Zacharie Segaoula

Julien Leclercq

Valérie Verones

Nathalie Flouquet

Lionel Ach

Nicolas Renault

Amélie Barczyk

Patricia Melnyk

Pascal Berthelot

Xavier Thuru

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

Keywords: Quinazolinone, benzothiadiazine, ABT-751, tubulin polymerization, vascular disrupting agent

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

, 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, 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.

Introduction

Microtubules provide an important framework supporting cellular morphology in interphase, they are the major components of the mitotic spindle which allows the controlled segregation of the chromosomes during mitosis. Spindle poisons are described to interfere with the dynamic instability of microtubules and to induce a cell cycle arrest in the G2/M phase, promoting a mitotic catastrophe and finally causing cell death via apoptosis. 1 Consequently, the microtubule has become an important target for the design of new antimitotic anticancer agents. Amongst these antitumor agents, two maJor groups have been well characterized, microtubule stabilizers such as paclitaxel [START_REF] Schiff | Promotion of microtubule assembly in vitro by taxol[END_REF] and microtubule destabilizers such as vinca alkaloids, 3 colchicine, [START_REF] Li | Chapter 14. Antimitotic agents[END_REF] combretastatin A-4 (CA-4), [START_REF] Nam | Combretastatin A-4 analogues as antimitotic antitumor agents[END_REF] and sulfonamide ABT-751 (1). [START_REF] Yoshino | Novel sulfonamides as potential, systemically active antitumor agents[END_REF] Compound 1 is an orally bioavailable sulfonamide molecule that interacts with the colchicine binding site on β-tubulin, inhibiting microtubule polymerization. It exhibits a broad-spectrum antitumor activity in vitro in a large panel of cancer cell lines including those showing resistance to other antimicrotubule agents. [START_REF] Yoshimatsu | Mechanism of action of E7010, an orally active sulfonamide antitumor agent: inhibition of mitosis by binding to the colchicine site of tubulin[END_REF] In addition, 1 displays antivascular activity, reducing tumor-associated blood flow and thereby enhancing cytotoxic effects with little influence on normal vasculature. [START_REF] Segreti | Tumor selective antivascular effects of the novel antimitotic compound ABT-751: an in vivo rat regional hemodynamic study[END_REF] Compound 1 has shown excellent antitumor activity against a variety of xenograft models, such as non-small cell lung cancer (NSCLC) and colon cancer. [START_REF] Jorgensen | Chemosensitization and radiosensitization of human lung and colon cancers by antimitotic agent, ABT-751, in athymic murine xenograft models of subcutaneous tumor growth[END_REF] As part of our program aiming at the identification of novel cytotoxic agents, a series of benzopyridothiadiazepine (2a) and benzopyridooxathiazepine (2b) derivatives showing antitubulin polymerization activities, that could be considered as constraint analogs of 1, and with potent in vitro and moderately in vivo antitumor activities have been earlier described (Figure 1). [START_REF] Lebegue | Novel benzopyridothiadiazepines as potential active antitumor agents[END_REF][START_REF] Gallet | Benzopyridooxathiazepine derivatives as novel potent antimitotic agents[END_REF] The relatively low in vivo antitumor activity of compound 2b1, has been explained by a lack of chemical and metabolic stability while it displayed good oral bioavailability (F,%: 54.2). [START_REF] Lecoeur | Structural elucidation of degradation products of a benzopyridooxathiazepine under stress conditions using electrospray orbitrap mass spectrometry -Study of degradation kinetic[END_REF][START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF][START_REF] Bourdon | Pharmacokinetic evaluation of a novel benzopyridooxathiazepine derivative as a potential anticancer agent[END_REF] In order to improve the ADME properties, we have then extended our studies to the synthesis of compounds bearing heterocyclic privileged structures which were important scaffolds in medicinal chemistry and in agreement with the structure-activity relationships previously established. [START_REF] Gallet | Benzopyridooxathiazepine derivatives as novel potent antimitotic agents[END_REF] A beneficial approach in medicinal chemistry consists of exploration of privileged structures as new tolls in drug discovery. [START_REF] Evans | Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists[END_REF][START_REF] Horton | The combinatorial synthesis of bicyclic privileged structures or privileged substructures[END_REF] Privileged scaffolds enhance the hit rates of therapeutic targets, leading to compounds with improved drug-like properties and could contribute to the discovery of other biologically targets of interest. [START_REF] Horton | The combinatorial synthesis of bicyclic privileged structures or privileged substructures[END_REF][START_REF] Desimone | Privileged structures: applications in drug discovery[END_REF] Quinazolinone is an heterocyclic core found in a variety of biological active agents with a broad range of activities (antibacterial, antifungal, antiviral, anticonvulsant, antitumor, sedative, antihypertensive…) that have been recently described. [START_REF] Sharma | Quinazolinone analogs as potential therapeutic agents[END_REF][START_REF] Khan | Synthetic approaches, functionalization and therapeutic potential of quinazoline and quinazolinone skeletons: the advances continue[END_REF] Moreover, Quinazolinone skeleton has been demonstrated to generate antimitotic agents, bearing their antitumor activity through the inhibition of the DNA repair processes or by the dysregulation of cell cycle progression of cancer cells. [START_REF] Hamel | Antitumor 2,3dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin[END_REF][START_REF] Yang | Selective induction of G2/M arrest and apoptosis in HL-60 by a potent anticancer agent, HMJ-38[END_REF] Among the various classes of antimitotics, quinazolin-4-ones constitute an important part of well-established pharmacologically active compounds as they are associated with inhibitory effects on tubulin polymerization and the anticancer activities of 2-styrylquinazolin-4-ones, [START_REF] Jiang | Synthesis and biological evaluation of 2-styrylquinazolin-4(3H)-ones, a new class of antimitotic anticancer agents which inhibit tubulin polymerization[END_REF] 2-aryl and 2,3dihydro-2-arylquinazolin-4-ones [START_REF] Hamel | Antitumor 2,3dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin[END_REF][START_REF] Yang | Selective induction of G2/M arrest and apoptosis in HL-60 by a potent anticancer agent, HMJ-38[END_REF][START_REF] Hour | 6-Alkylamino-and 2,3-dihydro-3'-methoxy-2-phenyl-4-quinazolinones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization[END_REF] and tetrahydropyrido [2,1-b]quinazolin-10-ones [START_REF] Liu | Privileged structure-based quinazolinone natural product-templated libraries: identification of novel tubulin polymerization inhibitors[END_REF] . From these considerations, the quinazolinone skeleton could be considered as a privileged structure.

Therefore, this paper describes the synthesis of five or six-membered rings containing nitrogen atoms based on 2,3-fused quinazolin-4-one systems (15-18) and their sulfonyl benzothiadiazine analogues (26-27) as novel, highly potent tubulin polymerization inhibitors.

Results and Discussion

Chemistry. The synthetic route to 2,3-fused quinazolin-4-one derivatives 15-18 is shown in Scheme 1. The 2-chloro-3H-quinazolin-4-one key intermediate 6 was synthesized by using the general procedure described by Hess and co-workers. [START_REF] Hess | Antihypertensive 2-amino-4(3H)-quinazolinones[END_REF] Commercial available anthranilamide 3 was first treated with potassium cyanate to yield quinazoline-2,4-dione 4 which reacted with POCl3 in the presence of N,N-dimethylaniline to provide 2,4-dichloroquinazoline 5 in 88% yield.

Selective hydrolysis of 5 with 2% aqueous sodium hydroxide solution at 30°C gave the 2-chloro-3H-quinazolin-4-one 6 in 83% yield. Displacement of the chlorine with amino acetaldehyde or 3aminopropionaldehyde diethyl acetal followed by treatment with concentrated sulfuric acid allowed the linear cyclisation to 1H-imidazo [2,1-b]quinazolin-5-one 9 and 1,2-dihydro-1Hpyrimidino [2,1-b]quinazolin-6-one 10 with respectively 60% and 89% yield. [START_REF] Murdoch | Synthesis of [1,2,4]triazoloquinazolinones and imidazoquinazolinones[END_REF] Arylethyl methanesulfonates 14a-d were obtained in three steps from the corresponding benzaldehydes by first, Wittig olefination with methyltriphenylphosphonium iodide followed by hydroboration leading to primary alcohols 13a-d which finally reacted with methanesulfonyl chloride.

Treatment of compounds 9 and 10 with sodium hydride in anhydrous DMF followed by addition of the arylethyl methanesulfonates 14a-d provided the corresponding alkylated derivatives 15a-d and 16a-d in low yields resulting in a competition between substitution and elimination reactions. Hydrogenation of the double bond but also hydrogenolysis of the benzyl protection of compounds 15d and 16d was carried out by decomposition of ammonium formate in the presence of palladium catalyst in ethanol leading to compounds 17a-d and 18a-d in moderate yields. Access to unsaturated -phenol derivatives 15e and 16e was performed by cleavage of the benzyl ether group in strong acid conditions of the acid-insensitive substrates 15d and 16d.

The benzothiadiazine analogues 26 and 27 were synthesized following the general procedures as detailed below (Scheme 2). 2-Methylthio-imidazolidine hydriodide 20 or 2-benzylthio-1,4,5,6-tetrahydropyrimidine hydriodide 21, obtained from the reaction of the iodide salts of 2imidazolidinethione or tetrahydro-2-pyrimidinethione with respectively methyl iodide or benzyl bromide, were reacted with 2-nitrobenzenesulfonyl chloride to give the corresponding sulfonamides 22 and 23. Reduction with zinc in acidic conditions of the nitro group followed by a simultaneous nucleophilic displacement of alkylthio group was described to conduct to the intramolecular cyclization with the resulting hydroxylamino group as maJor compound. 27 A modification of this previously procedure using tin (II) chloride in refluxing ethanol with a catalytic amount of hydrochloric acid caused the exclusive formation of the deoxygenated benzothiadiazines 24 and 25. Condensation of these tricyclic heterocycles with arylethyl methanesulfonates 14a-d and hydrogenolysis of the benzyl protections were performed as described in scheme 1 and provided the target compounds 26-27.

We planned to synthesize the corresponding unsaturated derivatives 28-29 by a key step of oxidative dehydrogenation using Pd/C in refluxing xylene but this reaction led only to recovery of starting material. We then decided to use DDQ as aromatization reagent to effect facile dehydrogenation of both simple and complex hydroaromatic heterocyclic compounds.

Compounds 26a was then refluxed in toluene with 2 eq. of DDQ and not conducted to the desired unsaturated derivative 28a but to single product which proved to be the cyclocondensed imidazobenzothiadiazines 30 bearing a chloro substituent and was isolated in only 25% yield.

Several substrates were then prepared and described recently to explore the scope of this reaction. [START_REF] Verones | Unexpected heteroannelation and chlorination of benzothiadiazine derivatives mediated by DDQ[END_REF] Biological Studies. Antiproliferative Activity in Cellular Assays. The in vitro cytotoxicity of all synthesized compounds against human colon adenocarcinoma HT29 cell line was investigated, in parallel with compound 2b1 and 1 as positive references, by the MTS assay after the incubation of cells with the compounds for 72 h. Data are expressed as percentage of cell proliferation inhibition at a high single dose (10 µM) or IC50 defined as the concentration at which the compounds reduce cell proliferation by 50% and are shown in Table 1. The results indicate that inhibition of cell proliferation was highly dependent upon the size of the fused ring and the presence of the double bond. Five-membered rings were less active than their sixmembered counterparts in which the presence of a double bond enhanced the antiproliferative activity. Indeed, compounds bearing a dihydro-imidazole as fused ring were not (17a-c) or poorly (17d) active while introduction of a double bond improved antiproliferative activities with IC50 values in the micromolar range (15a,e). Increasing the ring size by introduction of a tetrahydro-pyrimidine as six-membered ring led to better compounds (e.g., 18a,b vs 17a,b) in which the presence of a double bond enhanced the antiproliferative activity from the submicromolar to the nanomolar potency (e.g., 18a vs 16a). Replacement of the carbonyl group by its sulfonyl bioisostere generated a general decrease of the cytotoxicity as for example, the sulfonyl analogue 27a which was five-fold less potent than its direct carbonyl counterpart 16a.

Concerning the arylethyl side chain, the number and position of hydroxyl or methoxy substituents on the phenyl ring had a maJor influence on antiproliferative properties. The presence of a 3-hydroxy-4-methoxyphenyl substitution in both five-and six-membered ring series provided good antiproliferative activities at the micromolar range (15e, 16e, 17d, 18d) but the greatest activities occurred in the presence of a 4-methoxyphenyl substitution in the sixmembered series (16a, 18a). Introduction of a 3,4-dimethoxy-or 3,4-5-trimethoxyphenyl group, a typical pharmacophore found in many inhibitors of tubulin polymerization, [START_REF] Nguyen | A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach[END_REF] led to less active compounds (15b, 16b, 18b) or a total loss of antiproliferative activity (15c, 16c, 17c, 18c).

These results allowed to identify four new active antiproliferative compounds (16a, 16b, 18a and 27a) displaying low IC50 values ranging from 42 to 766 nM (Table 1). Structure-activity relationship (SAR) concluded that (1) a six-membered-ring bearing a double bond is the most tolerant of heterocyclic cores, (2) replacement of the carbonyl group by a sulfonyl moiety did not led to positive results, and (3) the 4-methoxy substitution on the phenylethyl side chain is more favorable than a 3-hydroxy-4-methoxy substitution. NCI's in vitro 60 cell screening. Compounds 16a, 18a and 27a were selected by the National Cancer Institute (NCI) for screening against 60 human tumor cell lines. [START_REF] Shoemaker | The NCI60 human tumour cell line anticancer drug screen[END_REF] They were initially tested at a high single dose (10 µM) in the full 60-cell panel and have progressed to the 5-dose screen in order to evaluate their GI50 values (Table 2). A mean graph midpoints (MG-MID) was calculated for each derivative selected for GI50 calculation, giving an average activity parameter over all cell lines for the tested compounds. These data indicated that quinazolinone 16a, with an average GI50 of 58.9 nM, was more active than its saturated analogue 18a (GI50 (MG-MID): 199.5 nM) and benzothiadiazine 27a (GI50 (MG-MID): 645 nM) which displayed weak growth inhibition potentials in several cancer cell lines as NSCL (NCI-H226), melanoma (UACC-62), ovarian (IGROV1, SK-OV-3) or renal (UO-31) cancers. Exhibiting the best activities comparable to our previously highlighted benzopyridooxathiazepine (2), compound 16a was then selected for further biological evaluations.

Cell Cycle Analysis. The effects of 16a, the most active compound on cell division process were evaluated using SK-Mel 28 melanoma cell line. The product was applied at varying concentrations, between 100 nM and 10 µM for 24h in parallel with vehicle (1% DMSO). The cells were then stained with propidium iodide (PI) and analyzed by flow cytometry. As represented in figure 2, cell populations treated with 16a were significantly arrested with an accumulation in the G2/M phase and an increase in subG1 apoptotic cells in a dose dependent manner and it was accompanied by a comparable reduction in the proportion of cells in both the G1 and S phases of the cell cycle. No effects on G2/M cell cycle blockade have been reported for the control populations which remained in a G0/ G1 state mainly. These results clearly indicated that the cell cycle arrest caused by 16a, as evidenced by the ability of the incubated cells to pass through the G2/M phase, was generally observed for antimitotic drugs that bind to tubulin. Immunocytochemical analysis. To determine the microtubule disrupting effects of the target compounds, we selected 16a as a representative compound in a cell-based phenotypic screening (Figure 3). SK-Mel 28 cells were used to examine the effect of 16a on the reorganization of microtubules during mitosis. Cells were treated for 6h with 16a or vehicle (1% DMSO), and microtubules were visualized by indirect immunofluorescence techniques. An antibody for βtubulin was used to visualize interphase and mitotic microtubule structures. Vehicle treated cells displayed a spider's web like network of fibrous filaments (green) wrapped around the cell nucleus (blue) (Figure 3A,B,C). Morphological aspects of untreated vs. 16a treated cells showed a significant difference in the microtubules structure between the two populations and a loss of polarity of 16a treated SK-Mel 28 cells which adopted a spherical configuration due to the disorganization of the microtubule network (Figure 3D,F).

Tubulin content. In order to evaluate whether our compounds affected polymerization or depolymerization of tubulin, an immunoblot analysis on protein extracts was performed to determine the polymerized and soluble tubulin fractions. Cells were treated for six hours with compound 16a (1 µM) or vehicle. The results showed a significant decrease in polymerized tubulin fractions between 16a treated group and the related controls (untreated, vehicle) (36% ± 6 vs. 100% ± 20, p value=0.0071, n=4) whereas no significant change have been reported in the soluble fractions between all groups (Figure . 4). Thus, these data confirmed that compound 16a induced its antimitotic effects by inhibiting tubulin polymerization since s/p tubulin ratio increased by a fold of two in 16a treated cells indicating more soluble protein form.

Inhibition of Tubulin Polymerization. Compounds 15-18 were then evaluated for their in vitro inhibition of tubulin polymerization in comparison with compound 2b1 and 1 (Table 1). In this assembly assay, the six-membered ring derivatives 16a,b,e, 18a, 27a,e displayed greater inhibitory activities than the reference compounds 2b1 and 1, with IC50 values ranging from 0.27 to 1.29 µM whereas the others seemed to be inactive as inhibitors of tubulin polymerization and did not inhibit tubulin assembly at concentrations as high as 10 µM. Inhibitions of tubulin polymerization of tested compounds were consistent with their antiproliferative activities.

Indeed, compounds bearing a 3,4,5-trimethoxy group were inactive at concentrations as high as 10 µM while other variations of substituents within the aryl moiety as 4-methoxy (16a, 18a, 27a), 3,4-dimethoxy (16b) and 3-hydroxy-4-methoxy groups (16e, 27e), led to significant activities with higher potencies than the reference compounds. In contrast with the cell proliferation assays, the carbonyl derivatives 16a,e were less potent than their counterparts 27a,e whose substitution with a sulfonyl group seemed to be most tolerant for tubulin polymerization inhibitory effect. Among all compounds tested, 16a, 18a and 27a were identified as the most active ones in both cell proliferation and tubulin assembly assays. The discrepancy between cytotoxicity and antitubulin activity of compounds 16e and 27e can be explained by the high resistance of HT29 cell line to most of tubulin polymerization inhibitors with a 3-hydroxy-4methoxy substitution as for CA-4. [START_REF] Beauregard | Differential sensitivity of two adenocarcinoma xenografts to the anti-vascular drugs combretastatin A4 phosphate and 5,6dimethylxanthenone-4-acetic acid, assessed using MRI and MRS[END_REF] It has been reported that glucuronidation of phenol derivatives as CA-4 is highly observed in HT29 colorectal cancer cells due to an overexpression of glucuronosyl transferase activity and then contributes to drug resistance phenomenon. [START_REF] Franklin | Glucuronidation associated with intrinsic resistance to mycophenolic acid in human colorectal carcinoma cells[END_REF] Molecular Modeling. Molecular docking tools are used for the structure-based analysis in order to elucidate the interactions of best compounds 16a and 27a of each class of quinazolinone and benzothiadiazine derivatives. The structural similarity between reference compound 1 and 16a and 27a made consistent the docking of these two new molecules into the cavity of the 1bound tubulin crystal structure (PDB ID 3HKC). [START_REF] Dorléans | Variations in the colchicine-binding domain provide insight into the structural switch of tubulin[END_REF] Using AutodockVina 34 with a 50 Å edged cubic box including one β-tubulin subunit and centered onto compound 1 binding site allowed to retrieve the first ranked docking pose of each compound very close to the co-crystallized compound 1 (Figure 5) suggesting they could be really considered as constraint analogs of 1.

According to previous studies, [START_REF] Nguyen | A common pharmacophore for a diverse set of colchicine site inhibitors using a structure-based approach[END_REF][START_REF] Wang | Structures of a diverse set of colchicine binding site inhibitors in complex with tubulin provide a rationale for drug discovery[END_REF] the main receptor-ligand interactions were hydrophobic contacts with Leu240, Leu255, Ala314, Val316 and Ile378 of β chain. In comparison with compound 1, an additional interaction was observed between carbonyl of 16a and sulfonyl of 27a and thiol group of Cys239 side chain. Interestingly, this amino acid was shown to be involved in the conformational changes of the various transition states during tubulin polymerization. [START_REF] Bai | Mapping the binding site of colchicinoids on -tubulin[END_REF] Arguing for a prediction of inhibition of polymerization by alpha/beta tubulin disruption rather than the prediction of cellular anti-proliferative activity, it emphasizes an inhibition of polymerization (0,812 μM and 0,27 μM) in the same order of magnitude by compounds 16a and 27a. By keeping suitable enthalpic contributions in term of intermolecular specificity and decreasing entropic contribution by a strategy of rigidification of compound 1, these docking results support the original design aiming at increase the potency of constraint analogs of 1.

HUVEC Cell invasion and endothelial tube formation assays.

To continue its growth and expansion, a neoplastic tissue requires oxygen and metabolic supplies, that's why, the tumor microenvironment stimulates the development of additional blood vessels from already preexistent branches; this process is well known under the term of angiogenesis. [START_REF] Baeriswyl | The angiogenic switch in carcinogenesis[END_REF] Recent antitumor strategies are based on the use of chemotherapeutic agents with antiangiogenic or antivascular drugs in order to increase the efficacy of the treatment. Besides the ability to inhibit tumor cell proliferation, microtubule-targeting drugs have also been shown to be active against the tumor vasculature by inhibiting endothelial cell invasion and capillary tube formation. [START_REF] Tozer | Disrupting tumour blood vessels[END_REF][START_REF] Schwartz | Antivascular actions of microtubule-binding drugs[END_REF] Considering that tube formation and invasion are highly relevant properties in the process of tumor vasculature, we investigated the effects of compound 16a and antiangiogenic inhibitor cediranib (31, VEGFR, PDGFR and c-Kit inhibitor) [START_REF] Lindsay | Current status of cediranib: the rapid development of a novel anti-angiogenic therapy[END_REF] , as the reference substance at several concentrations (0.001, 0.01, 0.1 and 1 µM) on the invasiveness of human umbilical vein cord endothelial cells (HUVEC) in a Boyden chamber assay. Transwell instets were precoated with basement membrane matrix and the number of HUVEC that penetrated the membrane was quantified. As shown in Figure 6, treatment with compound 16a inhibited HUVEC cell invasion at nontoxic concentrations lower than 0.01 µM ( 74% ±0.01 inhibition, p value < 0.001) while 31 displayed similar effects at a more pronounced concentration of 1 µM ( 74% ±0.09, p value < 0.001). We also studied the effects of compound 16a on vascular tube formation. The antiangiogenic effect was evaluated on HUVEC model by treating the cells with either the reference compound 31 or compound 16a. Cells were seeded on a specific membrane matrix, which is rich in pro-angiogenic factors that stimulate single endothelial cells to assume an extended shape, in order to mimic in vivo HUVEC and treated with several concentrations of 31 or 16a (0.001, 0.01, 0.1 and 1 µM). The overall effect results in a reticular similar to a capillary network and offers an in vitro model of angiogenesis. [START_REF] Hotchkiss | Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center[END_REF] As shown in Figure 7, compound 16a significantly displayed vascular disrupting effects at 0.1 μM with total destruction of the cordlike structures at 1 μM and at which 31 was less effective. SK-Mel28 cell wound healing assay. The effect of compound 16a on cell migration, which is an important mechanism involved in tumor invasion, was also evaluated by scratching a melanoma SK-Mel28 cell line monolayer and monitoring the percentage of wound closure. As shown in Figure 8, compound 16a was very efficient in arresting cell motility. The effect was statistically significant after 9 h of incubation at the concentration of 1µM, in comparison with control cells, keeping the wound nearly intact (wound closure 3% ± 2, p value < 0.001).

Druglike properties. The hydrophobic character (logP) and the in vitro metabolic stability of 16a lead compound have been determined and compared with those of the reference compound 2b1. Quinazolinone 16a exhibited lower logP value (2.63  0.01 versus 3.10  0.01) and greater metabolic stability in rat liver microsome (t1/2 = 56 min versus 38 min) than benzopyridooxathiazepine derivative 2b1. The determination of Michaelis-Menten parameter (Km), which is reciprocally proportional to the enzyme affinity to the substrate, also confirmed the previous results with Km values of 5.2  1.0 µM and 27.2  5.6 µM for compounds 2b1 and 16a, respectively. Its improved druglike properties as well as its antiproliferative and antivascular activities suggest that compound 16a may be a better potential drug candidate than its oxathiazepine analogue 2b1.

Antitumor Activitiy In Vivo. To further investigate the in vivo effects of compound 16a on tumor growth and vascularization, we used a syngeneic melanoma mouse model (B16F10). [START_REF] Bouvard | Tie2-dependent deletion of α6 integrin subunit in mice reduces tumor growth and angiogenesis[END_REF] In in vitro preliminary experiments, we demonstrated that compound 16a showed potent cytotoxic activity against melanoma cell lines. C57BL/ 6 mice were then subcutaneously inJected with B16F10 murine melanoma cell lines and when tumor bulging reached a measurable size (about 100 mm 3 ); mice were divided and randomly assigned to six groups. Three different doses ranging from 5 mg/ kg /day to 50 mg/ kg /day were tested for 16a compared to a single 50 mg/ kg /day dose for reference 1. [START_REF] Koyanagi | In vivo tumor growth inhibition produced by a novel sulfonamide, E7010, against rodent and human tumors[END_REF] Treatment was delivered continuously using subcutaneous osmotic pumps and in accordance with the pharmacokinetic data. As depicted in figure 9 and 10, product 16a showed a significant decrease in tumor growth and angiogenesis at the starting 5 mg/ kg dose as compared with the administration of vehicle (1000mm 3 ± 280 vs. 2400mm 3 ± 310, p value = 0.009) and a 60% decrease in the percentage of microvessel number with no significance to the standard compound 1 treatment (p value <0.005). At the lower dose, no significant difference was observed between 16a and 1 on tumor growth inhibition and angiogenesis, but a slight difference was shown at the higher 50 mg/ kg/ day with no statistical significance (760mm 3 ± 160 vs. 1200mm 3 ± 390, p value = 0.25 and 80% decrease in microvessel number) in comparison to vehicle (2400mm 3 ± 310, p value = 0.0007). Mice monitoring showed no significant difference in mice average weight between all groups before, during and after treatment initiation. Hence, compound 16a showed strong antitumor and antivascular activities on a well-tolerated dose schedule and was as effective as 10-fold lower dose of reference compound 1.

Conclusion

By structural modifications on the thiadiazepine and oxathiazepine moieties in prior leads 1 and 2, a series of 2,3-fused quinazolin-4-one systems (15-18) and their sulfonyl benzothiadiazine analogues (26-27) were synthesized and evaluated in cytotoxicity and tubulin inhibition assays and led to a new class of inhibitors of tubulin polymerization. The most potent compound, 16a exhibited the best in vitro cytotoxic activity in cellular assays with a mean GI50 value of 58.9 nM on the NCI60 human tumour cell line and significant potency against tubulin assembly with an IC50 value of 0.812 μM. In mechanism studies, 16a was shown to block cell cycle in G2/M phase, inhibited cell invasion and migration and displayed antiangiogenic effect, thus providing evidence that this tubulin polymerization inhibitor acts as vascular disrupting agents (VDAs).

SAR and molecular modeling studies suggested that the presence of a carbonyl or a sulfonyl group allowed additional interactions with thiol group of Cys239 side chain which might be responsible for the greater potency of 16a and 27a than the reference 1. Following druglike property assessments, 16a was evaluated in parallel with reference 1 in C57BL/6 mouse melanoma B16F10 xenograft models to evaluate their in vivo activities. Compound 16a exhibited strong in vivo antitumor and antivascular activities, suppressing tumor growth and angiogenesis equally to 1 but at a 10-fold lower dose. In summary, these newly tricyclic quinazolinone or benzothiathiazine derivatives displayed marked biological activities in vitro and in vivo as a novel class of anticancer agents and have potential for further development.

Experimental Section

1. General Chemistry. All reagents and solvents were purchased and used without further purification. Melting points were determined on a BÜCHI B-540 apparatus and are uncorrected.

NMR spectra were recorded on a Bruker Avance 300 spectrometer operating at 300MHz ( 1 H) or 75MHz ( 13 C). Chemical shifts are in parts per million (ppm) and were referenced to the residual proton peaks in deuterated solvents. Mass spectra were recorded with an LCMS (Waters Alliance Micromass ZQ 2000). LCMS analysis was performed using a Waters XBridge C18 column (5 µm particle size column, dimensions 50 mm x 4.6 mm). A gradient starting from 98% H2O/formate buffer 5 mM (pH 3.8) and reaching 100% CH3CN/ formate buffer 5 mM (pH 3.8) within 4 min at a flow rate of 2 mL/min was used followed by a return to the starting conditions within 1 min. Purities of tested compounds reached at least 95% and were determined by HPLC using the following instruments and conditions: Waters HPLC system was equipped with a HPLC 600 pump and a 2996 photodiode array detector. The HPLC analysis were performed on a LiChroprep Hibar® column (25-40 µm particle size column, dimensions 250 mm x 4.6 mm), detection at 254 nm, and injection volume of 20 μL. Mobile elution was conducted with a mixture of petroleum ether (PE):ethyl acetate (EA) (see sup info).

Procedure for synthesis of compound 4. Anthranilic acid 3 (15 g, 0.109 mol) was suspended in water (500 mL) and glacial acetic acid (6.75 mL) at 35 °C. A freshly prepared solution of potassium cyanate (11.5 g, 0.142 mol) in water (100 mL) was added dropwise to the stirred mixture. After 2 h, sodium hydroxide (144 g 3.6 mol) was added in portions, keeping the temperature below 40 °C. A clear solution was obtained momentarily before precipitation of the hydrated sodium salt. After cooling, the precipitate was filtered off and dissolved in hot water then acidified to pH 5, causing precipitation of 1H-quinazoline-2,4-dione which was filtered and dried.

1H-quinazoline-2,4-dione (4). Yield 74%, 13 g. White solid, mp 360-362 °C (Lit. [START_REF] Palazzo | Synthesis of 5H-[1,2]-benzisothiazolo[2,3a]quinazolin-5-one[END_REF] Procedure for synthesis of compound 5. A mixture of 1H-quinazoline-2,4-dione (12 g, 0.074 mol), trichlorophosphate (41.5 mL, 0.445 mol) and N,N-dimethylaniline (6.1 mL, 0.048 mol) is refluxed for 5h. The excess of trichlorophosphate is evaporated and ice is added to the residue.

After standing at room temperature overnight, the mixture was filtered to give a solid which was recrystallized from methanol.

2,4-dichloroquinazoline (5). Yield 88%, 12.95 g. Off-white solid, mp 119-121 °C (Lit. [START_REF] Sun | Solvent-free or low-solvent large-scale preparation of chloropyrimidine and analogues[END_REF] 118-120°C). 1 H NMR (DMSO-d6, 300 Hz): 7.91 (1H, td, 4 J = 1.2 Hz and 3 J = 6.9 Hz, CH quina), 8.05 (1H, dd, 4 J = 1.2 Hz and 3 J = 8.3 Hz, CH quina), 8.18 (1H, td, 4 J = 1.5 and 3 J = 6.9 Hz, CH quina), 8.31 (1H, dd, 4 J = 1.5 Hz and 3 J = 8.5 Hz, CH quina). C8H4N2Cl2; MW= 199.03 g/mol.

Procedure for synthesis of compound 6. A suspension of 2,4-dichloroquinazoline (2.8 g, 14.1 mmol) was stirred in 2% aqueous sodium hydroxide solution (50 mL) for 24 h at 30 °C. The reaction mixture was diluted with water (100 mL) and filtered to remove unreacted 2,4dichloroquinazoline. The filtrate was neutralized with dilute acetic acid, the precipitate thus obtained was filtered and washed with water. (6). Yield 83%, 2.1 g. Off-white solid, mp 218-220 °C (Lit. [START_REF] Prashad | Process development of a large-scale synthesis of TKA731: A tachykinin receptor antagonist[END_REF] 219-221 °C). 1 is refluxed in ethanol (35 mL) for 4h. After cooling, the reaction mixture is filtered and the filtrate evaporated under vacuum. The residue is precipitated in water, filtered and recrystallized from ethanol. (7). Yield 71%, 2.0 g. Yellow solid, mp 176-178 °C (Lit. [START_REF] Murdoch | Synthesis of [1,2,4]triazoloquinazolinones and imidazoquinazolinones[END_REF] 174-180°C). 1 H NMR (DMSO-d6, 300 Hz): 1.12 (6H, t, 3 J = 7.5 Hz, CH3), 3.44 (2H, t, 3 J = 5.5 Hz, CH2), 3.51 (2H, m, OCH2), 3.67 (2H, m, OCH2), 4.64 (1H, t, 3 J = 5.5 Hz, CH), 6.17 (1H, s, NH), 7.10 (1H, t, 3 J = 7. 8 1H-imidazo [2,1-b]quinazolin-5-one (9). Yield 60%, 1.1 g. Off-white solid, mp 300-301 °C (Lit. [START_REF] Murdoch | Synthesis of [1,2,4]triazoloquinazolinones and imidazoquinazolinones[END_REF] 281-284 °C). 1 H NMR (DMSO-d6, 300 Hz): 7.14 (1H, td, 4 J = 1.1 Hz and 3 J = 6.9 Hz, CH quina), 7.32 (1H, d, 3 (10). Yield 89%, 1.75 g. Yellow solid, mp > 400 °C. 1

2-chloro-3H-quinazolin-4-one

2-(2,2-diéthoxyéthylamino)-3H-quinazolin-4-one

1,2-dihydro-1H-pyrimidino[2,1-b]quinazolin-6-one

General Procedure for synthesis of compounds 12b-d.

To a stirred solution of methyltriphenylphosphonium iodide (20.7 g, 0.051 mol) in THF (100 mL) at 0 °C was added potassium tert-butoxide (8.5 g, 0.076 mol), and the mixture was stirred for another 1 h.

Commercial available (11b-c) or synthesized [START_REF] Shen | Total synthesis and structural revision of engelhardione[END_REF] (11d) benzaldehyde (0.026 mol) in THF (25 mL) was added, and the reaction mixture was warmed to room temperature. After 24 h, saturated aqueous ammonium chloride solution (150 ml) was added, the aqueous layer was extracted with EtOAc (3 × 150 mL), and the combined organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM 100%).

2-(3,4-dimethoxyphenyl)ethylene (12b

). Yield 87%, 3.7 g. Yellow oil. 1 H NMR (CDCl3, 300 Hz): 3.90-3.92 (6H, 2s, OCH3), 5.16 (1H, dd,3 J = 0.8 Hz and 3 J = 10.9 Hz, CH), 5.63 (1H, dd, 3 J = 0.8 Hz and 2 J = 17.5 Hz, CH), 6.66 (1H, dd, 3 J = 10.9 Hz and 2 J = 17.5 Hz, CH), 6.82 (1H, d,

General Procedure for synthesis of compounds 13b-d.

To a stirred solution of 12b-d (0.012 mol) in THF (30 mL) under a nitrogen atmosphere at 0 °C is added BH3• THF (12 mL, 0.012 mol, 1.0 mol/L), and the mixture is stirred for 3h at room temperature. The reaction mixture is cooled to 0 °C and water (1.7 mL) is added under magnetic stirring for 10 min before addition of 10% aqueous sodium hydroxide solution (15 mL, 0.0375 mol) and 35% H2O2 solution (15 mL).

The reaction mixture is stirred for another 30 min, quenched with saturated ammonium chloride solution (100 mL) and then extracted with diethyl ether (3 × 50 mL). The combined organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (DCM 100%).

2-(3,4-dimethoxyphenyl)ethanol (13b).

Yield 87%, 1.9 g. Clear oil. 1 H NMR (CDCl3, 300 Hz): 2.84 (2H, t, 3 J = 6.5 Hz, CH2), 3.84 (2H, t, 3 J = 6.6 Hz, CH2), 3.85 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.75-6.83 (3H, m, ArH). C10H14O3; MW= 182.21 g/mol.

2-(3,4,5-trimethoxyphenyl)ethanol (13c).

Yield 77%, 1.95 g. Clear oil. 1 H NMR (CDCl3, 300 Hz): 2.81 (2H, t, 3 J = 6.5 Hz, CH2), 3.81 (3H, s, OCH3), 3.85 (8H, m, CH2, CH3), 6.44 (2H, s, ArH). C11H16O4; MW= 212.24 g/mol.

2-(3-benzyloxy-4-methoxyphenyl)ethanol (13d).

Yield 75%, 2.3 g. White solid, mp 42-44 °C (Lit. [START_REF] Sasaki | The Glycosides of Martynia louisiana MILL. A new phenylpropanoid glycoside, martynoside[END_REF] 68-69°C). 1 H NMR (CDCl3, 300 Hz): 2.76 (2H, t, 3 J = 6.5 Hz, CH2), 3.78 (2H, t, 3 J = 6.5

2-(4-methoxyphenyl)ethylmethanesulfonate (14a).

Yield 72%, 0.8 g. White solid, mp 35-36 °C (Lit. [START_REF] Lebegue | Novel benzopyridothiadiazepines as potential active antitumor agents[END_REF] 35-36 °C). 1 H NMR (CDCl3, 300 Hz): 2.82 (3H, s, OSO2CH3), 3.05 (2H, t, 3 J = 6.5 Hz, CH2), 3.80 (3H, s, OCH3), 4.38 (2H, t, 3 J = 6.5 Hz, CH2), 6.90 (2H, d, 3 J = 8.1 Hz, ArH), 7.15 (2H, d, 3 J = 8.1 Hz, ArH). C10H14O4S; MW= 230.28 g/mol.

2-(3,4-dimethoxyphenyl)ethylmethanesulfonate (14b).

Yield 76%, 1.0 g. White solid, mp 42-43°C (Lit. [START_REF] Gupton | The application of vinylogous iminium salt derivatives to the synthesis of Ningalin B hexamethyl ether[END_REF] bp 168-169°C at 0.2 mm). 1 H NMR (CDCl3, 300 Hz): 2.89 (3H, s, OSO2CH3), 3.01 (2H, t, 3 J = 6.5 Hz, CH2), 3.86 (3H, s, OCH3), 3.88 (3H, s, OCH3), 4.41 (2H, t, 3 J = 6.5 Hz, CH2), 6.76 (1H, dd, 4 J = 1.7 Hz and 3 J = 8.0 Hz, ArH), 6.80 (1H, d, 3 J = 1.7 Hz, ArH), 6.84 (1H, d, 3 J = 8.0 Hz, ArH). C11H16O5S; MW= 260.30 g/mol.

2-(3,4,5-trimethoxyphenyl)ethylmethanesulfonate (14c).

Yield 83%, 1.2 g. White solid, mp 76-77 °C (Lit. [START_REF] Lebegue | Novel benzopyridothiadiazepines as potential active antitumor agents[END_REF] 76-77 °C). 1 H NMR (CDCl3, 300 Hz): 3.02 (3H, s, OSO2CH3), 3.10 (2H, t, 3 J = 6.5 Hz), 3.85 (3H, s, OCH3), 3.88 (6H, s, OCH3), 4.43 (2H, t, 3 J = 6.5 Hz, CH2), 6.46 (2H, s, ArH). C12H18O6S; MW= 290.33 g/mol.

2-(3-benzyloxy-4-methoxyphenyl)ethylmethanesulfonate (14d).

Yield 69%, 1.2 g. White solid, mp 95-97 °C (Lit. [START_REF] Mcdonald | Design of the substrate for oxidative phenol coupling. An efficient synthesis of the C-homoerythrinan skeleton[END_REF] 106-107 °C). 1 H NMR (CDCl3, 300 Hz): 2.78 (3H, s, OSO2CH3), 2.95 (2H, t, 3 J = 7.5 Hz, CH2), 3.89 (3H, s, OCH3), 4.34 (2H, t, 3 J = 7.5 Hz, CH2), 5.17 .07 (1H, dd, 4 J = 1.2 Hz and 3 J = 7.9 Hz, CH quina). [START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF]

N-[2-(3,4-dimethoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16b).

Yield 20%, 72 mg. White solid, mp 144-146 °C. 1

General Procedure for synthesis of compounds 15e and 16e.

A solution of 15d or 16d (0.5 mmol) in a 33% solution of hydrobromic acid (3 mL) and glacial acetic acid (5 mL) is stirred at room temperature until starting material disappeared (followed by TLC). After cooling, the reaction mixture was diluted with water (50 mL) and the resulting precipitate was extracted with EtOAc (3 x 20 mL). The organic layers were combined, washed with an aqueous 10% NaHCO3 solution, dried (MgSO4), and evaporated to give a white solid purified by recrystallization from ethanol.

N-[2-(3-hydroxy-4-methoxy-phenyl)ethyl]imidazo[2,1-b]quinazoline-5-one (15e

General Procedure for synthesis of compounds 17a-d and 18a-d.

A solution of 15a-d or 16ad (0.5 mmol) in EtOH (100 mL) is stirred with 10% Pd/C catalyst and ammonium formate (15 mmol) at 60°C until starting material disappeared (followed by TLC). The warm solution was filtered on a pad of celite and the filtrate was evaporated. The residue was recrystallized from methanol. and the mixture is allowed to stir at room temperature for 2h. The reaction mixture is hydrolyzed, extracted with DCM (2 x 50 mL) and washed with 10% aqueous sodium thiosulfate solution.

N-[2-(4-methoxyphenyl)ethyl]-2,3-dihydro-imidazo-[2,1-b]quinazolin-5-one (17a

The organic layers were combined, dried (MgSO4), and evaporated to give a colorless solid purified by recrystallization from methanol. (22). Yield 62%, 3.0 g.

N-(2-nitrophenylsulfonyl)-2-methylsulfanyl-4,5-dihydro-imidazole

Colorless solid, mp 184-185 °C (Lit. [START_REF] Friary | Dihydroimidazobenzothiadiazine dioxides[END_REF] 2. Biological Evaluation.

Cell Proliferation on HT29 Cell Line. HT 29 Colon cancer cells were obtained from

European Collection of Cell Cultures. FBS, medium, penicillin-streptomycin, and other agents used in cell culture studies were purchased from Invitrogen. Cancer cell lines were cultured, at 37 °C in a CO2 incubator, respectively in RPMI-1640, MEM and DMEM + Glutamax-I medium supplemented with 10% fetal bovine serum, penicillin (100 IU/mL) and streptomycin (100 mg/mL). Briefly, HT29 colon cancer cells were plated at a density of 3 x 10 3 cells/well in 96well plates for 24 h. Then the medium was removed, and cells were treated with either DMSO as a control or at a 10µM single dose or various concentrations of compounds. The final concentration of DMSO in the medium was <0.1% (v/v). The plates were incubated for 72 h and cell proliferation was estimated by MTS colorimetric assay.

Cell Growth Inhibition.

The compounds were tested against a panel of 60 human cancer cell lines at the National Cancer Institute (Rockville, MD). [START_REF] Shoemaker | The NCI60 human tumour cell line anticancer drug screen[END_REF] The cytotoxicity studies were conducted using a 48 h exposure protocol using the sulforhodamine B assay. [START_REF] Skehan | New colorimetric cytotoxicity assay for anticancerdrug screening[END_REF][START_REF] Boyd | The NCI in vitro anticancer drug discovery screen[END_REF] 2.3. Cell Cycle Analysis. Sk-Mel28 cells were seeded in complete RPMI medium and at a final concentration of 5 x 10 5 cells per well, 24h prior to compound application. The following concentrations were applied for compound 16a; 10 -7 , 10 -6 and 10 -5 M. The vehicle solution (DMSO) was applied at 0.1 and 1% final concentration. After 24h incubation, the cells were washed twice with PBS and fixed with cold ethanol 70% and stored at -20°C until analysis. DNA content was evaluated by PI staining. The cells were stained with 50 µg/mL PI solution supplemented with 5 µg RNAse stock. Analysis was carried out on a flow cytometer according to the presented protocols. Fluorescence of the PI-stained cells was measured using CyAn TM ADP Analyzer (Beckman Coulter, Inc.) and Summit® software. After excluding cell debris and clumps from analysis by using an appropriate dot-plot gating area, the software program provided the estimate percentage of cells with fractional DNA and cells in G1, S, and G2/M phases of the cycle.

2.4. Immunocytochemistry. SK-Mel28 cells were grown on precoated poly-Llysine-4-well chamber slides (Lab-Tek, Nunc®, Roskilde,Denmark). Approximately, 2.5 x 10 4 cells per well were seeded in complete RPMI medium (containing 10% FBS, 1% antibiotics and 1% sodium pyruvate) and exposed to compound 16a (1 µM) or vehicle. After 6 h, the cells were fixed in cold acetone for 15 min at room temperature, washed three times with PBS containing 1% BSA and permeabilized for 10 -15 min at room temperature with 0.025% Triton-X-100 (Sigma-Aldrich). Nonspecific binding sites were blocked for 30 min at room temperature with a PBS solution containing 3% BSA, 7% FBS and 10% mouse specific IgG. The cells were then incubated with a monoclonal anti-β-tubulin antibody (clone TUB2.1, 2 μg/ mL, Sigma-Aldrich)

for 1 h at room temperature, washed three times, and incubated for 1 h at room temperature with goat anti-mouse Alexa Fluor 488 (2 μg/ mL; Molecular Probes, Invitrogen®) in 1% BSA.

Fluorescent microscopic analysis was performed with Leica DMRB microscope (Leica Microsystems), using a PL Plan-Leica Fluotar 40×/1.00 oil obJective. Pictures were taken with a DFC345FX digital camera and processed with Leica Application Suite (LAS v 3.7 software) (Leica Microsystems).

Tubulin Content Analysis.

Cell content of the polymerized and soluble tubulin fraction was investigated by western blot analysis. Sk-Mel28 cells were seeded in T25 cm 2 culture flasks at a final concentration of 5 x 10 6 cells per flask 48h prior to treatment. Control and compound 16a (1 µM) were applied for 6h. Cells were then washed twice with PBS, harvested and protein extracts were prepared for western blot. Briefly, twenty micrograms of proteins was subJected to 4-12% SDS-NuPAGE polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Thermo Fisher Scientific Inc., Waltham, MA) then incubated with antibodies directed against tubulin and HSC-70 (Santa-Cruz Biotechnology, Santa Cruz, CA). Finally, blots were visualized using the Amersham ECL Prime Western Blotting System on a Las 4000 biomolecular imager and further analyzed by ImageQuant® tools (GE Healthcare Europe GmbH, Velizy-Villacoublay, France) The experiment was performed four times (n=4).

In Vitro Microtubule Polymerization Assay.

In-vitro tubulin polymerization assays were performed on a fluorescence spectrometer (Varioskan flash, Thermo Scientific, Courtaboeuf, France), equipped with a half area 96 wells microplate (Corning Costar). Excitation and emission wavelengths were 360 and 420 nm, respectively. All reagents were purchased from Cytoskeleton (Denver, CO, USA). For each inhibitor investigated, a series of ten concentrations (from 0.1 to 10 µM) was prepared in 100 µg purified bovine tubulin and buffer containing 20% glycerol, 1 mM GTP, 80 mM PIPES (pH 6.9), 2.0 mM MgCl2, 0.5 mM EGTA and 5 nM fluorescent reporter and kept at 0°C. Then, the medium was incubated in the plate reader, at 37°C, for 1h. A mixture without inhibitor exhibited an increase in fluorescent intensity as a function of incubation time corresponding to the inclusion of the probe inside the tubulin polymer. On the contrary, compounds which strongly inhibited microtubule formation were identified by a fluorescent signal remaining negligible along the experiment. The effect of inhibitor concentration on tubulin polymerization rate was studied and the IC50 value was defined as the concentration of product which inhibits the polymerization rate by a 50-fold decrease. It was determined from three independent assays using GraphPad Prism ® software.

Molecular Modeling Studies.

Docking was performed into one β subunit of compound 1bound tubulin crystal structure (PDB ID 3HKC [START_REF] Dorléans | Variations in the colchicine-binding domain provide insight into the structural switch of tubulin[END_REF] ) using AutodockVina 34 integrated into UCSF Chimera suite. [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF] With a search grid of 80 x 80 x 60 Å included the whole β subunit, the docking process was achieved onto a MacBook Pro OS X 10.9.5 (Apple Inc.) using an energy range of 3, an exhaustiveness of 8 and 9 retained binding modes.

2.8. Cell Invasion Assay. ECGM medium + SupplementMix were purchased from Promocell.

In vitro invasion assay was carried out using transwell chambers, precoated with Matrigel® matrix, with 6.4 mm diameter PET membrane (8 micron pore size, BD Biocat). The bottom chambers were filled with ECGM medium with 20 ng/mL VEGF and the top chambers were seeded with ECGM (without growth factors) and HUVEC (1 x 105 cells per well). The top chamber contained vehicle or compound at various concentrations (0.001, 0.01, 0.1 or 1 µM).

Cells were allowed to migrate for 24 h. Non migrated cells were scraped with a cotton swab, and migrated cells were fixed with 100% methanol and stained with 0.05% crystal violet. The number of HUVEC that penetrated the membrane was quantified by manual counting and photographed with Moticam®(Motic microscopy, Hong-Kong). The percentage of migrated cells was expressed on the basis of vehicle control wells.

2.9. In Vitro Endothelial Tube Formation Assay. Geltrex ® was thawed overnight at 4 °C in an ice bath, and then 100 μL of solution was used to coat 24-well plates. The plates were then incubated at 37 °C for 30 min to ensure complete gelation of the matrix. HUVEC cells were then seeded into 96-well plates at a cell density of 50 000 cells/well and allowed to incubate for 10 h at 37 °C. Cells were treated with different concentrations of compound 16a and 31 (0.001, 0.01, 0.1 and 1 μM). The morphological changes on the cell structure and the formed tubes were observed under inverted microscope and photographed with Moticam ® .

Cell Migration Assay.

Tumor cell motility was investigated using the "wound healing" assay of a confluent monolayer. Rapidly, Sk-Mel28 cells were seeded in complete medium and in 12-well plates at a final concentration of 8 x 10 4 cells per well until confluent monolayer was formed. Wound generation was performed using a 200 µL pipet tips and each well was rinsed twice with PBS to remove the floating/ detached cells. Compound 16a or vehicle were then applied to each appropriate well and photographs of each well were taken at different time intervals (t+3, t+6 and t+9h) under a light microscope. Area analysis was performed using Image J software and the wound width was measured in three different wells. The reported area value was normalized to the initial width. The experiment was performed three times, in triplicates (n =

3), at different time passages.

Microsomal Stability Assay.

Stock solutions of test compounds (5 mM) were prepared in ACN and stored at 4°C. Before performing the assay, the stock solutions were diluted with H2O to 100 µM. Kinetic of compounds 2b1 and 16a metabolism and the determination of Michaelis-Menten parameters were performed according to the protocol described by Bourdon et al. [START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF] Briefly, compounds 2b1 and 16a were incubated at 10 µM (for the kinetic of assay) or at various concentrations ranging from 0.4 to 20 µM (for the Km determination), in rat liver microsomes (RLM), using a microsomal incubation assays kit from BDbiosciences. The composition of microsomal incubation mixture was 0.5 mg.mL -1 microsomal protein, 1.55 mM NADP + , 3. [START_REF] Mirrlees | Direct measurement of octanol-water partition coefficients by high-pressure liquid chromatography[END_REF] The test compounds were analyzed on C18 stationary phase (150 x 4.6 mm i.d., 5 µm) using various compositions of ACN/H2O mobile phase delivered at a flow rate of 1 mL.min -1 . All other chromatographic conditions were the same as those described in microsomal stability assay section. NaCl containing 5% polyethylene glycol 400 and 0.5% Tween 80) 57 and the pumping rate and pumping duration were 0.5 µl/h and 14 days respectively. All the protocols were approved by the

Antitumor Assay In

ASSOCIATED CONTENT

Supporting Information

1 H NMR, [START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF] C NMR of original compounds and HPLC purity data for tested compounds.

Molecular formula strings in csv format. This material is available free of charge via the Internet at http://pubs.acs.org.

Accession Codes

PDB code 3HKC was used for model docking of compound 16a and 27a. Authors will release the atomic coordinates and experimental data upon article publication.

14a-d R 1 = H, OBn, OCH 3 R 2 = OCH 3 R 3 = H, OCH 3 15a-d: n=0 16a-d: n=1 a: R 1 , R 3 = H, R 2 = OCH 3 b: R 1 , R 2 = OCH 3 , R 3 = H c: R 1 , R 2 , R 3 = OCH 3 d: R 1 = OBn, R 2 = OCH 3 , R 3 = H 15e: n=0 16e: n=1 j ( ) n OCH 3 R 1 R 2 R 3 R 1 R 2 R 3 f g h 11b-d R 1 = H, OBn, OCH 3 R 2 = OCH 3 R 3 = H, OCH 3 12b-d R 1 = H, OBn, OCH 3 R 2 = OCH 3 R 3 = H, OCH 3 13a-d R 1 = H, OBn, OCH 3 R 2 = OCH 3 R 3 = H, OCH 3 a 4
( ) n R 1 R 2 R 3 k 3

Table of Contents Graphic

  355-358°C).1 H NMR (DMSO-d6, 300 Hz): 7.15 (2H, m, CH quina), 7.62 (1H, td, 4 J = 1.5 Hz and 3 J = 7.3 Hz, CH quina), 7.88 (1H, dd, 4 J = 1.5 Hz and 3 J = 8.2 Hz, CH quina), 11.2 (2H, s, NH). C8H6N2O2; MW= 162.14 g/mol.

  H NMR (DMSO-d6, 300 Hz): 7.57 (2H,m, 2H, CH quina), 7.83 (1H, td, 4 J = 1.8 Hz and 3 J = 7.3 Hz, CH quina), 8.09 (1H, dd, 4 J = 1.8 Hz and 3 J = 7.7 Hz, CH quina), 13.25 (1H, s, NH). C8H5ClN2O; MW= 180.59 g/mol. General Procedure for synthesis of compounds 7 and 8. A mixture of 6 (1.8g, 0.01 mol) and 2,2-diéthoxyethylamine (4.35 mL, 0.03 mmol) or 3,3-diethoxypropylamine (4.85 mL, 0.03 mol)

  J = 2.25 Hz, CH), 7.42 (1H, d, 3 J = 8.65 Hz, CH quina), 7.57 (1H, d, 3 J = 2.25 Hz, NCH), 7.63 (1H, td, 4 J = 1.6 Hz and 3 J = 8.65 Hz, CH quina), 8.11 (1H, dd, 4 J = 1.6 Hz and 3 J = 8.25 Hz, CH quina), 9.32 (1H, s, NH). 13 C NMR (DMSO-d6, 75 Hz): 106.7, 112.6, 121.1, 122.4, 124.1, 127.1, 134.2, 146.0, 146.9, 157.7. C10H7N3O, MW= 185.18 g/mol; tR,LCMS = 1.43 min; MS (ESI+): m/z = 186 [M+H]+.

  H NMR (DMSO-d6, 300 Hz): 4.03 (2H, m, CH2), 5.58 (1H, m, CH), 7.07 (1H, td, 4 J = 1.1 Hz and 3 J = 7.05 Hz, CH quina), 7.15 (1H, dd, 4 J = 1.1 Hz and 3 J = 8.25 Hz, CH quina), 7.32 (1H, dd, 4 J = 2.05 Hz and 3 J = 8.5 Hz, NCH), 7.54 (1H, td, 4 J = 1.75 Hz and 3 J = 8.15 Hz, CH quina), 7.69 (1H, s, NH), 7.86 (1H, dd, 4 J = 1.65 Hz and 3 J = 7.85 Hz, CH quina). 13 C NMR (DMSO-d6, 75 Hz): 39.8, 110.0, 116.7, 119.3, 122.1, 124.3, 127.1, 135.1, 148.2, 150.3, 158.6. C11H9N3O, MW= 199.20 g/mol; tR,LCMS = 1.40 min; MS (ESI+): m/z = 200 [M+H]+.

3 J

 3 = 8.0 Hz, ArH), 6.94 (1H, d, 4 J = 1.85 Hz, ArH), 6.98 (1H, dd, 4 J = 1.85 Hz and 3 J = 8.0 Hz, ArH). C10H12O2; MW= 164.20 g/mol. 2-(3,4,5-trimethoxyphenyl)ethylene (12c). Yield 90%, 4.5 g. Yellow oil. 1 H NMR (CDCl3, 300 Hz): 3.90 (9H, m, 9H, OCH3), 5.23 (1H, dd, 3 J = 1.2 Hz and 3 J = 11.2 Hz, CH), 5.67 (1H, dd, 3 J = 1.2 Hz and 2 J = 18.0 Hz, CH), 6.65 (3H, m, CH, ArH). C11H14O3; MW= 194.22 g/mol.2-(3-benzyloxy-4-methoxyphenyl)ethylene (12d).Yield 71%, 4.4 g. White solid, mp 67-69 °C (Lit. 49 68-69°C). 1 H NMR (CDCl3, 300 Hz): 3.85 (3H, s, OCH3), 5.08 (1H, dd, 3 J = 0.85 Hz and 1.8 Hz and 3 J = 8.45 Hz, ArH), 6.99 (1H, d, 4 J = 1.8 Hz, ArH), 7.20-7.69 (5H, m, ArH benz). C16H16O2; MW= 240.29 g/mol.

  Hz, CH2), 3.88 (3H, s, OCH3), 5.16 (2H, s, OCH2),6.80 (2H, m, ArH), 6.86 (1H, d, 3 J = 8.8 Hz, ArH), 7.30-7.50 (5H, m, ArH benz). C11H16O4; MW= 212.24 g/mol General Procedure for synthesis of compounds 14a-d. A solution of methanesulfonyl chloride (1 mL, 0.01 mol) in DCM (5 mL) is added dropwise to a solution of the commercial available alcohol 13a or synthesized alcohol 13b-d (0.005 mol) and triethylamine (1.7 mL, 0.0125 mol) in DCM (15 mL) at 0 °C and stirred for 8 h at room temperature. The reaction mixture is first washed with 1N aqueous hydrochloric acid solution (10 mL), then saturated sodium bicarbonate solution (10 mL) and finally water (10 mL). The organic layer is dried, filtered, concentrated in vacuo, and crystallized from cyclohexane.

  (2H, s, OCH2), 6.75-6.90 (3H, m, ArH), 7.30-7.50 (5H, m, ArH benz). C17H20O5S; MW= 336.40 g/mol.General Procedure for synthesis of compounds 15a-d and 16a-d. A solution of compound 9or 10 (1 mmol) in anhydrous DMF(5 mL) is added dropwise at room temperature under nitrogen to a suspension of sodium hydride (0.06 g, 1.5 mmol, 60% dispersion in mineral oil) in anhydrous DMF (5 mL) and then stirred for 1 h at 60 °C. A solution of synthesized arylalkyl methanesulfonate 14a-d (1.5 mmol) in anhydrous DMF (5 mL) is added dropwise to the previous solution and stirred for 24 h at 60 °C. The reaction mixture is cooled in an ice bath and hydrolyzed with water (85 mL). The resulting precipitate is filtered and recrystallized in EtOH/water (80/20).N-[2-(4-methoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15a). Yield 23%, 74 mg. White solid, mp 133-135 °C. 1 H NMR (CDCl3, 300 Hz): 3.13 (2H, t, 3 J = 7.4 Hz, CH2), 3.79 (3H, s, OCH3), 4.34 (2H, t, 3 J = 7.5 Hz, CH2), 6.57 (1H, d, 3 J = 2.85 Hz, NCH), 6.84 (2H, d, 3 J = 8.9 Hz, ArH), 7.07 (2H, d,3 J = 8.9 Hz, ArH), 7.28 (1H, t, 3 J = 7.6 Hz, CH quina), 7.45 (1H, d, 3 J = 2.7 Hz, CONCH), 7.65-7.72 (2H, m, CH quina), 8.37 (1H, d, 3 J = 8.15 Hz, CH quina). 13 C NMR (CDCl3, 75 Hz): 34.4, 46.7, 55.2, 105.0, 114.2, 114.6, 119.2, 122.0, 125.5, 126.9, 129.5, 129.8, 134.0, 144.7, 150.0, 158.0, 158.6. C19H17N3O2; MW= 319.35 g/mol; tR,LCMS = 2.38 min; MS (ESI+): m/z = 320 [M+H]+. HPLC purity 98.3%. N-[2-(3,4-dimethoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15b). Yield 20%, 69 mg. White solid, mp 160-162 °C. 1 H NMR (CDCl3, 300 Hz): 3.12 (2H, t, 3 J = 7.2 Hz, CH2), 3.82 (3H, s, OCH3), 3.85 (3H, s, OCH3), 4.36 (2H, t, 3 J = 7.1 Hz, CH2), 6.59 (1H, d, 3 J = 2.5 Hz, NCH), 6.68 (2H, m, ArH), 6.78 (1H, d, 3 J = 8.75 Hz, ArH), 7.28 (1H, t, 3 J = 7.3 Hz, CH quina), 7.45 (1H, d, 3 J = 2.35 Hz, CONCH), 7.63-7.73 (2H, m, CH quina), 8.36 (1H, d, 3 J = 8.0 Hz, CH quina).[START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF] C NMR (CDCl3, 75 Hz): 34.8, 46.5, 55.9, 105.0, 111.3, 111.8, 114.6, 119.2, 120.9, 122.0, 125.5, 126.9, 129.9, 134.0, 144.7, 147.9, 149.1, 150.0, 157.9. C20H19N3O3; MW= 349.38 g/mol; tR,LCMS = 2.17 min; MS (ESI+): m/z = 350[M+H]+. HPLC purity 99.4%.N-[2-(3,4,5-trimethoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15c). Yield 22%, 83 mg.Off-white solid, mp 172-174 °C.1 H NMR (CDCl3, 300 Hz): 3.12 (2H, t, 3 J = 7.2 Hz, CH2),3.80 (9H, s, OCH3), 4.39 (2H, t, 3 J = 7.1 Hz, CH2), 6.38 (2H, s, ArH), 6.67 (1H, d, 3 J = 2.6 Hz, NCH), 7.29 (1H, t, 3 J = 7.5 Hz, CH quina), 7.50 (1H, d, 3 J = 2.6 Hz, CONCH), 7.63-7.74 (2H, m, CH quina), 8.36 (1H, d, 3 J = 7.5 Hz, CH quina). 13 C NMR (CDCl3, 75 Hz): 35.7, 46.2, 56.1, 60.9, 105.2, 105.7, 114.6, 119.1, 122.1, 125.4, 127.0, 133.2, 134.1, 136.9, 144.8, 149.9, 153.4, 157.9. C21H21N3O4; MW= 379.41 g/mol; tR,LCMS = 2.07 min; MS (ESI+): m/z = 380 [M+H]+. HPLC purity 99.1%.N-[2-(3-benzyloxy-4-methoxyphenyl)ethyl]imidazo[2,1-b]quinazolin-5-one (15d). Yield 26%, 238 mg. Off-white solid, mp 104-106 °C.1 H NMR (CDCl3, 300 Hz): 3.07 (2H, t, 3 J = 7.2 Hz, CH2), 3.87 (3H, s, OCH3), 4.28 (2H, t, 3 J = 7.2 Hz, CH2), 5.12 (2H, s, OCH2), 6.36 (1H, d, 3 J = 2.9 Hz, NCH), 6.65 (1H, dd, 4 J = 1.8 Hz and 3 J = 8.1 Hz, ArH), 6.71 (1H, d, 4 J = 1.8 Hz, ArH), 6.79 (1H, d, 3 J = 8.12 Hz, ArH), 7.26-7.42 (7H, m, CONCH, CH quina, ArH benz), 7.65 (1H, d, 3 J = 7.4 Hz, CH quina), 7.71 (1H, td, 4 J = 1.6 Hz and 3 J = 6.8 Hz, CH quina), 8.37 (1H, dd, 4 J = 1.3 Hz and 3 J = 8.4 Hz, CH quina); 13 C NMR (CDCl3, 75 Hz): 34.6, 46.4, 56.0, 71.0, 104.9, 112.0, 114.6, 114.8, 119.2, 121.6, 122.0, 125.5, 126.9, 127.2, 127.9, 128.5, 129.8, 134.0, 137.0, 144.7, 148.0, 148.7, 150.0, 157.9. C26H23N3O3; MW= 425.48 g/mol; tR,LCMS = 2.69 min; MS (ESI+): m/z = 426 [M+H]+. N-[2-(4-methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one (16a). Yield 23%, 77 mg. White solid, mp 114-116 °C. 1 H NMR (CDCl3, 300 Hz): 3.00 (2H, t, 3 J = 7.6 Hz, CH2), 3.80 (5H, m, CH2, OCH3), 4.01 (2H, dd, 3 J = 2.1 Hz and 3 J = 3.5 Hz, NCH2), 5.34 (1H, td, 3 J = 3.5 Hz and 3 J = 8.5 Hz, CH2CH), 6.87 (2H, d, 3 J = 8.6 Hz, ArH), 7.13 (1H, td, 4 J = 1.05 Hz and 3 J = 7.15 Hz, CH quina), 7.22 (2H, d, 3 J = 8.6 Hz, ArH), 7.36 (1H, d, 3 J = 8.5 Hz, CONCH), 7.49-7.59 (2H, m, CH quina), 8

  Vivo. In-vivo cytotoxic antitumor activity of the selected compounds was evaluated using a syngeneic murine melanoma model (B16F10) in C57BL/6 mice purchased from Charles River (Wilmington, Massachusetts, USA). Briefly, animals were randomly divided into six groups (A: Control (n=5), B: Vehicle (n=5), C: 1 (n=5), D1, D2 and D3: 16a: 5, 25 and 50 mg/Kg/day (n=10)) and one million B16F10 melanoma cells were suspended in 100 μl of PBS and inJected subcutaneously in the right flank of 8-week-old male mice. Tumor growth was quantified by Vernier caliper measurements every 3 or 4 days. Tumor volume was calculated as 0.5 x length x width 2 . Seven days after inJection, individuals were chronically exposed to treatment by the use of Alzet® micro-osmotic pumps (200 µL total volume, model 2002, Charles River Laboratories, (Wilmington, Massachusetts, USA). Filling and preparation of the pumps for implantation was done as described by the manufacturer. The micro-osmotic pumps were engrafted on the upper-right dorsal skin area and under general anesthesia; each individual was administered with a single subcutaneous dose of Ketamine (80 mg/kg) and Xylazine (16 mg/kg). Each molecule was dissolved in the vehicle solution (0.9%

Figure 1 .

 1 Figure 1. Rational design of compounds 15-18 and 26-27.

  -d: n=0 18a-d: n=1 a: R 1 , R 3 = H, R 2 = OCH 3 b: R 1 , R 2 = OCH 3 , R 3 = H c: R 1 , R 2 , R 3 = OCH 3 d: R 1 = OH, R 2 = OCH 3 , R 3 = H a Reagents and conditions: (a) KOCN, AcOH, H 2 O, 35°C, 74%; (b) POCl

Scheme 2 .a

 2 Scheme 2. Synthesis of Benzothiadiazine Analogues (26-27) a

Figure 2 .

 2 Figure 2. Typical DNA content frequency histograms representing the effect of 16a vs. vehicle

Figure 3 .

 3 Figure 3. Effects of 16a vs. vehicle on cytoskeleton organization showing decreased microtubule

Figure 4 .

 4 Figure 4. Tubulin content analysis. (A) The effects of compound 16a on tubulin polymerization

Figure 5 .

 5 Figure 5. Predicted binding mode of (a): 16a (green stick) and (b): 27a (purple) with tubulin

Figure 7 .

 7 Figure 7. Effect of reference 31 and compound 16a on tube formation. Images depicting the

Figure 8 .

 8 Figure 8. Inhibition of SK-Mel28 cell migration in a wound healing assay. Cells were wounded

Figure 9 .

 9 Figure 9. Anticancer activity of compound 16a in a xenograft model of B16F10 murine

Figure 10 .

 10 Figure 10. In vivo effects of 16a on tumor angiogenesis. B16F10 murine melanoma cells were

General Procedure for synthesis of compounds 9 and 10

  Hz, CH quina), 7.22 (1H, d, 3 J = 8.65 Hz, CH quina), 7.56 (1H, t, 3 J = 7.8 Hz, CH quina), 7.87 (1H, d, 3 J = 7.8 Hz, CH quina), 10.80 (1H, s, CONH). 1H, s, NH), 7.08 (1H, t, 3 J = 8.2 Hz, CH quina), 7.22 (1H, d, 3 J = 8.0 Hz, CH quina), 7.55 (1H, t, 3 J = 7.15 Hz, CH quina), 7.86 (1H, d, 3 J = 7.8 Hz, CH quina), 10.10 (1H, s, NH); 13 C NMR (DMSO-d6, 75 Hz): 15.8, 33.4, 36.8, 61.2, 101.1, 117.8, 122.0, 125.0, 126.3, 134.6, 150.9, . A solution of compound 7 (2.8 g, 0.01 mol) or 8 (2.9 g, 0.01 mol) dissolved in sulfuric acid (40 mL) is stirred at room temperature for 12h. The reaction mixture is poured in ice, alkalinized with 10N aqueous sodium hydroxide solution (pH 10) and then acidified with diluted acetic acid solution to pH 5. The resulting precipitate is filtered and recrystallized in ethanol.

	6.31 (151.5, 162.4. C15H21N3O3, MW= 291.34 g/mol; tR,LCMS = 1.73 min; MS (ESI+): m/z = 292
	[M+H]+.
	C14H19N3O3; MW= 277.31 g/mol.
	2-(3,3-diéthoxypropylamino)-3H-quinazolin-4-one (8). Yield 88%, 2.55 g. Yellow solid, mp
	99-101 °C. 1 H NMR (DMSO-d6, 300 Hz): 1.15 (6H, t, 3 J = 7.5 Hz, CH3), 1.72 (2H, m, CH2),
	3.33 (2H, m, NCH2), 3.46 (2H, m, OCH2), 3.65 (2H, m, OCH2), 4.59 (1H, t, 3 J = 5.5 Hz, CH),

  C NMR (CDCl3, 75 Hz): 31.5, 46.9, 51.3, 55.3, 107.2, 114.0, 116.5, 120.2, 122.4, 125.2, 127.2, 129.8, 131.2, 134.6, 146.2, 149.5, 158.2, 159.5. C20H19N3O2, MW= 333.38 g/mol; tR,LCMS = 2.93 min; MS (ESI+): m/z = 334 [M+H]+. HPLC purity 97.9%.

  OCH2), 5.29 (1H, td, 3 J = 3.5 Hz and 3 J = 7.15 Hz, CH2CH), 6.84 (3H, m, ArH), 7.13 (1H, t, 3 J = 7.5 Hz, CH quina), 7.29-7.38 (4H, m, CH quina, ArH benz), 7.42-7.50 (3H, m, CONCH, ArH benz), 7.56 (1H, td, 4 J = 1.45 Hz and 3 J = 8.5 Hz, CH quina), 8.08 (1H, dd, 4 J = 1.25 Hz and 3 J =

	H NMR (CDCl3, 300 Hz): 3.01 (2H, t, 3 J = 7.6 7.9 Hz, CH quina); 13 C NMR (CDCl3, 75 Hz): 20.3, 32.7, 40.4, 46.3, 51.4, 55.9, 70.4, 113.3,
	Hz, CH2), 3.80-3.90 (8H, m, CH2, OCH3), 4.01 (2H, dd, 3 J = 2.0 Hz and 3 J = 3.3 Hz, NCH2), 114.4, 116.2, 121.1, 121.6, 124.8, 126.6, 128.2, 128.8, 132.8, 134.4, 137.7, 146.6, 148.9, 149.5,
	5.35 (1H, td, 3 J = 3.5 Hz and 3 J = 7.7 Hz, CH2CH), 6.82 (3H, m, ArH), 7.14 (1H, t, 3 J = 7.3 Hz, 149.8, 161.9. C27H25N3O3, MW= 439.50 g/mol; tR,LCMS = 2.95 min; MS (ESI+): m/z = 440
	CH quina), 7.33 (1H, d, 3 J = 8.15 Hz, CH quina), 7.51-7.59 (2H, m, CONCH, CH quina), 8.09 [M+H]+.
	(1H, d, 3 J = 7.95 Hz, CH quina). 13 C NMR (CDCl3, 75 Hz): 32.1, 46.9, 51.1, 55.9, 55.9, 107.1,
	111.3, 112.0, 116.5, 120.3, 120.7, 122.5, 125.1, 127.3, 131.8, 134.6, 146.2, 147.6, 149.0, 149.4,
	159.5 . C21H21N3O3, MW= 363.41 g/mol; tR,LCMS = 2.65 min; MS (ESI+): m/z = 364 [M+H]+.
	HPLC purity 97.4%.
	Yield 22%, 86 mg. White solid, mp 156-158 °C. 1 H NMR (CDCl3, 300 Hz): 3.00 (2H, t, 3 J = 7.4
	Hz, CH2), 3.84 (5H, m, CH2, OCH3), 3.88 (6H, s, OCH3), 4.04 (2H, dd, 3 J = 1.95 Hz and 3 J =
	3.25 Hz, NCH2), 5.37 (1H, td, 3 J = 3.55 Hz and 3 J = 8.3 Hz, CH2CH), 6.53 (2H, s, ArH), 7.14
	(1H, td, 13 C NMR
	(CDCl3, 75 Hz): 32.9, 46.9, 50.9, 56.1, 60.9, 105.6, 107.1, 116.5, 120.3, 122.5, 125.0, 127.3,
	134.7, 135.0, 136.5, 146.2, 149.4, 153.3, 159.5. C22H23N3O4, MW= 393.43 g/mol; tR,LCMS = 2.67
	min; MS (ESI+): m/z = 394 [M+H]+. HPLC purity 97.7%.
	N-[2-(3-benzyloxy-4-methoxyphenyl)ethyl]-1,2-dihydro-pyrimidino[2,1-b]quinazolin-6-one
	(16d). Yield 26%, 114 mg. White solid, mp 139-141 °C. 1 H NMR (CDCl3, 300 Hz): 2.94 (2H, t,

N-

[2-(3,4,5-trimethoxyphenyl)

ethyl]-1,2-dihydro-pyrimidino[2,

1-b]

quinazolin-6-one (16c).

[START_REF] Li | Chapter 14. Antimitotic agents[END_REF]

J = 1.0 Hz and 3 J = 8.1 Hz, CH quina), 7.32 (1H, d, 3 J = 8.1 Hz, CH quina), 7.51-7.60 (2H, m, CONCH, CH quina), 8.08 (1H, dd, 4 J = 1.15 Hz and 3 J = 7.8 Hz, CH quina). 3 J = 7.6 Hz, CH2), 3.74 (2H, t, 3 J = 7.15 Hz, CH2), 3.88 (5H, m, NCH2, OCH3), 5.16 (2H, s,

General Procedure for synthesis of compounds 22 and 23. To a mixture of compound 20 or

  6H, s, OCH3), 3.91 (2H, t, 3 J = 7.0 Hz, CH2), 4.06 (2H, t, 3 J = 5.95 Hz, CH2), 6.49 (2H, s, ArH), 7.11 (1H, td, 4 J = 1.15 Hz and 3 J = 8.0 Hz, CH quina), 7.33 (1H, d, 3 J = 8.2 Hz, CH quina), 7.56 (1H, td, 4 J = 1.7 Hz and 3 J = 8.45 Hz, CH quina), 8.09 (1H, dd, 4 J = 1.25 Hz and 3 J = 8.0 Hz, CH

	NMR (CDCl3, 75 Hz): 33.1, 40.4, 45.2, 46.2, 55.9, 110.8, 114.9, 117.8, 120.1, 122.4, 124.9,
	126.4, 131.9, 134.2, 145.4, 145.7, 151.0, 152.4, 161.3. C15H19N3O3; MW= 337.37 g/mol; tR,LCMS
	= 1.87 min (96.6%); MS (ESI+): m/z = 338 [M+H]+. HPLC purity 98.4%.
	N-[2-(4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one quina). 13 C NMR (CDCl3, 75 Hz): 20.7, 33.8, 40.1, 46.8, 51.7, 56.1, 60.9, 105.7, 116.3, 121.8, (18a).
	Yield 69%, 115 mg. White solid, mp 242-244 °C. 1 H NMR (CDCl3, 300 Hz): 1.97 (2H, m, CH2), 124.6, 126.9, 134.1, 135.2, 136.5, 148.3, 149.5, 153.3, 162.6. C22H25N3O4; MW= 395.45 g/mol;
	2.99 (2H, t, 3 J = 7.5 Hz, NCH2), 3.26 (2H, t, 3 J = 6.9 Hz, NCH2), 3.80 (3H, s, OCH3), 3.86 (2H, t, 3 J tR,LCMS = 2.38 min (97.3%); MS (ESI+): m/z = 396 [M+H]+. HPLC purity 98.1%.
	= 7.1 Hz, CH2), 4.05 (2H, t, 2 J = 6.8 Hz, CH2), 6.85 (2H, d, 3 J = 8.65 Hz, ArH), 7.11 (1H, td, 4 J = 1.3 N-[2-(3-hydroxy-4-methoxyphenyl)ethyl]-1,2,3,4-tetrahydropyrimidino[2,1-b]quinazolin-6-one
	(18d). Yield 51%, 89 mg. White solid, mp 153-155 °C. 1 H NMR (DMSO-d6, 300 Hz): 1.93 (2H, m, CH2), 2.81 (2H, t, 3 J = 7.7 Hz, NCH2), 3.31 (2H, t, 3 J = 5.3 Hz, NCH2), 3.73 (5H, m, CH2, Hz and N-[2-(3,4-dimethoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one OCH3), 3.93 (2H, t, 3 J = 5.8 Hz, CH2), 6.65 (1H, dd, 90 (1H, s, OH); 13 C
	(18b). Yield 52%, 95 mg. White solid, mp 132-134 °C. 1 H NMR (CDCl3, 300 Hz): 1.98 (2H, m, NMR (CDCl3, 75 Hz): 20.3, 32.3, 40.3, 46.4, 51.8, 56.1, 112.8, 116.2, 116.5, 119.7, 121.6,
	CH2), 2.99 (2H, t, 3 J = 7.5 Hz, NCH2), 3.26 (2H, t, 3 J = 5.8 Hz, NCH2), 3.87-3.91 (8H, m, CH2, 124.9, 126.6, 132.4, 134.4, 146.5, 146.9, 148.9, 149.8, 161.9. C20H21N3O3; MW= 351.39 g/mol;
	). Yield 65%, 104 mg. White solid, mp 98-100 °C. 1 H NMR (DMSO-d6, 300 Hz): 2.85 (2H, t, 3 J = 7.45 Hz, CH2), 3.60 (4H, m, NCH2), 3.71 (3H, s, OCH3), 4.02 (2H, t, 3 J = 7.9 Hz, CH2), 6.87 (2H, d, 3 J = 8.9 OCH3), 4.04 (2H, t, CH quina), 8.09 (1H, dd, 4 J = 1.5 Hz and 3 J = 8.0 Hz, CH quina). 13 C NMR (CDCl3, 75 Hz): 20.7, 21 (0.02 mol) and triethylamine (5.6 mL, 0.04 mol) in DCM (200 mL) is added under ice cooling
	Hz, ArH), 7.12 (1H, t, 3 J = 7.45, CH quina), 7.22 (2H, d, 3 J = 8.9 Hz, ArH), 7.29 (1H, d, 3 J = 7.9 Hz, 33.0, 40.0, 46.9, 51.9, 55.8, 55.9, 111.2, 112.1, 116.3, 120.9, 121.7, 124.6, 126.8, 132.0, 134.1, and dropwise 2-nitrobenzenesulfonyl chloride 19 (3.5 g, 0.016 mol) dissolved in DCM (50 mL)
	CH quina), 7.57 (1H, t, 3 J = 8.4 Hz, CH quina), 7.92 (1H, d, 3 J = 7.95 Hz, CH quina). 13 C NMR 147.6, 148.3, 148.9, 149.6, 162.6. C21H23N3O3; MW= 365.42 g/mol; tR,LCMS = 2.38 min (98.2%);
	(DMSO-d6, 75 Hz): 32.2, 44.8, 46.1, 55.4, 114.3, 117.8, 122.3, 125.0, 126.2, 130.1, 131.2, 134.4, MS (ESI+): m/z = 366 [M+H]+. HPLC purity 98.5%.
	151.3, 153.0, 158.2, 160.6. C19H19N3O2, MW= 321.37 g/mol; tR,LCMS = 2.21 min; MS (ESI+): N-[2-(3,4,5-trimethoxyphenyl)ethyl]-1,2,3,4-tetrahydro-pyrimidino[2,1-b]quinazolin-6-one
	m/z = 322 [M+H]+. HPLC purity 99.0%.

3 J = 7.1 Hz, CH quina), 7.18 (2H, d, 3 J = 8.4 Hz, ArH), 7.36 (1H, dd, 4 J = 0.5 Hz and 3 J = 8.

3

Hz, CH quina), 7.57 (1H, td, 4 J = 1.6 Hz and 3 J = 7.0 Hz, CH quina), 8.09 (1H, dd, 4 J = 1.15 Hz and 3 J = 8.0 Hz, CH quina).

[START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF]

C NMR (CDCl3, 75 Hz): 20.7, 32.5, 40.0, 47.0, 52.2, 55.3, 113.9, 116.3, 121.7, 124.7, 126.8, 129.9, 131.4, 134.1, 148.3, 149.6, 158.2, 162.7. C20H21N3O2; MW= 335.39 g/mol; tR,LCMS = 2.59 min (96.7%); MS (ESI+): m/z = 336 [M+H]+. HPLC purity 99.1%.

2 J = 5.85 Hz, CH2), 6.68 (3H, m, ArH), 7.11 (1H, td, 4 J = 1.1 Hz and 3 J = 8.0 Hz, CH quina), 7.34 (1H, d, 3 J = 8.15 Hz, CH quina), 7.56 (1H, td, 4 J = 1.6 Hz and 3 J = 8.3 Hz, (18c). Yield 43%, 85 mg. White solid, mp 136-138 °C. 1 H NMR (CDCl3, 300 Hz): 2.01 (2H, m, CH2), 2.99 (2H, t, 3 J = 7.45 Hz, NCH2), 3.31 (2H, t, 3 J = 5.8 Hz, NCH2), 3.82 (3H, s, OCH3), 3.84 (4 J = 1.9 Hz and 3 J = 8.1 Hz, ArH), 6.73 (1H, d, 4 J = 1.9 Hz, ArH), 6.84 (1H, d, 3 J = 8.3 Hz, ArH), 7.08 (1H, t, 4 J = 1.1 Hz and 3 J = 8.1 Hz, CH quina), 7.27 (1H, dd, 4 J = 1.1 Hz and 3 J = 7.75 Hz, CH quina), 7.57 (1H, t, 4 J = 1.6 Hz and 3 J = 8.4 Hz, CH quina), 7.89 (1H, dd, 4 J = 1.15 Hz and 3 J = 7.95 Hz, CH quina), 8.tR,LCMS = 2.10 min (99.2%); MS (ESI+): m/z = 352 [M+H]+. HPLC purity 96.6%.

  185-186 °C).1 H NMR (CDCl3, 300 Hz): 2.47 (3H, s, SCH3), 3.91 (2H, t, 3 J = 7.5 Hz, NCH2), 4.10 (2H, t, 3 J = 7.5 Hz, NCH2), 7.68-7.82 (3H, m, CH2), 3.28 (2H, t, 3 J = 5.8 Hz, NHCH2), 3.80 (2H, t, 3 J = 5.4 Hz, NCH2), 7.06-7.11 (2H, m, CH thidia), 7.53 (1H, td, 1H, 4 J = 1.55 Hz and 3 J = 7.95 Hz, CH thiadia), 7.66 (1H, dd, 4 J = 1.55 Hz and 3 J = 7.95 Hz, CH thiadia), 8.09 (1H, s, NH). C10H11N3O2S; MW= 237.27 g/mol; tR,LCMS = Yield 54%, 250 mg. White solid, mp 75-77 °C.1 H NMR (300 MHz, CDCl3): 2.88 (2H, t, 3 J = 7.15 Hz, PhCH2), 3.23 (2H, t, 3 J = 6.3 Hz, PhCH2CH2), 3.64 (2H, m, 3 J = 7.15 Hz, NCH2), 3.88-3.94 (5H, m, OCH3, SO2NCH2), 6.77-6.86 (3H, m, ArH), 7.16 (1H, td, 4 J = 1.15 Hz and 3 J = 8.05 Hz, CH thiadia), 7.29-7.43 (6H, m, CH thiadia, ArH benz), 7.53 (1H, td, 4 J = 1.5 Hz and 3 J = 8.3 Hz, CH thiadia), 7.83 (1H, dd, 4 J = 1.25 Hz and 3 J = 8.0 Hz, CH thiadia). pressure of hydrogen for 72h. The catalyst is separated by filtration, the solvent evaporated under reduced pressure and the crude product is crystallized in EtOH. = 1.95 Hz and 3 J = 8.1 Hz, ArH), 6.81 (1H, d, 3 J = 8.2 Hz, ArH), 6.86 (1H, d, 4 J = 2.0 Hz, ArH), 7.17 (1H, td, 4 J = 1.1 Hz and 3 J = 8.25 Hz, CH thiadia), 7.31 (1H, dd, 4 J = 0.7 Hz and 3 J = 8.3 Hz, CH thiadia), 7.54 (1H, td, 4 J = 1.5 Hz and 3 J = 8.3 Hz, CH thiadia), 7.84 (1H, dd, 4 J = 1.25 Hz and 3 J = 7.9 Hz, CH thiadia).[START_REF] Bourdon | In vitro pharmacokinetic profile of a benzopyridooxathiazepine derivative using rat microsomes and hepatocytes: Identification of phases I and II metabolites[END_REF] C NMR (75 MHz, CDCl3): 32.9, 38.9, 44.5, 46.4, 56.0,

	ArH), 8.28 (1H, dd, 4 J = 2.4 Hz and 3 J = 7.3 Hz, ArH). C10H11N3O4S2; MW= 301.34 g/mol. N-(2-nitrophenylsulfonyl)-2-benzylsulfanyl-1,4,5,6-tetrahydropyrimidine (23). Yield 91%, 5.7 g. Colorless solid, mp 68-70 °C (Lit. 27 85 °C). 1 H NMR (CDCl3, 300 Hz): 1.99 (2H, m, CH2), 3.63 (2H, m, NCH2), 3.89 (2H, m, NCH2), 4.12 (2H, s, SCH2), 7.16-7.31 (5H, m, ArH benz), 7.59-7.71 (3H, m, ArH), 8.27 (1H, dd, 4 J = 2.4 Hz and 3 J = 7.65 Hz, ArH). C17H17N3O4S2; MW= 391.47 g/mol. 2,3-dihydroimidazo[1,2-b]1,2,4-benzothiadiazine 5,5-dioxyde (24). Yield 47%, 1.05 g. Yellow solid, mp 244-246 °C (Lit. 27 267 °C). 1 H NMR (DMSO-d6, 300 Hz): 3.57 (2H, m, NHCH2), 4.03 (2H, m, NCH2), 7.10-7.20 (2H, m, CH thiadia), 7.58 (1H, td, 4 J = 1.55 Hz and 3 J = 7.15 Hz, CH thiadia), 7.78 (1H, dd, 4 J = 1.55 Hz and 3 J = 7.9 Hz, CH thiadia), 8.24 (1H, s, NH); 13 C NMR (DMSO-d6, 75 Hz): 39.0, 41.3, 122.2, 122.7, 123.0, 125.3, 134.7, 146.2, 154.9. C9H9N3O2S; MW= 223.25 g/mol; tR,LCMS = 1.53 min; MS (ESI-): m/z = 222 [M-H]+. 1,2,3,4-tetrahydropyrimido[1,2-b]1,2,4-benzothiadiazine-6,6-dioxyde (25). Yield 42%, 1.0 g. 1.57 min; MS (ESI-): m/z = 236 [M-H]+. General Procedure for synthesis of compounds 26a,d and 27a-d. A solution of benzothiadiazine 24 or 25 (1 mmol) in anhydrous DMF (10 mL) is added dropwise at room temperature under nitrogen to a suspension of sodium hydride (0.06 g, 1.5 mmol, 60% dispersion in mineral oil) in anhydrous DMF (5 mL) and then stirred for 1 h at 60 °C. A solution of synthesized arylalkyl methanesulfonate 14a-d (1.5 mmol) in anhydrous DMF (5 mL) is added dropwise to the previous solution and stirred for 24 h at 60 °C. The reaction mixture is cooled in an ice bath and hydrolyzed with water (85 mL). The resulting precipitate is filtered and recrystallized from methanol. 1-[2-(4-Methoxyphenyl)-ethyl]-2,3-dihydroimidazo[1,2-b]1,2,4-benzothiadiazine 5,5-dioxide (26a). Yield 69%, 246 mg. White solid, mp 76-78 °C. 1 H NMR (300 MHz, CDCl3): 2.96 (2H, t, 3 J = 7.3 Hz, PhCH2), 3.42 (2H, t, 3 J = 7.2 Hz, PhCH2CH2N), 3.69 (2H, m, 3 J = 7.3 Hz, NCH2), 3.88 (3H, s, OCH3), 4.00 (2H, m, SO2NCH2), 6.87 (2H, d, 3 J = 8.6 Hz, ArH), 7.18 (3H, m, ArH, CH thiadia), 7.37 (1H, dd, 4 J = 2.0 Hz and 3 J = 7.8 Hz, CH thiadia), 7.55 (1H, td, 4 J = 2.0 Hz and 3 J = 7.8 Hz, CH thiadia), 7.84 (1H, dd, 4 J = 0.8 Hz and 3 J = 7.5 Hz, CH thiadia). 13 C NMR (75 MHz, CDCl3) 32.6, 38.9, 44.5, 46.6, 55.3, 114.1, 122.1, 122.5, 122.9, 125.8, 129.7, 130.5, 134.1, 145.7, 152.0, 158.4. C18H19N3O3S; MW= 357.42 g/mol; tR,LCMS = 2.61 min (98.6%); MS (ESI+): m/z = 358 [M+H]+. HPLC purity 99.3%. 1-[2-(3-benzyloxy-4-Methoxyphenyl)-ethyl]-2,3-dihydroimidazo[1,2-b]1,2,4-benzothiadiazine 5,5-dioxide (26d). 13 C NMR (75 MHz, CDCl3): 33.0, 38.9, 44.6, 46.3, 56.1, 70.9, 112.0, 114.7, 121.4, 122.1, 122.5, 122.9, 125.7, 127.3, 127.8, 128.5, 130.9, 134.0, 137.1, 145.7, 148.1, 148.5, 152.0. C25H25N3O4S; MW= 463.54 g/mol; tR,LCMS = 2.93 min (100%); MS (ESI+): m/z = 464 [M+H]+. 1-[2-(4-methoxyphenyl)-ethyl]-1,2,3,4-tetrahydropyrimidino[1,2-b]1,2,4-benzothiadiazine 6,6-dioxyde (27a). Yield 63%, 234 mg. Off-white solid, mp 105-107 °C. 1 H NMR (300 MHz, CDCl3): 2.02 (2H, m, CH2), 3.00 (2H, t, 3 J = 7.3 Hz, PhCH2), 3.19 (2H, t, 3 J = 6.25 Hz, PhCH2CH2), 3.80 (3H, s, OCH3), 3.84 (4H, m, SO2NCH2, NCH2), 6.86 (2H, d, Hz and 3 J = 7.95 Hz, CH thiadia). 1 H NMR (75 MHz, CDCl3): 22.7, 32.5, 39.1, 47.3, 52.3, 55.3, 113.9, 121.1, 121.5, 123.8, 125.7, 129.9, 131.2, 133.5, 144.8, 146.9, 158.0. C19H21N3O3S; MW= 371.45 g/mol; tR,LCMS = 2.83 min (98.6%); MS (ESI+): m/z = 372 [M+H]+. HPLC purity 99.4%. 1-[2-(3,4-dimethoxyphenyl)-ethyl]-1,2,3,4-tetrahydropyrimidino[1,2-b]1,2,4-benzothiadiazine 6,6-dioxyde (27b). Yield 66%, 265 mg. White solid, mp 109-111 °C. 1 H NMR (300 MHz, CDCl3): 2.00 (2H, m, CH2), 3.01 (2H, t, 3 J = 7.05 Hz, PhCH2), 3.15 (2H, t, 3 J = 6.1 Hz, PhCH2CH2), 3.83 (4H, m, SO2NCH2, NCH2), 3.87 (3H, s, OCH3), 3.88 (3H, s, OCH3), 6.77-6.84 (3H, m, ArH), 7.12 (1H, t, 3 J = 8.0 Hz, CH thiadia), 7.30 (1H, d, 3 J = 8.5 Hz, CH thiadia), 7.52 (1H, td, 4 J = 1.5 Hz and 3 J = 8.45 Hz, CH thiadia), 7.73 (1H, dd, 4 J = 1.4 Hz and 3 J = 7.95 Hz, 5,5-dioxide (26e). Yield 54%, 101 mg. White solid, mp 172-173°C. 1 H NMR (300 MHz, CDCl3): 2.90 (2H, t, 3 J = 7.1 Hz, PhCH2), 3.43 (2H, t, 3 J = 7.1 Hz, PhCH2CH2), 3.70 (2H, t, 3 J = 7.1 Hz, NCH2), 3.89 (3H, s, OCH3), 4.00 (2H, t, 3 J = 7.6 Hz, SO2NCH2); 5.65 (1H, s, OH), 6.73 (1H, dd, 4 J 1-[2-(3-hydroxy-4-methoxyphenyl)-ethyl]-1,2,3,4-tetrahydropyrimidino[1,2-b]1,2,4-benzothiadiazine 6,6-dioxyde (27e ). Yield 51%, 243 mg. White solid, mp 149-151°C. 1 H NMR (300 MHz, CDCl3): 2.04 (2H, m, CH2), 2.97 (2H, t, 3 J = 7.35 Hz, PhCH2), 3.22 (2H, t, 3 J = 6.0 Hz, PhCH2CH2), 3.79-3.87 (4H, m, SO2NCH2, NCH2), 3.88 (3H, s, OCH3), 5.66 (1H, s, OH), 6.72 (1H, dd, 4 J = 1.9 Hz and 3 J = 8.15 Hz, ArH), 6.80 (1H, d, 3 J = 8.15 Hz, ArH), 6.86 (1H, d, 4 J = 1.9 Hz, ArH), 7.12 (1H, t, 3 J = 8.1 Hz, CH thiadia), 7.29 (1H, d, 3 J = 8.4 Hz, CH thiadia), 7.52 (1H, td, 4 J = 1.35 Hz and 3 J = 8.4 Hz, CH thiadia), 7.74 (1H, dd, 4 J = 1.2 Hz and 3 J = 7.9 Hz, CH thiadia). 1 H NMR (75 MHz, CDCl3): 22.7, 32.8, 39.1, 47.3, 52.2, 56.0, 110.7, 115.0, CH thiadia). 1-[2-(3-hydroxy-4-Methoxyphenyl)-ethyl]-2,3-dihydroimidazo[1,2-b]1,2,4-benzothiadiazine 120.5, 121.1, 121.5, 123.7, 125.7, 132.3, 133.5, 144.7, 145.2, 145.6, 146.9. C19H21N3O4S; MW=

General Procedure for synthesis of compounds 24 and 25. A mixture of compound 22 or 23 (0.01 mol), tin(II) chloride (0.05 mol) and a few drops of concentrated hydrochloric acid is refluxed for 12 in EtOH (100 mL). After cooling, the reaction mixture is alkalinized with 20% aqueous ammonia solution and evaporated under vacuum. The brown solid is then extracted with EtOAc (250 mL) using a soxhlet apparatus for 2 days. The organic layer is evaporated under vacuum to provide a yellow solid which is washed with diethyl ether.

Yellow solid, mp 246-247 °C (Lit. 27 245-248 °C).

1

H NMR (DMSO-d6, 300 Hz): 1.97 (2H, m, 3 J = 8.75 Hz, ArH), 7.11 (1H, t, 3 J = 8.05 Hz, CH thiadia), 7.17 (2H, d, 3 J = 8.75 Hz, ArH), 7.29 (1H, d, 3 J = 8.5 Hz, CH thiadia), 7.52 (1H, td, 4 J = 1.5 Hz and 3 J = 8.5 Hz, CH thiadia), 7.74 (1H, dd, 4 J = 1.5 1 H NMR (75 MHz, CDCl3): 22.7, 33.1, 39.2, 47.5, 52.4, 55.9, 111.3, 112.1, 120.8, atmospheric 110.8, 114.8, 120.2, 122.1, 122.5, 122.9, 125.8, 131.7, 134.0, 145.4, 145.7, 152.0. C18H19N3O4S; MW= 373.42 g/mol; tR,LCMS = 2.23 min (98.5%); MS (ESI+): m/z = 374 [M+H]+. HPLC purity 95.4%. 387.45 g/mol; tR,LCMS = 2.35 min (96.2%); MS (ESI-): m/z = 386 [M+H]+. HPLC purity 98.5%.

  Regional Ethics Committee on Animal Experimentation and all the experiments complied withEuropean Parliament Directive 2010/63/EU. The adequacy of anesthesia was confirmed by the lack of the tail pinch response. Results are expressed as means ± SEM. Data were analyzed using ANOVA followed by Fisher's PLSD post hoc test implemented with Graphpad Prism® and

	2.15. Statistical Analysis. StatView software (SAS, Cary, NC, USA). Significance was assumed at P<0.05 (*; p<0.05, **;
	p<0.01, ***; p<0.005, ****; p<0.001).
	2.14. Analysis of In Vivo Tumor Vascularization. Immunohistochemistry was performed
	using an automated protocol developed for the DISCOVERY XT automated slide staining
	system (Ventana Medical Systems, Inc.,). Tumors were harvested, weighted and formalin fixed
	paraffin embedded for immunohistochemical analysis. Histological 6-μm thick tumor sections
	were deparafinized and incubated for 1 h with a rabbit anti-mouse CD31 polyclonal antibody
	(clone ab28364, Abcam ® ), then with Discovery UltraMap anti-Rabbit HRP (760-4315, Roche ® )
	and Discovery ChromoMap DAB kit (760-159, Roche ® ). Ten fields were examined per section.
	The percentage of vessel surface area and the number of vessels per square milimeter were
	quantified with Image J. Results were expressed as the percentage of vessel surface area (%) and
	the number of vessels/mm 2 .

Table 1 .

 1 Structures, inhibition of tubulin polymerization and in vitro proliferation inhibition percentage against human colon adenocarcinoma HT29 cell line of compounds 15a-

e, 16a-e, 17a-d, 18a-d, 26a,e and 27a-e.

  % of inhibition at 10 µM or concentration inhibiting HT29 cell proliferation relative to untreated controls after 72 h of drug exposure. .b Inhibition of tubulin polymerization. c Data reported previously.

	27c	SO2	CH2-CH2	1	OCH3 OCH3	OCH3	0 %	>10
	27e	SO2	CH2-CH2	1	OH	OCH3	H	32 %	0.461 ± 0.046
	2b1 c								0.010	2.4
	1 c								0.21	2.2
						A	N	R 1
						N	N	( ) n	R 2
									R 3
	Compd	A		n	R1		R2	R3	% inhibition or IC50 (µM) a	IC50 TPI (µM) b
	15a	C=O	CH=CH	0	H	OCH3	H	7.77	>10
	15b	C=O	CH=CH	0	OCH3 OCH3	H	7 %	>10
	15c	C=O	CH=CH	0	OCH3 OCH3	OCH3	0 %	>10
	15e	C=O	CH=CH	0	OH	OCH3	H	4.73	>10
	16a	C=O	CH=CH	1	H	OCH3	H	0.042	0.812 ± 0.11
	16b	C=O	CH=CH	1	OCH3 OCH3	H	0.766	0.373 ± 0.07
	16c	C=O	CH=CH	1	OCH3 OCH3	OCH3	0 %	>10
	16e	C=O	CH=CH	1	OH	OCH3	H	4.05	0.65 ± 0.08
	17a	C=O	CH2-CH2	0	H	OCH3	H	0 %	>10
	17b	C=O	CH2-CH2	0	OCH3 OCH3	H	0 %	>10
	17c	C=O	CH2-CH2	0	OCH3 OCH3	OCH3	0 %	>10
	17d	C=O	CH2-CH2	0	OH	OCH3	H	7.78	>10
	18a	C=O	CH2-CH2	1	H	OCH3	H	0.16	1.29 ± 0.18
	18b	C=O	CH2-CH2	1	OCH3 OCH3	H	47 %	>10
	18c	C=O	CH2-CH2	1	OCH3 OCH3	OCH3	0 %	>10
	18d	C=O	CH2-CH2	1	OH	OCH3	H	5.73	>10
	26a	SO2	CH2-CH2	0	H	OCH3	H	0 %	>10
	26e	SO2	CH2-CH2	0	OH	OCH3	H	21 %	>10
	27a	SO2	CH2-CH2	1	H	OCH3	H	0.2	0.27 ± 0.03
	27b	SO2	CH2-CH2	1	OCH3 OCH3	H	0 %	>10

a

J = 10.8 Hz, CH), 5.25 (2H, s, OCH2), 5.51 (1H, d, 3 J = 0.85 Hz and 2 J = 11.75 Hz, CH), 6.58 (1H, dd, 3 J = 10.8 Hz and 2 J = 11.75 Hz, CH), 6.82 (1H, d, 3 J = 8.3 Hz, ArH), 6.92 (1H, dd,

J =

The authors gratefully acknowledge the National Cancer Institute (NCI) for biological evaluation of compounds on their 60-cell panel: the testing was performed by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (the URL to the Program's website: http://dtp.cancer.gov). We are also thankful to Anna LokaJczyk and team UMR-S1140 Inserm for the graciously provided B16F10 cells, the flow cytometry core facility of BiCEL and Jean-Pierre Decavel, PLETHA, Institut Pasteur de Lille for their technical assistance. ABBREVIATIONS ADME, Absorption Distribution Metabolism and Excretion; BSA, bovine serum albumin; CA-4, combretastatin A-4; DCM, dichloromethane; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; DMF, N,N-dimethyl-formamide; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; ECGM, endothelial cell growth medium; EGTA, ethylene glycol tetraacetic acid; EtOAc, ethyl acetate; EtOH, ethanol; FBS, fetal bovine serum; GTP, guanosine triphosphate; MG-MID, mean graph midpoints; NCI, national cancer institute; NSCLC, non-small cell lung cancer; PBS, phosphate-buffered saline; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid; RPMI medium, Roswell Park Memorial Institute medium; SAR, structure-activity relationships; THF, tetrahydrofurane; TLC, thin layer chromatography; VDA, vascular disrupting agent.

AUTHOR INFORMATION

Corresponding Author

*For N.L.: phone, +33 (0)320 964 977; fax, +33 (0) 320 964 913; e-mail, nicolas.lebegue@univ-lille2.fr. Websites: http://medchem-u1172.univ-lille2.fr/.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT