Shannon Pecnard

Abdallah Hamze
email: abdallah.hamze@universite-paris-saclay.fr

Jérome Bignon

Bastien Prost

Alain Deroussent

Laura Gallego-Yerga

Rafael Peláez

Ji Yeon Paik

Marc Diederich

Mouad Alami
email: mouad.alami@universite-paris-saclay.fr

Olivier Provot
email: olivier.provot@universite-paris-saclay.fr

Anticancer Properties of Indole Derivatives as IsoCombretastatin A-4 analogues

Keywords: Cancer, cytotoxicity, indole, isoCA-4, oxazinoindole, pyridoindole, quinazoline, tubulin. 2. Results and discussion 2.1. Chemistry

In this study, a variety of original ligands related to Combretastatin A-4 and isoCombretastatin A-4, able to inhibit the tubulin polymerization into microtubules, was designed, synthesized, and evaluated. Our lead compound 15d having a quinazoline as A-ring and a 2-substituted indole as B-ring separated by a N-methyl linker displayed a remarkable subnanomolar level of cytotoxicity (IC 50 < 1 nM) against 9 human cancer cell lines.

Introduction

Isolated from the African willow tree, Combretum caffrum by G. Pettit [1] in 1989, Combretastatin A-4 (CA-4 1, Figure 1) is a very potent cytotoxic agent having nanomolar IC 50 values against many human cancer cell lines. [START_REF] Mc Gown | Differential cytotoxicity of combretastatins A1 and A4 in two daunorubicin-resistant P388 cell lines[END_REF] This (Z)stilbene acts by binding to the colchicine site on -tubulin, thus interrupting the polymerization of tubulin into microtubules. [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] Moreover, it has been demonstrated that a moderate single dose of CA-4P 2, a water-soluble phosphate pro-drug of CA-4, causes almost complete cessation of the blood circulation in tumour vessels without vascular damage in normal cells. [4,[START_REF] Tozer | Combretastatin A-4 phosphate as a tumor vascular-targeting agent: Early effects in tumors and normal tissues[END_REF][START_REF] Tozer | The biology of the combretastatins as tumour vascular targeting agents[END_REF] This discovery was the source of a spectacular craze from the scientific community for this small molecule which was then the subject of numerous structure-activity relationships studies. [7,[START_REF] Siebert | Synthesis of Combretastatin A-4 Analogs and their Biological Activities Anti[END_REF][START_REF] Shan | Developments of combretastatin A-4 derivatives as anticancer agents[END_REF] The majority of these structural modifications consisted in introducing the (Z)-double bond into cyclic or heterocyclic systems since it has been shown that, despite its pronounced efficacy in clinical trials, the (Z)-double bond of CA-4 isomerizes during storage, administration and metabolism leading to the more stable but less active E-isomer. [ 10 , 11 , 12 ] In the continuation of our work dedicated to the synthesis of non-isomerizable analogues of CA-4 [13], we discovered that the stable synthetic compound isoCA-4 3, displayed the same anti-cancer activities as its natural isomer CA-4 but without the risk of isomerization. [14,[START_REF] Alvarez | Isocombretastatins A: 1, 1-diarylethenes as potent inhibitors of tubulin polymerization and cytotoxic compounds[END_REF][START_REF] Hamze | Synthesis, Biological Evaluation of 1,1-Diarylethylenes as a Novel Class of Antimitotic Agents[END_REF][START_REF] Soussi | Metabolites of isoCombretastatin A-4 in Human Liver Microsomes: Identification, Synthesis and Biological Evaluation[END_REF] We next demonstrated that several modifications of the phenolic B-ring of CA-4 and isoCA-4 were authorized by introducing, for example -NH 2 , -alkynes, -alkenyl groups, or a fluorine atom in place of the C3'-OH substituent. [START_REF] Hamze | B-Ring-Modified isoCombretastatin A-4 Analogues Endowed with Interesting Anticancer Activities[END_REF][START_REF] Alami | Developments of isoCombretastatin A-4 Derivatives as Highly Cytotoxic Agents[END_REF] We and the Pinney's group have also showed that the ethylene double bond of isoCA-4 can be reduced [START_REF] Messaoudi | Discovery of Novel isoErianin Analogues as Promising Anticancer Agents[END_REF] or inserted into cycles and heterocycles [START_REF] Rasolofonjatovo | Conformationnally restricted naphthalene derivatives type isocombretastatin A-4 and isoerianin analogues: synthesis, cytotoxicity and antitubulin activity[END_REF][START_REF] Renko | Rapid synthesis of 4arylchromenes from ortho-substituted alkynols: A versatile access to restricted isocombretastatin A-4 analogues as antitumor agents[END_REF][START_REF] Rasolofonjatovo | Design, synthesis and anticancer properties of 5-arylbenzoxepins as conformationally restricted isocombretastatin A-4 analogs[END_REF][START_REF] Sriram | Design, synthesis and biological evaluation of dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in cancer chemotherapy[END_REF][START_REF] Herdman | Synthesis and biological evaluation of benzocyclooctene-based and indene-based anticancer agents that function as inhibitors of tubulin polymerization[END_REF][START_REF] Tanpure | An amino-benzosuberene analogue that inhibits tubulin assembly and demonstrates remarkable cytotoxicity[END_REF] without loss of anti-cancer properties. Structure-activity relationships (SARs) also revealed that various non-isomerizable linkers of small size are welcome [13] of which the N-Methyl group predominated (e.g., azaFisoerianin 4). [27] Finally, it was demonstrated that the classical 3,4,5-trimethoxyphenyl (TMP) A-ring of CA-4 and isoCA-4 could be advantageously replaced by heterocycles as quinazolines [START_REF] Soussi | IsoCombretaQuinazolines: Potent Cytotoxic Agents with Antitubulin Activity[END_REF] or quinolines [START_REF] Khelifi | IsoCombretaQuinolines: Novel Microtubule Assembly Inhibitors with Potent Antiproliferative Activity[END_REF] to promote highly cytotoxic analogues 6 [START_REF] Naret | Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] and 7 [START_REF] Khelifi | N-bis-Heteroaryl Methylamines: Potent Anti-Mitotic and Highly Cytotoxic Agents[END_REF] having in structures a carbazole nucleus as B-ring. Herein, we were interested in synthesizing and evaluating the cytotoxicity of target molecules of type pyrido [1,2-a]indoles 11 [START_REF] Yao | Recent advances in the synthesis of pyrido[1,2a]indoles[END_REF] depicted in Scheme 1, which could be seen as analogues of carbazole compounds 5-7 [START_REF] Jimenez | Exploring the size adaptability of the B ring binding zone of the colchicine site of tubulin with para-nitrogen substituted isocombretastatins[END_REF][START_REF] Bzeih | A general synthesis of arylindoles and (1arylvinyl)carbazoles via a one-pot reaction from N-tosylhydrazones and 2-nitro-haloarenes and their potential application to colon cancer[END_REF] by connecting the nitrogen atom N1 to the carbon atom C2 in the carbazole system.

We will also evaluate the cytotoxicity of their 6,9-dihydropyrido [1,2-a]indole precursors 10 to understand the structureactivity relationships (SARs) better. Next, the insertion of an oxygen atom in the pyridine part of the pyrido [1,2a]indoles ring of compounds 11 leading to oxazino [4,3-a]indoles 12 (Scheme 2) and their "opened" analogues 15 (Schemes 3 and 4) will be presented by establishing a comprehensive SAR study.

Figure 1.

Structures of CA-4 1, CA-4P 2 and a selection of analogues 3-7 accompanied by target molecules pyrido [1,2a]indoles.

We also prepared for biological comparisons, a series of 1,2-disubstituted indole derivatives 15a-l as "opened analogues" of oxazino [4,3-a]indoles 12 (Schemes 3 and 4). Diarylethylene compounds 15a,b and 15f,g were prepared in two steps from the Barluenga's coupling reactions between the brominated platforms 14b or 14c with the required Ntosylhydrazones followed by a O-desylilation reaction using TBAF in THF (Scheme 3). Diarylmethylamines 15c-e were prepared in three steps according to a Buchwald coupling reaction using above reaction conditions with 14b and N-ethylindoles, and the required secondary amines were then N-methylated and O-deprotected without prior purification (Scheme 4). N-methylindole 15h having on C2 a CH 2 OH-substituent was prepared similarly after a three steps sequence (Buchwald-Hardwig coupling, N-methylation and desilylation). N-methylindoles 15j,k having on C2 a CH 2 OMe group were prepared according to a Buchwald-Harding coupling reaction followed by a N-methylation reaction as above. Finally, indole derivative 15i having on C2 a CH 2 OMOM-group was prepared in two steps from 5bromo-2-(methoxymethyl)-1-methyl-1H-indole 14e and 4-aminoquinaldine. It is important to note that all secondary amine and silylated intermediates were used without needing to be purified.

Cytotoxicity of new compounds against HCT116 cells

In vitro cytotoxicity of the synthesized 6,9-dihydropyrido [1,2-a]indoles 10a-d, pyrido [1,2-a]indoles 11a-d, dihydro-1H- [1,4]oxazino [4,3-a]indoles 12a-g as well as 2-substituted indoles 15a-l was evaluated against HCT116 human colon carcinoma (HCT116) cell line. A fluorimetry-based assay was used to determine of the drug concentration required to inhibit cell growth by 50% after incubation in the culture medium for 72 h. IsoCA-4 [START_REF] Giraud | One-pot hydrosilylation-protodesilylation of functionalized diarylalkynes: A highly selective access to Z-stilbenes. Application to the synthesis of combretastatin A-4[END_REF] was included as the reference compound for comparisons. Examination of compounds 10a-d in which the 6,9-dihydropyridino [1,2-a] (IC 50 = 5.2 nM) showed that the aromatization of the 6,9-dihydropyridino [1,2-a]indole nucleus decreased the cytotoxicity level. We can also note that pyrido [1,2-a]indole 11b was found to be significantly less cytotoxic than its carbazole analogue 7b. [START_REF] Khelifi | IsoCombretaQuinolines: Novel Microtubule Assembly Inhibitors with Potent Antiproliferative Activity[END_REF] However, we were pleased to observe that, in the pyrido [1,2-a]indoles series, the quinaldine A-ring replacement with a quinazoline motif increased the cytotoxic activity 11-fold (compare 11b with 11c). Next, compounds 12a-g were examined and compared from a biological point of view. Again, in this class of compounds having a 3,4-dihydro-1H- [1,4]oxazino [4,3-a]

Microsomal stability of studied compounds

To determine their metabolic stability, expressed as the percentage (%) of remaining parent compound concentration over time, kinetic monitoring was performed by HPLC-MS/MS of independent incubations of each compound with rat liver microsomes (RLM) and human liver microsomes (HLM). Concentrations of each compound in microsomal incubations were backcalculated using their calibration curves. After quantification of the ratio of the residual concentration to its initial value, the half-life time was 18 h for isoCA-4, 70 h for 15b, and 140 h for 15d with rat liver microsomes (Table 2). The half-life time was 30 h for isoCA-4, 89 h for 15b and 145 h for 15d with human liver microsomes (as shown in Table 2). It is of note that these in vitro t½ values for each drug are observed in good agreement between rat and human liver microsomes. The natural logarithms (ln) of the remaining concentrations (%)

were plotted against incubation times (Figures 2a and2b). Using the value 3.912 of (ln 50), the in vitro half-lives (t½)

were found with HLM in the following ascending order, isoCA-4 < 15b < 15d, in agreement with this obtained with RLM. However, a slower metabolism was observed with HLM than with RLM. The quinazoline compound 15d

showed the best metabolic stability when compared with isoCA-4. To investigate whether cytotoxic compounds 15b and 15d were exerting their activities by interacting with microtubules, their effects on in vitro polymerization of tubulin were examined using porcine brain tubulin, which was isolated following Shelanski's procedure. [START_REF] Shelanski | Microtubule Assembly in the Absence of Added Nucleotides[END_REF] As it can be seen in Figure 3, the selected compounds inhibited tubulin polymerization with micromolar IC 50 values. It is of note that the concentrations required to inhibit tubulin polymerization using 15b and 15d (IC 50 at a micromolar level) are much higher than those required for cytotoxicity and similar observations have been previously noticed in many other classes of antimitotic agents, including epithilones, [START_REF] Lichtner | Subcellular distribution of epothilones in human tumor cells[END_REF] paclitaxel, [START_REF] Jordan | Mitotic Block Induced in HeLa Cells by Low Concentrations of Paclitaxel (Taxol) Results in Abnormal Mitotic Exit and Apoptotic Cell[END_REF] and isoCA-4 analogues prepared in our group.

Inhibition of tubulin polymerization for selected compounds

Cytotoxicity of 15d against eight other human cancer cells

According to the metabolism and TPI results, we next decided to evaluate our lead compound 15d on eight other tumor cell lines: human glioblastoma (U87-MG), human lung epithelial (A549), human breast adenocarcinoma (MDA-MB231), Human pancreatic carcinoma (MiaPACA2), Human lung cancer (HT1080), Chronic myeloid leukemia cells (K562), Doxorubicin-resistant chronic myeloid leukemia cells (K562R) [START_REF] Pan | Nicotinamide increases the sensitivity of chronic myeloid leukemia cells to doxorubicin via the inhibition of SIRT1[END_REF] and Human colorectal adenocarcinoma cells (HT29). The results of this study are depicted in Table 3. HT29 Human colorectal adenocarcinoma cells.

Effect of 15d on cell cycle.

Indole derivative 15d which displayed against all cancer cell lines a sub-nanomolar cytotoxicity level was next tested in dose-response experiments on K562 cell cycle distribution. K562 cells were treated for 24 h with increasing concentrations of 15d and DMSO was used as control. As seen in Fig. 4, 15d, at a concentration of 0.5 nM has arrested the entire population of K562 cells in the G2/M phase of the cellular cycle. It is important to note that the cellular cycle blockade in the G2/M phase at such a low concentration has never been reported for isoCA-4 and analogues.

Effects of 15d 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. [START_REF] Vakifahmetoglu-Norerg | The role of mitochondria in metabolism and cell death[END_REF] Mitochondrial dysfunction 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 Figure 5

Inhibition of colony formation in K562 and imatinib-resistant K562 cells by 15d

We next quantified the inhibition of the clonogenic potential by 15d in a 3D culture environment using Methocult colony formation assays (CFA). 15d significantly reduced the number, total surface area and average size of K562 and imatinib resistant K562IR cell colonies (Figure 6). Statistical analysis was performed by two-way ANOVA, followed by Tukey's multiple comparisons test. Differences were considered significant when * p < 0.05, ** p < 0.01, *** p < 0.001 compared to control. n/s: not significant.

Molecular modelling

Figure 7a presents the results of molecular docking calculations for compound 15d within the colchicine binding site of tubulin β subunit (the structure obtained from X-ray crystal structure with accession code 6H9B). [START_REF] Naret | Diheterocyclic Ethylenes Derived from Quinaldine and Carbazole as New Tubulin Polymerization Inhibitors: Synthesis, Metabolism, and Biological Evaluation[END_REF] The overall binding mode observed match that previously reported for isoCA-4 (see overlay on Figure 7b) and for compound 6b, [START_REF] Soussi | IsoCombretaQuinazolines: Potent Cytotoxic Agents with Antitubulin Activity[END_REF] where the quinazoline nucleus was accommodated in the lipophilic pocket ordinarily occupied by the trimethoxyphenyl A-ring. Interactions that can be expected given this binding mode hypothesis include notably (see

Conclusion

Overall, we have presented efficient syntheses of various original isoCA-4 analogues having as B-rings pyridoindoles, oxazinoindoles, and indoles nucleus. Among the 26 compounds synthesized, a large majority showed excellent cytotoxicity levels that can be compared to CA-4 and isoCA-4 as references. Compound 15d, which has a quinazoline as ring A, a 2-substituted indole as ring B, and a N-Methyl linker has proven to be the most promising compound of these different series. Indole-quinazoline compound 15d shows excellent cytotoxicity on 9 human cancer cell lines at a sub-nanomolar level that has never been observed in this research area. Moreover, it was observed that this compound presents a remarkable efficacy on K562R Doxorubicin-resistant chronic myeloid leukemia cell lines. This compound, which inhibits tubulin assembly into microtubules at a classic micromolar level, shows a very interesting metabolic stability, making it a promising drug candidate. Cell cycle studies have revealed that 15d blocks this cycle in the G2/M phase at a very low concentration of 0.5 nM, a concentration at which 15d also induced an important mitochondrial dysfunction. Interestingly, indole 15d was showed to significantly reduced the number, total surface area and average size of K562 and K562IR cell colonies in a dose-dependent manner. Finally, docking studies show that 15d adopts a similar orientation to that of isoCA-4 when bound to -tubulin by establishing 3 stabilizing hydrogen bonds.

From a therapeutic point of view, it is important to note that compound 15d is certainly too cytotoxic to be used as in vivo. It is therefore appropriate to use this compound, due to its very high cytotoxicity (IC 50 << 1 nM) as a payload in an antibody-drug conjugate (ADC) strategy, as reported recently with CA-4. [START_REF] Huang | Combretastatin A4-derived payloads for antibody-drug conjugates[END_REF] Moreover, 15d could also be included in squalene nanoparticles, as shown in our lab with isoCA-4 [START_REF] Maksimenko | Therapeutic Modalities of Squalenoyl Nanocomposites in Colon Cancer: An Ongoing Search for Improved Efficacy[END_REF] for an improved efficacy, or in liposomal formulations.

Further studies are currently underway in the lab to determine this compound's full potential for further development.

Experimental

General considerations

Solvent peaks were used as reference values, with CDCl 3 at 7.26 ppm for 1 A sealed tube under argon atmosphere was charged at room temperature with 1 eq of appropriate 5-bromo-indole derivative (0.2 mmol) (9, 13, 14b, 14c and 14d), 1.2 eq of corresponding N-tosylhydrazone (0.24 mmol), Pd 2 dba 3 .CHCl 3 (5 mol%), XPhos (10 mol%) and 2.4 eq of LiOtBu (0.5 mmol) in dry dioxane. The mixture was then heated at 100 °C until disappearance of bromo indole derivative evaluated by silica TLC (8/2 cyclohexane/AcOEt). The crude mixture was then allowed to room temperature, filtered through Celite pad with ethyl acetate, and 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 crude was then concentrated under vacuum to give a solid (10a, 10d, 12a, 12b). For compounds having a O-Si group, the crude residue was dissolved in dry THF with 1.1 eq of TBAF (1M in THF) for 3 h at room temperature. Reaction completion was evaluated by silica TLC (6/4 cyclohexane/AcOEt). After completion, the solvent is evaporated under reduced pressure and the crude residue was purified by silica gel chromatography (90/10 to 70/30 of cyclohexane/EtOAc) to give the product as a solid (15a, 15b, 15f, 15g and 15l).

4.2.2.

General procedure for the Buchwald-Hartwig coupling reaction (protocol B) followed by a N-methylation reaction for 10b, 10c, 11c, 12c, 12d, 12e, 12f, 12g, 15i, 15j and 15k and, for 15c, 15d, 15e and 15h a N-methylation reaction and a desilylation step.

A sealed tube under argon atmosphere was charged at room temperature with 1 eq of corresponding 5-bromo-indole derivative (0.2 mmol) (9, 13, 14b, 14c, 14d, 14e), 1.2 eq of appropriate primary amine (0.24 mmol), Pd 2 dba 3 .CHCl 3 (5 mol%), XPhos (10 mol%) and 1.5 eq (0.3 mmol) of NaOtBu (or Cs 2 CO 3 for 12e) in dry dioxane. The mixture was then heated at 100 °C until disappearance of bromo indole derivative evaluated by silica TLC (7/3 cyclohexane/AcOEt). The crude mixture was then allowed to cool down to room temperature, filtration through Celite, and the solvents were evaporated under reduced pressure. The crude residue was dissolved in freshly distilled DMF (4mL) at 0 °C and 2 eq of NaH were added. After 20 min., 1.2 eq of MeI was slowly added and the mixture was stirred at room temperature for 6 h. Reaction completion was evaluated by silica TLC (6/4 cyclohexane/EtOAc). The reaction was cooled to 0 °C, saturated aqueous NH 4 Cl solution was added by small portions and the mixture was extracted with EtOAc. The organic layer was washed with saturated aqueous NH 4 Cl solution (2x), saturated aqueous NaHCO 3 solution and brine. The combined organic layers were dried over MgSO 4 and concentrated in vacuum. The crude product was purified by silica gel chromatography (95/5 to 80/20 cyclohexane/EtOAc). The product was then concentrated under vacuum to give a solid (10b, 10c, 12c, 12d, 12e, 12f, 12g, 15i, 15j and 15k). For compounds having a OTBS group, the crude residue was dissolved in dry THF with 1.1 eq of TBAF (1M in THF) at room temperature until disappearance of starting materiel evaluated by silica TLC (6/4 cyclohexane/AcOEt). After completion the solvent was evaporated under reduced pressure and the crude residue was purified by silica gel chromatography (90/10 to 60/40 of cyclohexane/EtOAc) to give the product as a solid (15c, 15d, 15e and 15h).

General aromatization procedure leading to 11a-c (protocol C).

A sealed tube under argon atmosphere was charged at room temperature with 1 eq of corresponding dihydropyrido indole 10a or 10b and Pd/C (20 mol%) in p-xylene. The mixture was then heated at 145 °C for 48 h. Reaction completion was evaluated by analytical HPLC. The crude mixture was then allowed to cool down to room temperature, Filtration through Celite, and the solvent was evaporated under reduced pressure. The crude product was purified by inverse phase column on preparative HPLC. The product was then concentrated under vacuum to give compounds 11a or 11b. Compound 11c was obtained after a Buchwald-Hartwig coupling reaction (protocol B) between 9 and 4aminoquinazoline followed by a N-methylation reaction and an oxidation step (as for 11a,b; protocol C). 4,vinyl)-6,9-dihydropyrido [1,2-a] 5, 139.2, 139.1, 136.5, 134.8, 134.0, 129.2, 122.8, 121.7, 121.5, 120.2, 112.3, 109.3, 107.0 (2C), 98.3, 60.6, 56.4 (2C), 42.5, 24.5; IR neat (cm -1 ): 2934, 1579, 1504, 1412, 1343, 1235, 1125, 1005 indol-2-amine 10b

Description of new compounds

Protocol B followed by a N-Methylation reaction. Column chromatography on silica gel afforded 46 mg of the product as yellow solid (0.13 mmol, yield 71%); TLC (SiO 2 ,9/1 DCM/MeOH ); R f = 0.58 ; m.p = 84 °C; 1 H NMR (300 MHz, CDCl 3 ) δ 7.94 (d, J = 8.4 Hz, 1H), 7.58 (t,J = 7.4 Hz,1H),1H),1H),1H),7.02 (dd,J = 8.5,6.8 Hz,1H),2H),6.20 (d,J = 15.9 Hz,1H),6.04 (s,1H),1H),4.59 (dd,J = 5.6,3.5 Hz,2H),2H),3.49 (s,3H), 2.72 (s, 3H); 13 C NMR (75 MHz, Acetone-d 6 ) δ 164. 2, 158.2, 156.5, 146.9, 144.2, 135.8, 130.4, 126.8, 126.5, 124.8, 122.7, 121.4, 119.9, 118.4, 115.9, 111.1, 109.2, 100.1, 98.3, 45.3, 42.5, 24.5, 23.4; IR (cm -1 ): 2932, 2920, 1486, 1466, 1362, 1212, 1088, 805, 787 4,indol-2-amine 10c

Protocol B followed by a N-Methylation reaction. Column chromatography on silica gel afforded 58 mg of the product as a yellow oil (0.16 mmol, yield 57%); TLC (SiO 2 , 7/3 cyclohexane/EtOAc); 1 H NMR (300 MHz, Acetone-d 6 ) δ 7. 35 -7.27 (m, 2H), 6.97 (dd, J = 8.6, 2.2 Hz, 1H), 6.61 (dt, J = 9.9, 1.9 Hz, 1H), 6.31 (s, 1H), 6.12 -5.99 (m, 3H), 4.13 (t, J = 7.1 Hz, 2H), 3.67 (s, 6H), 3.64 (s, 3H), 3.28 (s, 3H), 2.66 (tdd, J = 6.9, 4.4, 1.9 Hz, 2H); 13 C NMR (75 MHz, Acetone- ), 148.2, 142.9, 136.5, 135.6, 132.2, 130.5, 125.6, 121.4, 120.8, 117.5, 110.4, 100.0 (2C), 95.2, 60.7, 56.3 (2C), 41.6, 40.4, 24.9; IR (cm -1 ): 3001,2955,2254,1655,1504,1412,1235,1125,1005,797 2, 152.3, 149.1, 148.8, 137.0, 135.5, 133.1, 130.7, 129.9, 128.5, 127.6, 127.0, 126.8, 123.8, 123.1, 121.5, 120.1, 119.2, 115.1, 109.9, 98.6, 42.7, 24.71, 24.65; IR (cm -1 ): 3044,2924,1592,1485,1463,1446,1393,1366,882,767 4,vinyl)pyrido [1,2-a]indole 11a

d 6 ) δ 154.6 (2C
Protocol C. A sealed tube under argon atmosphere was charged at room temperature with 1 eq of 10a and Pd/C (20 mol%) in p-xylene. The mixture was then heated at 145°C for 48 h. Reaction completion was evaluated by analytical HPLC. The crude mixture was then allowed to cool down to room temperature, filtration through Celite, and the solvent was evaporated under reduced pressure. The crude product was purified by inverse phase column on preparative HPLC to give compound 11mg 11a as brown oil (0.03 mmol, yield 37%); TLC (SiO 2 ,8/2 Cyclohexane/EtOAc ); R f = 0.70; pyrido [1,2-a]indol-2-amine 11b

Protocol C. A sealed tube under argon atmosphere was charged at room temperature with 1 eq of 10b and Pd/C (20 mol%) in p-xylene. The mixture was then heated at 145°C for 48 h. Reaction completion was evaluated by analytical HPLC. The crude mixture was then allowed to cool down to room temperature, filtration through Celite, and the solvent was evaporated under reduced pressure. The crude product was purified by inverse phase column on preparative HPLC to give compounds 8 mg of 11b as a brown solid (0.02 mmol, yield 32%); TLC (SiO 2 ,9/1 DCM/MeOH ); R f = 0.65; m.p. = 103 °C; 1 H NMR (300 MHz, Methanol-d 4 ) δ 8.66 -8.45 (m, 2H), 8.12 (d, J = 9.0 Hz, 1H), 7.78 (d,J = 8.4 Hz,4]oxazino [4,3-a] 7, 162.3, 152.6, 142.1, 135.5, 135.3, 131.9, 129.4, 127.9, 127.0, 124.0, 120.2, 118.3, 115.4, 110.4, 96.6, 65.3, 65.1, 43.6, 42.5, 26.7 Bromoindole 14a was N-alkylated as above (using 2 eq of NaH and 1.2 eq of EtI or MeI) and the crude product was then charged in a round bottom flask under argon with 1.1 eq of TBDMsCl and 1.1 eq of imidazole in dry DMF at room temperature overnight. A solution of saturated NH 4 Cl was added by small portion and the mixture was extracted with EtOAc (3x). The organic layers were then washed twice with brine, dried over MgSO 4 , concentrated in vacuum and purified by silica gel chromatography to give 14b or 14c as solids.

4.

oxy)methyl)-1-ethyl-1H-indole 14b

Column chromatography on silica gel afforded 715 mg of the product as a white solid (1.94 mmol, yield 97%); TLC (SiO 2 , 7/3 Cyclohexane/EtOAc ); R f = 0.14 ; m.p = 76 °C; 1 H NMR (200 MHz, Chloroform-d) Hz, 3H), 0.92 (s, 9H), 0.09 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 139. 6, 135.5, 129.2, 124.2, 123.2, 112.5, 110.6, 100.4, 58.0, 38.5, 25.9 (3C), 18.3, 15.3, -5.3 (2C); IR (cm

oxy)methyl)-1-methyl-1H-indole 14c

Column chromatography on silica gel afforded 115 mg of the product as a white solid (1.13 mmol, yield 90%); TLC (SiO 2 , 7/3 Cyclohexane/EtOAc ); R f = 0.13 ; m.p = 82 °C; 1 3, 123.1, 112.5, 110.4, 100.2, 58.0, 30.1, 25.8 (3C), 18.2, -5.3 (2C); IR (cm -1 ): 2948,2925,2853,1462,1388,1248,1142,1052,854; HRMS (ESI + ) for C 16 H 25 BrNOSi [M + H] + : calcd 354.0883 found 354.0885.

Bromoindole 14a was charged in a round bottom flask under argon with 2.5 eq of NaH in dry DMF at 0 °C for 30 min.

Then, 3 eq of MeI were slowly injected and the mixture was stirred at room temperature for 2 h. At 0 °C a small quantity of water and a solution of saturated NH 4 Cl were added by small portions before extraction with EtOAc (3x).

The organic layers were then washed twice with brine and dried over MgSO 4. After concentration, the crude product was purified by silica gel chromatography (95/5 to 85/15 cyclohexane/EtOAc). Column chromatography on silica gel afforded 550 mg of the product as a pink pale solid (2.17 mmol, yield 87%); TLC (SiO 2 , 6/4 Cyclohexane/EtOAc ); R f = 0.37 ; m.p = 77 °C; 1 H NMR (300 MHz, Methanol-d 4 ) δ 7.64 (d, J = 1.9 Hz, 1H), 7.28 (d,J = 8.7 Hz,1H),7.24 (dd,J = 8.7,1.8 Hz,1H),6.42 (s,1H),4.61 (s,2H),3.74 (s,3H),3.35 (s,3H).; 13 C NMR (75 MHz, MeOD) δ 138.7, 138.3, 135.5, 125.5, 123.9, 113.4, 111.9, 103.2, 67.2, 57.8, 30.2; IR (cm -1 ): 2926, 1690, 1471, 1400, 1333, 1142, 1088,

methyl)-1-methyl-1H-indole 14e

Bromoindole 14a was N-methylated using 2 eq of NaH and 1.2 eq of MeI as above and the crude product was next charged in a round bottom flask under argon with 3 eq of DIPEA in dry DCM at room temperature for 20 min. Then, 2

(s, 1H), 6.94 -6.90 (m, 1H), 6.90 -6.85 (m, 1H), 6.29 (s, 1H), 4.66 (s, 2H), 4.63 (s, 2H), 3.70 (s, 3H), 3.45 (s, 3H), 3.36 (s, 3H), 2.65 (s, 3H); 13 C NMR (75 MHz, MeOD) δ 160. 1, 156.5, 149.5, 144.8, 138.4, 137.2, 130.1, 129.3, 127.8, 127.1, 124.7, 122.2, 120.4, 117.0, 111.4, 110.0, 103.9, 96.2, 61.9, 55.8, 44.9, 30.2, 24.5; IR (cm -1 ): 2940, 2828, 1578, 1480, 1083, 763, 731 9, 154.6, 149.0, 143.8, 136.8, 135.4, 128.7, 128.3, 127.7, 125.3, 123.9, 121.6, 118.9, 114.9, 110.3, 110.1, 102.9, 66.4, 57.5, 44.1, 29.9, 25.2 2, 141.7, 136.5, 135.8, 130.7, 127.9, 120.9, 117.1, 110.0, 102.9, 93.6 (2C), 66.5, 61.0, 57.5, 56.0 (2C), 41.4, 29.9; IR (cm -1 ): 2930,2581,1506,1485,1236,1124,1082,975,729 4, 9, 138.0, 133.6, 128.1, 123.1, 121.2, 112.4, 109.7, 107.0 (2C), 103.6, 66.9, 60.6, 57.5, 56.4 (2C); IR (cm -1 ): 2935,2828,2358,1578,1503,1450,1410,1365,1235,1174,1084 presence of a NADPH-generating system consisting in 0.6 mM NADPNa4, 6.4 mM glucose-6-phosphate and 2 U/mL glucose-6-phosphate dehydrogenase, the incubation started by the addition of substrate at a final 100µM concentration (2% of total incubation volume) and the solution was shaken at 37 °C. After the incubation time of 0, 1,[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][START_REF] Tozer | The biology of the combretastatins as tumour vascular targeting agents[END_REF][START_REF] Sriram | Design, synthesis and biological evaluation of dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in cancer chemotherapy[END_REF][START_REF] Maksimenko | Therapeutic Modalities of Squalenoyl Nanocomposites in Colon Cancer: An Ongoing Search for Improved Efficacy[END_REF]72,96 h (up to 144 h), 50µL aliquots (in triplicate) were drawn in polypropylene tubes containing 100µL of internal standard solution (isoCA-4, 2500 ng/mL acetonitrile) and mixed with 1 mL of ethyl acetate to stop the reaction. After centrifugation at 13,000 rpm for 4 min. and evaporation of the recovered organic phase under vacuum centrifugation at 40 °C, the residue was dissolved in 0.1 mL of acetonitrile and analysed by HPLC-MS/MS.

Tubulin Binding Assay

Porcine brain tubulin was isolated following Shelanski procedure. [START_REF] Shelanski | Microtubule Assembly in the Absence of Added Nucleotides[END_REF] Tubulin was solved in 0.1 M MES buffer, 1.5 mM GTP, 1 mM EGTA, 1mM β-ME, 1 mM MgCl 2 , pH 6.7 buffer and centrifuged at 50000 rpm for 30 min in a TLA-100.3 rotor (Beckman Optima TLX centrifuge). Tubulin was then diluted to 1.5 mg/mL. Samples, containing the ligand or DMSO (negative control), were incubated 30 min at 20 ºC, followed by cooling on ice for 10 min. Tubulin polymerization was assessed by the UV absorbance increase at 450 nm (in Helios Alpha spectrophotometer, Thermo

Fisher Scientific) due to the turbidity caused by a temperature shift from 4 ºC to 37 ºC. When a stable absorbance value was reached and maintained for at least 20 min, the temperature was switched back to 4 ºC to ascertain the return to the initial absorption values, to confirm the reversibility of the process. The degree of tubulin assembly for each experiment was calculated as the difference in amplitude between the stable plateau (absorbance at 37 ºC) and the initial baseline of the curves (absorbance at 4 ºC). Control experiments in identical conditions but with the absence of ligand were taken as 100% tubulin polymerization. The IC 50 value of tubulin polymerization was determined by measuring the tubulin polymerization inhibitory activity at different ligand concentrations. The obtained values of the mole ratio of total ligand to total tubulin in solution were fitted to mono exponential curves and the IC 50 values of tubulin polymerization inhibition were calculated from the best-fitting curve of three independent experiments using Graphpad Prism 7.0 software.

Cell Cycle Analysis

Exponentially growing cancer K562 were incubated with 15d at different concentrations (0.5 and 1 nM) or in DMSO alone for 24 h. Cell-cycle profiles were determined by flow cytometry on a FC500 flow cytometer (Beckman-Coulter, France) as described previously. [START_REF] Venot | The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression[END_REF]

Mitochondrial membrane potential assay

One of the hallmark 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 indole 15d for 48 h. 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).

Colony Formation Assays

For colony formation assays, human chronic myeloid leukemia K562 cells (ATCC, CCL-243, Manassas, VA, USA)

were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Lonza, Basel, Switzerland), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Biowest, Riverside, CA, USA) and 1% penicillin-streptomycin solution (100×) (GenDEPOT, Katy, TX, USA). Imatinib-resistant K562 (K562IR) cells were a gift of the Catholic University of Seoul, South Korea and cultured in RPMI 1640 medium with 25 mM HEPES (Lonza) supplemented with 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotics. K562IR cells were cultured with 1 µM of imatinib and washed three times before each experiment. Cells were maintained at 37 °C and 5% of CO 2 in a humified atmosphere.

Mycoplasma detection by Mycoalert TM (Lonza) was performed every 30 days, and cells were used within three months after thawing. For the colony formation assay, 10 3 K562 or K562IR cells were counted and grown in a semisolid methylcellulose medium (Methocult H4230, StemCell Technologies Inc., Vancouver, BC, Canada) supplemented with 10% FBS in absence or presence of indicated concentrations of 15d. Colonies were detected after 10 days of culture by adding 1mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich) and were analyzed by Image J 1.8.0 software (U.S. National Institute of Health, Bethesda, MD, USA). Data of three independent experiments are expressed as the mean ± SD and significance was estimated by using two-way ANOVA (analysis of variance) followed by Tukey's multiple comparison test, unless otherwise stated, using GraphPad Prism 8

Software (La Jolla, CA, USA). p-values were considered statistically significant when p < 0.05. Legends are represented as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Molecular modeling

Atomic coordinates for tubulin α,β-dimer were retrieved from the Protein Data Bank (accession code 6H9B). 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 15d were obtained using the Conformers function from MarvinSketch v19.12 software package [ 51 ] 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].

  Reagents and conditions: (a) Pd2dba3.CHCl3, Xphos, NaOtBu, dioxane, 100 °C. (b) NaH, CH3I, DMF, rt (c) TBAF, THF, rt.

Scheme 4 .

 4 Scheme 4. Synthesis of target indole compounds 15 having a N-methyl linker

  indole B-ring was constant showed that derivative 10b (IC 50 = 5.2 nM) having a quinoline as A-ring and a N-Methyl linker was undoubtedly the most active compound in this series. Replacing the quinoline with the isosteric TMP nucleus considerably reduced the cytotoxicity level against HCT116 cancer cells (10c, IC 50 = 959 nM), similarly, the replacement of the N-methyl linker present in 10b by a 1,1-ethylene linker decreased cytotoxicity by a five-fold factor (10d, IC 50 = 25.7 nM). Pyrido[1,2-a]indoles 11a-c, which were the first compounds targeted in this study as structural analogues of carbazoles (Figure 1) displayed varying toxicity levels. Comparison of 11b (IC 50 = 78.4 nM) with 10b

Figure 2 .

 2 Figure 2. Metabolic stability profiles of isoCA-4, 15d and 15b derivatives in rat liver microsomes (A) and in human liver microsomes (B).

Figure 3 .

 3 Figure 3. Effect of compounds 15b (blue line) and 15d (red line) on tubulin polymerization. Tubulin polymerization inhibition (TPI) is indicated in percentage. The polymerization percentage was calculated, for each concentration, by comparing the amplitude of tubulin-ligand curves from 4 ºC to 37 °C with the amplitude observed for the negative control, considered as 100% of polymerization. Results are expressed as mean ± SEM of three independent experiments.

Figure 4 .

 4 Figure 4. Effect of 15d on cell cycle distribution in K562 cells determined by flow cytometry analysis. DNA content was assessed via propidium iodide staining.

Figure 5

 5 Figure 5 Compound 15d induced mitochondrial dysfunction in K562 leukemia cells. Cells were incubated with 15d at concentrations from 0.5 to 50 nM for 48 h at 37 °C. The portion of mitochondria dysfunction was measured using the JC1 assay.

Figure 6 .

 6 Figure 6. Representative pictures from three independent experiments of clonogenic assays after treatment with 15d (A) K562, (B) K562IR. Quantifications (number of colonies and the total surface area of colonies) are indicated.

Figure

  Figure 6a) three potential hydrogen bonds between (i) the side-chain SH group of the cysteine β241 residue and the N1 atom of the quinazoline moiety (ii) the primary O-H function (hydrogen bond donor) of the indole with the carbonyl of backbone's asparagine β350 (iii) the O-H (hydrogen bond acceptor) with the NH of backbone's valine β315.

Figure 7a

 7a Figure 7aPutative binding mode of 15d (green) within colchicine binding site of tubulin X-ray structure (accession code 6H9B) showing expected hydrogen bonds between ligand atoms and cysteine β241 and valine β315.

Figure 7b

 7b Figure 7b Docked pose of 15d (green) overlayed with isoCA-4 (fushia) in the tubulin binding site.

  H NMR (300 MHz, Chloroform-d) δ 7.62 (d, J = 1.8 Hz, 1H), 7.21 (dd, J = 8.7, 1.9 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.25 (s, 1H), 4.75 (s, 2H), 3.69 (s, 3H), 0.84 (s, 9H), -0.00 (s, 6H); 13 C NMR (75 MHz, CDCl 3 ) δ 140.2 (2C), 129.0, 124.

Table 1

 1 Cytotoxicity against HCT116 cells a and ITP of new compounds 10

, 11, 12 and 15.

	Compounds
		10a	10b	10c	10d
	IC 50 b [nM]	970 ± 39.7	5.2 ± 0.1	959 ± 37	25.7 ± 1.24
	Compounds
		11a	11b	11c	12a
	IC 50 b [nM]	213 ± 17.5	78.4 ± 9.6	6.87 ± 0.34	109 ± 2.9
	Compounds
		12b	12c	12d	12e
	IC 50 b [nM]	23.5 ± 11.2	3.22 ± 0.48	29.2 ± 4.76	2.80 ± 0.96
	Compounds
		12f	12g	15a	15b
	IC 50 b [nM]	361 ± 8.6	10.4 ± 0.2	1.47 ± 0.03	0.52 ± 0.01
	Compounds
		15c	15d	15e	15f
	IC 50 b [nM]	21.6 ± 0.9	0.08 ± 0.007	987 ± 32	1.25 ± 0.09
	Compounds
		15g	15h	15i	15j
	IC 50 b [nM]	2.12 ± 1	16.4 ± 3.8	44.3 ± 5.8	10.2 ± 0.39
	Compounds
				isoCA-4
		15k	15l
	IC 50 b [nM]	47.5 ± 2.42	72.4 ±2.2	0.64 ± 0.03

a HCT116 human colon carcinoma b IC50 is the concentration of compound needed to reduce cell growth by 50% following 72 h cell treatment with the tested drug (average of three experiments).

  indole as B-ring, the two more potent drugs 12c (IC 50= 3.22 nM) and 12e(IC 50 = 2.80 nM) have in structure a 2-substituted quinoline as ring A and an N-methyl linker. Examining the cytotoxicity level of 12g (IC 50 = 10.4 nM) revealed that a 2-methylquinazoline nucleus as A-ring was also welltolerated in this series. The replacement of these heterocycles by the "classical" TMP A-ring present in 12b (IC 50= 23.5

	nM) and 12d (IC 50 = 29.2 nM) caused a slight decrease in cytotoxicity against HCT116 cells. Finally, results depicted in
	Table 1 suggest that compounds 15 having a 2-substituted indole as B-ring are the more cytotoxic compounds of this
	study, with five compounds displaying a cytotoxicity level ranging from 0.08 nM to 2.12 nM. Examination of Table 1
	also revealed that compounds having as B-ring a C2-hydroxymethylindole and an ethylene linker tolerated well as A-
	ring a quinaldine (15a IC 50 = 1.47 nM) or a TMP nucleus (15b IC 50 = 0.52 nM; 15g IC 50 = 2.12 nM). With compounds
	having the same indolic B-ring and a N-methyl linker, results depicted in Table 1 showed that the best A-ring was a
	quinazoline, with our lead compound 15d having a remarkable IC 50 value of 0.08 nM. The replacement of a quinazoline
	by a quinaldine in these compounds led to a slight decrease in cytotoxicity (15c IC 50 = 21.6 nM; 15h IC 50 = 16.4 nM)
	and led to a dramatic loss of activity by changing these heterocycles by a TMP ring (15e IC 50 = 978 nM).[40]
	Comparison of the cytotoxicity level of compounds 15h, 15i and 15j revealed that the methylation of the alcohol
	function led to equipotent drugs (compare 15h IC 50 = 16.4 nM and 15j IC 50 = 10.2 nM) whereas a -CH 2 OMOM
	substituent on C2 of the indole decreased the activity (15i IC 50 = 44.3 nM). In this preliminary screening, we found 16
	compounds of different structures having a cytotoxicity level inferior to 30 nM. Before studying the mechanism of
	action of the most promising compounds in this series, we performed comparative metabolization studies of the two
	most cytotoxic compounds 15b and 15d against HCT116 cells in the presence of rats and human microsomes.
	2.2.2 In vitro metabolisation study of 15b and 15d vs isoCA-4.[17]
	2.2.2.1 Characterization of 15d, 15b, isoCA-4 and metabolites by HPLC-MS.
	High resolution electrospray mass spectra (HRMS/ESI + ) of compounds 15b, 15d and isoCA-4 provided their protonated
	molecular ions [M+H] + at m/z 347.1863 (347.1872 calculated for C 21 H 23 N 4 O), m/z 368.1854 (368.1862 calculated for
	C 22 H 26 NO 4 ) and m/z 317.1384 (317.1389 calculated for C 18 H 21 O 5 ), respectively. After 96 h incubation with rat or
	human liver microsomes, compound 15d could produce minor N-demethylated metabolites and hydroxylated
	metabolites, although 15b compound and isoCA-4 were mainly metabolized into O-demethylated (M-14) and
	hydroxylated metabolites as described before.[30]

Table 2 .

 2 Metabolic stability of isoCA-4, quinazoline 15d and 15b in rat liver microsomes (RLM) and human liver The metabolic stability is expressed as the in vitro half-life time, based on the 3,912 value (ln 50) corresponding to the remaining concentration.

	microsomes (HLM)
	Compound structure	Half-life time (h) a (Rat liver microsomes) (Human liver microsomes) Half-life time (h) a
	15d	140 ± 15	145 ± 15
	15b	70 ± 5	89 ± 5
	isoCA-4	18 ± 2	30 ± 3

a

Table 3 .

 3 Cytotoxicity against, U87-MG, A549, MDA-MB231, MiaPACA2, K562, K562R and HT29 human cancer cell

	lines.
	Compounds	U87-MG b	A549 c	MDA-MB231 d MiaPACA2 e HT1080 f	K562 g	K562R h	HT29 i
	IC 50 a [nM] 15d	0.51 ± 0.009 0.70 ± 0.02 0.25 ± 0.02	0.09 ± 0.005 0.08 ± 0.1 0.26 ± 0.03 0.10 ± 0.02 0.84 ± 0.14
	IC 50 [nM] isoCA-4	8.24 ± 0.9 52.7 ± 6.7	1.98 ± 0.6	3.01 ± 0.1 0.91 ± 0.07 4.26 ± 0.5 5.13 ± 0.04	> 100

a IC 50 is the concentration of 15d needed to reduce cell growth by 50% following 72 h cell treatment with the tested drug (average of three experiments). b U87 Human glioblastoma cells. c A549, Human lung epithelial cells. d MDA-MB231 Human breast adenocarcinoma cells. e MiaPACA2 Human pancreatic carcinoma cells. f HT1080 Human lung cancer cells. g K562 Chronic myeloid leukemia cells. h K562R Doxorubicin-resistant chronic myeloid leukemia cells. i

  H NMR and 77.16 ppm for13 C NMR, with (CD 3 ) 2 CO at 2.05 ppm for1 H NMR and 29.84 ppm for13 C NMR, and with DMSO-d 6 at 2.50 ppm for1 H NMR and 39.5 ppm for13 C NMR. Chemical shifts δ are given in parts per million, and 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). Reaction courses and product mixtures were routinely monitored by TLC on silica gel, and compounds were visualized with UV light (254nm) or by solution of phosphomolybdic acid/Δ, anisaldehyde/Δ, ninhydrine/Δ or vanillin/Δ. Flash chromatography was performed using silica gel 60 (40-63 mm, 230-400 mesh) at medium pressure (200 mbar). Fluorobenzene was used as received, and dioxane, dichloromethane, cyclohexane, and tetrahydrofuran were classically dried. Organic extracts were, in general, dried over MgSO 4 or Na 2 SO 4 . High-resolution mass spectra were recorded on a Bruker Daltonics micrOTOF-Q II instrument. All products reported showed

1 H and 13

C NMR spectra in agreement with the assigned structures. LCMS were done with H2O/ACN/0.1%Formic Acid gradient 1-30% 15 min with column Sunfire C18 -2.1x150mm -3.5m. IR spectra were measured on a Brucker Vector 22 spectrophotometer (neat, cm -1 ).

4.2 Procedure for the synthesis of new compounds 4.2.1 General procedure for the Barluenga coupling reaction (protocol A) if necessary followed by a desilylation reaction used for: 10a, 10d, 12a, 12b, 15a, 15b, 15f, 15g and 15l.

  ; HRMS (ESI + ) for C 23 H 24 NO 3 [M + H] + : calcd 362.1756 found 362.1753.

  ; HRMS (ESI + ) for C 23 H 22 N 3 [M + H] + : calcd 340.1808 found 340.1805.

  ; HRMS (ESI + ) for C 22 H 25 N 2 O 3 [M + H] + : calcd 365.1865 found 365.1858.

	4.2.4.4. 2-(1-(2-Methylquinolin-4-yl)vinyl)-6,9-dihydropyrido[1,2-a]indole 10d
	Protocol A. Column chromatography on silica gel afforded 71 mg of the product as a light brown solid (0.21 mmol,
	yield 53%); TLC (SiO 2 , 6/4 cyclohexane/EtOAc); R f = 0.66; m.p. = 129 °C 1 H NMR (300 MHz, Methanol-d 4 ) δ 7.95
	(d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.60 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.34 (s, 1H), 7.27 (d, J = 1.6 Hz, 1H),
	7.24 (dd, J = 8.4, 1.4 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.09 (dd, J = 8.6, 1.8 Hz, 1H), 6.06 (s, 1H), 6.00 (s, 2H), 5.95

(d, J = 1.3 Hz, 1H), 5.27 (d, J = 1.3 Hz, 1H), 4.54 -4.42 (m, 2H), 3.54 -3.43 (m, 2H), 2.72 (s, 3H); 13 C NMR (75 MHz, Methanol-d 4 ) δ 160.

  indol-8-amine 12gProtocol B followed by a N-Methylation reaction. Column chromatography on silica gel afforded 35 mg of the product as a light brown solid (0.10 mmol, yield 35%); TLC (SiO 2 , 8/2 Cyclohexane/EtOAc ); R f = 0.50 ; m.p = 191 °C;1 H NMR (300 MHz, CD 2 Cl 2 ) δ 7.65 (dd, J = 8.4, 1.4 Hz, 1H),7.44 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H),7.31 (dd, J = 8.6, 0.8 Hz, 1H), 7.01 (dd, J = 8.6, 2.1 Hz, 1H), 6.93 (dd, J = 8.6, 1.5 Hz, 1H), 6.80 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 1H), 4.95 (d, J = 1.2 Hz, 2H), 4.16 (td, J = 4.7, 3.9, 1.5 Hz, 2H), 2H), 3.62 (s, 3H), 2.65 (s, 3H);13 C NMR (75 MHz, CD 2 Cl 2 ) δ 163.

  δ 7.70(d, J = 1.8 Hz, 1H), 1H), 7.19 (d, J = 8.7 Hz, 1H), 6.32 (s, 1H), 4.82 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.40(t, J = 7.2

  866; HRMS (APCI) for C 11 H 13 BrNO [M + H] + : calcd 254.0181 found 254.0174.

  ; HRMS (ESI + ) for C 23 H 26 N 3 O 2 [M + H] + : calcd 376.2025 found 376.2021.

	4.2.4.28. N-(2-(Methoxymethyl)-1-methyl-1H-indol-5-yl)-N,2-dimethylquinolin-4-amine 15j
	Protocol B followed by a N-methylation reaction. Column chromatography on silica gel afforded 660 mg of the product
	as a yellow solid (1.74 mmol, yield 84%); TLC (SiO 2 , 9/1 DCM/MeOH ); R f = 0.55; m.p = 156 °C; 1 H NMR (300
	MHz, CDCl 3 ) δ 7.95 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.26 -7.15 (m, 2H), 7.02 (t,
	J = 7.7 Hz, 1H), 6.94 (dd, J = 8.7, 1.8 Hz, 1H), 6.87 (s, 1H), 6.35 (s, 1H), 4.55 (s, 2H), 3.73 (s, 3H), 3.48 (s, 3H), 3.35
	(s, 3H), 2.72 (s, 3H); 13 C NMR (75 MHz, CDCl 3 ) δ 158.

  ; HRMS (ESI + ) for C 21 H 27 N 2 O 4 [M + H] + : calcd 371.1971 found 371.1959.

Acknowledgments

The authors gratefully acknowledge support of this project by CNRS and University Paris-Saclay. S. Pecnard thank La Ligue contre le Cancer for its Ph.D. funding. L. Gallego-Yerga and R. Peláez acknowledge the support by the Spanish Ministry of Science, Innovation and Universities (RTI2018-099474-BI00), co-funded by the EU's European Regional Development Fund FEDER. Ji Yeon Paik and Marc Diederich thank the National Research Foundation (NRF) of Korea [Grant Number 019R1A2C1009231], the Brain Korea (BK21) FOUR program and the Creative-Pioneering Researchers Program at Seoul National University [Funding number: 370C-20160062]. Marc Diederich thanks the "Recherche Cancer et Sang" Foundation, "Recherches Scientifiques Luxembourg", "Een Häerz fir kriibskrank Kanner", Action Lions "Vaincre le Cancer", and Télévie Luxembourg.

Experiments were performed in triplicate. After 72 h of incubation, 100 μL of CellTiter 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).

Metabolization study of 15b and 15d

Chemicals

The two synthesized 15b and 15d were characterized by NMR spectroscopy and HRMS spectrometry with a HPLC-UV purity of >99%.

Glucose-6-phosphate, glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide phosphate tetra sodium (NADPNa4) were provided by Sigma-Aldrich (St Louis, MO, USA). LC/MS-grade acetonitrile, LC/MS-grade water, methanol and formic acid were purchased from Merck (Darmstadt, Germany). Stock solutions of the two compounds were prepared at 1600 µg/mL (about 4 mM) in methanol/dichloromethane (96/4, v:v) and stored at 4°C until the in vitro metabolism study.

High performance liquid chromatography-mass spectrometry (HPLC-MS)

HPLC-MS chromatograms were performed using an Alliance 2695 HPLC system (Waters, Milford, USA) fitted with a XBridge C18 column (2.1 × 150 mm, 3.5 μm). Gradient elution was achieved with a flow rate of 0.25 mL/min using water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) from A:B (95/5, v:v) to A:B (0/100, v:v) in 20 min. Analytes were detected with a LCT ® Premier time-of-flight mass spectrometer (Waters, Milford, USA) operating in positive electrospray ionization mode scanning from 100 to 1000 m/z with 10 spectra/s. MS parameters settings were: capillary and cone voltage at 3500 V and 25 V. High resolution mass spectra (HRMS) were processed using MassLynx ® software.

High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

HPLC-MS/MS analyses were performed using a HPLC system (Ultimate 3000, Dionex, USA) fitted with a Hypersil ® C18 column (2.1 × 100 mm, 5 µm) Thermo Fischer Scientific Inc, Waltham, MA, USA. Isocratic elution was achieved with a flow rate of 200 µL/min of the mobile phase, acetonitrile/water (60:40, v/v) with 0.2% formic acid. The total run time was 5 min. After injection of 10µL, HPLC-MS/MS analyses were performed using the LTQ-Orbitrap™ Velos Pro hybrid mass spectrometer (Thermo Fischer Scientific Inc, Waltham, MA, USA), controlled by the Xcalibur ® software operating in positive electrospray ionization at unit mass resolution by scanning over m/z 150 to 450 using collisioninduced dissociation of the selected precursor ions. Quantification of compounds was performed by selective reaction monitoring with the following ion transitions: m/z 347.1 → 332.1 for 15d, m/z 368.1 → 338.1 for 15b and m/z 317.1→ 302.1 for IS (isoCA-4). Collision energy was optimized at 45 eV for 15d and at 25 eV for 15b and isoCA-4 using helium as collision gas. HPLC-MS/MS analyses were processed using Xcalibur ® software. Calibration curves of each compound were fitted with 1/c weighted least-squares regression by plotting the peak area ratio of each analyte to IS against the analyte concentration over the calibration range (1-50 µg/mL or 3-140 nM/mL) in buffer (PH 7.4). The ESI-MS parameters were set as follows: spray voltage at 3400 V, source heater and capillary temperature at 300 °C and 350 °C, respectively with drying gas flowrate at 40 L/h.

Incubation of CH compounds with rat and human liver microsomes

Rat liver microsomes (RLM) and human liver microsomes (HLM) were provided at a protein concentration of 20 mg/mL by Thermofischer Scientific Inc, Waltham, MA, USA. Incubations were performed at a final protein concentration of 1 mg/mL for microsomal suspensions in 10 mM buffer (pH 7.4). After pre-warming at 37 °C, in the