Synthesis and Biological Evaluation of N-[2-(4-Hydroxyphenylamino)-pyridin-3-yl]-4-methoxy-benzenesulfonamide (ABT-751) Tricyclic Analogues as Antimitotic and Antivascular Agents with Potent in Vivo Antitumor Activity.

Benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) were modified to produce tricyclic quinazolinone 15-18 or benzothiadiazine 26-27 derivatives. These compounds were evaluated in cytotoxicity and tubulin inhibition assays and led to potent inhibitors of tubulin polymerization. N-[2(4-Methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a) exhibited the best in vitro cytotoxic activity (GI50 10-66.9 nM) against the NCI 60 human tumor cell line and significant potency against tubulin assembly (IC50 0.812 µM). In mechanism studies, 16a was shown to block cell cycle in G2/M phase and to disrupt microtubule formation and displayed good antivascular properties as inhibition of cell migration, invasion, and endothelial tube formation. Compound 16a was evaluated in C57BL/6 mouse melanoma B16F10 xenograft model to validate its antitumor activity, in comparison with reference ABT-751 (1). Compound 16a displayed strong in vivo antitumor and antivascular activities at a dose of 5 mg/kg without obvious toxicity, whereas 1 needed a 10-fold higher concentration to reach similar effects.

Synthesis of triazoloquinazolinone based compounds as tubulin polymerization inhibitors and vascular disrupting agents.

A series of 1-phenyl-[1,2,4]triazolo[4,3-a]quinazolin-5-ones designed as conformationally restricted CA-4 analogues, were tested for their tubulin polymerization and growth inhibitory activities. The 3-hydroxy-4-methoxy derivatives 11d and 12d are potent inhibitors of tubulin assembly but only the N-methylated amid counterpart 12d possesses potent anticancer activity in a large panel of cancer cell lines. Upon treatment with compound 12d, remarkable cell shape changes as cell migration and tube formation were elicited in HUVECs, consistent with vasculature damaging activity.

In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: identification of phases I and II metabolites.

In the present study, the in vitro metabolic behavior of a benzopyridooxathiazepine (BZN), a potent tubulin polymerization inhibitor, was investigated by liquid chromatography-UV detection (LC-UV). First, simple and fast LC-UV methods have been optimized and validated to evaluate the pharmacokinetic profile of BZN using rat liver microsomes or hepatocytes primary cultures suspensions. Whatever the medium investigated, baseline resolution between the internal standard and BZN was achieved in a run time less than 15min using a Symmetry ODS column (150mm×4.6mm i.d., 5µm) and a mobile phase consisting of acetonitrile/water/formic acid 60:40:0.1 (v/v/v). Linearity was assessed in the 0.1-50µM and in the 0.05-5µM concentration ranges, respectively, in microsomal and hepatocyte matrix. According to the novel strategy based on the build of the accuracy profile, total error of the developed methods was included within the ±10% limits of acceptance. Then, from incubation of BZN with both liver microsomes and or hepatocytes, structural informations on phase I and phase II metabolites were acquired using liquid chromatography coupled to electrospray orbitrap mass spectrometer (LC-MS). Mass spectrum, double bond equivalent and elemental composition were useful data to access to the chemical structure of each metabolite. In microsomal suspension, four main metabolites were observed including monohydroxylation and dihydroxylation of the benzopyridooxathiazepine core, demethylation of the methoxyphenyl moiety, as well as their combinations. The phase II metabolites detected in hepatocytes suspension were the glucuronide adducts of both demethylated BZN and mono-oxygenated BZN. Based on the structural elucidation of the metabolites detected, we proposed an in vitro metabolic pathway of BZN, a new tubulin polymerization inhibitor.

Synthesis, antiproliferative activity and tubulin targeting effect of acridinone and dioxophenothiazine derivatives.

The synthesis of new acridinone and dioxophenothiazine derivatives along with their tubulin polymerization inhibitory and antiproliferative activities is reported. The analysis of correlation for cytotoxic and antitubulin potential of tested compounds showed that 4-methoxyphenylethyl derivatives 18a and 19a were highly cytotoxic but were regarded to have no significant antitubulin activity. However, the introduction of a 3-hydroxy substituent leading to compounds 18e and 19e, strongly increased the antitubulin potential but was associated with a loss of the antiproliferative activity. Modeling studies, topoisomerase inhibition assays and cell cycle analysis have been performed to better investigate the mechanism of action of such compounds.

Structural elucidation of degradation products of a benzopyridooxathiazepine under stress conditions using electrospray orbitrap mass spectrometry - study of degradation kinetic.

1-(4-Methoxyphenylethyl)-11H-benzo[f]-1,2-dihydro-pyrido[3,2,c][1,2,5]oxathiazepine 5,5 dioxide (BZN) is a cytotoxic derivative with very promising in vitro activity. Regulatory authority for registration of pharmaceuticals for human use requires to evaluate the stability of active compound under various stress conditions. Forced degradation of BZN was investigated under hydrolytic (0.1M NaOH, 0.1M HCl, neutral), oxidative (3.3% H(2)O(2)), photolytic (visible light) and thermal (25 °C, 70 °C) settings. Relevant degradation took place under thermal acidic (0.1M HCl, 70 °C) and oxidative (3.3% H(2)O(2)) conditions. Liquid chromatography-mass spectrometry (LC-MS) analyses revealed the presence of ten degradation products whose structures were characterized by electrospray ionization-orbitrap mass spectrometry. The full scan accurate mass analysis of degradation products was confirmed or refuted using three tools furnished by the MS software: (1) predictive chemical formula and corresponding mass error; (2) double bond equivalent (DBE) calculation; and (3) accurate mass product ion spectra of degradation products. The structural elucidation showed that the tricycle moiety was unstable under thermal acidic and oxidative conditions since four degradation products possess an opened oxathiazepine ring. Then, a simple and fast HPLC-UV method was developed and validated for the determination of the degradation kinetic of BZN under acidic and oxidative conditions. The method was linear in the 5-100 µg mL(-1) concentration range with a good precision (RSD=2.2% and 2.7% for the repeatability and the intermediate precision, respectively) and a bias which never exceeded 1.6%, whatever the quality control level. With regards to the BZN concentration, a first-order degradation process was determined, with t(1/2)=703 h and 1140 h, under oxidative and acidic conditions, respectively.

Synthesis, biological evaluation and docking studies of 4-amino-tetrahydroquinazolino[3,2-e]purine derivatives.

The synthesis of new 4-amino-tetrahydroquinazolino[3,2-e]purine derivatives along with their activity in cell-free enzymatic assays on Src is reported. Some compounds emerged as moderately active inhibitors of the enzyme and showed antiproliferative effects on the murine leukemia L1210 cell line. Docking studies have been also performed to analyze the binding mode of compounds under study and to identify the structural determinants of their interaction. Therefore, this study provides a new promising scaffold with moderate enzymatic inhibitory activities for further development of new anticancer drugs targeting Src tyrosine kinase.