an acceptable yield (50 %). Finally, the reduction of 7 using sodium borohydride (NaBH4) afforded the putative metabolite 8, which was found to be unstable and oxidizes to p-quinone 7 in air. This compound must be prepared carefully just before use and stored under an inert atmosphere at 40 8C. isoCA-4 was incubated in human liver microsomal (HLM) fractions in the presence of a NADPH regenerating system. isoCA-4 and its metabolites were separated by HPLC using a C18 reverse-phase column and a mixture of water and acetonitrile acidified with 1 % formic acid as the eluent. The LC/MS [a] M. A. Soussi, Dr. S. Messaoudi, Dr. O. Provot, Prof. Dr. J.-D. Brion, Dr. M. Alami Univ Paris-Sud, Facult de Pharmacie, CNRS, BioCIS-UMR 8076 Laboratoire de Chimie Th rapeutique 5 Rue J.-B. Cl ment, Ch tenay-Malabry 92296 (France) E-mail : mouad.alami@u-psud.fr [b] Dr. S. Aprile, Dr. E. Del Grosso, Dr. G. Grosa Dipartimento di Scienze Chimiche Alimentari Farmaceutiche e Farmacologiche and Drug and Food Biotechnology Center Universit degli Studi del Piemonte Orientale “A. Avogadro” Largo Donegani 2, 28100 Novara (Italy) E-mail : Giorgio.Grosa@pharm.unipmn.it [c] Dr. J. Bignon, Dr. J. Dubois Institut de Chimie des Substances Naturelles, UPR 2301, CNRS Avenue de la Terrasse, 91198 Gif-sur-Yvette (France) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100193.

Metabolic fate of combretastatin A-1: LC-DAD-MS/MS investigation and biological evaluation of its reactive metabolites.

Combretastatin A-1, an antineoplastic agent characterized by a remarkable cytotoxicity and a strong antitubulinic activity, is currently under investigation in phase I clinical trials. Yet a comprehensive metabolic study of CA-1 in human and animal models has so far not been reported in the literature. We have therefore investigated, through LC-UV and LC-MS analysis the in vitro/in vivo metabolism of CA-1, have synthesized its reactive quinone metabolite Q1, and have evaluated its cytotoxic and antitubulinic activities. In vitro CA-1 metabolism was studied in rat and human liver microsomes in the presence of the nucleophilic trapping agent GSH were performed while urine samples of the CA-1-treated rats were analyzed to establish the in vivo metabolic pathways. The metabolite Q1, that was synthesized in good yield using a polymer supported oxidant, displayed a significant cytotoxicity but was devoid of significant antitubulinic activity. Finally the chemical interaction of Q1 and the other combretastatin quinone metabolites with sulphydryl groups of tubulin was measured by Ellmans method: the results suggested the haptenization of the tubulin through the formation of a covalent bond.

In vitro metabolism study of 2-isopropyl-9H-thioxanthen-9-one (2-ITX) in rat and human: evidence for the formation of an epoxide metabolite

2-Isopropyl-9H-thioxanthen-9-one (2-ITX) is one of the most extensively used photoinitiators in inks found in paper and/or packaging materials for foodstuffs. Recently, traces of 2-ITX as a contaminant were discovered in baby milk and other foodstuffs at a level sufficient to pose a risk to human health. However, little is known about the toxicological profile of 2-ITX. The high lipophilicity of this substance would suggest that it could be a good substrate for hepatic metabolizing enzymes and that these metabolites could have a role in the toxicological properties of 2-ITX. The metabolism of 2-ITX, using both rat and human subcellular preparations, was studied and has resulted in the formation of eight polar metabolites (M1–M8) as revealed in the liquid chromatograpy/ultraviolet (LC/UV) and liquid chromatograpy/mass spectrometry (LC/MS) analyses. Their structures highlight a marked regioselectivity in the metabolism of 2-ITX; it is directed mainly toward the isopropyl moiety and the sulfur atom. The unsaturated metabolite 6 causes the formation of a reactive epoxide metabolite 7. This finding was supported by identification in microsomal incubations of 1,2-diol metabolite 8 arising from the epoxide by hydrolysis and it was validated by incubating in the same conditions the synthetic epoxide 7: the formation of metabolite 8 was again observed. On the basis of these data, we propose that the metabolite 6 could be included in toxicological studies of 2-ITX.

Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

Solution-Phase Parallel Synthesis of Aryloxyimino Amides via a Novel Multicomponent Reaction among Aromatic (Z)-Chlorooximes, Isocyanides, and Electron-Deficient Phenols

A library of 41 aryloxyimino amides was prepared via solution phase parallel synthesis by extending the multicomponent reaction of (Z)-chlorooximes and isocyanides to the use of electron-deficient phenols. The resulting aryloxyiminoamide derivatives can be used as intermediates for the synthesis of benzo[d]isoxazole-3-carboxamides, dramatically reducing the number of synthetic steps required by other methods reported in literature.

In vitro metabolic fate of alizapride: evidence for the formation of reactive metabolites based on liquid chromatography-tandem mass spectrometry.

The study of the formation of reactive metabolites during drug metabolism is one of the major areas of research in drug development since the link between reactive metabolites and drug adverse effects was well recognized. In particular, it has been shown that acrolein, a reactive carbonyl species sharing carbonylating and alkylating properties, binds covalently to nucleophilic sites in proteins, causing cellular damage. Alizapride, (±)-6-methoxy-N-{[1-(prop-2-en-1-yl)-pyrrolidin-2-yl]methyl}-1H-benzotriazole-5-carboxamide, is a N-allyl containing dopamine antagonist with antiemetic properties for which no data concerning its metabolic fate are so far reported. The study of the in vitro metabolism of alizapride showed the formation of acrolein during the oxidative N-deallylation. Moreover, the formation of an epoxide metabolite has been also described suggesting its role as a putative structural alert. The reactivity of the acrolein and the epoxide generated in alizapride metabolism was demonstrated by the formation of the corresponding adducts with nucleophilic thiols. Overall, ten metabolites have been identified and characterized by electrospray ionization tandem mass spectrometry analysis allowing to propose an in vitro metabolic scheme for alizapride. At the best of our knowledge, this is the second case of a drug involved in the generation of acrolein during its metabolism being the first represented by cyclophosphamide.