Silvio Aprile

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.

Regioselective Suzuki Coupling of Dihaloheteroaromatic Compounds as a Rapid Strategy To Synthesize Potent Rigid Combretastatin Analogues

Combretastatin A-4 (CA-4) is a potent tubulin depolymerizing agent able to inhibit tumor growth and with antivascular effects. Although it is in clinical trials, the search for novel analogues that may display better/different features is still ongoing. In this manuscript we describe the synthesis of novel constrained analogues of CA-4 obtained in only two synthetic steps exploiting a regioselective Suzuki coupling of dihalogenated heteroaromatic and alicyclic compounds. All the compounds synthesized have been evaluated for cytotoxicity and for their ability to inhibit tubulin assembly. One of them, 38, displayed low nanomolar cytotoxicity and proved to have a pharmacodynamic profile similar to that of CA-4 and a better pharmacokinetic profile, but most important of all, this synthetic strategy may pave the way for the easy and rapid generation of novel rigid analogues of combretastatins.

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.

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.

Design, Synthesis, and Biological Evaluation of Combretabenzodiazepines: A Novel Class of Anti-Tubulin Agents

In the present manuscript, starting from the 1,4-benzodiazepin-2-one nucleus, a privileged structure in medicinal chemistry, we have synthesized a novel class of cis-locked combretastatins named combreatabenzodiazepines. They show similar cytotoxic and antitubulin activity compared to combretastatin A-4 in neuroblastoma cells, showing a better pharmacokinetic profile. This class of compounds has therefore the potential for further development as antitubulin agents.

A Concise Synthesis of Pyrazole Analogues of Combretastatin A1 as Potent Anti‐Tubulin Agents

Combretastatin A1 (CA1) binds to the β‐subunit at the colchicine binding site of tubulin and inhibits polymerization. As such, it is both an antitumor agent and a vascular disrupting agent. It has been shown to be at least tenfold more potent than combretastatin A4 (CA4) in terms of vascular shutdown, which correlates with its metabolism to reactive ortho‐quinone species that are assumed to be directly cytotoxic in tumor cells. A series of 3,4‐diarylpyrazoles were concisely synthesized, one of which, 3‐methoxy‐6‐[4‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazol‐3‐yl]benzene‐1,2‐diol (27), proved to be a cytotoxic anti‐tubulin agent with low nanomolar potency. We also report that combretastatins, including CA1, CA4, and 27, are effective against mesothelioma cell lines and therefore have significant clinical promise. Metabolism experiments demonstrate that 27 retains the ability to form ortho‐quinone species, while the pyrazole ring shows high metabolic stability, suggesting that this compound might result in better pharmacokinetic profiles than CA1, with similar pharmacodynamic properties and clinical potential.

Development and validation of a stability-indicating HPLC-UV method for the determination of alizapride and its degradation products

A stability-indicating high-performance liquid chromatography procedure has been developed for the determination of alizapride (AL) and its main degradation products alizapride carboxylic acid (AL-CA) and alizapride N-oxide (AL-NO2) in drug substance and product. The method was developed based on forced degradation data obtained by HPLC-MS analysis. Indeed AL underwent chemical degradation by acid/base catalyzed hydrolysis and oxidation the main degradation products being AL-CA and AL-NO2 respectively. The separation and quantisation were achieved on a 150-mm reverse phase column with a hydrophilic linkage between silica particles and hydrophobic alkyl chains. The mobile phase was constituted (flow rate 1.5mLmin(-1)) of eluant A: aqueous acetate buffer (pH 4.0; 20mM) and eluant B: CH(3)OH using a gradient elution and detection of analytes at 225nm. The method showed good linearity for the AL, AL-CA, AL-NO2 mixture in the 25-75, 1-15 and 1-15microgmL(-1) ranges respectively, being all the square of the correlation coefficients greater than 0.999. The precision, determined in terms of intra-day and inter-day precisions and expressed as RSDs were 0.8, 1.3 and 2.1% and 1.0, 1.7, 4.8% for AL, AL-CA and AL-NO2 respectively. The method demonstrated also to be accurate; indeed the average recoveries, at 100% and 0.2% of the target assay concentration, were 100.5, 98.6, and 96.8% for AL, AL-CA and AL-NO2 respectively. The robustness was also evaluated by variations of mobile phase composition and pH. Finally, the applicability of the method was evaluated in commercial dosage form analysis as well as in stability studies.

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 and in vivo phase II metabolism of combretastatin A-4: Evidence for the formation of a sulphate conjugate metabolite

Combretastatin A-4 (CA-4), is a natural compound with a potent tubulin polymerization and cell growth inhibitor properties. For these reasons CA-4 is one of the most potent anti-vascular agents that shows strong cytotoxicity against a variety of human cancer cells, including multi-drug-resistant cancer cell lines. In order to complete the knowledge of metabolic fate of CA-4, the in vitro and in vivo phase II metabolism was investigated. Both in incubation with rat and human liver S9 preparation in the presence of 39-phosphoadenosine-5´-phosphosulfate (PAPS) as a cofactor the formation of a previously no reported sulphate metabolite was demonstrated through liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) data and comparison with a synthetic reference sample. In incubation of CA-4 using rat and human liver microsomes, the formation of CA-4 glucuronide was observed and chromatographic and mass spectral properties of the metabolite were achieved and compared with those of a synthetic reference sample. Incubation of CA-4 with rat and human liver S9 preparation in the presence of uridine-5´-diphosphoglucuronic acid trisodium salt (UDPGA) and an β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system as cofactors resulted in the formation of glucuronides arising from phase I CA-4 metabolites. When CA-4 was administered intraperitoneally to rat at a dose of 30 mg kg−1, both glucuronide and sulphate metabolites were observed in LC-ESI-MS-MS chromatograms and their mass spectral data were identical to those obtained from synthetic standards.