Natural product derivatives for inhibiting cellular necroptosis, ferroptosis and oxytosis

Abstract

The present invention relates to a compound of the following general formula (I): ##STR00001##
or a pharmaceutically acceptable salt and/or solvate thereof, for use as drug, particularly intended for inhibiting a programmed cell death route selected from the group consisting of ferroptosis, oxytosis and cellular necroptosis. The present invention also relates to a compound of general formula (I) for use as a drug for neuroprotection as well as for preventing and/or treating disorders associated with cellular necroptosis or ferroptosis. The present invention also relates to a pharmaceutical composition comprising a compound of general formula (I), or a pharmaceutically acceptable salt and/or solvate thereof. The present invention also encompasses the use of a compound of the general formula (I) for organs preservation.

Claims

1. A pharmaceutical composition comprising at least one compound of the following general formula (I): ##STR00006## or a pharmaceutically acceptable salt and/or solvate thereof, wherein: X.sub.1 represents a (C.sub.1-C.sub.6)alkyl an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group or an OR.sub.X group, wherein R.sub.X is selected from a (C.sub.1-C.sub.6)alkyl, an aryl and an aryl-(C.sub.1-C.sub.6)alkyl group, X.sub.2 and X.sub.3 each represent, independently of each other, a hydrogen atom or a (C.sub.1-C.sub.6)alkyl group, Y.sub.1, Y.sub.2 and Y.sub.3 each represent, independently of each other, a hydrogen atom, a (C.sub.1-C.sub.6)alkyl, an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group, an OH or an OR.sub.Y group, with at least one of Y.sub.1, Y.sub.2 and Y.sub.3 representing a (C.sub.1-C.sub.6)alkyl, an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group or an OR.sub.Y group, wherein R.sub.Y is selected from a (C.sub.1-C.sub.6)alkyl, an aryl and an aryl-(C.sub.1-C.sub.6)alkyl group, and at least one pharmaceutically acceptable excipient.

2. The pharmaceutical composition according to claim 1, wherein the compound is of the following general formula (II): ##STR00007## or a pharmaceutically acceptable salt and/or solvate thereof, wherein R.sub.X represents a (C.sub.1-C.sub.6)alkyl group and R.sub.Y represents an aryl-(C.sub.1-C.sub.6)alkyl group.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutically acceptable excipient is selected from the group consisting of long-term stabilizers, drug absorption enhancers, viscosity reducers and solubility enhancers.

4. The pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable excipient is a solubility enhancer.

5. The pharmaceutical composition according to claim 1, wherein it further comprises at least one other active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the other active ingredient is selected from the group consisting of another cellular necroptosis inhibitor, an apoptosis inhibitor, an autophagy inhibitor, a ferroptosis inhibitor, an inhibitor of mitochondrial permeability transition (MPT) pore-dependent necrosis, a cyclophilin inhibitor, a Cyclin-dependent kinase 5 (CDK5) inhibitor, a parthanatos inhibitor, a thrombin inhibitor, an antioxidant an inflammatory inhibitor and combinations thereof.

7. The pharmaceutical composition according to claim 6, wherein the other active ingredient is an antioxidant.

8. A combination product comprising: (i) at least one compound of the following general formula (I): or a pharmaceutically acceptable salt and/or solvate thereof, wherein: X.sub.1 represents a (C.sub.1-C.sub.6)alkyl an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group or an OR.sub.X group, wherein R.sub.X is selected from a (C.sub.1-C.sub.6)alkyl, an aryl and an aryl-(C.sub.1-C.sub.6)alkyl group, X.sub.2 and X.sub.3 each represent, independently of each other, a hydrogen atom or a (C.sub.1-C.sub.6)alkyl group, Y.sub.1, Y.sub.2 and Y.sub.3 each represent, independently of each other, a hydrogen atom, a (C.sub.1-C.sub.6)alkyl, an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group, an OH or an OR.sub.Y group, with at least one of Y.sub.1, Y.sub.2 and Y.sub.3 representing a (C.sub.1-C.sub.6)alkyl, an aryl, an aryl-(C.sub.1-C.sub.6)alkyl group or an OR.sub.Y group, wherein R.sub.Y is selected from a (C.sub.1-C.sub.6)alkyl, an aryl and an aryl-(C.sub.1-C.sub.6)alkyl group, and (ii) at least another active ingredient selected from the group consisting of another cellular necroptosis inhibitor, an apoptosis inhibitor, an autophagy inhibitor, a ferroptosis inhibitor, an inhibitor of mitochondrial permeability transition (MPT) pore-dependent necrosis, a cyclophilin inhibitor, a Cyclin-dependent kinase 5 (CDK5) inhibitor, a parthanatos inhibitor, a thrombin inhibitor, an antioxidant an inflammatory inhibitor and combinations thereof, separate from the at least one compound of formula (I), for simultaneous, separate or sequential administration.

9. The combination product according to claim 8, wherein the compound is of the following general formula (II): or a pharmaceutically acceptable salt and/or solvate thereof, wherein R.sub.X represents a (C.sub.1-C.sub.6)alkyl group and R.sub.Y represents an aryl-(C.sub.1-C.sub.6)alkyl group.

10. The combination product according to claim 8, wherein the other active ingredient is an antioxidant.

11. The pharmaceutical composition according to claim 3, wherein the pharmaceutically acceptable excipient is a drug absorption enhancer.

Description

BRIEF SUMMARY OF THE FIGURES

(1) FIG. 1 represents the dose-dependent inhibition by compound 1 of necroptosis induced by TNF-α in human T lymphocyte (Jurkat FADD deficient cell line);

(2) FIG. 2 represents the dose-dependent inhibition by compound 1 of the cell membrane permeabilization induced by TNF-α in human T lymphocyte (Jurkat FADD deficient cell line) (the light grey curve is obtained when cells are treated with compound 1 only);

(3) FIG. 3 represents the dose-dependent inhibition by compound 1 of the ATP depletion induced by TNF-α in human T lymphocyte (Jurkat FADD deficient cell line) (the light grey curve is obtained when cells are treated with compound 1 only);

(4) FIG. 4 represents the viability of human primary blood leukocytes treated with increasing concentrations of compound 1;

(5) FIG. 5 represents the viability of human retinal pigment epithelial cells treated with increasing concentrations of compound 1;

(6) FIG. 6 represents the dose-dependent inhibition of RIPK1 autophosphorylation by compound 1;

(7) FIG. 7 represents the inhibition of RIPK1 autophosphorylation by compound 1 at high ATP concentrations (up to 1 mM);

(8) FIG. 8 represents the analysis of compound 1 selectivity against a large panel of kinases;

(9) FIG. 9 represents the determination of binding constant (Kd) of compound 1 for its major cellular target RIPK1 at two different temperatures (4° C. and Room Temperature, r.t);

(10) FIG. 10 represents the protection from hypoxic injury of human artery endothelial cells (HAEC) by compound 1 during hypoxic cold storage; and

(11) FIG. 11 represents the protection of human artery endothelial cells by compound 1 during both hypoxic cold storage and reoxygenation step.

(12) FIG. 12 represents the predictive orientation for compound 1 within the theoretical RIPK1-6E11 complex.

(13) FIGS. 13a et 13b represent the binding sites of compound 1 and Nec1s on RIPK1.

(14) FIG. 14 represents the dose-dependent protection from ferroptosis (induced by erastin) and Glutamate-induced oxidative toxicity of murine hippocampal neuronal cell line HT22 by compound 1.

(15) FIGS. 15a and 15b represent the dose-dependent protection of compound 1 from the NaIO.sub.3-induced retinal cell death (ARPE-19, a human retinal pigment epithelial cell line).

(16) FIGS. 16a and 16b represent the dose-dependent protection of compound 1 from the cold-induced cell death (LLC-PK1 cells, porcine kidney proximal tubule cell line).

(17) FIG. 17 represents the protection of rat embryonic myoblastic H9C2 cells by compound 1 from necrosis induced by H.sub.2O.sub.2.

(18) In the above-mentioned figures, “6E11” refers to compound 1.

EXAMPLES

(19) The following abbreviations, commonly used in this field of art, have been used in the following examples: AGC: Protein kinase A, G, and C families (PKA, PKC, PKG) BSA: Bovine Serum Albumin CAMK: Ca.sup.2+/calmodulin-dependent protein kinases CMGC: CDKs, MAP kinases, GSK and CDK-like kinases CDK: Cyclin-dependent kinase CK1: Cell Kinases 1 (originally known as Casein Kinase 1) DMF: Dimethylformamide DMSO: Dimethylsulfoxide DTT: Dithiothreitol EC.sub.50: Half maximal effective concentration EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N-tetraacetic acid Et: Ethyl (CH.sub.2CH.sub.3) EtOAc: Ethyl acetate FACS: Fluorescence-activated cell sorting FADD: Fas-Associated Death Domain GSH: glutathione GSK: Glycogen synthase kinases GST: Glutathione S-transferase h: hour HAEC: Human Artery Endothelial Cells HB: hydrogen bond hPBLs: human Peripheral Blood Lymphocytes hRPE-1: human Retinal Pigment Epithelial cell line IC.sub.50: Half maximal inhibitory concentration Kd: dissociation constant kg: kilogram M: Molar MAP: Mitogen-Activated Protein kinases MD: Molecular dynamic Me: Methyl (CH.sub.3) mg: milligram MHz: MegaHertz min: minute(s) ml: milliliter mM: millimolar mmol: millimole MOPS: 3-(N-morpholino)propanesulfonic acid MTS: 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium n: number of replicates in an experiment N: number of independent experiments NMR: Nuclear Magnetic Resonance PBS: Phosphate buffered saline PCR: Polymerase Chain Reaction RIPK1: Receptor-Interacting Protein Kinase 1 ROS: reactive oxygen species r.t: Room temperature SD: Standard Deviation STE: STE Kinases (Homologs of yeast STErile kinases) TK: Tyrosine Kinases TKL: Tyrosine Kinases-Like TNF-α: Tumor Necrosis Factor α μg: microgram μl: Microliter μM: Micromolar

(20) I. Synthesis of the Compounds According to the Invention

Example 1: Synthesis of the Compounds of General Formula (I)

(21) Compound 1 was prepared according to the method disclosed in Hauteville et al. Tetrahedron 1980, 37, p. 377-381.

(22) Said method can be generalized to obtain a compound of general formula (I), according the following reaction scheme:

(23) ##STR00005##

(24) The starting materials are commercially available, or can be easily prepared according to methods well-known of the one skilled in the art. Optionally, and if necessary, additional protection or deprotection steps well-known of the skilled person might be incorporated within the general procedure represented above.

(25) II. Biological Tests of the Compounds According to the Invention

Example 2: Cell-Based Screening of Chemical Libraries for Characterization of Necroptosis Inhibitors

(26) TNF-α can induce necroptosis in Jurkat cells (human T lymphocytes) when FADD is deleted. This model was used to screen various libraries of chemical compounds for characterization of new inhibitors of cellular necroptosis. Details on this cell-based assay can be found in Miao and Degterev (Methods Mol. Biol. 2009, 559, 79-93). The Jurkat FADD-deficient I 2.1 cell line used was purchased from ATCC and was maintained in RPMI 1640 medium (Gibco) containing Glutamax and 15% fetal calf serum (Life Technology). Necroptosis was induced by addition of 10 ng/ml of human recombinant TNF-α (Life Technology). Necrostatin-1 (Nec-1, Enzo Life Sciences) was used as model necroptosis inhibitor. Cells were maintained in 75 cm.sup.2 flask and passed every 2 or 3 days. Chemical collections analysed were formatted in 96-well plates with 80 molecules per plate at 10 mM in 100% DMSO. For each collection plate, two plates were prepared: one corresponding to necroptosis-induced with TNF-α, and the other without TNF-α to evaluate the intrinsic toxicity of the tested compound. Cells were seeded at 20000 cells/well, in 40 μl of medium, in a 96-well clear, flat bottom plate (CytoOne, Starlab) before treatment. Then, 40 μl of medium with or without TNF-α at 25 ng/ml were added to all wells in the corresponding plate. Immediately after TNF-α addition, 20 μl of diluted compound at 50 μM were added to the plates. Final concentration of each chemical compound was 10 μM at 0.1% DMSO. Eight positives (Nec-1 at 10 μM final) and eight negative (DMSO) controls were used in each plate to validate the assay. Cells were incubated at 37° C., 5% CO.sub.2 for 24 hours before performing MTS viability assay, described hereafter. Compounds were diluted before to treat cells. Liquid handling was performed using the Nimbus Microlab liquid handler (Hamilton Robotics) under microbiological safety workbench. The 10 mM compounds were diluted at 50 μM directly in cell medium.

(27) Compound 1 has emerged from this screening to be a very efficient necroptosis inhibitor, as discussed below.

Example 3: Anti-Necrotic Effect of Compound 1

(28) Effect on cell viability: Jurkat FADD-deficient I 2.1 cells were treated by TNF-α (10 ng/ml) and increasing concentrations of compound 1 (0.01-0.05-0.10-0.50-1.00-2.50-5.00-10.00-25.00-50.00 μM). Cells were incubated at 37° C., 5% CO.sub.2 for 24 hours before performing MTS viability assay. Cell viability was monitored using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI, USA), based on the water-soluble tetrazolium compound MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) according to the manufacturers instructions. As it appears from FIG. 1, compound 1 protects cells from death induced by TNF-α.

(29) Effect on two hallmarks of necroptosis: Jurkat FADD-deficient I 2.1 cells were treated by TNF-α (10 ng/ml) and increasing concentrations of compound 1 (0, 1, 5, 10, 20 and 50 μM). Cells untreated by TNF-α are used as control (light grey curves). Cells were incubated at 37° C., 5% CO.sub.2 for 24 hours before performing the measurements of both plasma membrane permeabilization and intracellular ATP levels. Dead cells were detected by FACS analysis of Propidium Iodide-stained nuclei (FIG. 2). ATP Quantification is performed using the CellTiter-Glo® Luminescent Cell Viability Assay. The luminescent signal produced by a luciferase reaction is proportional to the amount of ATP present and the amount of ATP is directly proportional to the number of metabolically active cells (n=4) (FIG. 3). As reported on FIGS. 2 and 3, compound 1 inhibits two major hallmarks on necroptosis induced by TNF-α.

Example 4: Compound 1 Cytotoxicity Assays

(30) On Human Peripheral Blood Lymphocytes (hPBLs): hPBLs were treated with increasing concentrations of compound 1 (0, 1, 5, 10, 20, 50 and 100 μM) for 24 hours. Viability was assessed by cell proliferation assay (MTS). % of cell viability was determined using the CellTiter 96® AQ.sub.ueous Non-Radioactive Cell Proliferation Assay (Promega). Data represent the quantitative analysis of six independent experiments with means±SD (n=6 individuals).

(31) As it appears from FIG. 4, compound 1 is not cytotoxic towards hPBLs up to a concentration of 20 μM.

(32) On the Human Retinal Pigment Epithelial Cell Line (hRPE-1): hRPE-1 cells were treated with increasing concentrations of compound 1 (0, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 25 and 50 μM) for 24 hours. A colorimetric MTS assay was used to calculate the percentage of cell viability.

(33) As it appears from FIG. 5, compound 1 is not cytotoxic towards hRPE-1 cells at the tested concentrations.

Example 5: RIPK1 Autophosphorylation Assay and Binding Assays

(34) RIPK1 Autophosphorylation Assay: Human RIPK1 full length GST-tagged was baculovirally expressed in Sf9 cells according to manufacturer's instructions (Bac-to-Bac expression system, Invitrogen) and purified using gluthation-sepharose beads (GE Healthcare). The elution was made in 50 mM Tris-HCl, pH 8.0 buffer supplemented with 30 mM reduced gluthathione (Sigma). The protocol used to detect the enzymatic activity is adapted from Miao and Degterev (Methods Mol. Biol. 2009, 559, 79-93). Kinase reaction was initiated mixing 5 μl of eluted RIPK1, 5 μl of 3X kinase reaction buffer (5 mM MOPS pH 7.2, 2.5 mM β-glycerophosphate, 4 mM MgCl.sub.2, 2.5 mM MnCl.sub.2, 1 mM EGTA, 0.4 mM EDTA, 50 μg/ml BSA, 0.05 mM DTT), 2 μl H.sub.2O and 3 μl of the tested molecule. The mixture was kept on ice for 10 minutes. During the incubation, the ATP solution was prepared by mixing 5 μl of 3X kinase reaction buffer, 4 μl H.sub.2O, 6 μl cold ATP at 150 μM and 2 μCi of [γ-.sup.32P] ATP. The ATP solution and the tested inhibitor were added to the kinase and incubated for 30 minutes at 30° C. To stop the enzymatic reaction, 5 μl of loading buffer were added and solution was heated for 3 minutes at 95° C. 25 μl of each reaction were loaded per well in pre-cast NuPage 12% Bis-Tris gel (Life Technology). Necrostatin-1, a well-described inhibitor of RIPK1, was used as an internal control. Coomassie staining was performed in order to estimate the total amount of protein loaded on polyacrylamide gel. Autophosphorylated RIPK1 band was visualized on radiographic film after 6h exposition at −80° C.

(35) The results of this test obtained with compound 1 are indicated in FIG. 6. The decrease of the amount of radioactively labeled RIPK1 indicates that compound 1 inhibits the RIPK1 autophosphorylation in a dose-dependent way.

(36) Besides, as shown in FIG. 7, the inhibition of RIPK1 auto-phosphorylation by compound 1 is not affected by high ATP concentrations (e.g. 1 mM), which suggests that compound 1 is a non-ATP competitive inhibitor.

(37) Binding Assays:

(38) (i) Characterization of Kinase Targets of Compound 1 (“KINOMEscan Max”).

(39) This in vitro competition binding assay was used for the profiling of compound 1 against 456 kinases, including eight lipid kinases. This experimental approach quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase (e.g. RIPK1); immobilized ligand; and a test compound (here compound 1). The ability of compound 1 to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag. The codes reported on the FIG. 8 indicate the subclasses of protein kinases: CMGC for CDKs, MAP kinases, GSK and CDK-like kinases; AGC for Protein kinase A, G, and C families (PKA, PKC, PKG); CAMK for Ca2+/calmodulin-dependent protein kinases; CK1, Cell Kinases 1 (originally known as Casein Kinase 1); STE, STE Kinases (Homologs of yeast STErile kinases); TKL, Tyrosine Kinases-Like; TK, Tyrosine Kinases. Each kinase tested in the assay panel is marked with a circle. The hit kinase reported, RIPK1, is marked with a black circle. The size of the circle is proportional to the binding efficiency of compound 1 to the kinase of interest. Small grey dots represent only poor affinity for the tested kinase (as over 30% of the tested kinase are still on the affinity matrix after competition with the tested compound, here compound 1). For RIPK1, only 0.15% of the initial amount of kinase is still on the affinity matrix after competition with compound 1. The graphic representation of the human kinome phylogenetic tree (TREEspot™ Kinase dendrogram,© DiscoveRx, Fremont, USA), reported on FIG. 8, illustrates the high specificity of compound 1 for RIPK1 among the large panel of tested kinases.

(40) (ii) Determination of Dissociation Constant (Kd) of Compound 1 for RIPK1 Kinase.

(41) KdELECT is a service of DiscoveRx Corporation, Fremont, USA. This assay is based on a competition binding assay described hereabove. An 11-point 3-fold serial dilution of compound 1 was prepared in 100% DMSO in order to determine the dissociation constant (Kd) at two different temperatures, r.t and 4° C. Kd was then calculated with a standard dose-response curve (reported on FIG. 9) using the Hill equation. The calculated Kd of compound 1 for RIPK1 is 128 nM (n=2) at r.t and 136 nM (n=2) at 4° C. It validates compound 1 as a true ligand of RIPK1 kinase. Indeed, since the Kd value is low (nM range), the interaction between RIPK1 and compound 1 is strong. Moreover, the high affinity of compound 1 for RIPK1 is not affected by low temperature, conditions occurring during cold storage of grafts.

Example 6: In Vitro “Hypoxic Cold Storage” Viability Assays

(42) Human endothelial cells (HAEC) were grown to confluence, then synchronized using depleted media for 16h. For hypothermia/hypoxia, cells were washed twice with PBS then incubated in University of Wisconsin (UW) solution in 95% N.sub.2/5% CO.sub.2 atmosphere at 4° C. for 24 hours. Compound 1, Nec1 or Nec1s were added to the preservation solution at the indicated dilution (04) during hypoxia (FIG. 10) or during both hypoxia and reoxygenation steps (FIG. 11). Shown are mean+/−SD, n=3. UW are cells treated only with UW preservation solution. Controls are cells not subjected to this protocol (cells are continuously oxygenated) but cultivated for the same amount of time in regular culture conditions. The treatment with compound 1 during hypoxia or during hypoxia and reoxygenation brings measurable benefits on cell survival. Compared to the control inhibitors of necroptosis (Nec-1 and Nec-1s), the effect of compound 1 is significantly better. It should be noted that the hypoxic cold storage mimics the process occurring during graft preservation.

Example 7: In Silicon Analysis of the Theoretical RIPK1-6E11 Complex

(43) The predictive orientation for compound 1 was studied by in silico analysis of the theoretical RIPK1-6E11 complex. Stable contact residues defining a pharmacophore and determined on the most representative structural model included six key amino acids Lys30, Val47, Leu60, Leu78, Tyr88 and Leu90 (FIG. 12). Surrounding residues (4.0 Å cut-off distance) describing the compound 1 binding pocket observed over the molecular docking simulation trajectory also comprised of Phe28, Val31, Lys45, Thr46, Ala59, Glu63, Val81, Ile83, Ser89 and Asp156. We should note that among these amino acids, three of them (Leu78, Leu90 and Asp156) have been already described to be involved in the interaction with necrostatins (Xie et al. Structure 2013, 21, 493-499). Molecular dynamic (MD) simulation of RIPK1-6E11 model allowed us to improve the preferential binding mode identified by docking calculations which is different from Nec-1s (FIGS. 13a,b). The binding site of Nec-1s is marked as “site #1” on FIG. 13a. From analysis of the most frequent contacts of the compound to the kinase, we are able to propose that compound 1 should bind RIPK1 kinase through tight hydrophobic interactions and a non-specific hydrogen bond (HB), as well as other transient HB interactions observed during the simulation (FIGS. 13a,b). The putative binding site #2 of compound 1 is marked as “site #2” on FIG. 13b. Our simulations suggest that compound 1 fits tightly in an alternative and putative cleft surrounded notably by the RIPK1 catalytic triad residues: Lys45, Glu63 and Asp156. This cleft of RIPK1 is mainly hydrophobic but richer in hydrogen bond acceptors than the kinase hinge within the ATP-binding site. Interestingly, this model shows that compound 1 does not make any interaction with the kinase hinge in this conformation of RIPK1 regardless of hydrogen bonds. Moreover, this proposed binding mode for compound 1 occupying a lipophilic pocket in a cleft near the substrate binding site of RIPK1 indicates that this compound is likely a type III kinase inhibitor. This binding mode is in line with the high selectivity of compound 1 detected by the KINOMEscan℠ Assay (FIG. 8) and also with the non-ATP competitive mode of inhibition (reported on FIG. 7).

Example 8: Effect of Compound 1 on Both Ferroptosis and Oxytosis Programmed Cell-Death Routes

(44) Murine hippocampal neuronal cell line HT22 was treated with 10 mM of (L)-glutamate (oxytosis initiator) or 1 μM erastin (ferroptosis initiator).

(45) In this assay, 5,000 cells were seeded per well and treated with increasing concentrations of compound 1 (2.50-5.00-10.00-25.00-50.00 μM) with or without 1 μM erastin or 10 mM L-glutamate. Cells were then incubated at 37° C., 5% CO.sub.2 for 24 hours before performing MTS viability assay. Cell viability was monitored using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI, USA), based on the water-soluble tetrazolium compound MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) according to the manufacturers instructions. As it appears from FIG. 14, >25 μM of compound 1 protects cells from death induced by both L-glutamate and erastin.

Example 9: Effect of Compound 1 on a Cellular Model of Age-Related Macular Degeneration (AMD)

(46) At it was previously shown (Hanus et al. Cell Death Discov. 2016, 2, 16054), NaIO.sub.3 is an oxidizing agent that induces necroptosis in retinal pigment epithelial cell line that can be inhibited by 200 μM of necrostatin-1. The experiment described in the cited literature was reproduced to detect the effect of compound 1. Human retinal pigment epithelial cell line, ARPE-19, was treated with 10 mM of NaIO.sub.3 as cellular model of age-related macular degeneration (AMD). In this assay, 10,000 cells were seeded per well and treated with 20 or 200 μM of compound 1 or Nec-1s (a specific RIPK-1-dependent necroptosis inhibitor) (FIG. 15a) or increasing concentrations of compound 1 (0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1.00, 2.00, 2.50, 5.00, 10.00, 20.00, 25.00 μM) (FIG. 15b) with or without 10 mM of NaIO.sub.3. Cells were then incubated at 37° C., 5% CO.sub.2 for 24 hours before performing MTS viability assay. Cell viability was monitored using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI, USA), based on the water-soluble tetrazolium compound MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) according to the manufacturers instructions. As it appears from FIGS. 15a and 15b, subtoxic concentration of compound 1 (+/−20 μM) protects significantly cells from death induced by NaIO.sub.3 (N=3, n=2, mean±SD, *** P<0.001).

Example 10: Effect of Compound 1 on Cold-Induced Cell Death (Cold-Stress Preservation)

(47) At it was previously shown (Ahlenstiel et al. Transplantation 2016, 81(2), 231-239), cold storage induces a cell-death process. Porcine LLC-PK1 cells (kidney proximal tubule cell line) were stored at approximately 4° C. during 24 hours with or without the tested molecules. In this assay, 5,000 cells were seeded per well and treated with increasing concentrations of compound 1 or Nec-1s (0.01, 0.05, 0.10, 0.50, 1.00, 2.50, 5.00, 10.00, 25.00 and 50.00 μM) and stored for 24 hours at 4° C. Cells were then incubated at 37° C., 5% CO.sub.2 before performing MTS viability assay. Cell viability was monitored using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Fitchburg, WI, USA), based on the water-soluble tetrazolium compound MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) according to the manufacturers instructions. As it appears from FIGS. 16a and 16b, compound 1 protects cells from death induced by cold storage with a maximal activity at 504. Nec-1s is inactive at the tested doses (FIG. 16a, N=2, n=6, mean±SD, *** P<0.05; FIG. 16b, n=2).

Example 11: Effect of Compound 1 on H.SUB.2.O.SUB.2.-Induced Necrosis (Anti-Oxidant Property)

(48) The rat embryonic myoblastic H9C2 cells were cultured at 10,000 cells/well in 96-well-plates for 24 hours at 37° C./5% CO.sub.2. Then, cells were treated or not (DMSO) with 800 μM H.sub.2O.sub.2 for 24 hours after a 2 hours pretreatment or not (NT) with 10 μM compound 1 (comp 1), 10 μM Necrostatin-1s (Nec-1s), or 10 μM Ferrostatin-1 (Fer-1). LB corresponds to cells treated with a lysis buffer (100% of cell death). Cell cytotoxicity was determined by a colorimetric assay quantitatively measuring lactate dehydrogenase (LDH) released into the cytosol. As it appears from FIG. 17, compound 1 protects from death induced by H.sub.2O.sub.2 in the same extent as Fer-1, Nec-1s being less effective (FIG. 17, N=2, n=6, mean±SD, ** P<0.005; * P<0.05).