Youzhi Li

Michel Nguyen

Laure Vendier

Anne Robert
email: anne.robert@lcc-toulouse.fr

Yan Liu
email: yanliu@gdut.edu.cn

Bernard Meunier

Yan Liu Conceptualization

X-Ray diffraction structure of Cu(II) and Zn(II) complexes of 8-aminoquinoline derivatives (TDMQ), related to the activity of these chelators as potential drugs against Alzheimer's disease

Keywords: Alzheimer's disease, Copper chelator, 8-Aminoquinoline, X-Ray crystallography, Zinc Methodology, investigation, validation, formal analysis, resources Conceptualization, validation, supervision,

come

X-Ray diffraction structure of Cu(II) and Zn(II) complexes of 8-aminoquinoline derivatives (TDMQ), related to the activity of these chelators as potential drugs against Alzheimer's disease

Introduction

The search for efficient drugs able to stop the cognitive decline of Alzheimer's disease (AD) at the early stages is one of the most challenging topics in medicinal chemistry [START_REF] Cummings | Alzheimer's disease drug-development pipeline: few candidates, frequent failures[END_REF]. The detrimental redox effect of the disruption of copper homeostasis in AD brain has been considered as a valuable target. Then, design of specific chelators able to inhibit the catalytic reduction of dioxygen produced by copper-loaded amyloids (Cu-Ab) is a promising way [2,[START_REF] Barnham | Biological metals and metal-targeting compounds in major neurodegenerative diseases[END_REF].

Such ligands must be specific for Cu(II) with respect to Zn(II) in order to avoid the neurotoxicity due to chelation of zinc, that was a feature of first generation of metal chelators like clioquinol or PBT2, based on a non-specific 8-hydroxyquinoline motif [START_REF] Cherny | Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice[END_REF]. We have identified a series of tetradentate ligands based on a 8-aminoquinoline scaffold bearing an aliphatic polyamine chelating side chain, named TDMQ (for the structures of some of these TDMQ ligands, see Scheme 1) [5]. One of these copper ligands, TDMQ20, is highly efficient to rescue the declarative memory impairment in a robust non-transgenic murine model of AD, and to reduce the oxidative stress in mouse cortex [START_REF] Zhao | TDMQ20, a specific copper chelator, reduces memory impairments in AD-mouse models[END_REF]. Many of these TDMQ ligands are also specific for Cu(II) with respect to Zn(II), their affinity for Cu(II) being 10 log higher than that for Zn(II) [5a].

In order to achieve high Cu(II) selectivity and suitable biological properties, the design of TDMQ chelators must meet some critical coordination chemistry requirements related to the structures of the Cu II -TDMQ complexes. The correlation between X-ray diffraction structures of Cu-and Zn-TDMQ complexes and their physicochemical properties should therefore help to draw pertinent correlations between structure and properties of the drugs, and to select the most promising ones for biological and pharmacological studies. Structures of TDMQ ligands. The X-ray structures of Cu-TDMQ36, Zn-TDMQ35, Zn-TDMQ36, and Zn-TDMQ22 are described in this report. Other structures, previously reported in References 5a, 5c and 7, are used for comparison (see Table 1).

We report here the X-ray structure of single crystals of Cu-TDMQ36, and that ones of the zinc complexes of TDMQ35, TDMQ36 and TDMQ22. Comparison with the already reported structures of other copper and zinc complexes of TDMQ [5a, 2b, 7] then will allow to specify the structure requirement for potentially efficient drugs.

Results and discussion

X-Ray structure of Cu-TDMQ36

Single crystals of Cu II -TDMQ36 (Figure 1 H … Cl depicted in Table S1 andFigure S1. This gives rise to an infinite sequence in the three directions and contributes to the supramolecular arrangement. This structure is consistent with the previously reported X-ray diffraction structures of the Cu(II) complexes of TDMQ20 [2b], TDMQ22 [5a] and TDMQ37 [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease[END_REF] (Table 1). Like TDMQ22, these ligands exhibit a side chain suitable perfect square planar coordination sphere of Cu(II) by the four nitrogen atoms of the ligand, that designed cycles Naniline-Cu-Npyridine, Npyridine-Cu-Nproximal, and Nproximal-Cu-Ndistal exhibiting 5, 6, and 5 bonds, respectively (Scheme 2a). This preferred coordination of Cu(II) consequently resulted in the high affinity of such ligands for Cu(II) (log Kapp = 16.5, and 17.1 for TDMQ20 [2b], and TDMQ37 [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease[END_REF], respectively). Conversely, TDMQ ligands having a shorter tether between Npyridine and Nproximal (TDMQ5 and TDMQ19) that resulted in a 5-membered ring Npyridine-Cu-Nproximal (Scheme 2b, [5a]) prevented efficient coordination of Ndistal whatever the Nproximal-Ndistal length was (m = 1 or 3). These latter complexes have a strained N3Cl coordination sphere around Cu(II) and the square plane around Cu(II) is consequently distorted, resulting in a lower affinity for Cu(II) (log Kapp = 9.8, and 10.2 for TDMQ5 and TDMQ19, respectively [5a]. The affinity of TDMQ36 has an intermediate value between these two series (log Kapp = 13.1 [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease[END_REF]).

Table 1. Summary of affinity constants of the Cu(II) and Zn(II) complexes of selected TDMQ chelators, correlated with their ability to inhibit the oxidative stress induced by Cu-Ab, and with the metal environment in the X-ray diffraction structures of single crystals of these complexes. The design of TDMQ ligands aimed to inhibit the oxidative stress induced by Cu-amyloid complexes. This property is indirectly evaluated as the ability of these ligands to inhibit ascorbate oxidation in the presence of Cu-Ab1-16 [START_REF] Nguyen | Transfer of copper from an amyloid to a natural copper-carrier peptide with a specific mediating ligand[END_REF]. Due to the high stability of the Cu(II) complexes of the N4-tetradentate TDMQ20, TDMQ22 and TDMQ37, these ligands completely inhibited the ROS production induced by Cu-Ab1-16 [5a, 7]. In similar conditions, the N3Cl ligands TDMQ5 and TDMQ19 were unable to fully resulted inhibit ROS production, and ascorbate oxidation was 81 % and 36 %, respectively, after 10 min. On this point of view, TDMQ36 did not efficiently inhibited ROS production, with an ascorbate oxidation at 40 % [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease[END_REF] UV In each ZnTDMQ35 motif, the metal ion was chelated by the two nitrogen atoms of the ligand side chain and two chloride ligands in a tetrahedral environment (Figure 2) that generally represents the optimal environment of Zn(II) [START_REF] Dudev | Tetrahedral vs octahedral zinc complexes with ligands of biological interest: A DFT/CDM study[END_REF]. The aminoquinoline nitrogen atoms did not take part in complexation. The tetrahedron S2, Figure S2).

The structures of these two zinc complexes are fully consistent with the already reported structures of Zn-TDMQ5, Zn-TDMQ19 and Zn-TDMQ20, with coordination of two nitrogen atoms of the side chain in a N2Cl2 environment ([5a], Table 1 and Scheme 4). S3 and Figures S3 andS4. These halogen-hydrogen bonds through the in situ generated (ZnCl4) 2-counterion might be a driving force of the complex crystallization.

The structure of Zn-TDMQ22 differs from the X-ray structures of all other Zn-TDMQ complexes that have been obtained, in which the aminoquinoline motif does not chelate zinc which is coordinated only by the nitrogen atoms of the aliphatic side chain, in a N2X2 environment. It might be a special feature due to the in situ formation of the counterion (Cu II Cl4) 2-which is itself due to the high concentration of the species in the crystallization solution (26 mM). This structure, close to that of several Cu II -TDMQ complexes, confirms the versatility of complexation modes of zinc, which have been reported for the 8-hydroxyquinoline derivative PBT2 [START_REF] Nguyen | Structures of copper and zinc complexes of PBT2, a chelating agent evaluated as potential drug for neurodegenerative diseases[END_REF].

Conclusion

We report here the X-ray structure of single crystals of Cu-TDMQ36, and also those of the zinc complexes of the tetradendate ligands TDMQ35, TDMQ36 and TDMQ22. Comparison with the already reported structures of other copper and zinc complexes of TDMQs [5a, 2b, 7] allow to specify the structure requirement for drugs potentially efficient to regulate copper homeostasis in the zinc-rich environment of human brain. Among the various possibilities offered by the diversity of metal chelators, we confirmed that the most suitable N4-tetradentate 8-aminoquinoline ligands to transfer copper from metal-amyloids to metal-carrier proteins, and capable of inhibiting the catalytic reduction of dioxygen produced by copper-loaded amyloids exposed to a reductant, generate strictly square planar Cu(II) complexes in vitro. On the other hand, the coordination of Zn(II) involving only the TDMQ side chain but not the quinoline nucleus, may explain the low affinity of these chelators for physiological concentrations of zinc.

Experimental Section

Chemicals and methods TDMQ5, TDMQ19, TDMQ20 and TDMQ22 were prepared according to Reference 5a. TDMQ35-37 were prepared according to Reference 7. Cu(II)-and Zn(II) chloride were used as sources of metal ions.

The crystals were kept in their mother liquor until they were dipped in perfluoropolyether oil and their structure determined. The chosen crystals were mounted on a Mitegen micromount and quickly cooled down to low temperature (100 K). The selected crystals were mounted on a Bruker Kappa APEX II using a micro-focus molybdenum Ka radiation (l = 0.71073 Å) with a graphite monochromator and equipped with an Oxford Cryosystems Cooler Device. The unit cell determination and data integration were carried out using APEX III [11]. The structures have been solved by Direct Methods using SHELXS [START_REF] Sheldrick | SHELX programmes[END_REF], and refined by means of least-squares procedures either on F with the aid of the program CRYSTALS [START_REF] Betteridge | CRYSTALS version 12: software for guided crystal structure analysis[END_REF] , or on F 2 with the aid of were obtained by the slow vapour diffusion of dichloromethane into the filtered solution placed at 4°C.

Zn II -TDMQ22. ZnCl2 in ethanol (0.97 M, 22.5 µL, 21.8 µmol) was added to TDMQ22 (10.0 mg, 21.8 µmol) dissolved in ethanol (0.8 mL) containing TEA (3 mole equiv). The resulting solution was filtered through a polyester syringe filter (ChromafilÒ GF/PET-45/25). Crystals suitable for X-ray analysis were grown by the slow vapour diffusion of dichloromethane into the filtered solution placed at 4°C.

Graphical abstract

  Scheme 1. Structures of TDMQ ligands. The X-ray structures of Cu-TDMQ36, Zn-TDMQ35, Zn-TDMQ36, and Zn-TDMQ22 are described in this report. Other structures, previously reported in References 5a, 5c and 7, are used for comparison (see Table1).

  ) were obtained by slow evaporation in air at room temperature of a DMF/ethanol (1/3, v/v) filtered solution containing an equimolecular mixture of CuCl2 and TDMQ36 (32 mM) in the presence of triethylamine (3 mol equiv). The space group was P21/c. In each Cu-TDMQ36 motif, Cu(II) was chelated by the four nitrogen atoms of the ligand: Npyridine, Naniline, and the Nproximal and Ndistal of the side chain. The aniline nitrogen was not deprotonated, and a chloride anion and a water molecule were bound as axial ligands (dCu-Cl = 2.810 Å, dCu-O = 2.730 Å, H2O-Cu-Cl angle = 164.04°). Electroneutrality of the unit cell was achieved by the presence of another chloride ion. The N4 square plane around copper was nearly perfect. The length of the bonds between Cu and nitrogens were quite similar, measured at 2.031, 2.024, 2.212, and 2.080 Å for Npyridine, Naniline, Nproximal and Ndistal, respectively. The N-Cu-N angles in the base plane were Npyridine-Cu-Naniline (g) = 89.43°, Nproximal-Cu-Ndistal (d) = 85.94°, Naniline-Cu-Ndistal (b) = 96.25°, and and Npyridine-Cu-Nproximal (a) = 93.99°. The dihedral angles between Npyridine-Cu-Naniline and Nproximal-Cu-Ndistal, Npyridine-Cu-Naniline and Npyridine-Cu-Nproximal, and Npyridine-Cu-Nproximal and Nproximal-Cu-Ndistal were 6.43°, 4.45°, and 4.96°, respectively. The copper was located at 0.084 Å above the mean plane of the four nitrogen atoms, slightly driven by the axial chloride. Noteworthy, the oxygen atom of the morpholine residue did not take part in metal chelation. Moreover, the three-dimensional structure of Cu II -TDMQ36 exhibits numerous standard hydrogen interactions of the N-H … O and O-H … O type, and also halogen-hydrogen bonds O-

Figure 1 .

 1 Figure 1. ORTEP drawing with ellipsoids at 30% probability of the X-ray diffraction structure of the Cu II -TDMQ36 complex.

Scheme 2 .a

 2 Scheme 2. Two different types of coordination of Cu(II) by TDMQ ligands, depending on the structure of the side chain.

  -visible measurement of affinity constants or ascorbate oxidation were carried out in drastically different conditions than single crystals preparation, particularly with regard to ligand concentration, 30 mM for single crystals preparation and 15-20 µM for UV-visible studies. Then, we propose that the steric hindrance generated by the morpholine residue resulted in an equilibrium between N3 and N4 chelation of Cu(II) in solution (Scheme 3), this equilibrium being dependent on the ligand concentration and/or the copper/ligand molar ratio. This feature might be responsible for a lower affinity of TDMQ36 for Cu(II) than that of TDMQ20 or TDMQ37, and for a poorly efficient inhibition of ROS production. X-Ray structure of Zn-TDMQ35 Slow vapor diffusion of dichloromethane into solution of TDMQ35 and ZnCl2 (TDMQ35/Zn mole ratio = 2/1) in ethanol at 4 °C afforded single crystals of Zn(II)-TDMQ35. The space group was P21/n.

Scheme 3 .

 3 Scheme 3. Possible equilibrium in solution between N4and N3Cl coordination of copper in the Cu II -TDMQ36 complex.

Figure 2 .

 2 Figure 2. ORTEP drawing with ellipsoids at 30% probability of the Zn II -TDMQ35 complex.

  by the 5-membered Nproximal-Zn-Ndistal ring, with the Nproximal-Zn-Ndistal and Cl-Zn-Cl angles measured at 86.29° and 116.72°, respectively. The length of the bonds between Zn and nitrogen atoms were measured at 2.104 and 2.122 Å for Nproximal and Ndistal, respectively. The length of the Zn-Cl bonds were 2.236 and 2.207 Å for the two chloride ligands. The dihedral angle between Nproximal-Zn-Ndistal and Cl-Zn-Cl planes was 87.21°. X-Ray structure of Zn-TDMQ36 Slow vapor diffusion of dichloromethane into solution of TDMQ36 and ZnCl2 (TDMQ36/Zn mole ratio = 1/1) in ethanol at 4 °C afforded single crystals of Zn II -TDMQ36. The space group was P21/c. The two Zn-TDMQ36 motifs contained in each unit cell were very close, and similar to that of Zn-TDMQ35, with the metal ion in a distorted tetrahedral N2Cl2 environment (Figure 3). The Nproximal-Zn-Ndistal and Cl-Zn-Cl angles measured 87.60° and 118.16° (motif A) or 87.82° and 103.34° (motif B), respectively. The distances between Zn and Nproximal and Ndistal were measured at 2.093 and 2.088 Å, respectively, in motif A), and 2.059 and 2.104 Å, respectively, in motif B. The length of the Zn-Cl bonds were 2.205 and 2.230 Å for the two chloride ligands (motif A), or 2.235 and 2.208 Å, respectively (motif B). The dihedral angles between Nproximal-Zn-Ndistal and Cl-Zn-Cl planes were 84.88° and 83.77°, close to the theoretical value of 90°, in motifs A and B, respectively. In addition, the three-dimensional structure of Zn-TDMQ36 exhibits intra-and inter-molecular hydrogen bonds, of the N-H … O and O-H … O types (Table

Figure 3 .Scheme 4 .

 34 Figure 3. ORTEP drawing with ellipsoids at 30% probability of the Zn II -TDMQ36 complex.

Figure 4 .

 4 Figure 4. ORTEP drawing with ellipsoids at 30% probability of the Zn II -TDMQ22 complex.

Funding

This work was supported by the NSFC (grant 21502023, YL), the Guangdong Province (Program for Innovative Research Teams and Leading Talents Introduction, grant 2050205, BM), GDUT (grant 220418037, BM), and the CNRS.

the program SHELXL2018 [START_REF] Sheldrick | SHELX programmes[END_REF] included in the software package WinGX version 1.63 [START_REF] Farrugia | WINGX -1.63 Integrated system of Windows programs for the solution, refinement and analysis of single crystal X-ray diffraction data[END_REF].

Atomic scattering factors were taken from the International Tables for X-ray crystallography [START_REF]International tables for X-ray crystallography[END_REF]. Absorption correction was done using multi-scan [START_REF] Blessing | An empirical correction for absorption anisotropy[END_REF]. Almost all hydrogen atoms were geometrically placed and refined using a riding model, excepted the hydrogen atoms H2Naniline, H2O, and HNdistal of Zn-TDMQ22, and H2Naniline, and HNdistal of Zn-TDMQ35, which were located by Fourier differences and isotropically refined.

All non-hydrogen atoms were anisotropically refined. Details of the structure solution and refinements are given in the Supporting Information (CIF files) as CCDC no. 1952669 (Cu-TDMQ36), 1952670 (Zn-TDMQ36), 2112483 (Zn-TDMQ22), and 2112484 (Zn-TDMQ35).

Drawing of molecules were performed with the program Mercury [START_REF] Macrae | Mercury: visualization and analysis of crystal structures[END_REF].

Preparation of single crystals for X-ray diffraction

Cu II -TDMQ36. CuCl2 in ethanol (0.97 M, 22.3 µL, 21.6 µmol) was added to TDMQ36 dissolved in a mixture of dimethylformamide and ethanol (DMF/EtOH = 0.15 mL / 0.5 mL, 10.7 mg, 21.6 µmol) containing triethylamine (TEA, 65 µmol, 3 mole equiv). The resulting solution was filtered through a polyester syringe filter (ChromafilÒ GF/PET-45/25). Crystals suitable for X-ray analysis were obtained by the slow evaporation of the filtered solution in air at room temperature.

Zn II -TDMQ35. TDMQ35 was first neutralized extraction with dichloromethane of a solution containing TDMQ35 (hydrochloride salt, 4.95 mg, 10 µmol) and TEA (3.3 mole equiv) in water (0.6 mL). The organic layer was dried over Na2SO4, filtered and concentrated to give a yellow residue of the base form of TDMQ35 base, which was dissolved in ethanol (0.45 mL). Then, ZnCl2 in ethanol (0.97 M, 5.15 µL, 5 µmol, 0.5 mol equiv) was added and the resulting solution was filtered through a polyester syringe filter (ChromafilÒ GF/PET-45/25). Crystals suitable for X-ray analysis were obtained by the slow vapour diffusion of dichloromethane into the filtered solution placed at 4°C.

Zn II -TDMQ36. TDMQ36 was neutralized by extraction with dichloromethane of a solution containing TDMQ36 (hydrochloride salt, 5.0 mg, 10 µmol) and TEA (3.3 mole equiv) in water (0.6 mL). The organic layer was dried over Na2SO4, filtered and concentrated to give a yellow residue of the base form of TDMQ36 which was dissolved in ethanol (0.45 mL). Then, ZnCl2 in ethanol (0.97 M, 10.3 µL, 10 µmol) was added and the resulting solution was filtered through a polyester syringe filter (ChromafilÒ GF/PET-45/25). Crystals suitable for X-ray analysis


Myriam Le Roch

Damien Afonso

Egor Chirkin

Xavier Guillory

François-Hugues Porée

Structure simplification of the Securinine skeleton reveals the importance of BCD ring system for the cytotoxic activity on HCT116 and HL60 cell lines

Keywords: Securinega, simplified analogs, molecular editing, cytotoxicity, HL60, HCT116

Function-oriented molecular editing of the polycyclic scaffold of securinine led to the preparation of a library of simplified analogs that have been evaluated for their cytotoxicity potential against HCT116 and HL60 human cell lines. Chemical diversity at the C14 position (securinine numbering) was generated through the site-selective -iodination followed by Pdcatalyzed Sonogashira and Suzuki-Miyaura reactions. To explain the selectivity in the iodination step, a reaction mechanism has been proposed. Surprisingly, the piperidine ring (ring A) of the securinine skeleton has been found to be irrelevant for the cytotoxic activity. Based on this finding, the pharmacophoric core of securinine could be simplified to the key BCD motif. The nature of the substituent at the nitrogen can vary from a methyl or an isobutyl group to a benzyl or a carbamate moiety. Interestingly, the N-benzyl substituted simplified analog exhibited the same cytotoxic activity as the parent compound securinine. This functional group tolerance paves the way for the installation of reactive handles for the synthesis of molecular probes for target identification.

Introduction

Securinega alkaloids represent a unique class of plant metabolites whose botanical distribution is limited to Securinega (= Flueggea), Phyllanthus, Margaritaria and Breynia genera (Phyllanthaceae family) with the exception of Zygogynum pauciflorum species (Winteraceae). To date, more than 60 compounds have been isolated and characterized allowing their classification in four groups according to the unique bridged tetracyclic scaffold which includes the securinane, the norsecurinane, the neosecurinane and the norneosecurinane. [START_REF] Perez | Synthesis and biological evaluation of new securinine analogues as potential anticancer agents[END_REF] Recent phytochemical investigations led to the isolation of original minor alkaloids displaying complex and rearranged structures which in turn provided insights into some biosynthetic pathways. [START_REF] Fan | Discovery and biosynthesis of ascorbylated Securinega alkaloids[END_REF] In addition, extensive analytical work was conducted over the last years to assign the absolute stereochemistry of new derivatives, leading to the revision or the structure clarification of some already known alkaloids. [START_REF] Wu | Absolute Configurations and Stereochemical Inversion Mechanism of Epimeric Securinega Alkaloids from Flueggea suffruticosa[END_REF] However, further work is still needed to elucidate the whole biosynthetic machinery for the Securinega alkaloids. The original and complex architecture of the Securinega alkaloids prompted the chemist's community to develop versatile and efficient strategies to access these compounds. [START_REF] Weinreb | Total synthesis of the Securinega alkaloids[END_REF][START_REF] Wehlauch | Securinega Alkaloids: Complex Structures, Potent Bioactivities, and Efficient Total Syntheses[END_REF] Of note, most of the efforts were focused on securinine (1), the first isolated and most emblematic representant of this alkaloid family. [START_REF] Murav'eva | Chemical study of alkaloids of Securinega suffruticosa[END_REF] The biological potential of these compounds has also been profiled with a particular emphasis on securinine (1) which displays interesting properties as a central nervous system (CNS) and anticancer agent. 7 Because of its CNS stimulant and antispasmodic properties the nitrate salt of securinine (1) was clinically used in the USSR as a substitute for strychnine until the early 1990s. [START_REF]Russian drug compedium[END_REF] In addition, antimicrobial properties of securinine and its congeners have been reported. 7a Despite these compelling activities and a safe pharmacological profile, the structure-activity relationships (SAR) studies on the securinine scaffold have been to this day confined to easily accessible reactive positions. Thus far, derivatization at the C12, C14 9,10 and C15 11,12 positions has been explored and has shown to increase in some cases the overall potential of securinine (1) (Scheme 1). Because of the complexity of diverted total synthesis of 1, all previous studies exploited the semi-synthetic approach starting from the natural alkaloid. Scheme 1. C14-functionalized simplified Securinega analogs.

Interest for securinine (1) as a hit for further development as anticancer agent came mainly from Wald et al. who described its potential on both HCT116 and HL60 cancer cell lines. 13 In particular, a significant effect of securinine (1) in a xenograft mouse model for acute myeloid leukemia. Later, the cytotoxic activity was extended to others cancer cell lines such as MCF-7 14 and SW480 [START_REF] Chen | Virosecurinine induces apoptosis by affecting Bcl-2 and Bax expression in human colon cancer SW480[END_REF] . Some pharmacological investigations have been carried out to elucidate the mechanism of action of securinine (1). Thus, securinine (1) was shown to downregulate in a dose-and time-dependent manner the PI3K/AKT/mTOR signaling pathway [START_REF] Han | L-securinine induces apoptosis in the human promyelocytic leukemia cell line HL-60 and influences the expression of genes involved in the PI3K/AKT/mTOR signaling pathway[END_REF] and to activate the JNK-ERK signaling pathway 17 in HL60 cells line, respectively. Action on these pathways provokes the differentiation of the HL60 cells to monocytic lineage which ultimately causes cell death. Other effects including ROS generation and mitochondrial dysfunction were also observed, all of these contributing to the apoptosis activation upon exposure to securinine (1). Furthermore, securinine (1) was identified to inhibit protein disulfide isomerase (PDI), a redoxdependent folding assistant whose overexpression is linked to cancer development. [START_REF] Kaplan | Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase[END_REF] The , unsaturated lactone motif of securinine (1) was found to be crucial for the PDI inhibition by acting as a Michael acceptor. Despite its apparent risk profile due to the presence of a conjugated butenolide, securinine (1) was not identified as a pan-assay interference compound (PAIN) by on-line PAINS filters. [START_REF]New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays[END_REF] It is also worth mentioning that securinine (1) was used as a CNS drug for several decades and its in vitro metabolic stability was confirmed recently. [START_REF] Kaplan | Structural Elucidation of a Small Molecule Inhibitor of Protein Disulfide Isomerase[END_REF] Therefore, securinine could be rationally considered as a good starting point for SAR exploration. Recent semi-synthetic studies on securinine (1) conducted separately by Wald et al. and Vidal et al. highlighted the C14 position as an interesting location for late-stage functionalization. Their strategy consisted in introducing at this position an iodine atom to subsequently carry out a Suzuki-Miyaura or a Sonogashira coupling reaction. These studies illustrated the interest in modulating the C14 position of securinine (1) to increase its antiproliferative properties against HL60 cell line and, to a lesser extent, HCT116 cell line. Unfortunately, as demonstrated by Vidal et al., the Sonogashira adducts bearing a conjugated triple bond displayed low plasmatic stability. The C15 position was also subject to medicinal chemistry efforts. This position is synthetically advantageous as it can be directly functionalized either by Michael addition 10b,11 or by Heck coupling 10a . Of note, in the Michael addition, the conjugated -unsaturated--lactone is lost and the C15-derivatives obtained were devoid of cytotoxic activities, thus establishing the importance of the conjugated unsaturated system in the pharmacophore model. Outside of the semi-synthetic modifications listed above, SAR studies employing the analogs resulting from the molecular editing of the securinine skeleton are critically missing. In particular, the importance of the piperidine ring (cycle A) for the cytotoxic activity of 1 is yet unknown.

Results and discussion

In order to fill this lacuna of knowledge, we sought to access the first-in-class library of simplified analogs of 1 lacking the piperidine ring and to investigate their antiproliferative potential against HCT116 and HL60 cancer cell lines. The rational for the choice of these two cancer cell lines is linked to the previous results reported separately by the groups of Wald and Vidal, which showed a promising activity of securinine (1) and its C14-analogs In our previous work, we have reported a straightforward route to the critical BCD core 2 of securinine (1) and related alkaloids (Scheme 2). [START_REF] Chirkin | Viability of a [2+2+1] Hetero-Pauson-Khand cycloaddition strategy toward Securinega alkaloids: synthesis of the BCD-ring core of securinine and related alkaloids[END_REF] The tricyclic core 2 can be considered on its own as a simplified securinine analog which would allow to study the contribution of the cycle A to the biological activity of 1. Moreover, BCD core 2 can be further explored as a starting point for the synthesis of analogs that are not otherwise accessible through the semi-synthetic route. Central to this approach is generating a great deal of structural complexity through the strategic application of a [2+2+1]-hetero-Pauson-Khand reaction. The pivotal enyne 3 could be accessed from Lhydroxyproline through a Z-selective olefination. Next, we envisaged that the C14-selective iodination of 2 should be possible as it had been demonstrated earlier by Chen et al. for securinine (1). [START_REF] Li | Unexpected ring contraction and oxidation rearrangement reactions of securinine[END_REF] Then, either a Suzuki-Miyaura or a Sonogashira Pd-catalyzed cross coupling reaction would be utilized to generate structural diversity. Acquiring 2 in large quantities was the major limitation for preparation of analogs through the late-stage functionalization approach. Some modifications to the previous report were made to secure the preparation of 2 in acceptable yields on a higher scale. In particular, the modified Julia olefination reaction to install the Z-enyne motif could be carried out on the gram scale with high reproducibility. [START_REF] Chiummiento | Direct Preparation of Z-1,3-Enyne Systems with a TMS-Propargylic Sulfone: Application of a One-Pot Julia Olefination[END_REF] In this case, the premetallated conditions i.e. treatment of the sulfone with NaHMDS prior to the aldehyde addition, were preferred and the reaction went to completion within 1.5 h (TLC monitoring) at -55°C with a good yield (82%). Also, the conditions for the key hetero-Pauson-Khand cyclization were simplified: the reaction was performed at a 1.7 mmol scale in the absence of CO atmosphere. Under these conditions, the reaction was very fast (1 h), albeit the overall yields could not be improved beyond 49% despite numerous efforts. To the best of our knowledge, the use of molybdenum hexacarbonyl as a donor of CO in hetero-Pauson-Khand reaction is without precedent. [START_REF] Tao | Formal synthesis of (±)-Aplykurodinone-1 through a hetero-Pauson-Khand cycloaddition approach[END_REF] Scheme 2. hetero-Pauson-Khand strategy to the BCD-core of securinine (1) and related alkaloids (right); synthetic plan for simplified analogs via scaffold simplification (left).

We then turned our efforts to the site-selective -iodination reaction. This reaction was serendipitously discovered by Chen et al. for securinine (1) and is known to give the C14iodinated derivative in a modest 40% yield together with minor by-products resulting from the A-ring contraction. [START_REF]New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) from Screening Libraries and for Their Exclusion in Bioassays[END_REF] We speculated that the tricyclic core 2 lacking the A-ring should exhibit even a higher level of site-selectivity. To our surprise, exposure of 2 to N-iodosuccinimide (NIS) in dry methanol resulted in recovery of the starting material with no trace of the iodinated derivative formed. To circumvent the unexpected lack of reactivity of 2, we explored several other iodination conditions: I2, K2CO3 [START_REF] Lin | Enantioselective approach to (-)-Hamigeran B and (-)-4-Bromohamigeran B via catalytic asymmetric hydrogenation of racemic ketone to assemble the chiral core framework[END_REF] , ICl 25 , I2, CF3CO2Ag [START_REF] Zhou | Anti-AIDS agents 79. Design, synthesis, molecular modeling and structure-activity relationships of novel dimethyldihydropyranochromone (DCP) analogs as potent anti-HIV agents[END_REF] . In addition, the preparation of the corresponding bromo analog was attempted either by dibromation followed by elimination of HBr 27 , or via photocatalysis [START_REF] Wang | Regioselective synthesis of α-bromo-α,β-unsaturated carbonyl compounds via photocatalytic α-bromination reactions[END_REF] . None of these reaction conditions furnished the expected halogenated compound. At this point, we postulated that the nature of the substituent at the nitrogen atom of 2 could play a dramatic role in the reactivity of the conjugated system. From compound 2, N-methyl-(4), N-isobutyl- (5) and N-benzyl-(6) derivatives were prepared by acid promoted Boc removal followed by direct N-alkylation or reductive amination 29 of the amine trifluoroacetic salt. These derivatives were further treated with NIS in dry methanol according to the initial procedure (Scheme 3). Under these conditions, N-methyl substituted 4 was almost unreactive and only traces of the corresponding iodo compound were observed in the 1 H NMR spectrum. In the case of a N-isobutyl substituent (5), a conversion of 20% was observed by [START_REF] Perez | Synthesis and biological evaluation of new securinine analogues as potential anticancer agents[END_REF] H NMR and the iodo compound 7 was isolated in less than 10% yield by preparative TLC. The N-benzyl derivative 6 was the most reactive with 70% of conversion observed by [START_REF] Perez | Synthesis and biological evaluation of new securinine analogues as potential anticancer agents[END_REF] H NMR and compound 7 was isolated in 53% yield.

The energies of the highest occupied molecular orbitals (HOMO) of securinine (1) and compounds 2 (Boc), 4 (Me), 5 (iBu) and 6 (Bn) were calculated by DFT at the B3LYP-D3_6-311++G(d,p) level. Among all compounds, the Boc-protected analog 2 showed the lowest HOMO level (-6.91 eV, see 4.2 Computational Details Fig 1). The electron-withdrawing substituent at the nitrogen impacted the electron density distribution in 2, in particular at the C14 position, leading to a higher conjugation of the C14-15 double bond to the butenolide system. This observation could explain the lack of reactivity of 2 in the iodination reaction.

Scheme 3. C14-Iodination of the BCD scaffold

In the report of Chen et al., the mechanistic origin of the high site-selectivity in the iodination of securinine (1) had not been revealed. To explain the formation of the iodo adducts 7 and 8, we put forward the mechanism outlined in Scheme 4. NIS is known to be a source of Lewisacidic I + and may activate carbonyl group for nucleophilic attack (intermediate II). 30 Then, the electron-deficient tricycle II undergoes conjugate addition of methanol to the reactive C15 position to form a transient zwitterion III. Electrophilic capture of III by NIS gives rise to a vicinal iodo-methoxy derivative IV which in turn undergoes subsequent elimination to provide iododerivative V. The similar Morita-Baylis-Hillman-type mechanistic pathways have been reported for the -iodination of -unsaturated ketones 31 , acrylate esters 32 and nitroalkenes [START_REF] Pamarthi | -Iodonitroalkenes as potential antifungal and antitubercular agents[END_REF] . It is interesting to note that NIS may play a dual role, as an electrophile and as a catalyst for the nucleophilic addition of methanol. In the case of N-alkyl substituted derivatives (compounds 1, 4-6), the observed difference of reactivity could be rationalized in term of steric hindrance around the nitrogen atom. One could postulate that the lone pair of the nitrogen reacts with the iodonium to form a quaternary ammonium salt thus preventing the iodination step. With a small substituent such as a methyl, the iodonium sequestration is fast and and the yield of the iodo derivative is poor, whereas the presence of a bulkier group such as an isobutyl or a benzyl favors the C14-selective iodination by blocking the access to the nitrogen by steric hindrance.

Scheme 4.

Proposed mechanism for the iodination reaction.

Chemical diversity at the C14 position was introduced by Pd-mediated Suzuki-Miyaura and Sonogashira reactions (Scheme 5). For the Sonogashira reaction, three different partners were selected (phenyl, n-propyl and p-dimethylaminophenyl) based on the most active securinine analogs reported in the previous studies. Application of phosphine-free conditions enabled the preparation of the adducts in very good yields. However, these unstable compounds were unsuitable for storage and could not be investigated for their biological properties. 34 . Two different reaction conditions were applied in the Suzuki-Miyaura reaction depending on the nature of the boronic acids or esters used. Phosphine-free conditions could be used with boronic acids and esters possessing an electron-donating group whereas the presence of an electro-attracting group prompted us to envisage palladium tetrakis as the Pd source. [START_REF] Trost | Palladium-catalyzed asymmetric allylic fluoroalkylation/ trifluoromethylation[END_REF] The overall yields were good to excellent.

Scheme 5. C14-Functionalization by Pd coupling. Taking these results into account, compounds 6, 8 and 18 were selected for determination of their IC50 and compared to securinine (1), the parent compound (Table 2). As previously observed, securinine (1) was more active against HL60 cell line than HCT116 cell line with an IC50 of 0.1 ± 0.006 M and 8.6 ± 0.28 M, respectively. Interestingly, the benzyl analog 6 demonstrated on HL60 cell line an IC50 value that was nearly similar to securinine (1). This finding highlights in a quantitative manner that the piperidine ring is not crucial for the cytotoxic activity. Moreover, presence of an aromatic substituent at the nitrogen position is well tolerated and could pave the way for the introduction of a reactive or fluorescent tag for further target identification or biological exploration on this cell line. On the other hand, IC50 determination on p-nitrophenyl derivative 18 was less conclusive. Despite an IC50 value comparable to securinine (1) on HCT116 cell line, the strong effect of 18 on the viability of HL60 cell line could not be correlated with an increased IC50 value (above 10 M).

Conclusion

In conclusion, the first-in-class library of simplified securinine analogs built on the BCD core of the securinine skeleton and functionalized at the C14 position has been prepared. Molecular editing approach provided new insights into the functional dissection of the polycyclic architecture of the Securinega alkaloids. It has been found that the piperidine ring A of securinine ( 1) is not essential for its cytotoxic activity. The simplification of the pharmacophoric core of 1 to the key BCD motif will greatly facilitate the synthesis of analogs for further SAR exploration of the Securinega alkaloids. Several conclusions can be drawn from the analysis of the prepared library. The Sonogashira adducts were found to be not stable and could not be considered for further development. The substituents at the nitrogen atom ranging from a benzyl to a carbamate moiety were tolerated without impacting the overall cytotoxic activity. This functional group tolerance represents an opportunity to install reactive handles for the synthesis of molecular probes for target identification. This would open the way to a structurebased drug design approach and the rational design of novel analogs. Finally, new mechanistic details on the C14-selective -iodination were revealed. The reaction outcome was found to be influenced by the nature of the substituent at the nitrogen. We hypothesize that the presence of a Boc group prevents the nucleophilic attack through electronic effects, whereas a benzyl group furnished the best results as it closely mimics the parent alkaloid securinine (1). We also propose for the first time a reaction mechanism for the formation of the C14 iodo adducts that relies on a Morita-Baylis-Hillman-type reaction. Taken together, we believe that the reported results would accelerate the research on securinine (1) and related compounds as leads for anticancer therapy development.

Experimental

Chemistry

All reactions were carried out under argon atmosphere with anhydrous solvents under anhydrous conditions, unless otherwise stated. Commercially available reagents were used without further purification, unless otherwise stated. Yields refer to isolated yields, unless otherwise noted. NMR spectra were recorded on Bruker Avance 300 instrument in CDCl3 or CD2Cl2. The signal of the residual non-deuterated solvent was used as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm). The following abbreviations are used for multiplicities: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; td, triplet of doublets; dt, doublet of triplets; ddd, doublet of doublets of doublets; m, multiplet. Coupling constants (J) are reported in Hertz (Hz) and were processed with MNova software. IR spectra were recorded on Perkin Elmer FT-IR instrument (spectrum two); only selected absorbances are reported. HRMS analyses of new compounds were performed by the "Centre Regional de Mesures Physiques de l'Ouest" (CRMPO, Rennes), and were obtained using a Bruker Maxis 4G or an Agilent 6510 Q-TOF. Optical rotations () were recorded on a Perkin Elmer Model were determined from the following formula [α] T D = α x 100 / c (c concentration, g/100 mL).

Boc-butenolide 2.

Part 1: modified Julia-Kocienski olefination. To a stirred solution of 2-[(3-(trimethylsilyl)prop-2yn-1-yl)sulfonyl]benzo [d]thiazole (2.195 g, 7.09 mmol, 1.1 eq) in dry THF (200 mL) at -78°C was added dropwise NaHMDS (7.1 mL, 1M solution in THF). The reaction mixture was stirred for 15 min before addition of a solution of tert-butyl (2S,4R)-4-[(tert-butyldimethylsilyl)oxy]-2formylpyrrolidine-1-carboxylate (2.125 g, 6.45 mmol, 1.0 eq) in dry THF (22 mL). The resulting mixture was stirred at -55°C for 1.5 h, then allowed to warm to -10°C. Brine (250 mL) and diethyl ether (250 mL) were added. After 0.5 h, the layers were separated and the aqueous phase was extracted with diethyl ether (2x200 mL). The combined organic layers were washed with brine (200 mL), dried (MgSO4), filtered and concentrated in vacuo to afford a brown oil. Chromatography on silica gel (cyclohexane/EtOAc, 9:1) gave the title compound as a paleyellow oil (2.245 g, 82 % yield). Part 2: hetero-Pauson-Khand cyclization. A Schlenk-tube was charged with Mo(CO)6 (1.15 g, 4.35 mmol, 2.5 eq) and dry DMF (19 mL). Then, a solution of (S)-tert-Butyl 2-[(Z)-But-1-en-3ynyl]-4-oxopyrrolidine-1-carboxylate (410 mg, 1.74 mmol, 1 eq) in dry toluene (33 mL) was added. The resulting mixture was stirred for 1h at 140 °C and then cooled to rt. The contents were transferred into a round bottom flask and concentrated in vacuo. The resulting black residue was suspended into ethyl acetate and filtered through celite then washed thoroughly with ethyl acetate. The combined organic layer was concentrated in vacuo to afford a black oil which was subjected to chromatography on silica gel (CH2Cl2/Et2O, 95:5) to give Bocbutenolide 2 as a pale-yellow solid (223 mg, 49 % yield). The analytical data were identical to those reported by Chirkin et al.

Methyl-butenolide 4

To a solution of boc-butenolide 2 (100 mg, 0.38 mmol, 1 eq) in dry CH2Cl2 (3.4 mL) at 0 °C was added trifluoroacetic acid (0.6 mL). The resulting solution was stirred allowing to warm to rt for 2 h. Volatiles were removed in vacuo to afford the corresponding TFA-salt as a brown solid. The residue was suspended in dry CH2Cl2 (4 mL) and cooled in an ice-water bath. Then DIPEA (146 µL, 0.84 mmol, 2.2 eq) and methyl iodide (26 µL, 0.42 mmol, 1.1 eq) were successively added and the resulting mixture was stirred overnight allowing to warm to rt. EtOAc was added to reaction mixture and washed with a solution of 10% aq NaHCO3 (3 mL).

Aqueous phase was extracted twice with EtOAc (5 mL) and the combined organic layer was washed with brine (2 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel (CH2Cl2/Acetone 2:8) to give methyl-butenolide 4 as a cream solid (34.0 mg, 50% yield).

[α]

Isobutyl-butenolide 5

To a solution of Boc-butenolide 2 (65.8 mg, 0.25 mmol, 1 eq) in dry CH2Cl2 (2.2 mL) at 0 °C was added trifluoroacetic acid (0.4 mL). The resulting solution was stirred allowing to warm to rt for 2 h. Volatiles were removed in vacuo to afford the corresponding TFA-salt as a brown solid. Then, isobutyraldehyde (23 µL, 0.25 mmol, 1 eq) and chlorodicarbonyl rhodium (I) dimer (1 mg, 2.5 µmol, 1 mol %) were added to the solid-salt suspended in dry CH2Cl2 (2.5 mL) with NaHCO3 (32 mg, 0.375 mmol, 1.5 eq). After addition of PhSiH3 (62 µL, 0.5 mmol, 2 eq) at 0 °C, the reaction mixture was slowly warmed to rt and stirred for 24 h. The reaction mixture was diluted with Et2O (8 mL) and washed with a solution of 10% aq NaHCO3 (5 mL). The layers were separated and the aqueous phase was extracted with Et2O (2x8 mL). The combined organic phase was washed with a 10% aq NaHCO3 solution (5 mL), brine (5 mL), dried (MgSO4), filtered and concentrated in vacuo to afford an orange oil. Chromatography on silica gel (petroleum ether/Et2O, 85:15) furnished isobutyl-butenolide 5 as a white solid (36.8 mg, 67% yield).

[α] [START_REF] Chirkin | Viability of a [2+2+1] Hetero-Pauson-Khand cycloaddition strategy toward Securinega alkaloids: synthesis of the BCD-ring core of securinine and related alkaloids[END_REF]

Bn-Butenolide 6

To a solution of Boc-butenolide 2 (400 mg, 1.52 mmol, 1 eq) in dry CH2Cl2 (13.6 mL) at 0 °C was added trifluoroacetic acid (2.4 mL). The resulting solution was stirred allowing to warm to rt for 2 h. Volatiles were then removed in vacuo to afford the corresponding TFA-salt as a brown solid. The residue was dissolved in dry CH2Cl2 (16 mL) and cooled to 0 °C. Then DIPA (685 µL, 4.86 mmol, 3.2 eq) and benzylbromide (235 µL, 1.98 mmol, 1.3 eq) were successively added. The mixture was stirred overnight allowing to warm to rt. A solution of 10% aq NaHCO3 (20 mL) and ethyl acetate (40 mL) were added. The layers were separated and the aqueous phase was extracted with ethyl acetate (15 mL). The combined organic layer was washed with brine twice (15 mL), dried (MgSO4), filtered and concentrated in vacuo to afford a brown oil. Chromatography on silica gel (CH2Cl2/Et2O, 9:1) gave Bn-butenolide 6 as a cream solid (366 mg, 95% yield).

[α] [START_REF] Chirkin | Viability of a [2+2+1] Hetero-Pauson-Khand cycloaddition strategy toward Securinega alkaloids: synthesis of the BCD-ring core of securinine and related alkaloids[END_REF]

Iodo-butenolide (Isobutyl) 7

According to the iodation procedure of Bn-butenolide 6 (see below), iodo-butenolide (isobutyl) 7 was obtained after preparative TLC (CH2Cl2/Et2O, 95:5).

Iodo-butenolide (Bn) 8

To a suspension of Bn-butenolide 6 (366 mg, 1.44 mmol, 1 eq) in dry MeOH (9.5 mL) at 0 °C was added NIS (585 mg, 2.6 mmol, 1.8 eq). The resulting suspension was stirred overnight away of light allowing to warm to rt. Volatiles were removed in vacuo and the residue was dissolved in ethyl acetate (40 mL). A solution of 10% aq NaHCO3 (20 mL) was added and the mixture stirred for 10 min. Layers were separated and the aqueous phase was extracted with ethyl acetate (2x20 mL). The combined organic layer was successively washed with a solution of 10% aq Na2S2O3 (20 mL) and brine (20 mL), dried (MgSO4), filtered and concentrated in vacuo. Chromatography on silica gel (CH2Cl2/Et2O, 95:5) furnished iodo-butenolide (Bn) 8 as a brown solid (290 mg, 53% yield).

[α] [START_REF] Chirkin | Viability of a [2+2+1] Hetero-Pauson-Khand cycloaddition strategy toward Securinega alkaloids: synthesis of the BCD-ring core of securinine and related alkaloids[END_REF]

General procedure for Sonogashira coupling reaction

In a glass Schlenk-tube, iodobutenolide 8 (50 mg, 0.13 mmol, 1 eq), palladium on activated charcoal 10% wt/Pd (7 mg, 6.59 µmol, 5 mol %) and copper iodide (2.5 mg, 0.013 mmol, 10 mol %) were successively introduced. Then, 2 mL of dry tetrahydrofuran were added. The mixture was degassed by 3 vacuum/argon cycles. DIPA (37 µL, 0.26 mmol, 2 eq) and 1.3 eq of alkyne were successively introduced and the mixture was stirred at r. t. After the iodobutenolide has been consumed (TLC moitoring), the reaction mixture was filtered through a pad of celite eluting with EtOAc (10 mL). The organic layer was washed with a saturated aq NaHCO3 solution (10 mL), brine (10 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel.

(6S,8aS)-7-benzyl-4-(phenylethynyl)-7,8-dihydro-6,8a-methanofuro[2,3-c]azepin-2 (6H)-one 9

to the general procedure, compound 9 was obtained after purification on silica gel (CH2Cl2/Et2O, 95:5) as an orange yellow gum (38.0 mg, 81%). The product decomposed upon storing even in the freezer (air and/or light sensitive).

1

H NMR (300 MHz, CD2Cl2): δ (ppm) 7.57-7.51 (m, 2H, Ho'), 7.43-7.24 (m, 8H, Ho, m, m', p, p'), 6.91 (d, J = 5.6 Hz, 1H, H5), 5.90 (s, 1H, H2), 3.93-3.89 (m, 1H, H6), 3.81 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.65 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.52 (d, J = 9.9 Hz, 1H, H8A), 2.71 (dd, J = 9.9 Hz, J = 4.4 Hz, 1H, H10A), 2.46 (d, J = 9.9 Hz, 1H, H8B), 1.93 (d, J = 9.9 Hz 1H, H10B) According to the general procedure, compound 10 was obtained after purification on silica gel (CH2Cl2/Et2O, 96:4) as a yellow oil (40.0 mg, 95%). The product decomposed upon storing even in the freezer (air and/or light sensitive). According to the general procedure, compound 11 was obtained after purification on silica gel (petroleum ether/EtOAc, 75:25) as an orange yellow gum (48.6 mg, 93%). The product decomposed upon storing even in the freezer (air and/or light sensitive).

General procedure for Suzuki-Miyaura coupling reaction

Method A: To a Schlenk-tube charged with iodo-butenolide (Bn) 8 (50 mg, 0.13 mmol, 1 eq), boronic acid (or boronic pinacol ester) (2 eq), Na2CO3 (28 mg, 0.26 mmol, 2 eq) and 10% wt Pd/C (7 mg, 6.59 µmol, 5 mol %) was added DME and water (2 mL, 1/1, v/v). The reaction mixture was stirred at 85 °C for 2.5 h, cooled to rt and then filtered through celite. The celite pad was washed with DME and ethyl acetate. The organic layer was successively washed with 1M aq NaOH, brine, dried (MgSO4), and concentrated in vacuo. The residue was purified by chromatography on silica gel. Method B: To a Schlenk-tube charged with iodo-butenolide (Bn) 8 (50 mg, 0.13 mmol, 1 eq), boronic acid (or boronic pinacol ester) (1.5 eq) and tetrakis(triphenylphosphine) palladium (7.6 mg, 6.59 µmol, 5 mol %) were added dry toluene, EtOH and 2M aq Na2CO3 (2.08 mL, 62.5/12.5/25, v/v/v). The reaction mixture was stirred at 110 °C for 3 h, then diluted with water and extracted with ethyl acetate. The organic phase was washed with brine, dried (MgSO4), filtered and concentrated under reduce pressure. The residue was purified by chromatography on silica gel. [α] 20 D = -428 (c 0.49, CH2Cl2) 1 H NMR (300 MHz, CDCl3): δ (ppm) 7.43 (dd, J = 4.9 Hz, J = 2.9 Hz, 1H, H5'), 7.39 (dd, J = 2.9 Hz, J = 1.4 Hz, 1H, H2'), 7.33-7.25 (m, 5H, Ho, m, p), 7.19 (dd, J = 4.9 Hz, J = 1.4 Hz, 1H, H4'), 6.67 (d, J = 5.6 Hz, 1H, H5), 5.87 (s, 1H, H2), 3.94-3.91 (m, 1H, H6), 3.76 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.63 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.60 (d, J = 9.9 Hz, 1H, H8A), 2.75 (dd, J = 9.7 Hz, J = 4.3 Hz, 1H, H10A), 2.49 (d, J = 9.9 Hz, 1H, H8B), 1.95 (d, J = 9.7 Hz, 1H, H10B)

Computational Details

All calculations were carried with Gaussian 16 program. Structure optimizations of molecular structures in ground states were carried out at the B3LYP-D3 level in combination with the 6-31+G(d,p) basis set and using the PCM methanol solvent model. The vibrational frequencies were computed at the same level to check whether each optimized structure is an energy minimum (no imaginary frequency) or a transition state (single imaginary frequency). Singlepoint energies, molecular orbitals and Fukui indices of geometrically optimized structures were calculated using the 6-311++G (d,p) Cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA) and were cultured according to the supplier's instructions. Human HCT-116 colorectal carcinoma cells were grown in Gibco McCoy's 5A supplemented with 10% fetal calf serum (FCS) and 1% glutamine. HL60 myelogenous leukemia cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 1% glutamine. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell viability was determined by a luminescent assay according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, the cells were seeded in 96-well plates (2.5 × 10 3 cells/well) containing 100 μL of growth medium. After 24 h of culture, the cells were treated with the tested compounds at two different final concentrations (10 M and 1 M). 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 IC50 values were calculated using the Graph Prism software from polynomial curves (four or five-parameter logistic equations). Doxorubicin was used as an internal standard.

1 H

 1 D = -974 (c 0.49, CH2Cl2); m.p.: 75-76 °C NMR (300 MHz, CDCl3): δ (ppm) 6.65 (dd, J = 9.2 Hz, J = 1.3 Hz, 1H, H4), 6.59 (dd, J = 9.2 Hz, J = 4.9 Hz, 1H, H5), 5.59 (s, 1H, H2), 3.87 -3.84 (m, 1H, H6), 3.54 (d, J = 9.6 Hz, 1H, H8A), 2.65 (dd, J = 9.6 Hz, J = 4.4 Hz, 1H, H10A), 2.33 -2.15 (m, 3H, H11A, H8B, H11B), 1.82 (d, J = 9.6 Hz, 1H, H10B), 1.66 -1.53 (m, 1H, H12), 0.90 (d, J = 6.6 Hz, 3H, H13 or H14), 0.87 (d, J = 6.6 Hz, 3H, H13 or H14) 13 C NMR (75 MHz, CDCl3): δ (ppm) 173.2, 169.8, 144.6, 121.9, 106.2, 88.8, 62.1, 59.5, 56.5, 42.9, 27.6, 21.0, 20.7 HRMS (ESI) calcd for [M+Na] + (C13H17NO2Na) 242.1152, found 242.1151 IR (UATR): ῡ (cm -1 ) 3065 (=CH), 1744 (C=O), 1626 (C=C)

1 H

 1 D = -843 (c 0.5, CH2Cl2); m.p.: 112-113°C NMR (300 MHz, CDCl3): δ (ppm) 7.35-7.23 (m, 5H, Ho, m, p), 6.71 (dd, J = 9.1 Hz, J = 0.8 Hz, 1H, H4), 6.58 (dd, J = 9.1 Hz, J = 5.3 Hz, 1H, H5), 5.63 (s, 1H, H2), 3.86-3.83 (m, 1H, H6), 3.70 (Syst AB, JAB= 13.0 Hz, 1H, H11A), 3.59 (Syst AB, JAB= 13.0 Hz, 1H, H11B), 3.54 (d, J = 9.8 Hz, 1H, H8A), 2.69 (dd, J = 9.7 Hz, J = 4.4 Hz, 1H, H10A), 2.36 (d, J = 9.8 Hz, 1H, H8B), 1.86 (d, J = 9.7 Hz 1H, H10B) C NMR (75 MHz, CDCl3): (ppm) 173.

1 H

 1 NMR (300 MHz, CDCl3): δ (ppm) 7.16 (d, J = 5.6 Hz, 1H, H5), H5), 5.63 (s, 1H, H2), 3.75 -3.72 (m, 1H, H6), 3.55 (d, J = 9.8 Hz, 1H, H8A), 2.68 (dd, J = 9.9 Hz, J = 4.2 Hz, 1H, H10A), 2.37 -2.21 (m, 3H, H11A, H8B, H11B), 1.89 (d, J = 9.9 Hz, 1H, H10B), 1.65 -1.60 (m, 1H, H12), 0.91 (d, J = 6.6 Hz, 3H, H13 or H14), 0.88 (d, J = 6.6 Hz, 3H, H13 or H14) HRMS (ESI) calcd for [M+H] + (C13H17NO2I) 346.0299, found 346.0301

1 H

 1 D = -528 (c 0.58, CH2Cl2); m.p.: 92-93°C NMR (300 MHz, CDCl3): δ (ppm) 7.36-7.24 (m, 5H, Ho, m, p), 7.14 (d, J = 5.7 Hz, 1H, H5), 5.66 (s, 1H, H2), 3.73-3.70 (m, 1H, H6), 3.74 (Syst AB, JAB= 13.0 Hz, 1H, H11A), 3.65 (Syst AB, JAB= 13.0 Hz, 1H, H11B), 3.55 (d, J = 10.0 Hz, 1H, H8A), 2.72 (dd, J = 10.0 Hz, J = 4.3 Hz, 1H, H10A), 2.43 (d, J = 10.0 Hz, 1H, H8B), 1.92 (d, J = 10.0 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3): δ (ppm) 171.

13C

  NMR (75 MHz, CD2Cl2): δ (ppm) 172.2, 169.6, 147.9, 138.8, 132.1, 129.5, 129.0, 128.8, 128.8, 127.7, 122.4, 118.5, 107.8, 92.9, 88.4, 83.3, 58.9, 57.7, 56.3, 42.3 HRMS (ESI) calcd for [M+Na] + (C24H19NO2Na) 376.1308, found 376.1310 IR (UATR): ῡ (cm -1 ) 2207 (CC), 1752 (C=O), 1627 (C=C)

1 H

 1 NMR (300 MHz, CD2Cl2): δ (ppm) 7.34-7.22 (m, 5H, Ho, m, p), 6.71 (d, J = 5.6 Hz, 1H, H5), 5.77 (s, 1H, H2), 3.85-3.81 (m, 1H, H6), 3.76 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.59 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.47 (d, J = 9.8 Hz, 1H, H8A), 2.65 (dd, J = 9.8 Hz, J = 4.4 Hz, 1H, H10A), 2.41-2.36 (m, 3H, H8B, H14), 1.86 (d, J = 9.8 Hz, 1H, H10B), 1.62 (h, J = 7.4 Hz 2H, H15), 1.04 (t, J = 7.4 Hz 3H, H16) 13 C NMR (75 MHz, CD2Cl2): δ (ppm) 172.3, 170.3, 146.4, 138.9, 128.8, 128.7, 127.6, 118.9, 107.5, 94.6, 88.4, 74.9, 58.8, 57.7, 56.2, 42.4, 22.4, 21.6, 13.7 HRMS (ESI) calcd for [M+Na] + (C21H21NO2Na) 342.1464, found 342.1465 IR (UATR): ῡ (cm -1 ) 2227 (CC), 1752 (C=O), 1627 (C=C)

1 H

 1 NMR (300 MHz, CD2Cl2): δ (ppm) 7.40-7.23 (m, 7H, Ho, o', m, p), 6.79 (d, J = 5.6 Hz, 1H, H5), 6.67 (d, J = 9.0 Hz, 1H, Hm'), 5.88 (s, 1H, H2), 3.90-3.87 (m, 1H, H6), 3.80 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.63 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.50 (d, J = 9.8 Hz, 1H, H8A), 3.00 (s, 6H, N(Me)2), 2.69 (dd, J = 9.8 Hz, J = 4.4 Hz, 1H, H10A), 2.44 (d, J = 9.8 Hz, 1H, H8B), 1.91 (d, J = 9.8 Hz 1H, H10B) 13 C NMR (75 MHz, CD2Cl2): δ (ppm) 172.3, 170.2, 151.1, 145.6, 138.9, 133.2, 128.8, 128.7, 127.6, 119.0, 112.1, 108.5, 107.6, 94.7, 88.4, 81.6, 59.0, 57.7, 56.3, 42.4, 40.3 HRMS (ESI) calcd for [M+Na] + (C26H24N2O2Na) 419.1730, found 419.1734 IR (UATR): ῡ (cm -1 ) 2197 (CC), 1754 (C=O), 1627 (C=C)

4. 1 . 1 H 1 H 1 H

 1111 12. (6S,8aS)-7-benzyl-4-phenyl-7,8-dihydro-6,8a-methanofuro[2,3-c]azepin-2(6H)one 12 Method A. After purification (CH2Cl2/Et2O, 95:5) compound 12 was obtained as a cream solid (40.0 mg, 92% yield). [α] 20 D = -490 (c 0.49, CH2Cl2); m.p.: 120-121°C NMR (300 MHz, CDCl3): δ (ppm) 7.49-7.24 (m, 10H, Ho, o', m, m', p, p'), 6.62 (d, J = 5.5 Hz, 1H, H5), 5.75 (s, 1H, H2), 3.96 -3.93 (m, 1H, H6), 3.77 (Syst AB, JAB= 13.0 Hz, 1H, H11A), 3.64 (Syst AB, JAB= 13.0 Hz, 1H, H11B), 3.63 (d, J = 9.9 Hz, 1H, H8A), 2.76 (dd, = 9.7 Hz, J = 4.3 Hz, 1H, H10A), 2.50 (d, J = 9.9 Hz, 1H, H8B), 1.97 (d, J = 9.7 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3): δ (ppm) 172.7, 169.8, 140.5, 138.3, 136.4, 136.1, 129.1, 129.0, 128.6, 128.6, 127.5, 127.5, 107.5, 89.2, 58.3, 57.7, 56.1, 42.5 HRMS (ESI) calcd for [M+Na] + (C22H19NO2Na) 352.1308, found 352.1308 IR (UATR): ῡ (cm -1 ) 1741 (C=O), 1618 (C=C) 4.1.13. (6S,8aS)-7-benzyl-4-(4-fluorophenyl)-7,8-dihydro-6,8a-methanofuro[2,3-c]azepin-2(6H)-one 13 Method A. After purification (CH2Cl2/EtOAc, 96:4) compound 13 was obtained as cream solid (34.0 mg, 74% yield). [α] 20 D = -451 (c 0.51, CH2Cl2); m.p.: 132-133°C NMR (300 MHz, CDCl3): δ (ppm) 7.41-7.24 (m, 7H, Ho, o', m, p, p'), 7.18 -7.10 (m, 2H, Hm'), 6.58 (d, J = 5.5 Hz, 1H, H5), 5.71 (s, 1H, H2), 3.95 -3.92 (m, 1H, H6), 3.76 (Syst AB, JAB= 13.0 Hz, 1H, H11A), 3.64 (Syst AB, JAB= 13.0 Hz, 1H, H11B), 3.63 (d, J = 9.9 Hz, 1H, H8A), 2.76 (dd, J = 9.8 Hz, J = 4.3 Hz, 1H, H10A), 2.49 (d, J = 9.9 Hz, 1H, H8B), 1.96 (d, J = 9.8 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3) : δ (ppm) 172.5, 169.7, 163.1 (d, J 1 C-F = 248.8 Hz), 140.5, 138.2, 135.1, 132.4 (d, J 4 C-F = 3.4 Hz), 129.3 (d, J 3 C-F = 8.2 Hz), 128.6, 128.6, 127.6, 116.1 (d, J 2 C-F = 21.6 Hz), 107.6, 89.2, 58.3, 57.7, 56.1, 42.4 HRMS (ESI) calcd for [M+Na] + (C22H18NO2FNa) 370.1214, found 370.1215 IR (UATR): ῡ (cm -1 ) 3093 (=CH), 1741 (C=O), 1613 (C=C) After purification (CH2Cl2/EtOAc, 92:8) compound 14 was obtained as pale-yellow solid (34.7 mg, 73% yield). [α] 20 D: -434 (c 0.49, CH2Cl2); m.p: 139-140°C NMR (300 MHz, CDCl3): δ (ppm) 7.37-7.23 (m, 7H, Ho, o', m, p, p'), 6.97 (d, J = 8.8 Hz, 1H, Hm'), 6.54 (d, J = 5.5 Hz, 1H, H5), 5.74 (s, 1H, H2), 3.93 -3.90 (m, 1H, H6), 3.86 (s, 3H, OMe), 3.76 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.63 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.62 (d, J = 9.9 Hz, 1H, H8A), 2.75 (dd, J = 9.7 Hz, J = 4.3 Hz, 1H, H10A), 2.48 (d, J = 9.9 Hz, 1H, H8B), 1.95 (d, J = 9.7 Hz 1H, H10B)

4. 1 . 1 H

 11 16. (6S,8aS)-7-benzyl-4-(pyridin-4-yl)-7,8-dihydro-6,8a-methanofuro[2,3-c]azepin-2 (6H)-one 16 Method B. After purification (CH2Cl2/Acetone, 80:20) compound 16 was obtained as a beige solid (30.3 mg, 70% yield). [α] 20 D: -529 (c 0.49, CH2Cl2); m.p.: 172-173°C NMR (300 MHz, CDCl3): δ (ppm) 8.72-8.70 (m, 2H, Hm'), 7.36 -7.24 (m, 7H, Ho, o', m, p), 6.75 (d, J = 5.5 Hz, 1H, H5), 5.77 (s, 1H, H2), 3.98 -3.95 (m, 1H, H6), 3.76 (Syst AB, JAB= 13.0 Hz, 1H, H11A), 3.65 (Syst AB, JAB= 13.0 Hz, 1H, H11B), 3.64 (d, J = 10.0 Hz, 1H, H8A), 2.79 (dd, J = 9.9 Hz, J = 4.3 Hz, 1H, H10A), 2.52 (d, J = 10.0 Hz, 1H, H8B), 1.97 (d, J = 9.9 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3): δ (ppm) 172.1, 168.0, 150.7, 143.8, 143.0, 138.0, 133.9, 128.7, 128.5, 127.7, 122.1, 107.8, 89.0, 58.2, 57.7, 56.1, 42.2 HRMS (ESI) calcd for [M+Na] + (C21H18N2O2Na) 353.1260, found 353.1256 IR (UATR) : ῡ (cm -1 ) 3127 (=CH), 1748 (C=O), 1618 (C=C) 4.1.17. (6S,8aS)-7-benzyl-4-(thiophen-3-yl)-7,8-dihydro-6,8a-methanofuro[2,3-c]azepin-2 (6H)-one 17 Method B. After purification (CH2Cl2/EtOAc, 9:1) compound 17 was obtained as amorphous pale-yellow solid (38.0 mg, 86% yield).

1 H 1 H

 11 Method After purification (CH2Cl2/EtOAc, 9:1) compound 18 was obtained as an amorphous orange solid (39.5 mg, 80% yield).[α] 20 D = -474 (c 0.51, CH2Cl2) NMR (300 MHz, CDCl3): δ (ppm) 8.32 (d, J = 8.9 Hz, 1H, Hm'), 7.58 (d, J = 8.9 Hz, 1H, Ho'), 7.36 -7.25 (m, 5H, Ho, m, p),6.74 (d, J = 5.5 Hz, 1H, H5), 5.72 (s, 1H, H2), 4.00-3.97 (m, 1H, H6), 3.77 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.67 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.65 (d, J = 10.0 Hz, 1H, H8A), 2.80 (dd, J = 9.9 Hz, J = 4.3 Hz, 1H, H10A), 2.54 (d, J = 10.0 Hz, 1H, H8B), 1.99 (d, J = 9.9 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3): δ (ppm) 172.0, 168.3, 148.1, 143.3, 142.6, 137.9, 134.3, 128.7, 128.5, 128.5, 127.7, 124.4, 108.0, 89.1, 58.2, 57.8, 56.1, 42.2 HRMS (ESI) calcd for [M+Na] + (C22H18N2O4Na) 397.1159, found 397.1161 IR (UATR): ῡ (cm -1 ) 1748 (C=O), 1619 (C=C), 1515 and 1344 (NO2)4.1.19. N-[4-((6S,8aS)-7-benzyl-2-oxo-2,6,7,8-tetrahydro-6,8a-methanofuro[2,3-c]azepin-4-yl)phenyl]acetamide 19 Method B. After purification (petroleum ether/EtOAc, 2:8) compound 19 was obtained as an amorphous cream solid (48.0 mg, 94% yield). [α] 20 D = -398 (c 0.52, CH2Cl2) NMR (300 MHz, CDCl3): δ (ppm) 7.60 (d, J = 8.5 Hz, 1H, Hm'), 7.47 (bs, 1H, NH), 7.38-7.23 (m, 7H, Ho, o', m, p), 6.58 (d, J = 5.5 Hz, 1H, H5), 5.73 (s, 1H, H2), 3.94-3.91 (m, 1H, H6), 3.75 (Syst AB, JAB= 13.1 Hz, 1H, H11A), 3.63 (Syst AB, 13.1 Hz, 1H, H11B), 3.62 (d, J = 9.9 Hz, 1H, H8A), 2.75 (dd, J = 9.7 Hz, J = 4.3 Hz, 1H, H10A), 2.49 (d, J = 9.9 Hz, 1H, H8B), 2.22 (s, 3H, NHCOMe), 1.95 (d, J = 9.7 Hz 1H, H10B) 13 C NMR (75 MHz, CDCl3): δ (ppm) 172.8, 170.0, 168.6, 140.1, 138.7, 138.2, 135.3, 132.1, 128.6, 128.6, 128.1, 127.6, 120.2, 107.4, 89.3, 58.3, 57.7, 56.1, 42.5, 24.8 HRMS (ESI) calcd for [M+Na] + (C24H22N2O3Na) 409.1523, found 409.1526 IR (UATR): ῡ (cm -1 ) 3313 (NH), 1735 (C=O lactone), 1693 (C=O amide), 1670 (C=C)

1 :

 1 DFT-generated HOMO representation of securinine (1) and compounds 2 (Boc), 4 (Me), 5 (iBu) and 6 (Bn) 4.3. Cell Culture and Proliferation Assay Securinine (1) and compounds 2, 4, 5, 6, 8, 12-19 were evaluated by the CIBI platform (Plateforme des cibles biologiques, Institut de Chimie des Substances Naturelles, Gif/Yvette, France) on HL60 and HCT116 human cancer cell lines.

Table 1 .

 1 Cytotoxic activity of simplified securinine analogs on colon cancer HCT116 and acute leukemia HL60 cell lines (% viability).All derivatives showed low activity levels against the HCT116 cancer cell line nearly similar to those reported previously by Vidal et al. for the tetracyclic Securinega analogs, with the exception of the iodinated compound 8 which displayed interesting properties.10 It is worth mentioning that the tetracyclic securinine equivalent of iodo derivative 8 described by Vidal et al. also demonstrated a high percentage of growth inhibition at 1 M (80.9%) on the same cell line. This result may serve as a compelling argument to suggest that the ring A is not important while the presence of the iodine is crucial for the antiproliferative activity. A few examples of marketed drugs containing an iodine such as levothyroxine or amiodarone are limited to aromatic compounds. The chemical stability of nontrivial vinyl iodo motif of 8 in biological media should be carefully evaluated prior to any further development. In context of this study, we urge caution on this point and consider compound 8 as an intermediate to access C14functionalized derivatives rather than an optimizable hit. Regarding the effects on HL60 cell line, most of the analogs displayed an activity close to that of securinine at 1 M. It is thus worth noting that the removal of the piperidine A ring and its replacement by a bulky(2, Boc)

	Compound	HCT116 (% viability) 10 M 1 M	HL60 (% viability) 10 M 1 M
	Securinine (1)	13 ± 0.5	98 ± 5	0.4 ± 0.2	8 ± 0.3
	Boc-butenolide 2	11 ± 1	94 ± 4	0.2 ± 0.1	7 ± 1
	Me-butenolide 4	92 ± 4	94 ± 5	4 ± 0.6	25 ± 7
	Isobutyl-butenolide 5	78 ± 4	83 ± 4	1 ± 0.3	15 ± 0.4
	Bn-butenolide 6	86 ± 3	100 ± 5	3 ± 0.8	11 ± 1
	14-iodo Bn-butenolide 8	1 ± 0.05	5 ± 1	0 ± 0.1 a	4 ± 1
	14-phenyl Bn-butenolide 12	89 ± 2	92 ± 2	2 ± 0.7	7 ± 0.3
	14-pF-phenyl Bn-butenolide 13	90 ± 5	100 ± 4	1 ± 0.2	7 ± 0.7
	14-pOMe-phenyl Bn-butenolide 14	100 ± 2	100 ± 1	4 ± 0.3	18 ± 1
	14-pN(CH3)2-phenyl Bn-butenolide 15	84 ± 5	95 ± 4	1 ± 0.06	7 ± 3
	14-pyridin-4-yl Bn-butenolide 16	100 ± 1	100 ± 4	9 ± 1	9 ± 3

or a hydrophobic (6, benzyl) substituent didn't significantly alter the cytotoxic activity compared to securinine

(1)

. Other structural modifications at the C14 position didn't improve the cytotoxicity and had a low to moderate impact on the activity. Despite being moderate, these results are interesting as they highlight the opportunity to simplify the skeleton of securinine without losing the overall effect. Of note, the p-nitrophenyl derivative was the most potent of this series

Table 2 .

 2 IC50 (M) of selected compounds on HCT116 and HL60 cell lines.

	IC50 (M)

  1, 169.6, 144.1, 138.3, 128.6, 128.6, 127.5, 122.3, 106.6, 88.7, 58.6, 57.6, 56.2, 42.7 HRMS (ESI) calcd for [M+Na] + (C16H15NO2Na) 276.0995, found 276.0999 IR (UATR): ῡ (cm -1 ) 3137 (=CH), 1755 (C=O), 1635 (C=C)

  C NMR (75 MHz, CDCl3): δ (ppm) 172.8, 170.2, 160.2, 139.2, 138.3, 135.5, 128.8, 128.7, 128.6, 128.6, 127.5, 114.4, 107.5, 89.3, 58.3, 57.7, 56.1, 55.6, 42.6 HRMS (ESI) calcd for [M+H] + (C23H22NO3) 360.1594, found 360.1596 IR (UATR): ῡ (cm -1 ) 1743 (C=O), 1622 (C=C), 1249 (Ar-OMe)

	4.1.15.	(6S,8aS)-7-benzyl-4-[4-(dimethylamino)phenyl]-7,8-dihydro-6,8a-methanofuro
	[2,3-c]azepin-2(6H)-one 15
	Method A. After purification (CH2Cl2/EtOAc, 9:1) compound 15 was obtained as an amorphous
	yellow solid (36.0 mg, 73% yield).
	[α] 20 D = -417 (c 0.50, CH2Cl2)
	1 H NMR (300 MHz, CDCl3): δ (ppm) 7.34-7.23 (m, 7H, Ho, o', m, p), 6.76 (d, J = 8.9 Hz, 1H, Hm'),
	6.50 (d, J = 5.6 Hz, 1H, H5), 5.79 (s, 1H, H2), 3.92-3.89 (m, 1H, H6), 3.75 (Syst AB, JAB= 13.1
	Hz, 1H, H11A), 3.62 (Syst AB, JAB= 13.1 Hz, 1H, H11B), 3.61 (d, J = 9.8 Hz, 1H, H8A), 3.02 (s,
	6H, N(Me)2), 2.73 (dd, J = 9.6 Hz, J = 4.3 Hz, 1H, H10A), 2.47 (d, J = 9.8 Hz, 1H, H8B), 1.94 (d,
	J = 9.6 Hz 1H, H10B)
	13 C NMR (75 MHz, CDCl3): δ (ppm) 173.0, 170.7, 150.9, 138.5, 137.4, 135.6, 128.6, 128.6,
	128.3, 127.4, 124.0, 112.3, 107.3, 89.4, 58.4, 57.6, 56.0, 42.7, 40.5
	HRMS (ESI) calcd for [M+Na] + (C24H24N2O2Na) 395.1730, found 395.1726
	IR (UATR): ῡ (cm -1 ) 1748 (C=O), 1609 (C=C)

  basis set.

	Compounds	Lowest	Frequency	EHOMO (eV) ELUMO (eV)	G298K/1atm (kcal/mol)
		(cm -1 )
	Securinine	105.77		-6.106	-2.309	-23.184
	(1)
	2 (Boc)	43.27		-6.915	-2.450	-26.334
	4 (Me)	105.13		-6.321	-2.347	-21.722
	5 (iBu)	54.41		-6.144	-2.273	-24.506
	6 (Bn)	49.35		-6.232	-2.343	-25.200

Acknowledgments

Université de Rennes 1 and CNRS are acknowledged for their financial support. XG is grateful to the 'Fondation ARC pour la recherche sur le cancer' for his post-doctoral fellowship (PDF20191209830).

Supporting Information.

Copies of the NMR data are given in the supporting information file.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.