Shannon Pecnard

Olivier Provot

Hélène Levaique

Jérome Bignon

Laurie Askenatzis

Francois Saller

Delphine Borgel

Sophie Michallet

Marie-Catherine Laisne

Laurence Lafanechère

Mouad Alami

Abdallah Hamze

Cyclic bridged analogs of isoCA-4: Design, synthesis and biological evaluation

Keywords: tubulin inhibitor, combretastatin A-4, quinaldine, pyridine, cancer

Introduction

Microtubules (MTs) are cytoskeletal filaments composed of α-and β-tubulin heterodimers, and they are essential for intracellular organization, organelle trafficking, and chromosome segregation. [1,2] In the context of anti-cancer small-molecule drug development, dynamic microtubules continue to be among the most successful cancer chemotherapeutic targets. [3] Microtubule-binding agents (MTAs) generally interact with one of five-primary tubulin binding sites: the laulimalide, maytansine, paclitaxel/epothilone, vinca alkaloid, and the colchicine sites. They perturb mitosis and arrest cells during the G 2 /M phase of the cell cycle. [4] MTAs also specifically perturb endothelial cell proliferation and migration. Thus, drugs that bind to the colchicine site undergo intensive investigation as vascular-targeting agents for cancer therapy. [5] Colchicine is not now in clinical use as an anti-cancer treatment owing to its narrow therapeutic index. [6] The combretastatins consist of a group of diaryl stilbenoid isolated by [START_REF] Pettit | Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4[END_REF] from the bark of the South African Bush tree Combretum caffrum. [START_REF] Pettit | Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4[END_REF] Combretastatin-A4 (CA-4) is the prototype of this series of vascular disrupting agents (VDA). [8,9] CA-4 has proven to be a significant cancer cell growth inhibitor and antimitotic agent through tubulin polymerization inhibition via binding to the colchicine binding site of tubulin. It causes rapid vascular shutdown and cell death in the tumor. [START_REF] Lin | Antimitotic natural products combretastatin A-4 and combretastatin A-2: studies on the mechanism of their inhibition of the binding of colchicine to tubulin[END_REF] The water-soluble phosphate prodrug of CA-4, the CA-4P (fosbretabulin, Fig. 1), was found to inhibit tumor blood flow at concentrations 10-fold lower than its maximum tolerated dose. This observation led to the first clinical trial of CA-4 as a VDA. [START_REF] Dark | Combretastatin A-4, an Agent That Displays Potent and Selective Toxicity toward Tumor Vasculature[END_REF] Fosbretabulin is in phase II/III clinical trials either alone or in combination with traditional chemotherapeutic agents or radiotherapy. [START_REF] Grisham | Clinical trial experience with CA4P anticancer therapy: focus on efficacy, cardiovascular adverse events, and hypertension management[END_REF][START_REF] Siemann | Realizing the Potential of Vascular Targeted Therapy: The Rationale for Combining Vascular Disrupting Agents and Anti-Angiogenic Agents to Treat Cancer[END_REF] CA-4 exists as cisand trans-stilbene isomer. Only the cis configuration of CA-4 possesses anti-cancer activity. Isomerization of cis CA-4 to less active trans-CA-4 is readily observed during storage, administration, and metabolism. [START_REF] Gaspari | Structural Basis of cis-and trans-Combretastatin Binding to Tubulin[END_REF][START_REF] Aprile | In Vitro Metabolism Study of Combretastatin A-4 in Rat and Human Liver Microsomes[END_REF] SAR studies on CA-4 demonstrated that the following elements are crucial for the biological activity and the inhibition of tubulin polymerization: 1) the presence of cis-double bond separating the two phenyl rings; 2) the 3,4,5-trimethoxyphenyl (TMP) A-ring play an important role to maintain the activity ; 3) more modifications in terms of SAR can be done on the C3' of B-ring. Concerning the deleterious isomerization issue of natural CA-4, several heterocyclic bridging CA-4 analogs have been prepared to restrict the cis configuration and provide optimal bioactivity. [START_REF] For Reviews | An Overview of Tubulin Inhibitors That Interact with the Colchicine Binding Site[END_REF] Our group has extensively reported a series of antiproliferative, tubulin-binding isoCA-4 compounds 3-5 (Fig. 1). We resolved the instability issue of CA-4, the new analogs such as isoCA-4, have similar inhibition of tubulin polymerization activity and cytotoxicity compared to CA-4 but show higher stability. [START_REF] Hamze | Synthesis, biological evaluation of 1,1diarylethylenes as a novel class of antimitotic agents[END_REF][18][19][20][START_REF] Maksimenko | Therapeutic modalities of squalenoyl nanocomposites in colon cancer: An ongoing search for improved efficacy[END_REF][START_REF] Hamze | How do we improve histone deacetylase inhibitor drug discovery?[END_REF] A few reports have attempted to modify the trimethoxyphenyl ring (ring A) with mixed outcomes. We have recently undertaken the challenge of optimizing isoCA-4 analogs with heterocycles, by replacing both traditional A-and B-rings. Thus, the 3,4,5-trimethoxyphenyl A ring was replaced by a quinolinyl (isoCoQuine, 6) [START_REF] Khelifi | Design, synthesis and anticancer properties of IsoCombretaQuinolines as potent tubulin assembly inhibitors[END_REF] or a quinazolinyl nuclei (isoCoQ, 7) [START_REF] Soussi | IsoCombretaQuinazolines: Potent Cytotoxic Agents with Antitubulin Activity[END_REF] successfully. Wang et al. developed a 3-atom linker containing nitrogen with a good antiproliferative activity (compounds 8 and 9). [START_REF] Zheng | Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents[END_REF] Recently, we found that classical ring B of CA-4 or isoCA-4 can be substituted effectively by a carbazolyl group (compound 10). [START_REF] Bzeih | A general synthesis of arylindoles and (1-arylvinyl)carbazoles via a one-pot reaction from Ntosylhydrazones and 2-nitro-haloarenes and their potential application to colon cancer[END_REF] Next, we noticed that the combination which associates a quinolinyl as ring A and carbazolyl group as ring B resulted in the highest potency. The Quinaldinyl-iso-Carbazolyl (QnisoCz) 11 compound was found more active than isoCA-4 against A549, U87-MG, and HUVEC cells. [START_REF] Naret | 1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] Also, QnisoCz 11 was 67-fold more cytotoxic than CA-4 against lung adenocarcinoma epithelial cells (A549). A similar replacement as in compound 11 was also achieved with a N(Me) linker in compound 12. [28] In the continuation of our work in the study of SARs of isoCA-4 series and encouraged by the exciting results obtained with quinolines and quinazolines, we undertake the current study to investigate the effect of novel variation of bridge structure on the antiproliferative activities of the resulting CA-4 analogs. Precisely, we have designed, synthesized, and evaluated a series of novel cyclic bridged analogs (CBAs) of CA-4 having a phenyl or a pyridine linker (Fig. 1). The use of a disconnection and reconnection approach of carbazole derivatives (10 or 11) led us to investigate the indole nucleus as B-ring equivalent. Simultaneously, for A-ring, different combinations were studied, including the use of 3,4,5-trimethoxyphenyl (TMP), and quinolinyl groups (Fig 1). We analyzed these novel heterocyclic structures' effect on cell viability, cell cycle, and tubulin polymerization in cells and in vitro, on pure tubulin.

Results and discussion

Chemistry

First, we prepared the series of compounds having a TMP group (ring A) as in the case of CA-4 and isoCA-4 and a phenyl group as a linker (Scheme 1). To explore the SARs, first, modifications were realized on the indole ring. The key N-tosylhydrazone (NTH) derivative 15 was prepared in a two steps reaction starting from commercial 5-bromo-1,2,3-trimethoxybenzene 13 (Scheme 1). We successfully realized a Suzuki-Miyaura coupling between reagent 13 and 3-acetylphenyl-boronic acid. Then, we converted the acetophenone derivative 14 into NTH 15 in excellent yield. Next, we used our established methodology to convert this NTH to indoles. [START_REF] Bzeih | A general synthesis of arylindoles and (1-arylvinyl)carbazoles via a one-pot reaction from Ntosylhydrazones and 2-nitro-haloarenes and their potential application to colon cancer[END_REF]29,30] We obtained the corresponding derivatives 16-19 in a moderate yield after two steps: the first consists of the formation of 1-nitro-2-(1-phenylvinyl)benzene intermediates by coupling 15 and the appropriate 1bromo-2-nitrobenzene derivative, whereas, in the second step, all the generated intermediates were the subject to Cadogan cyclization without prior purification. [31] To explore the importance of free NH-indole on the biological activity, we also realized the N-methylation of compound 16. We obtained the N-methylated indole 20 in a moderate yield (53%). As in our previous SAR studies, we demonstrated the possibility of the change of TMP part by a heterocyclic ring with similar or better activity. Next, we replaced the TMP ring present in compounds 16-20 by a quinoline nucleus to prepare a second series of bis-heterocyclic derivatives 28-31 (Scheme 2). First, the starting material 22 was prepared by a Negishi coupling starting from 2,4-dichloroquinoline 21 and Zn(CN) 2 under Pd-catalysis in excellent yield. We prepared acetophenone derivatives 24-25 by a Suzuki-Miyaura coupling between 22 or commercial 4-chloro-2methylquinoline 23 and 3-acetylphenyl-boronic acid. The conversion of these acetophenones to NTH 26-27 was realized easily by treatment with 4-methylbenzenesulfonohydrazide in refluxing EtOH. Again, we used our methodology [START_REF] Bzeih | A general synthesis of arylindoles and (1-arylvinyl)carbazoles via a one-pot reaction from Ntosylhydrazones and 2-nitro-haloarenes and their potential application to colon cancer[END_REF]29,30,[START_REF] Naret | One-Pot Selective Functionalization of Nitrogen-Containing Heterocycles with N-tosylhydrazones and Amines[END_REF][START_REF] Zhang | Pyrroloimidazo[1,2-a]pyridine Scaffolds through a Sequential Coupling of N-Tosylhydrazones with Imidazopyridines and Reductive Cadogan Annulation, Synthetic Scope, and Application[END_REF][START_REF] Zhang | Imidazodipyridines via DMAP Catalyzed Domino N-H Carbonylation and 6π-Electrocyclization: Synthetic Scope and Application[END_REF] to convert the NTH 26-27 to indoles in two steps, and the corresponding desired compounds 28-29 were obtained in moderate to good yield. To study the importance of free indole in the series, on the biological activity, we masked this amine either by a methyl or hydroxymethyl group, starting from compound 29. We obtained compound 30 in a good yield, by simple N-methylation. While compound 31 was obtained in a moderate yield after formylation and treatment in a basic media. As Wang et al. demonstrated the utility of the use of a pyridine bridge as a linker on the SAR study of CA-4 analogs, [START_REF] Zheng | Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents[END_REF] we, next, changed the linker between the A-and B-rings, from phenyl to pyridine nucleus (Scheme 3). We followed the same strategy depicted in Scheme 1 and 2. We obtained compounds 35-37 in moderate to good yield (Scheme 3). For the synthesis of compound 42, we first realized the synthesis of bromopyrdine derivative 40, and then, we performed the Suzuki-Miyaura coupling with 2-methylquinolin-4-yl)boronic acid. The indole formation was realized by Cadogan cyclization to give compound 42 in a 75% yield. Finally, N-methylation of the indole nucleus of 42 led to compound 43 in excellent yield.

Biological effects of the new compounds

Effects on cell viability

In a first approach, the effect of the new compounds on cell viability was evaluated on a human colon cancer cell line (HCT116), as mentioned in Table 1, in parallel with isoCA-4 and CA-4 as experimental controls. We determined their effect on cell viability by using the sensitive CellTiter-Glo® luminescent assay, a homogeneous-method to determine the number of viable cells in culture and based on the quantitation of the ATP (an indicator of metabolically active cells). The amount of ATP is directly proportional to the number of viable cells present in the culture.

As shown in Table 1, in the series of the compounds having a phenyl ring as a linker, compound 16 having the TMP ring demonstrates significant antiproliferative activity with an IC 50 of 70 nM. When a substituent such as methoxy (compound 17), nitrile (compound 18), or trifluoromethyl group (compound 19), was introduced on position C6 of indole nucleus, the cytotoxicity is no longer observed, suggesting that such a substitution of the indole ring does not favor binding of the compound to the colchicine site or impairs the ability of the compounds to penetrate the cells. When the NH of indole was methylated (compound 20), a less cytotoxic activity (drop by a factor 7) was obtained compared to compound 16, which has a free indole ring. Next, based on our previous SARs, we carried out the replacement of the TMP nucleus by a quinoline having a substituent in position C2 (compounds 28-31). 2-Quinolinecarbonitrile nucleus 28 displayed a modest antiproliferative activity (less active than 16). To our surprise, however, compound 29 having a quinaldine as A-ring showed about 2-fold higher activity in comparison to 16. Again, modifications of the indole ring reduce the cytotoxic activity as the N-methylated indole 30 or (indol-1-yl)methanol 31 derivatives were less potent than compound 29 having the free NH-indole. To better understand the influence of the nature of A-and B-rings on the cytotoxicity activity, in this series containing a phenyl ring as the linker, we compared reference compound 47 [35] having the same TMP (ring A) and 2methoxyphenol (ring B) as in the structure of CA-4 and isoCA-4. This compound showed low antiproliferative activity (around 60-and 114-fold less active then 16 and 29 respectively), suggesting that the quinaldine and indole nucleus interact better with the colchicine-binding site. Wang et al. have demonstrated that the use of a pyridine bridge between the TMP ring A and 2-methoxyphenol B of CA-4 induces an interesting antiproliferative activity. [START_REF] Zheng | Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents[END_REF] Therefore, we included in our SAR study the analysis of the effect of such pyridine linker. As shown in Table 1, this replacement led to a significant increase of the cytotoxicity activity in comparison with the phenyl linker: compound 35 having the TMP ring exhibited an IC 50 of 20 nM (3.5-fold higher than 16), and even better, compound 42 having quinaldine ring showed high antiproliferative activity with low single-digit nM (IC 50 value 2.2 nM, 18-fold more active than 29) in the HCT116 cell line. The activity obtained with 42 is comparable to those of prior lead isoCA-4 and CA-4 in the same assays, thus indicating that the A/Bring system is changeable and favorable bioisosteres include quinaldine and indole moieties. Again, we synthesized and tested compound 8, having pyridine linker and ring A and B as the natural CA-4. We found that this compound had low cytotoxicity against HCT-116 cells; this demonstrates the critical role played by the combination of quinaldine as ring A, pyridine as the linker, and indole as ring B in the same molecule, for the cytotoxic activity (Fig. 2). Table 1. Effect of designed compounds on HCT116 cell viability. a a HCT-116 human colon carcinoma cells. b Compound concentration required to decrease cell growth by 50%; values represent the average SD of three experiments. c The IC 50 value for CA-4 [36] and isoCA-4 [37,[START_REF] Lamaa | Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4[END_REF] were determined in this study.

After this first screening on HCT116 viability, the most active compound Quinaldinyl-Pyridyl-Indole (QnPyInd) 42 was selected for evaluation against six additional human cancer cell lines and compared with the reference compound isoCA-4 (Table 2 CA-4, respectively. The antiproliferative activity of 42 is as important in the MDR1 overexpressing K562R cell line as in the parental K562 cell line, indicating that this lead compound is not a substrate for P-glycoprotein (Pgp). Cytotoxic activities of compound 42 and isoCA-4 were equipotent against lung and breast cancer cells (A549 and MCF7). However, QnPyInd 42 was 25-and 17-fold more active than CA-4 against A549 and MCF7 cell lines, respectively. Next, we evaluated compound 42 on the colon-carcinoma cells HT-29, which are resistant to combretastatin A-4 because of the overexpression of a multidrug-resistance protein (MRP-1). [START_REF] Schobert | Stable combretastatin A-4 analogues with sub-nanomolar efficacy against chemoresistant HT-29 cells[END_REF] As depicted in Table 2, compound 42 displayed an intense antiproliferative activity against this resistant cell line, with a nanomolar IC 50 value of 9.1 nM, confirming that 42 is not a substrate of efflux pumps. The two reference compounds isoCA-4 and especially CA-4 were found inactive against HT-29 resistant cells, with IC 50 values of 275 and > 8000 nM, respectively. The differential activity of CA4, isoCA4, and compound 42 can be explained by the structural resemblance between iso-CA-4 and CA-4, which can be a substrate of MRP-1. In contrast, the new structure of 42 allows it to escape the detoxifying actions of these proteins overexpressed in HT-29 cells. Also, isoCA-4 and CA-4 share a meta-hydroxy group on the Bring, which is partially mediated by the MRP-1 transporter and can favorably eliminate glucuronidated phenols. [40] Compound 42, lacking the meta-OH group in the B-ring, was highly active in HT-29 cells.

Analyzing the physicochemical properties of compound 42 in comparison to the two references compounds CA4 and isoCA-4 shows that 42 is more lipophilic, with clogP of 5.2, compared to clogP of 3.3 for CA-4 and 3.15 for isoCA-4. Furthermore, in view of the powerful antiproliferative activity of 42 (Table 2), this compound could find application for the treatment of glioblastoma because of its potential ability to cross the blood-brain barrier.

Effect on Cell Cycle.

The cytotoxicity test probes all vital cell functions. To get further insights into the mechanism of action of the new active compound, we investigate the effect of 42 on cell cycle progression. Thus, we conducted a flow cytometry analysis and analyzed the effect of a 24 hour-treatment with different concentrations (20, 50, 100 nM) of compound 42 on the fate of K562 cells. As shown in Fig. 3, when we treated cells with compound 42, the number of cells in G 2 /M increases in a dosedependent manner. A large part of the cells (81 %) is blocked in G2/M when 42 is at a 100nM concentration. These results indicate that this compound induced a cell cycle arrest in mitosis. [START_REF] Hamze | Synthesis, biological evaluation of 1,1diarylethylenes as a novel class of antimitotic agents[END_REF]18], suggesting that this new compound targets tubulin.

G0/G1

Effect on cell microtubule network.

To analyze the effect of the compound on cell microtubules, we used a variant of a recently described sensitive cell-based assay, [START_REF] Ramirez-Rios | A New Quantitative Cell-Based Assay Reveals Unexpected Microtubule Stabilizing Activity of Certain Kinase Inhibitors, Clinically Approved or in the Process of Approval[END_REF] which quantifies the amount of microtubules present in cells. We compared the depolymerizing effect of 42 to CA-4 and iso-CA4. As shown in Fig. 4, after 2 h of incubation, we observed that 42 was indeed able to induce dose-dependent depolymerization of the microtubule network, with complete depolymerization observed for a 5 µM concentration. In the assay, compound 42 (IC 50 = 0.4 µM), was found less potent than CA-4 and iso-CA4 (IC 50 of 7 nM and 8 nM, respectively).

Fig. 4.

Comparative analysis of the effect of 42, CA-4, and iso-CA4 on microtubule dynamics in HeLa cells. Different doses of the compounds were applied to HeLa cells in microplates and the amount of residual microtubules was assessed after 2 hours, using a luminescent assay described in the material and methods section. Results are expressed as mean ± SEM of their independent experiments.

Effect on in vitro tubulin polymerization

The effect of compound 42 on in vitro tubulin polymerization (Fig. 5A) was compared to the effect of CA-4 (Fig. 5B) by monitoring absorbance at 350 nm. Indeed, light is scattered by microtubules to the extent that it is proportional to the microtubule polymer concentration. [START_REF] Lee | In vitro reconstitution of calf brain microtubules: effects of solution variables[END_REF] Tubulin (50 µM) was pre-incubated for 30 min with different concentrations of 42 or CA-4 at 4 °C. The formation of microtubules was induced by raising the temperature to 37 °C and adding GTP (1 mM).

As shown in Fig. 5A, compound 42 inhibited pure tubulin polymerization in a dose-dependent manner, showing that it can bind tubulin directly. In these experimental conditions, the estimated concentration to inhibit 50% of the polymerization was found twice fold higher for 42 (5 µM) than for CA-4 (2.5 µM). Thus, although 42 can bind tubulin, it is a little less active than CA-4.

Effects of compound 42 on Mitochondrial Dysfunction in K562 Cells

Mitochondria play critical roles in cellular metabolism, homeostasis, and stress responses by generating ATP for energy and regulating cell death. [43] Mitochondrial dysfunctions is usually caused by depolarization and is the early hallmark of toxicity mediated through caspase-induced apoptosis. [START_REF] Spinelli | The multifaceted contributions of mitochondria to cellular metabolism[END_REF] As shown in Fig. 6, compound 42-induced mitochondrial dysfunction was detected using a fluorescence-based mitochondria-specific voltage-dependent dye, JC-1. Thus, our results showed that 42 induced mitochondrial dysfunctions in a concentration-dependent manner, with significant effects beginning at a concentration of 20 nM. Our study provides convincing evidence indicating that compound 42 dose-dependently caused caspase-induced apoptosis of K562 cells through mitochondrial dysfunction.

Fig. 6 Compound 42 induced mitochondrial dysfunctions in K562 leukemia cells. Cells were incubated with 42 at concentrations of 20, 50, 100 nM for 24 h at 37 °C. The portion of mitochondria dysfunction was measured using the JC1 assay.

Effect of compound 42 and the reference compound isoCA-4 in peripheral blood lymphocytes (PBLs).

To evaluate the safety profile and the cytotoxic potential of compound 42 in human no cancer cells, we compared its cytotoxicity to that of the reference compound isoCA-4 in quiescent peripheral blood lymphocytes (PBLs) isolated from one healthy donor. Under our experimental conditions, dose-response curves (Fig. 7A) yielded IC 50 values of 137 nM and >10 μM for isoCA-4 and compound 42, respectively. This clearly indicated that compound 42 was far less cytotoxic than isoCA-4 in quiescent PBLs, with a difference of at least two orders of magnitude. The proliferation of PBLs from the same donor was also induced by the potent mitogen PHA, and in agreement with previous results, [START_REF] Naret | 1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] isoCA-4 was more cytotoxic in proliferating than in quiescent PBLs. However, as in quiescent PBLs, compound 42 was again less cytotoxic than isoCA-4 (IC 50 value of 114 nM and 4.3 nM for compound 42 and isoCA-4, respectively) (Fig. 7B). Together, these results very interestingly supported a better safety profile for compound 42 than for isoCA-4.

Docking study

We performed molecular docking for compound 42 within the colchicine binding site of tubulin β subunit (the structure obtained from the X-ray crystal structure with accession code 6H9B). [START_REF] Naret | 1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] The overall binding mode observed matched that previously known for isoCA-4 as well as with compound 10, with the quinaldine ring system being accommodated by the lipophilic pocket ordinarily occupied by the trimethoxyphenyl nucleus of isoCA4, as well as the indole ring sitting at the same place as the B ring of isoCA-4. Interactions that can be expected given this binding mode hypothesis include notably (see Fig. 8) a potential hydrogen bond between the side-chain SH group of the cysteine β241 residue and the endocyclic nitrogen atom of the quinaldine moiety. The contribution of this interaction to the overall binding is reinforced by the contact between the northern part of the pyridine linker and the hydrophobic part of the side chain of lysine β254 as well as the indole moiety being sandwiched between hydrophobic parts of the side chains of lysine β352 and that of asparagine β258.

Conclusion

In this work, we have designed and synthesized a series of Cyclic Bridged Analogs CBA derivatives of isocombretastatin A-4 with different aryl or heteroaryl cycles as A-ring and indole as B ring. After the SAR study, we found that the best combinations associate in their structures (i) quinaldine nucleus as an A ring (ii) connected to the pyridine as a linker and (iii) an indole nucleus as B-ring. The more interesting compound in these novel series is Quinaldinyl-Pyridyl-Indole (QnPyInd) 42, which displayed a promising antiproliferative activity against seven human cancer cell lines. Besides, the new tri heterocyclic derivative 42 demonstrated high cytotoxic activity in PGP overexpressing multidrug-resistant leukemia cells (K562-R) and highly multidrug-and CA-4-resistant HT-29 cells; this result is of considerable significance given that the expression of Pgp is frequently associated with clinical resistance to chemotherapy. Compound 42 directly targets tubulin, as indicated by its ability to inhibit pure tubulin polymerization in vitro. Interestingly, although its depolymerizing microtubules activity is less important than CA-4 and isoCA, both in vitro and in cells, compound 42 has a cytotoxic activity at least as high as that of CA-4 and isoCA. Although we cannot formally exclude that 42 has other targets than tubulin, our results support the conclusion that this small microtubule depolymerizing activity is sufficient to trigger cell cycle arrest and apoptosis. Finally, we demonstrated the cytotoxic potential of compound 42 in human no cancer cells compared to the reference compound, and it was far less cytotoxic than isoCA-4 in quiescent PBLs.

Experimental

General considerations

Solvents and reagents are obtained from commercial suppliers and were used without further purification. Analytical TLC was performed using Merck silica gel F254 (230-400 mesh) plates and analyzed by UV light or by staining upon heating with vanillin solution. For silica gel chromatography, the flash chromatography technique was used, with Merck silica gel 60 (230-400 mesh) and p.a. grade solvents unless otherwise noted. The A sealed tube under argon atmosphere was charged at room temperature with ArX (0.8 mmol) and 1.5 equivalents of boronic acid (1.2 mmol). [Pd] (5 mol%), ligand (10 mol%), and base (2.4 mmol) were then added. 5 mL of the solvent were added via syringe at room temperature. The sealed tube was put into a preheated oil bath (110°C) and stirred overnight. Reaction completion was evaluated by silica TLC (80/20 cyclohexane/EtOAc). The crude mixture was then allowed to cool down to room temperature. EtOAc was added to the mixture, which was filtered through Celite. The solvents were evaporated under reduced pressure and the crude residue was purified by silica gel chromatography (95/5 to 85/15 of cyclohexane/EtOAc). The product was then concentrated under vacuum to give the desired product.

1-(3',4',5'-Trimethoxy-[1,1'-biphenyl]-3-yl)ethan-1-one 14

14 was prepared according to the general method A from 5-bromo-1,2,3-trimethoxybenzene ( 13) and 3-acetylphenyl-boronic acid, using the following catalytic system: Pd(OAc) 2 (5 mol%), SPhos (10 mol%), K 3 PO 4 .H 2 O (3 equiv), CPME/H 2 O (5:1 V/V). Column chromatography on silica gel afforded 209 mg of the product as a white powder (0.73 mmol, yield 91%); TLC (SiO 2 , 80/20 cyclohexane/EtOAc); R f = 0.28; 1

4-(3-Acetylphenyl)quinoline-2-carbonitrile 24

24 was prepared according to the general method A from 4-chloroquinoline-2-carbonitrile ( 22) and 3-acetylphenyl-boronic acid, using the following catalytic system: Pd(PPh 3 ) 4 (5 mol%), K 3 PO 4 .H 2 O (3 equiv), dioxane/H 2 O (5:1 V/V). The crude residue was purified by silica gel chromatography (95/5 to 85/15 of cyclohexane/ EtOAc). Column chromatography on silica gel afforded 146 mg of the desired compound as a white solid (0.54 mmol, yield 67%). TLC (SiO 2 , 80/20 cyclohexane/EtOAc) R f = 071; m.p = 191-193 °C; 1

1-(3-(2-Methylquinolin-4-yl)phenyl)ethan-1-one 25

25 was prepared according to the general method A from 4-chloro-2-methylquinoline ( 23) and 3acetylphenyl-boronic acid, using the following catalytic system: Pd(PPh 3 ) 4 (5 mol%), K 3 PO 4 .H 2 O (3 equiv), dioxane/H 2 O (5:1 V/V). Column chromatography on silica gel afforded 146 mg of the desired compound as a white powder (0.56 mmol, yield 70%); TLC (SiO 2 , 85/15 cyclohexane/ EtOAc) R f = 0.15; m.p. = 125-126°C; 1

1-(6-(3,4,5-Trimethoxyphenyl)pyridin-2-yl)ethan-1-one 33

33 was prepared according to the general method A from 3,4,5-trimethoxyphenyl)boronic acid (32) and 1-(6-bromopyridin-2-yl)ethanone, using the following catalytic system: Pd(OAc) 2 (5 mol%), PPh 3 (10 mol%), K 2 CO 3 (3 equiv), dioxane/H 2 O (5:1 V/V). Column chromatography on silica gel afforded 225 mg of the desired compound as a white solid (0.784 mmol, yield 98%). TLC (SiO 2 , 85/15 cyclohexane/ EtOAc) R f = 0.60; m.p. = 91-93 °C; 1

2-Methyl-4-(6-(1-(2-nitrophenyl)vinyl)pyridin-2-yl)quinoline 41

41 was prepared according to the general method A from 2-bromo-6-(1-(2-nitrophenyl)vinyl)pyridine ( 40) and 2-methylquinolin-4-yl)boronic acid, using the following catalytic system: Pd 2 dba 3 .CHCl 3 (10 mol%), SPhos (20 mol%), K 2 CO 3 (3 equiv), dioxane/H 2 O (5:1 V/V). Column chromatography on silica gel afforded 88 mg of the desired compound as an orange/pink oil (0.24 mmol, yield 30%). TLC (SiO 2 , 7/3 cyclohexane/AcOEt) R f = 0.75; 1

3'-Bromo-4-methoxy-[1,1'-biphenyl]-3-yl acetate 45.

45 was prepared from commercial (3-bromophenyl)boronic acid [START_REF] Spinelli | The multifaceted contributions of mitochondria to cellular metabolism[END_REF] and 5-iodo-2-methoxyphenyl acetate according to the general method A using the following catalytic system: Pd(PPh 3 ) 4 (2 mol%), K 2 CO 3 (3 equiv), toluene/H 2 O (4:1 V/V). Column chromatography on silica gel afforded 158 mg of the desired compound 45 as a white solid (0.5 mmol, yield 30%). TLC (SiO 2 , 7/3 cyclohexane/AcOEt) R f = 0.47; 1 [START_REF] Prudent | Pharmacological Inhibition of LIM Kinase Stabilizes Microtubules and Inhibits Neoplastic Growth[END_REF] was prepared from the product 3'-bromo-4-methoxy-[1,1'-biphenyl]-3-yl acetate [START_REF] Naret | 1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] and (3,4,5trimethoxyphenyl)boronic acid (30) according to the general method A using the following catalytic system: Pd(PPh 3 ) 4 (2 mol%), K 2 CO 3 (3 equiv), toluene/H 2 O (4:1 V/V) to afforded 3'',4,4'',5''tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate. Column chromatography on silica gel afforded 30 mg of the desired compound 46 as a pale oil (0.07 mmol, yield 46%). TLC (SiO 2 , 5/5 cyclohexane/AcOEt) R f = 0.38; 1

3'',4,4'',5''-Tetramethoxy-[1,1':3',1''-terphenyl]-3-ol 47[35]

Deprotection of acetate group of 3'',4,4'',5''-tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate ( 46) with K 2 CO 3 (10 eq) in MeOH afforded 3'',4,4'',5''-tetramethoxy-[1,1':3',1''-terphenyl]-3-ol [START_REF] Peris | Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends[END_REF]. Column chromatography on silica gel afforded 16 mg of the desired compound 42 as a white beige solid (0.04 mmol, yield 73%). 1

2-Bromo-6-(3,4,5-trimethoxyphenyl)pyridine 49[25]

49 was prepared in three steep from commercial 2,6-dibromopyridine ( 48) and (3,4,5trimethoxyphenyl)boronic acid (30) according to the general method A using the following catalytic system: Pd(OAc) 2 (5 mol%), P(Cy 3 ).BF 3 (10 mol%), K 2 CO 3 (3 equiv), toluene/H 2 O (30:1 V/V). Column chromatography on silica gel afforded 35 mg of the desired compound 48 as a brown solid (0.1 mmol, yield 30%). TLC (SiO 2 , 7/3 cyclohexane/AcOEt) R f = 0.53; 1

2-(3-(Benzyloxy)-4-methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine 50

50 was prepared from 2-bromo-6-(3,4,5-trimethoxyphenyl)pyridine ( 49) and (3-(benzyloxy)-4methoxyphenyl)boronic acid according to the general method A using the following catalytic system: Pd(OAc) 2 (5 mol%), PPh 3 (10 mol%), K 2 CO 3 (3 equiv), dioxane/H 2 O (2:1 V/V) . Column chromatography on silica gel afforded 50 mg of the desired compound 49 as a pale white oil (0.11 mmol, yield 60%). TLC (SiO 2 , 7/3 cyclohexane/AcOEt) R f = 0.51; 1 ), 3.97 (s, 6H, 2 OCH 3 ), 3.95 (s, 3H, OCH 3 ), 3.92 (s, 3H, OCH 3 ). 13 C NMR (101 MHz, CDCl3) δ 156.1, 156.0, 153.3 (2C), 150.8, 148.3, 137.4 (2C), 137.0, 128.4 (2C), 127.7, 127.2 (2C), 120.2, 117.9 (2C), 112.9 (2C), 111.7 (2C), 104.4, 104.3, 71.0, 60.8, 56.1 (2C), 55.9. 50) with Pd/C (10 mol%), H 2 , in EtOH. Column chromatography on silica gel afforded 20 mg of the desired compound 8 as a white solid (0.05 mmol, yield 80%). 1 H NMR (300 MHz, CDCl 3 ) δ 7.83 -7.72 (m, 2H, H Ar ), 7.67 (dd, J = 8.4, 2.2 Hz, 1H, H Ar ), 7.59 (ddd, J = 7.6, 6.5, 0.9 Hz, 2H, H Ar ), 7.37 (s, 2H, H Ar ), 6.97 (d, J = 8.4 Hz, 1H, H Ar ), 5.71 (s, 1H, OH), 3.99 (s, 6H, 2 OCH 3 ), 3.96 (s, 3H, OCH 3 ), 3.91 (s, 3H, OCH 3 ); 13

General procedure for the preparation of N-tosylhydrazones derivatives (Method B): 15, 26, 27, 34 and 39

A round bottom flask was charged with 1 equivalent of ketone and 2 equivalents of 4methylbenzenesulfonohydrazide in 15mL of absolute ethanol was refluxed for 16 h at 95°C. Reaction completion was evaluated by silica TLC (85/15 cyclohexane/AcOEt). The crude mixture was then allowed to cool down to room temperature and put in an ice bath to form a solid which was filtered with absolute ethanol to give the product as a solid.

(E)-4-Methyl-N'-(1-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3yl)ethylidene)benzenesulfonohydrazide 15

15 was prepared according to the general method B from 1-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl) ethan-1-one (4 mmol) (15) and 4-methylbenzenesulfonohydrazide (8 mmol). Crystallization in cold bath and then filtration afforded 1.55 g of the product as an orange solid (3.43 mmol, yield 86%); TLC (SiO 2 , 8/2 cyclohexane/EtOAc) R f = 0.59; m.p. = 173-175 °C; 1

(E)-N'-(1-(3-(2-Cyanoquinolin-4-yl)phenyl)ethylidene)-4-methylbenzenesulfonohydrazide 26

26 was prepared according to the general method B from 4-(3-acetylphenyl)quinoline-2-carbonitrile (24) (1.9 mmol) and 4-methylbenzenesulfonohydrazide (3.86 mmol). Crystallization in cold bath and then filtration afforded 510 mg of the desired compound as a yellow powder (1.16 mmol, yield 60%). TLC (SiO 2 , 6/4 cyclohexane/EtOAc) R f = 0.62; m.p. = 182-184 °C; 1

(E)-4-Methyl-N'-(1-(3-(2-methylquinolin-4-yl)phenyl)ethylidene)benzenesulfonohydrazide 27

27 was prepared according to the general method B from 1-(3-(2-methylquinolin-4-yl)phenyl)ethan-1-one (25) (2.30 mmol) and 4-methylbenzenesulfonohydrazide (4.6 mmol). Crystallization in cold bath and then filtration afforded 909 mg of the desired compound as a white solid (2.12 mmol, yield 92%). TLC (SiO 2 , 6/4 cyclohexane/ EtOAc) R f = 0.29; m.p. = 144-146 °C; 1 (E)-4-Methyl-N'- (1-(6-(3,4,5-trimethoxyphenyl)pyridin-2yl)ethylidene)benzenesulfonohydrazide 34 [START_REF] Zhang | Imidazodipyridines via DMAP Catalyzed Domino N-H Carbonylation and 6π-Electrocyclization: Synthetic Scope and Application[END_REF] was prepared according to the general method B from 1-(6-(3,4,5-trimethoxyphenyl)pyridin-2yl)ethan-1-one [START_REF] Zhang | Pyrroloimidazo[1,2-a]pyridine Scaffolds through a Sequential Coupling of N-Tosylhydrazones with Imidazopyridines and Reductive Cadogan Annulation, Synthetic Scope, and Application[END_REF] (1.38 mmol) and 4-methylbenzenesulfonohydrazide (2.77 mmol). Crystallization in cold bath and then filtration afforded 440 mg of the desired compound as an orange solid (0.97 mmol, yield 70%). TLC (SiO 2 , 60/40 cyclohexane/EtOAc) R f = 0.60; m.p. = 186-187 °C; 1

(E)-N'-(1-(6-Bromopyridin-2-yl)ethylidene)-4-methylbenzenesulfonohydrazide 39

39 was prepared according to the general method B from 1-(6-bromopyridin-2-yl)ethan-1-one [START_REF] Lamaa | Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4[END_REF] (1.24 mmol) and 4-methylbenzenesulfonohydrazide (2.48 mmol). Crystallization in cold bath and then filtration 380 mg of the desired compound as a white powder (1,03 mmol, yield 83%). TLC (SiO 2 , 8/2 cyclohexane/EtOAc) R f = 0.74; m.p. = 230-231 °C; 1 A sealed tube under argon atmosphere was charged at room temperature with 1 equivalent of corresponding N-tosylhydrazone (0.2 mmol), 1 equivalent of appropriate 1-bromo-2-nitrobenzene derivative (0.2 mmol), Pd 2 dba 3 .CHCl 3 (5 mol%), XPhos (10 mol%) and 2.5 equivalent of LiOtBu (0.5 mmol) in dry dioxane. The mixture was then heated at 110 °C for 16h. Reaction completion was evaluated by silica TLC (8/2 cyclohexane/AcOEt). The crude mixture was then allowed to cool down to room temperature, Filtration through Celite, and evaporation to give an oily brown crude directly used for next step without purification. In a sealed tube the 1-nitro-2-(1-phenylvinyl)benzene derivative was treated with 4 equivalent of PPh 3 and 10 mol% of catalyst MoO 2 Cl 2 (DMF) 2 in 2mL of dry dioxane under argon and stirred in MW at 135 °C for 4h. The crude mixture was then allowed to cool down to room temperature. EtOAc was added to the mixture, which was filtered through Celite. The solvents were evaporated under reduced pressure and the crude residue was purified by silica gel chromatography (95/5 to 85/15 of cyclohexane/EtOAc). The product was then concentrated under vacuum to give the product as a white solid.

3-(3',4',5'-Trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 16

16 was prepared according to the general method C from (15) and 1-bromo-2-nitrobenzene. Column chromatography on silica gel afforded 40 mg of the product as an orange solid (0.11 mmol, yield 55%); TLC (SiO 2 , 8/2 cyclohexane/EtOAc) R f = 0.23; m.p. = 170-172 °C; 1

6-Methoxy-3-(3',4',5'-trimethoxy-[1,1'-biphenyl]-3-yl)-1H-indole 17

17 was prepared according to the general method C from (15) and 1-bromo-4-methoxy-2nitrobenzene. Column chromatography on silica gel afforded 27 mg of the product as a yellow oil (0.07 mmol, yield 35%); TLC (SiO 2 , 80/20 cyclohexane/AcOEt) R f = 0.6; 1

Biology

Cell culture and proliferation assay

Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier's instructions. K562R (doxorubicin-resistant) leukemia cells were a generous gift from JP Marie (France). Human HCT-116 colorectal carcinoma cells were grown in Gibco McCoy's 5A supplemented with 10% fetal calf serum (FCS) and 1% glutamine. A549 lung carcinoma, K562 and K562R leukemia cells were grown in RPMI 1640 supplemented with 10% fetal calf serum and 1% glutamine. MCF7 breast adenocarcinoma, HT29 colon carcinoma and Mia-Paca2 pancreatic carcinoma cells were grown in Gibco medium DMEM supplemented with 10% fetal calf serum (FCS) and 1% glutamine. Human HeLa cells were obtained from the American Type Culture Collection and cultured at 37 °C in 5% CO 2 in the recommended media supplemented with 10% fetal bovine serum (51810-500, Dutscher). Cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . Cell viability was determined by a luminescent assay according to the manufacturer's instructions (Promega, Madison, WI, USA). For IC 50 determination, the cells were seeded in 96-well plates (3 × 103 cells/well) containing 100 μL of growth medium. After 24 h of culture, the cells were treated with the tested compounds at 10 different final concentrations. Each concentration obtained from serial dilutions in culture medium starting from the stock solution. Control cells were treated with the vehicle. All the compounds were solubilized in DMSO and diluted in culture medium to obtain a final concentration of 0.1 % of DMSO in cell culture. Experiments were performed in triplicate. After 72 h of incubation, 100 μL of Cell Titer Glo Reagent was added for 15 min before recording luminescence with a spectrophotometric plate reader PolarStar Omega (BMG LabTech). The dose-response curves were plotted with Graph Prism software and the IC 50 values were calculated using the Graph Prism software from polynomial curves (four or five-parameter logistic equations).

Cell cycle analysis. After 24h of treatment with 42, K562 cells were fixed in 70% ethanol and stained with propidium iodide solution containing RNase A. DNA content was further determined by flow cytometry using FC500 (Beckman Coulter).

Cell-based assay for the quantification of the effect of drugs on microtubules

This procedure was adapted from Ramirez-Rios et al. [START_REF] Ramirez-Rios | A New Quantitative Cell-Based Assay Reveals Unexpected Microtubule Stabilizing Activity of Certain Kinase Inhibitors, Clinically Approved or in the Process of Approval[END_REF] 7,500 HeLa cells were seeded in 96-well microplates (Greiner #655083) in 100 µL of complete medium per well and then incubated at 37 °C in 5% CO 2 . 24 hours after seeding, cells were treated with compounds (1 concentration per column, 1 plate per molecule) at different concentrations or DMSO at 0.1%, which was used as a positive control. At the end of the incubation, the medium was aspirate. Cells were permeabilized for 10 minutes at 37 °C using 100 µL per well of OPT buffer (80 mM Pipes, 1 mM EGTA, 1 mM MgCl 2 , 0.5% Triton X-100, and 10% glycerol, pH 6.8) pre-warmed to 37 °C. After buffer aspiration, cells were fixed overnight at room temperature using 100 µL per well of 4% formaldehyde in PBS pH 7.2. Cells were washed 3 times in PBS pH 7.2, 0.1% Tween-20 (150 µL per well). Then 50 µl of α3A1 anti-tubulin antibody (1/5,000 in PBS pH 7.4, 0.3% BSA, 0.03% NaN 3 ) were added for 45 minutes. After washing of cells as described above, 50 µl of anti-mouse antibody coupled to HRP (1/2,000 in PBS pH 7.4, 0.3% BSA, 0.03% NaN 3 ) were added for 45 minutes. Then cells were washed again. The microplate was placed into the BMG FLUOstar OPTIMA Microplate Reader and 100 µl of ECL Western blotting substrate (Biorad Clarity) were injected to each well. The luminescent signal was read immediately after injection.

Immunofluorescence

Cells were fixed and processed for immunofluorescence analysis of the MT network as previously described, [START_REF] Prudent | Pharmacological Inhibition of LIM Kinase Stabilizes Microtubules and Inhibits Neoplastic Growth[END_REF] using anti-α-tubulin clone α3A1, [START_REF] Peris | Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends[END_REF] as primary antibody and a secondary antibody coupled to Alexa 488 (Jackson Immuno-Research Laboratories, 115-545-003). DNA was stained with 20 µM Hoechst 33342 (Sigma, #23491-52-3). Images were captured with a Zeiss AxioimagerM2 microscope equipped with the acquisition software AxioVision.

In vitro microtubule assembly assay

Pure tubulin (purchased at the Centro de Investigaciones Biológicas, CSIC, Madrid, Spain) was equilibrated in 100 mM MES, 1 mM EGTA, 1 mM MgCl 2 , pH 6.8 buffer and centrifuged at 75,000 rpm for 10 minutes in an TLA-100 rotor in a Beckman Optima TLX centrifuge. Tubulin was then diluted to 50 μM. The samples were supplemented with the desired drug (or the vehicle, i.e. DMSO) and incubated for 30 min at 4 °C. Microplates ((half area, 96 well-microplates, Greiner ref 675096)) were then transferred to a thermostatically controlled reader (37 °C, BMG FLUOstar OPTIMA). Assembly kinetics was initiated by addition of GTP 1 mM and followed by measuring the absorbance of the samples at 350 nm.

Mitochondrial membrane potential Assay

One of the hallmarks for apoptosis is the loss of mitochondrial membrane potential (ΔΨm). The changes in the mitochondrial potential were detected by 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocya-nine iodide/chloride (JC-1), a cationic dye that exhibits potential dependent accumulation in mitochondria, indicated by fluorescence emission shift from red (590 nm) to green (525 nm). In brief, K562 cells were treated with different concentrations of compound 42 for 24h. After treatment, cells were re-suspended in 1 ml of PBS containing 2 µM final concentration JC-1 probe and incubated at 37 °C for 15 min. Analysis of cells was performed on a FC500 flow cytometer (Beckman Coulter, France).

Cytotoxicity Evaluations in Peripheral Blood Lymphocytes (PBL)

The cytotoxicity of compound 42 was compared to that of isoCA-4 in quiescent and proliferating peripheral blood lymphocytes (PBLs). Peripheral blood mononuclear cells were obtained from one healthy donor by density gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare) and cultured overnight at 37 °C in RPMI-1640 medium containing 10% fetal calf serum (complete RPMI-1640 medium). Non-adherent cells were harvested, and diluted at 1.11x10 5 /mL in complete RPMI-1640 medium containing or not phytohemagglutinin (PHA, final concentration 2.5 μg/mL), which is a potent mitogen inducing activation and proliferation of lymphocytes. Untreated (quiescent) and PHAtreated (proliferating) PBLs (90 μL, 10.000 cells/well) were plated in white walled 96-well plates and cultured overnight at 37 °C.

Quiescent and proliferating PBLs were then treated in triplicate with various concentrations of isoCA-4 and compound 42 (final concentrations: 2.5x10 -5 M, 1x10 -5 M, 2.5x10 -6 M, 1x10 -6 M, 2.5x10 -7 M, 1x10 -7 M, 2.5x10 -8 M, 1x10 -8 M, 2.5x10 -9 M, 1x10 -9 M, 2.5x10 -10 M, 1x10 -10 M, 2.5x10 -11 M and 1x10 - 11 M) for 72 h at 37 °C. To this purpose, stock solutions of isoCA-4 and 42 were prepared by dissolving them in DMSO at a concentration of 10 mM, and both compounds were serially diluted in DMSO. Ten μL of each dilution or DMSO were diluted 1:100 in complete RPMI-1640 medium, and 10 μL of these dilutions were then added to quiescent and proliferating PBLs (90 μL). The final concentration of DMSO was 0.25% for each well. After 72 h of incubation, cell viability was determined by adding 100 μL of CellTiter Glo Reagent, as previously described for cancer cell lines.

Luminescence was recorded on a FLUOstar Omega (BMG LabTech), and IC 50 values were calculated from the corresponding dose-response curves using the Graph Pad Prism software.

Molecular docking

Atomic coordinates for tubulin α,β-dimer were retrieved from the Protein Data Bank (accession code 6H9B). [START_REF] Naret | 1,1-Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin-Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] Missing hydrogen atoms were added using the Dock Prep module from the UCSF Chimera v1.13 software package, [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF] and atoms from the ligand co-crystallized in the colchicine binding at the interface between chains C and D were deleted. Coordinates for a low-energy starting conformer of compound 42 were obtained using the Conformers function from MarvinSketch v19.12 software package[49] with default parameters. Molecular docking was performed using AutoDock Vina v1.1.2 software package [START_REF] Trott | AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading[END_REF] with default parameters and the binding site defined as the box circumscribed to all the protein residues in contact with the co-crystallized ligand. Analysis and depiction of poses were performed using UCSF Chimera v1.13 software package. [START_REF] Pettersen | UCSF Chimera-A visualization system for exploratory research and analysis[END_REF]

Fig. 1 .

 1 Fig. 1. Structures of CA-4, isoCA-4 derivatives, synthetic analogs and target Cyclic Bridged Analogs CBA

Scheme 1 .

 1 Scheme 1. Reagents and conditions: (a) 3-acetylphenyl-boronic acid, Pd(OAc) 2 , SPhos, K 3 PO 4 .H 2 O, cyclopentyl methyl ether (CPME)/H 2 O, 110 °C; (b) 4-methylbenzenesulfonohydrazide, EtOH, reflux; (c) Appropriate 1-bromo-2-nitrobenzene derivative, Pd 2 dba 3 .CHCl 3 (5 mol%), XPhos (10 mol%), LiOtBu, dry dioxane, 110 °C overnight, after filtration on Celite and evaporation, compounds were subject to Codagan cyclization; (d) MoO 2 Cl 2 (dmf) 2 (10 mol %), PPh 3 (4 equiv), in 3 mL of dioxane under microwave irradiation (MWI) at 135 °C; (e) NaH, CH 3 I, DMF, rt.

Scheme 2 .

 2 Scheme 2. (a) Pd(PPh 3 ) 4 , Zn(CN) 2 , DMF, 120 °C; (b) 3-acetylphenyl)boronic acid, Pd(PPh 3 ) 4 , K 3 PO 4 .H 2 O, Dioxane/H 2 O, 110 °C; (c) 4-methylbenzenesulfonohydrazide, EtOH, 95 °C; (d) 1-bromo-2nitrobenzene Pd 2 dba 3 .CHCl 3 (5 mol%), XPhos (10 mol%), LiOtBu, dry dioxane, 110 °C overnight, after filtration on Celite and evaporation, the crude was subject to Codagan cyclization; (e) MoO 2 Cl 2 (dmf) 2 (10 mol %), PPh 3 (4 equiv), ), in 3 mL of dioxane under MWI at 135 °C; (f): NaH, CH 3 I, DMF, rt; (g): NaOH aq , HCOH aq , EtOH, rt.

Scheme 4 .

 4 Scheme 4. (a) 5-iodo-2-methoxyphenyl acetate, Pd(PPh 3 ) 4 (2 mol%), K 2 CO 3 ,Toluene/H 2 O (4:1), 110 °C, 1 h; (b) (3,4,5-trimethoxyphenyl)boronic acid, Pd(PPh 3 ) 4 (2 mol%), K 2 CO 3 , Toluene/H 2 O (4:1), 110 °C, 1 h; (c) K 2 CO 3 (10 eq), MeOH, 50 °C, overnight; (d) (3,4,5-trimethoxyphenyl)boronic acid, Pd(OAc) 2 (5 mol%), P(Cy 3 ).BF 4 (10 mol%), K 2 CO 3 , Toluene/H 2 O (30:1), 110 °C, 20 min; (e) (3-(benzyloxy)-4methoxyphenyl)boronic acid, Pd(OAc) 2 (5 mol%), PPh 3 (10 mol%), K 2 CO 3 , Dioxane/H 2 O (2:1), 110 °C, 1 h; (f) Pd/C H 2 , (10 mol%), EtOH, rt, overnight.

Fig. 2 .

 2 Fig. 2. Summary of the SARs study of target compounds based on cell viability evaluated on a human colon cancer cell line.

5 >8000a

 5 Colon-carcinoma cells (HCT116); b Chronic myelogenous leukemia cells (K562); c Doxorubicinresistant chronic myeloid leukemia cells (K562R); d Human pancreatic carcinoma (MiaPaca2); e Adenocarcinoma human alveolar basal epithelial cells (A549); f Breast cancer cell line (MCF-7); Human colon cancer cell line (HT-29).

Fig. 3 .

 3 Fig. 3. Effect of compound 42 on the cell cycle of K562 cell lines. One representative replicate out of two is shown.

Fig. 5

 5 Fig. 5 Comparison of the effect of compound 42 and CA-4 on tubulin polymerization. Compound 42 (A) or CA-4 (B) were added at different concentrations to 50 µM tubulin and their effect on tubulin kinetics assembly was monitored. Black: DMSO (control); blue: 0.1 µM; green: 0.5 µM; orange: 1 µM; grey: 2.5 µM; red: 5 µM; brown: 7 µM. Representative curves of two independent experiments.

Fig. 7 .

 7 Fig. 7. Cytotoxicity of compound 42 and isoCA-4 in peripheral blood lymphocytes (PBLs). (A)Quiescent PBLs (10.000/well) from one healthy donor were treated in triplicate with various concentrations (10-11-2.5 x 10 -5 M) of isoCA-4 and compound 42 for 72 h at 37 °C, and cell viability was measured by a luminescent assay. A cell viability of 100% corresponds to the mean luminescence value obtained for vehicle (0.25% DMSO)-treated PBLs, and data represent the mean (± standard error of the mean, SEM). Dose-response curves were fitted to a log(inhibitor) vs response curve using the Graph Pad Prism software, yielding IC 50 values of 137 nM and >10 µM for isoCA-4 and compound 42, respectively. Compounds could not be tested at higher concentrations than 25 µM, precluding more accurate determination of the IC 50 value for compound 42. (B) PBLs from the same donor (10.000/well) were activated with phytohemagglutinin (PHA. 2.5 µg/mL) to induce their proliferation, and were subsequently treated in triplicate with various concentrations (10-11-2.5 x 10 -5 M) of isoCA-4 and compound 42 for 72 h at 37 °C. Cell viability was calculated as described for quiescent PBLs, and data represent the mean (± standard error of the mean, SEM). Dose-response curves were fitted to a log(inhibitor) vs response curve using the Graph Pad Prism software, yielding IC 50 values of 4.3 nM and 114 nM for isoCA-4 and compound 42, respectively.

Fig. 8 .

 8 Fig. 8. (a) Putative binding mode of compound 42 (green color) within colchicine binding site of tubulin X-ray structure (accession code 6H9B). (b) same figure with previously reported binding mode of reference compound 11 in overlay (magenta color). Showing expected hydrogen bonds between nitrogen of quinoline moiety and cysteine β241, and plausible hydrophobic interactions.

4. 2

 2 .4,pyridin-2-yl)phenol 8[START_REF] Zheng | Design, Synthesis, and Biological Evaluation of Novel Pyridine-Bridged Analogues of Combretastatin-A4 as Anticancer Agents[END_REF]

Table 2 .

 2 Effects on cell viability of compound 42 against a panel of seven human cancer cell lines, IC 50 values are reported in nM.

	Compd HCT116 a	K562	b	K562R	c	MiaPaca2	d	A549 e	MCF7 f	HT-29 h
	Tumor	Colon	Leukemia	Leukemia	Pancreas		Lung	Breast	Colon
	Type

C

  NMR spectra were recorded in either CDCl 3 , MeOD, or DMSO-d 6 on Bruker Avance 300 or 400 spectrometers. The chemical shifts of 1 H and 13 C are reported in ppm relative to the solvent residual peaks. Solvent peaks were used as reference values, with CDCl 3 at 7.26 ppm for The following abbreviations are used: singlet (s), doublet (d), doublet of doublet (dd), triplet (t), td (triplet of doublet), ddd (doublet of doublet of doublet) multiplet (m) and broad singlet (bs). IR spectra were measured on a Perkin Elmer spectrophotometer. High resolution mass spectra (HR-MS) were recorded on a MicroMass LCT Premier Spectrometer.

				1 H NMR and 77.16 ppm for	13 C NMR,
	with (CD 3 ) 2 CO at 2.05 ppm for	1 H NMR and 29.84 ppm for	13 C NMR, and with DMSO-d 6 at 2.50 ppm for	1 H NMR
	and 39.5 ppm for C NMR. 4.2. Chemistry 13
	4.2.1. General procedure for the Suzuki-Miyaura Coupling (Method A
					1 H NMR
	and	13

): compounds 14, 24, 25, 33, 41, 45, 46, 49 and 50.

  H NMR (300 MHz, CDCl 3 ) δ 8.18 -8.02 (m, 3H, H Ar ), 7.84 -7.55 (m, 4H, H Ar ), 7.45 (t, J = 7.5 Hz, 1H, H Ar ), 7.25 (s, 1H, H Ar ), 2.79 (s, 3H, CH 3 ), 2.66 (s, 3H, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 198.2, 159.1, 149.0, 147.9, 139.3, 138.1, 134.6, 130.1, 129.8, 129.4, 128.8, 126.6, 125.7, 125.4, 122.8, 77.2, 27.3, 25.9; IR ( max /cm -1 ) 3059, 2363, 1682, 1575, 1402, 1259, 122; HRMS (ESI+) for C 18 H 16 NO [M + H] + : calcd 262.1632, found 262.1636.

  H NMR (400 MHz, CDCl 3 ) δ 8.08 -7.98 (m, 2H, H Ar ), 7.97 -7.93 (m, 1H, H Ar ), 7.76 (t, J = 7.8 Hz, 1H, H Ar ), 7.61 (dtd, J = 16.3, 7.2, 1.3 Hz, 2H, H Ar ), 7.51 -7.40 (m, 4H, H Ar ), 7.36 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H, H Ar ), 7.33 (s, 1H, H Ar ), 6.34 (s, 1H, CH 2 ), 5.60 (s, 1H, CH 2 ),

	2.75
	(s, 3H, CH 3 ); 13 C NMR (101 MHz, CDCl 3 ) δ 158.8, 156.6, 156.3, 149.1, 148.9, 147.0, 146.3, 137.6, 136.4,
	133.7, 133.0, 129.5, 129.3, 129.1, 126.2, 125.9, 124.7, 124.7, 125.0, 122.8, 120.2, 119.1, 25.7; IR
	( max /cm -1 ) 3061, 2960, 2924, 2853, 2367, 1566, 1523, 1453, 1346; HRMS (ESI+) for C 23 H 18 N 3 O 2 [M +
	H]+: calcd 368.1399, found 368.1389.

  H NMR (300 MHz, CDCl 3 ) δ 7.68 (t, J = 1.9 Hz, 1H), 7.49 -7.42 (m, 2H), 7.42 -7.37 (m, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 4.1 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 3.88 (s, 3H), 2.34 (s, 3H). 13 C NMR (101 MHz, CDCl 3 ) δ 169.0, 151.1, 142.1, 140.2, 132.7, 130.4, 130.0, 129.9, 125.4 (2C), 123.0, 121.7, 112.8, 56.1, 20.7.

	4.2.1.7. 3'',4,4'',5''-Tetramethoxy-[1,1':3',1''-terphenyl]-3-yl acetate 46

  H NMR (200 MHz, CDCl 3 ) δ 7.68 (s, 1H, H Ar ), 7.54 -7.43 (m, 4H, H Ar ), 7.33 (d, J = 2.2 Hz, 1H, H Ar ), 7.07 (d, J = 8.5 Hz, 1H, H Ar ), 6.81 (s, 2H, H Ar ), 3.94 (s, 6H, 2 OCH 3 ), 3.91 (s, 3H, OCH 3 ), 3.89 (s, 3H, OCH 3 ), 2.35 (s, 3H, CH 3 ); 13 C NMR (75 MHz, DMSO) δ 168.5, 153.2, 150.5, 146.8, 141.2, 139.7, 139.5, 137.3, 136.1, 132.8, 129.2, 125.7, 125.2, 125.1, 124.7, 121.3, 113.1, 104.6 (2C), 60.0, 56.0 (2C), 55.9 , 20.4.

  H NMR (200 MHz, CDCl 3 ) δ 7.85 (d, J = 2.1 Hz, 1H, H Ar ), 7.78 (d, J = 7.8 Hz, 1H, H Ar ), 7.71 (dd, J = 8.2, 2.3 Hz, 1H, H Ar ), 7.59 (d, J = 2.2 Hz, 1H, H Ar ), 7.55 (dd, J = 2.8, 0.9 Hz, 1H, H Ar ), 7.53 -7.45 (m, 2H, H Ar ), 7.44 -7.31 (m, 5H, H Ar ), 7.02 (d, J = 8.5 Hz, 1H, H Ar ), 5.26 (s, 2H, CH 2

  C NMR (101 MHz, CDCl 3 ) δ 156.5, 156.4, 153.6 (2C), 147.7, 145.9, 139.3, 137.5, 135.5, 133.2, 119.1, 118.1 (2C), 113.3, 110.8, 104.6 (2C), 61.1, 56.5 (2C), 56.2; HRMS (ESI+) for C 21 H 22 NO 5 [M + H] + : calcd 368.1498, found 368.1502.

  H NMR (300 MHz, CDCl 3 ) δ 7.92 (d, J = 8.2 Hz, 2H, H Ar ), 7.79 (s, 1H, H Ar ), 7.61 (d, J = 7.6 Hz, 1H, H Ar ), 7.54 (d, J = 7.6 Hz, 1H, H Ar ), 7.47 (s, 1H, H Ar ), 7.41 (t, J = 7.7 Hz, 1H, H Ar ), 7.31 (d, J = 8.1 Hz, 2H, H Ar ), 6.75 (s, 2H, H Ar ), 3.94 (s, 6H, 2OCH 3 ), 3.91 (s, 3H, OCH 3 ), 2.41 (s, 3H, CH 3 ), 2.20 (s, 3H, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 154.

	1, 153.0 (2C), 144.8,
	142.1 (2C), 138.4, 137.4, 136.0, 130.1 (2C), 129.3 (2C), 128.9, 128.74, 125.8, 125.7, 105.2 (2C), 61.56,
	56.87 (2C), 22.17, 14.16; HRMS (ESI+) for C 24 H 27 N 2 O 5 S [M + H] + : calcd 455.1635, found 455.1673.

  H NMR (300 MHz, CDCl 3 ) δ 8.26 (d, J = 8.4 Hz, 1H, H Ar ), 7.92 (d, J = 8.9 Hz, 2H, H Ar ), 7.88 -7.83 (m, 3H, H Ar ), 7.80 (dt, J = 7.7, 1.6 Hz, 1H, H Ar ), 7.75 (d, J = 1.8 Hz, 1H, H Ar ), 7.67 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H, H Ar ), 7.60 (s, 1H, H Ar ), 7.56 (t, J = 7.7 Hz, 1H, H Ar ), 7.49 (dd, J = 7.7, 1.5 Hz, 1H, H Ar ), 7.24 (s, 1H, H Ar ), 2.39 (s, 3H, CH 3 ), 2.22 (s, 3H, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 151.5, 150.0, 149.0, 144.6, 138.3, 136.6, 135.4, 133.5, 131.2, 130.7, 130.5, 129.7 (2C), 129.2, 128.3 (2C), 128.1, 127.5, 127.4, 127.2, 125.91, 123.2, 117.8, 21.7, 13.6. IR ( max /cm -

	1 ) 3242, 3063, 2925, 2360, 1597, 1405, 1335, 1167, 1066; HRMS (ESI+) for C 25 H 20 N 4 O 2 S [M + H] + : calcd
	441.1385, found 441.1385.

  H NMR (300 MHz, CDCl 3 ) δ 8.17 (d, J = 8.5 Hz, 1H, H Ar ), 7.88 (d, J = 8.2 Hz, 2H, H Ar ), 7.79 (d, J = 8.0 Hz, 2H, H Ar ), 7.74 (s, 2H, H Ar ), 7.49 (p, J = 7.8 Hz, 3H, H Ar ), 7.23 (d, J = 5.3 Hz, 2H, H Ar ), 7.19 (s, 1H, H Ar ), 2.83 (s, 3H, CH 3 ), 2.36 (s, 3H, CH 3 ), 2.20 (s, 3H, CH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 158.3, 151.8, 146.0, 144.9, 144.2, 138.0, 137.8, 135.3, 131.3, 130.5, 129.8, 129.5 (2C), 128.7, 128.2 (2C), 127.4, 126.4, 126.2, 125.5, 125.0, 122.2, 24.9, 21.6, 13.5; IR ( max /cm -1 ) 2360, 2252, 1686, 1403, 1339, 1302, 1165, 905; HRMS (ESI+) for C 25 H 23 N 3 O 2 S [M + H] + : calcd 430.1589, found 430.1581.

	4.2.3.4.

16-19, 28, 29, 35, and 42

  H NMR (300 MHz, DMSO-d 6 ) δ 10.92 (s, 1H, NH), 7.85 -7.78 (m, 3H, H Ar ), 7.75 (td, J = 7.6, 0.9 Hz, 1H, H Ar ), 7.62 (dt, J = 7.4, 1.1 Hz, 1H, H Ar ), 7.41 (d, J = 8.0 Hz, 2H, H Ar ), 2.37 (s, 3H, CH 3 ), 2.19 (s, 3H, CH 3 ); 13 C NMR (75 MHz, DMSO-d 6 ) δ 155.7, 151.4, 143.5, 140.2, 139.9, 136.0, 129.5 (2C), 128.2, 127.5 (2C), 119.1, 21.0, 12.5. IR ( max /cm -1 ) 3225, 2425, 2412, 1675, 1610, 1480, 1375, 1212; HRMS (ESI+) for C 14 H 15 N 3 O 2 SBr [M + H] + : calcd 368.0068, found 368.0067.

	4.2.4. General procedure for the Barluenga coupling followed Cadogan cyclisation one pot (Method
	C):

  H NMR (300 MHz, CDCl 3 ) δ 8.40 (brs, 1H, NH), 7.98 (d, J = 7.7 Hz, 1H, H Ar ), 7.86 (s, 1H, H Ar ), 7.67 (d, J = 7.1 Hz, 1H, H Ar ), 7.54 (d, J = 7.6 Hz, 1H, H Ar ), 7.49 (d, J = 7.6 Hz, 2H, H Ar ), 7.44 (d, J = 5.4 Hz, 1H, H Ar ), 7.30 (d, J = 7.2 Hz, 1H, H Ar ), 7.25 -7.18 (m, 1H, H Ar ), 6.87 (s, 2H, H Ar ), 3.95 (s, 6H, 2 OCH 3 ), 3.93 (s, 3H, OCH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 153.5, 142.0, 137.5, 136.7, 136.1, 129.2, 126.6, 126.3, 125.8, 124.9, 122.5, 122.0, 120.4, 119.7, 118.2, 111.5, 104.6, 61.0, 56.2. IR ( max /cm -1 ) 2938, 1582, 1511, 1455, 1238, 1126, 1002; HRMS (ESI + ) for C 23 H 22 NO 3 [M + H] + calcd 360.1594 found 360.1653; LCMS 97% 20.2 min.

  IR ( max /cm -1 ) 2925, 1581, 1455, 1240, 1164, 1125, 1003; HRMS (ESI+) for C 24 H 24 NO 4 [M + H] + calcd 390.1700 found 390.1731; LCMS 95% 19,9 min.

	H NMR (300 MHz, CDCl 3 ) δ
	8.27 (brs, 1H, NH), 7.89 -7.77 (m, 2H, H Ar ), 7.64 (dt, J = 7.0, 1.5 Hz, 1H, H Ar ), 7.57 -7.42 (m, 2H, H Ar ),
	7.32 (d, J = 2.4 Hz, 1H, H Ar ), 6.93 (d, J = 2.1 Hz, 1H, H Ar ), 6.92 -6.81 (m, 3H, H Ar ), 3.94 (s, 6H, 2 OCH 3 ),
	3.93 (s, 3H, OCH 3 ), 3.87 (s, 3H, OCH 3 ); 13 C NMR (75 MHz, CDCl 3 ) δ 156.3, 153.2 (2C), 141.7, 137.8,
	137.7, 136.4, 135.7, 129.0, 126.2, 125.9, 124.4, 121.2, 120.0, 118.8, 117.3, 109.9, 104.5 (2C), 95.0,
	60.8, 56.1 (2C), 55.6;

Acknowledgments

The authors acknowledge support of this project by the CNRS and University Paris-Saclay. S. Pecnard thanks La Ligue Contre le Cancer for the financing of his Ph.D. Our laboratory is a member of the Laboratory of Excellence LERMIT supported by a grant (ANR-10-LABX-33).