WO2025078659A1

WO2025078659A1 - Method and kit for inducing cell differentiation, and regulated necrotic cell death inhibitor for use for regenerating damaged tissues

Info

Publication number: WO2025078659A1
Application number: PCT/EP2024/078791
Authority: WO
Inventors: Sophie BELAL; Morgane Rousselot; Romain LUCAS
Original assignee: Seabelife
Current assignee: Seabelife
Priority date: 2023-10-12
Filing date: 2024-10-11
Publication date: 2025-04-17
Anticipated expiration: 2026-04-12

Abstract

The present invention relates to methods, uses and kits for inducing and/or improving cell differentiation in vivo or ex vivo using a regulated necrotic cell death inducer and a regulated necrotic cell death inhibitor, as well as regulated necrotic cell death inhibitors useful in a method for regenerating a tissue or an organ damaged by a degenerative affection or disease.

Description

Method and kit for inducing cell differentiation, and regulated necrotic cell death inhibitor for use for regenerating damaged tissues

TECHNICAL FIELD

The present invention relates to methods and kits for inducing cell differentiation in vivo or ex vivo using inhibitors of regulated necrotic cell death.

TECHNICAL BACKGROUND

Recently, there has been significant interest for low molecular weight molecules able to selectively affect the proliferation, differentiation, and migration of adult stem cells within the tissues in which they exist. By inducing the selective regeneration of damaged tissues or organs, such agents would indeed open entirely new avenues for conditions with unmet medical needs, such as severe and degenerative diseases involving cell damage and loss. Indeed, replacement of damaged or lost mature cells with normal functioning mature cells through the differentiation of unspecialized cells could be a therapeutic treatment for severe and degenerative diseases, an alternative to a transplant of mature (generally cultured) cells into patients - or as a first curative therapy (such as when the cells concerned are neurons). Conditions that could be treated using such new treatment are cardiovascular decay, neurological disorders including neurodegenerative diseases such as Alzheimer’s disease, degenerative eye diseases, in particular retinal degenerative diseases such as macular degeneration.

Such molecules would further provide for more reliable and accurate models of the human system, which would be useful for research. Some methods and kits are already known in the art for inducing cell differentiation, however they require multiple culturing medium changes and are not easily implemented.

There is thus a need for compounds able to selectively induce cell differentiation, in vitro and in vivo. In particular, there is a need for compounds useful in the treatment of diseases associated with, caused by or causing the death of non-regenerative cells, such as degenerative diseases. There is also a need for more straightforward and reproducible methods and kits to generate homogenous and viable human differentiated cell cultures.

In particular, there is a need for compounds able to selectively induce cell differentiation, in a patient suffering from a disease or disorder associated with regulated necrotic cell death, in particular ferroptosis or a patient treated with inducer of regulated necrotic cell death, in particular ferroptosis such as a patient suffering from a cancer.

SUMMARY OF THE INVENTION Now, the Inventors have discovered that when regulated necrotic cell death is induced on different type of cell lines able to differentiate into specialized cells, the inhibition of the cell death pathway with a regulated necrotic cell death inhibitor induced a differentiation of the cells to specialized cells.

Indeed, without wishing to be bound by theory, the Inventors hypothesize that the cell differentiation is induced by cell death inhibitors in a context where regulated necrosis pathways have been activated, either naturally (for instance in the case of a disease or disorder associated with regulated necrotic cell death) or typically by addition of a regulated necrotic cell death inducer, in particular a ferroptosis inducer.

Therefore, in a first aspect, the invention concerns an in vitro or ex vivo use of a regulated necrotic cell death inhibitor, typically with a regulated necrotic cell death inducer, for inducing and/or improving cell differentiation, wherein the cell to be differentiated has preferably not been obtained by a method involving the destruction of human embryos.

In another aspect, the invention relates to a method for inducing and/or improving cell differentiation, comprising contacting in vitro or ex vivo at least one cell to be differentiated with an effective amount of a regulated necrotic cell death inducer and of a regulated necrotic cell death inhibitor, to obtain a differentiated cell, wherein the cell to be differentiated has preferably not been obtained by a method involving the destruction of human embryos.

In another aspect, the invention concerns a kit for inducing cell differentiation, comprising: a regulated necrotic cell death inducer, a regulated necrotic cell death inhibitor, and instructions for use.

In another aspect, the invention relates to a regulated necrotic cell death inhibitor for use as a medicament for inducing differentiation of pre-necrotic cells of a patient in need thereof.

In another aspect, the invention relates to a regulated necrotic cell death inhibitor for use for treating an affection or disease associated with ferroptosis by inducing differentiation of pre- necrotic cells of a patient in need thereof.

In another aspect, the invention relates to a kit comprising: a) A first therapeutic agent, which is a regulated necrotic cell death inducer, and b) A second therapeutic agent, which is a regulated necrotic cell death inhibitor, for separate, simultaneous, or sequential use of the first and the second therapeutic agents, as medicament, in particular for inducing differentiation of pre-necrotic cells of a patient in need thereof or for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, advantageously by inducing differentiation of pre-necrotic cells.

In another aspect, the invention concerns a regulated necrotic cell death inhibitor for use in a method for regenerating a tissue or an organ damaged by a degenerative affection or disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of the regulated necrotic cell death inhibitor, thereby inducing cell differentiation of cells to be differentiated into cells suitable to regenerate said tissue or organ.

DETAILED DESCRIPTION

As used herein, the term “a” or “an”, as in the expression “a compound” or “an inhibitor” for example, means one or more than one.

L Method for inducing and/or improving cell differentiation

The invention thus relates to a method for inducing and/or improving cell differentiation, comprising contacting in vitro or ex vivo at least one cell to be differentiated with an effective amount of a regulated necrotic cell death inducer and of a regulated necrotic cell death inhibitor, to obtain a differentiated cell, wherein the cell to be differentiated has preferably not been obtained by a method involving the destruction of human embryos.

In other words, the present invention relates to the in vitro or ex vivo use of a regulated necrotic cell death inhibitor, optionally with a regulated necrotic cell death inducer, for inducing and/or improving cell differentiation”.

As used herein, “inducing cell differentiation” is understood as inducing a cell change from one type to another, e.g., from one non-specialized type to a specialized type, or from one specialized type to another specialized type by modifying at least one phenotype or morphotype, such as the cell size, shape, membrane potential, metabolic activity, responsiveness to signals, membrane protein expression, etc. This cell change is usually due to epigenetic modifications in gene expression. It may affect the proliferation, behavior and/or migration of the at least one cell to be differentiated. The term “differentiation” as used herein, includes “transdifferentiation”.

As used herein, “improving cell differentiation” is understood as inducing a cell change toward a more specialized phenotype or morphotype. For example, if the cell to be differentiated is not a omni- nor pluripotent cell but an “intermediary” differentiated cell (i.e. a cell which is not pluri- or omnipotent but is already specialized in a particular cell subtype), then “improving cell differentiation” would lead to complete or resume the differentiation process of these intermediary differentiated cells into fully specialized or finally differentiated cell.

In one embodiment, the method of the invention is for inducing and/or improving neuronal differentiation.

Cell to be differentiated

The cell to be differentiated can be any cell of interest, including a cell line or a primary cell, that is not fully specialized or differentiated, and that can be even more differentiated. It may be available from the ATCC or it may be a primary cell culture obtained from an animal, preferably from a mammal. In particular, it can be an intermediary differentiated cell as defined above, e.g. a cardiopoietic mesenchymal cell, a myoblast cell. In one embodiment, the cell to be differentiated is a neuroblast.

Alternatively, the at least one cell to be differentiated in the method I use of the invention can be a non-specialized cell or unspecialized cell, i.e. , a pluripotent or a multipotent cell that may give rise to several different differentiated I specialized cell types or intermediary differentiated cell subtypes. Typically, said cell to be differentiated can be a stem cell or any cell that is known to give rise to at least one specialized or intermediary differentiated cell. This cell to be differentiated can be for example a cardiac, a bone marrow or a blood CD34+ stem cell. It can also be a neural stem cell collected from the brain or spinal cord of an animal, that can eventually be differentiated into neuron or glial cells. It can also be a progenitor cell, that can eventually be differentiated into a retinal pigment epithelial (RPE) cell, such as a fetal retinal progenitor cell (preferably allogenic). In another embodiment, the at least one cell to be differentiated is a specialized cell, and said cell may differentiate (by transdifferentiation) into another specialized type of cell; in this case, the cell to be differentiated is for example a retinal pigment epithelium cell.

Preferably, the cell to be differentiated is a mammal cell, preferably a human cell.

Advantageously, the cell to be differentiated is not a human embryonic cell. However, the cell to be differentiated can be derived from parthenogenetically activated human oocytes.

In other embodiments, the cell to be differentiated is not an (animal such as a mammal) embryonic cell.

In a preferred embodiment, the cell to be differentiated is an induced pluripotent stem cell (IPS) that has been derived from an animal sample, preferably from a human sample.

In one embodiment, the cell to be differentiated is any cell able to differentiate into neurons, including neural progenitor cells, multipotent neural stem cells, specialized cell able to transdifferentiate into neurons, in particular neuroblast and RPE cell.

In some instances, the cell to be differentiated is a RPE or a neuroblast cell. As shown in the examples below with the two tested cell lines SHSY-5Y and ARPE 19, these cells advantageously differentiate into specialized neuron-like cells once contacted with the compounds of the invention.

The SH-SY5Y cell line is available from the ATCC. It is a thrice cloned subline of the neuroblastoma cell line SK-N-SH (ATCC HTB-11), which was established in 1970 from a metastatic bone tumor from a 4-year-old cancer patient. Applications include use as a transfection host or in immunology, neuroscience, and toxicology research.

ARPE-19 is a spontaneously arising retinal pigment epithelia (RPE) cell line derived from the normal eyes of a 19-year-old male who died from head trauma in a motor vehicle accident.

It can also be other tumoral leukemic cells (e.g., from the cell lines THP1 or K562) or other solid tumor cells (e.g., from the cell line HEP G2) that are known to be able to differentiate into other cell types (respectively for the cited cell lines, macrophages, erythrocytes, megakaryocytes, hepatocytes).

Differentiated Cell

Typically, the differentiated cell obtained by means of the method I use of the invention is an intermediary differentiated cell or a fully differentiated cell, an intermediary or fully specialized cell (either immature or mature), advantageously a neuron, a retinal pigment epithelium cell, a glial cell, specialized blood cells, hepatocytes, etc.

In one embodiment, the differentiated cell is a specialized cell, in particular a neuron, more particularly a retinal neuron or a dopaminergic neuron.

Culture Steps

In the context of the present invention, the terms “in vitro" as well as “ex vivo" mean outside of the organism from which the biological material derives.

In particular embodiments, the method I use of the invention comprises a first step of contacting in vitro or ex vivo at least one cell to be differentiated with a regulated necrotic cell death inducer, and a second step of contacting the at least one cell to be differentiated with an effective amount of a regulated necrotic cell death inhibitor, to obtain a differentiated cell. These two steps can be simultaneous or separated in time.

In other particular embodiments, the method I use of the invention comprises contacting in vitro or ex vivo at least one cell to be differentiated with a composition containing a combination of a regulated necrotic cell death inducer and a regulated necrotic cell death inhibitor.

The step(s) of “contacting” typically comprise(s) incubating the cell to be differentiated in a culture medium comprising - or to which is added - the regulated necrotic cell death inhibitor and/or regulated necrotic cell death inducer, respectively.

Preferably, the method comprises or consists of:

- culturing in vitro or ex vivo the cells to be differentiated in a culture medium, until more than 50%, more preferably more than 80% of the cells become adherent (the duration and culture conditions of this culturing step depends on the cell to be differentiated, and is performed until appropriate adhesion according to classical protocols known in the art for this particular cell),

- once the cells are mostly adherent, adding to the culture medium, the regulated necrotic cell death inducer, together with (/.e. in combination) or, in a second step, the regulated necrotic cell death inhibitor, to induce differentiation of the at least one cell to be differentiated,

- recovering the at least one differentiated cell.

Interestingly, the use/method of the invention does not require changing the culture medium prior to the addition of the regulated necrotic cell death inducer or combination of a regulated necrotic cell death inducer and a regulated necrotic cell death inhibitor. Therefore, the regulated necrotic cell death inducer (either alone or in combination with the regulated necrotic cell death inhibitor), is preferably added directly to the culture medium containing the cells to be differentiated, preferably when they are reached adherent stage.

The regulated necrotic cell death inducer(s) and/or regulated necrotic cell death inhibitor(s) (collectively referred to as “the compounds of the invention”) can be added (together or separately) as soon as more than 50%, preferably more than 80% of the cells have become adherent. They can be added also afterwards, typically in a time range of 1h - 48h after appropriate adhesion is observed. Preferably, the compounds are added in a time range of 1 h-24h after appropriate adhesion is observed.

The cells are then cultured with the compounds of the invention during several days, until differentiation markers (morphogenic or genotypic) can be perceived. The duration of this step depends on the cell type. Some treated cells can be sufficiently differentiated and can therefore be collected 24h, two days, three days, four days, five days, six days, seven days or even more, after the compounds are put in the culture medium. For the cell lines used in the examples below, the differentiation markers (apparition of neurites that are typical of neuron-like cells) were observed as of day 1 after treatment, but the cells were collected at day 5 after the compounds have been added.

The culturing step can be performed on classical 2D supports (petri dishes, plates, flasks, ...) or on a 3D polymer matrix or tissue-like structures (e.g., hydrogels, agarose gels, spheroids, clusteroids, etc.).

Regulated necrotic cell death inducer

For the purpose of the invention, the expression “regulated necrotic cell death inducer” (hereinafter “the inducer”) refers to a compound which allows to trigger at least partially regulated necrotic cell death. In other words, a regulated necrotic cell death inducer is a compound comprising means for inducing regulated necrotic cell death (in particular ferroptosis), and/or a compound comprising means for triggering at least partially regulated necrotic cell death (in particular ferroptosis).

The inducer of regulated necrotic cell death is advantageously a ferroptosis and/or a necroptosis inducer. Preferably, the inducer of regulated necrotic cell death is a ferroptosis inducer, and optionally a necroptosis inducer.

Ferroptosis is a type of non-apoptotic regulated cell death that was first described in 2012, and usually involves high intracellular levels of free iron and lipid peroxidation. This death pathway is directly linked to the ability of the cell to regulate its internal oxidative stress, notably via the activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4). The failure of the glutathione-dependent antioxidant defenses causes an accumulation of lipid-based reactive oxygen species (ROS), which result notably from lipid peroxidation by Fe2+, through the Fenton’s reaction, leading to membrane damage and cell death.

Therefore, as used herein, a “ferroptosis inducer” is a compound known in the art to induce cell-death through ferroptosis.

Necroptosis, a programmed cell death route, is clearly distinct from apoptosis as it does not involve key apoptosis regulators, such as caspases, Bcl-2 family members or cytochrome c release from mitochondria. “Necroptosis” is a specialized biochemical pathway of programmed necrosis that depends notably on the serine/threonine kinase activity of RIPK1 (Receptor-Interacting Protein Kinase 1). The ground-breaking finding that necroptosis is a genetically controlled process led to the hypothesis that this programmed cell-death is 'druggable', an emerging breakthrough that carries the potential to revolutionize every day clinical medicine [Linkermann and Green, N. Eng. J. Med. 2014, 370(5), 455-465], Indeed, molecular targets, including RIPK1 (Receptor Interacting Protein 1), RIPK3 and MLKL (Mixed Lineage Kinase domain-Like), have convincingly been shown to contribute to multiple disorders where necroptosis is of central pathophysiological relevance.

Therefore, as used herein, a “necroptosis inducer” is a compound known in the art to induce cell-death through necroptosis.

Examples of ferroptosis inducers are described in particular in Cell Death Discov. 8, 501 (2022) (https://doi.org/10.1038/s41420-022-01297-7). Sodium iodate, erastin, RSL3, ML162, ML210, 6-hydroxydopamine (6-OHDA), rotenone, 1-methyl-4-phenylpyridinium (MPP+), cisplatin, FIN56 (CAS n° 1083162-61-1), Sorafenib (CAS n°284461-73-0), buthionine sulfoximine (BSO, CAS n°83730-53-4), a salt or solvate thereof, or mixtures thereof. Preferably, the ferroptosis inducer is sodium iodate, erastin, RSL3, a salt thereof, a solvate thereof, or mixtures thereof, even more preferably, inducer is sodium iodate, erastin, RSL3, a salt thereof, a solvate thereof, or mixtures thereof.

Examples of necroptosis inducers are tumor necrosis factor cytokines (TNF cytokines) including TNF-a, factor-associated suicide ligand (FASL) tumor necrosis factor-related apoptosis inducing ligand (TRAIL); lipopolysaccharide, caspases inhibitors including carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-FMK), Quinoline- Val-Asp-Difluorophenoxymethylketone (Q-VD-Oph), IDN-6556... ; smac mimetics also known as Inhibitors of apoptosis protein (IAP) antagonists including BV6, birinapant, xevinapant (AT406), LCL161 , .... Or mixtures thereof such as TNF-a and z-VAD-FMK, TNF- a and Z-VAD-FMK and BV-6, TNF-a and Q-VD-Oph, TNF-a and Q-VD-Oph and BV6, FASL and zVAD, TRAIL and zVAD.

Examples of inducers of both ferroptosis and necroptosis are for instance mixtures ferroptosis and necroptosis inducers such as RSL3 and lipopolysaccharide, erastin and TNF- a, RSL3 and TNF-a and Z-VAD-FMK and BV-6, or molecules that can induce both ferroptosis and necroptosis including 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP+), cisplatin, sodium iodate...

Regulated necrotic cell death inhibitor

The regulated necrotic cell death inhibitor may be as described below under section IV.

IL Kit for inducing cell differentiation The invention further relates to a kit useful for inducing cell differentiation, comprising a regulated necrotic cell death inducer, a regulated necrotic cell death inhibitor, and advantageously instructions for use.

The invention also relates to a use of a kit for inducing cell differentiation in vitro or ex vivo, the kit comprising a regulated necrotic cell death inducer, a regulated necrotic cell death inhibitor, and advantageously instructions for use.

The kit may further contain any classical agents useful for cell culture, such as antibiotics, serum albumin, cell culture medium, conventional buffers, amino acids, vitamins, ITS, hormones (hydrocortisone, estradiol, insulin, prostaglandin, dexamethasone, erythropoietin, glucagon, etc.), carbohydrates, growth factors, cytokines, etc. The skilled person well knows which reagent and reagent quantity should be used for favoring cell culture for a defined type of undifferentiated cell.

Preferably, the kit of the invention contains cell adhesion agents (such as poly-L-lysine, extracellular matrix molecules, basement membrane extracts, etc.).

In said kit, the regulated necrotic cell death inducer and the cell death inhibitor are contained or not in the same recipient. The inhibitor(s) and inducer(s) are preferably under liquid I powder form so as to be easily added in the cell culture medium.

The regulated necrotic cell death inducer may be as described above in section I.

HL Therapeutic applications

The invention relates to a regulated necrotic cell death inhibitor for use as a medicament for inducing differentiation of pre-necrotic cells of a patient in need thereof.

The regulated necrotic cell death inhibitor may also be useful for regenerating a tissue or an organ damaged by a degenerative affection or disease.

Without wishing to be bound by theory, it is postulated that the regulated necrotic cell death inhibitor induces cell differentiation of cells to be differentiated, into cells suitable to regenerate said tissue or organ, thus leading to improvement of the tissue or organ.

In some embodiments, the invention relates to a regulated necrotic cell death inhibitor for use for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, in particular a degenerative affection or disease, by inducing differentiation of pre-necrotic cells of a patient in need thereof.

As used herein, “pre-necrotic cells” are understood as cells wherein regulated necrosis pathways have been activated, either naturally (for instance in the case of an affection or disease associated with necrotic cell death in particular associated with ferroptosis) or typically by addition of a regulated necrotic cell death inducer, in particular a ferroptosis inducer. Preferably, the pre-necrotic cell is a pre-necrotic non-specialized cell or a pre- necrotic neuroblast or a pre-necrotic retinal pigment epithelium cell Alternatively, the pre- necrotic cell may be a pre-necrotic specialized cell and said cell may differentiate (by transdifferentiation) into another specialized type of cell; in this case, the cell to be differentiated is for example a pre-necrotic retinal pigment epithelium cell. In some embodiments, the pre-necrotic cell is a cancerous cell, such as a cancerous pre-necrotic non-specialized cell or a cancerous pre-necrotic neuroblast or a cancerous pre-necrotic retinal pigment epithelium cell. It may also be a cancerous pre-necrotic specialized cell, and said cell may differentiate (by transdifferentiation) into another specialized type of cell; in this case, the cell to be differentiated is for example a cancerous pre-necrotic retinal pigment epithelium cell.

In some embodiments, the invention relates to a method for inducing differentiation of pre- necrotic cell(s) of a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a regulated necrotic cell death inhibitor.

In some embodiments, the invention relates to a method for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, more particularly a degenerative affection or disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of a regulated necrotic cell death inhibitor, thereby inducing differentiation (in particular neuronal differentiation) of pre-necrotic cell(s) of the patient.

The invention also relates to a method for regenerating a tissue or an organ damaged by a degenerative affection or disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of the regulated necrotic cell death inhibitor, thereby inducing cell differentiation of cells to be differentiated, notably into cells suitable to regenerate said tissue or organ, or pre-necrotic cells.

The invention also concerns the use of a regulated necrotic cell death inhibitor, in particular a ferroptosis inhibitor, for use a medicament for inducing differentiation, in particular for inducing neuronal differentiation, of pre-necrotic cell(s) of a patient in need thereof.

The invention also concerns the use of a regulated necrotic cell death inhibitor, in particular a ferroptosis inhibitor, for use a medicament for differentiating pre-necrotic cell(s) of a patient in need thereof into differentiated cell(s), wherein the differentiated cell is a specialized cell, advantageously a neuron.

The invention also concerns the use of a regulated necrotic cell death inhibitor for manufacturing a drug intended for inducing differentiation, in particular for inducing neuronal differentiation, of pre-necrotic cell(s) of a patient in need thereof. The invention also concerns the use of a regulated necrotic cell death inhibitor for manufacturing a drug intended for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, in particular a degenerative affection or disease, by inducing differentiation of pre-necrotic cell(s) of a patient in need thereof. The invention also concerns the use of a regulated necrotic cell death inhibitor for manufacturing a drug intended for regenerating a tissue or an organ damaged by a degenerative affection or disease.

The patient may be a mammal and is preferably a human being.

The patient typically suffers from an affection or disease associated with ferroptosis, notably with both ferroptosis and necroptosis. In some embodiments, the patient suffers from a degenerative affection or disease in particular associated with ferroptosis, notably with both ferroptosis and necroptosis.

In some embodiments, the patient is treated (or co-treated) or pre-treated with a regulated necrotic cell death inducer. In these embodiments, the patient typically suffers from cancer, such as solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells. In one embodiment, the patient is pretreated or co-treated with a regulated necrotic cell death inducer targeting tumor cells, the patient being suffering from cancer. In some embodiments, the cancer is a resistant or drug-resistant cancer, i.e. in particular a cancer that does not respond to usual treatment involving apoptosis mechanisms.

As used herein, “a patient treated with a regulated necrotic cell death inducer” is a patient under treatment with a regulated necrotic cell death inducer. In contrast, “a patient pretreated with a regulated necrotic cell death inducer” is a patient which was treated with a regulated necrotic cell death inducer, and whose treatment treated with the regulated necrotic cell death inducer has been completed. A “patient co-treated with a regulated necrotic cell death inducer” is a patient under treatment with both a regulated necrotic cell death inducer and another drug, such as a regulated necrotic cell death inhibitor.Examples of such treatments with a regulated necrotic cell death inducer are for instance disclosed in Cells. 2020; 9(12): 2709 (doi: 10.3390/cells9122709) or in Sei. Immunol. 2019;4 doi: 10.1126/sciimmunol.aaw2004, or in Cancer Discov. 2019;9:1673-1685 (doi: 10.1158/2159- 8290.CD-19-0338), or Mol Cancer 21 , 47 (2022). (doi.org/10.1186/s12943-022-01530-y). The inducer of regulated necrotic cell death is preferably as described herein in section I. Preferably, it is a ferroptosis inducer. In some embodiments, regulated necrotic cell death inhibitor is used in combination with a regulated necrotic cell death inducer, in particular for a simultaneous, separate or sequential use.

The invention thus concerns a regulated necrotic cell death inhibitor for use as a medicament for inducing differentiation of pre-necrotic cells of a patient in need thereof, in combination with a regulated necrotic cell death inducer (in particular targeting tumor cells), in particular for a simultaneous, separate or sequential use. In these embodiments, the patient typically suffers from cancer, such as solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer advantageously targets tumor cells. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells.

The invention also relates to a combination of a regulated necrotic cell death inhibitor and a regulated necrotic cell death inducer for use for treating cancer by inducing differentiation of pre-necrotic cells of a patient in need thereof. In these embodiments, the cancer is typically a solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer advantageously targets tumor cells. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells.

The invention also relates to a method for inducing differentiation of pre-necrotic cell(s), the method comprising administering simultaneously, separately or sequentially to a patient in need thereof a therapeutically effective amount of a regulated necrotic cell death inhibitor and a regulated necrotic cell death inducer.

The invention also concerns the use of a combination of a regulated necrotic cell death inhibitor and a regulated necrotic cell death inducer, for inducing differentiation of pre- necrotic cell(s) of a patient in need thereof. In these embodiments, the patient typically suffers from cancer, such as solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer advantageously targets tumor cells. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells.

The invention also concerns the use of a combination of a regulated necrotic cell death inhibitor and a regulated necrotic cell death inducer, for manufacturing a drug intended for treating cancer by inducing differentiation of pre-necrotic cell(s) of a patient in need thereof. In these embodiments, the cancer is typically a solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer advantageously targets tumor cells. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells.

The invention thus further relates to a (therapeutic) kit comprising: a) a first therapeutic agent, which is a regulated necrotic cell death inducer, and b) a second therapeutic agent, which is a regulated necrotic cell death inhibitor, for separate, simultaneous, or sequential of the first and the second therapeutic agents, as medicament, in particular for inducing differentiation of pre-necrotic cells of a patient in need thereof or for treating an affection or disease associated with ferroptosis, advantageously by inducing differentiation of pre-necrotic cells. In these embodiments, the patient typically suffers from cancer, such as solid cancer, in particular hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas. In these embodiments, the regulated necrotic cell death inducer (and preferably regulated necrotic cell death inhibitor) advantageously targets tumor cells. I The inducer of regulated necrotic cell death may be as described herein in section I. Preferably, it is a ferroptosis inducer.

Typically, the affection or disease is associated with (for instance involves or causes or is caused by) regulated necrotic cell death, such as ferroptosis, and optionally necroptosis.

In a particular embodiment, the affection or disease is associated with ferroptosis, notably with both ferroptosis and necroptosis.

Hallmarks of ferroptosis were used as key elements to define ferroptosis-associated disease biomarkers. Ferroptosis is an iron-dependent regulated tissue necrosis mainly caused by unrestricted lipid peroxidation and subsequent membrane damage. The modifications of the physiological levels of the following components were reported as associated with ferroptosis: iron, reactive oxygen species, ROS (including lipid ROS such as 4- Hydroxynonenal (4-HNE) and malondialdehyde (MDA), and oxidized phospatidylethanolamine (oxPE) species followed by oxidized phosphatidylserine (oxPS) and oxidized phosphatidylinositol (oxPI) [Wiernicki et al., Cell Death Dis., 2020, 11(922)]) and related peroxide detoxification molecules (including the thiol-containing compound gluthation, GSH, or Coenzyme Q10, also known as ubiquinone), and the long-chain-fatty- acid-CoA ligase 4 (ACSL4) [Chen X. et a!., Front. In Cell and Dev. Biol., 2021 , 9(637162)]. These key biochemical ferroptosis biomarkers can be measured and quantified by assays in bodily fluids (blood, plasma, serum, urine, cerebrospinal fluid) or highlighted by immunohistochemistry labeling on biopsies of damaged tissues.

Depending on both the pathology and the damaged organ, several among ferroptosis- associated biomarkers could vary (increase > or decrease <) in quantity and/or activity relative to normal physiological thresholds. Here we only described reference values for serum:

(1) Iron metabolism (by measuring iron and ferritin levels in serum) is over the physiological thresholds (serum iron, in male > 180 pg/dl, in female > 160 pg/dl, ferritin, in male >300 ng/ml, in female > 200 ng/ml, [Wang et al., Biochim Biophy. Acta, 2010, 1800(8): 760-769]);

(2) Glutathione redox status (by measuring reduced glutathione (GSH) and oxidized glutathione (GSSG) as well as glutathione peroxidase activity (GPx) in plasma using ELISA) (GSH < 717 pmol/L, GSSG > 5.32 pmol/L; ratio GSH/GSSG < 156; GPx, in male < 20 UI/gHb, in female < 26 UI/gHb), [Haleng J. et al., Rev. Med. Liege, 2007]);

(3) Oxidative stress (by measuring levels of total Q10 and reduced and active form of Q10 (Q10H2) in plasma (in male Q10 < 3.44 pmol/l and Q10H2 < 3.04 pmol/l; in female Q10 < 1.88 pmol/l and Q10H2 < 1.64 pmol/l, [Kaikkonen et al., Scand J Clin Lab Invest, 1999, 59: 457-466]);

(4) Lipid peroxidation (measured by detection of 4-Hydroxynonenal (4-HNE) and malondialdehyde (MDA) adducts) is over the physiological thresholds (> 10 pmol/L for 4- HNE [Chen and Niki, IUBMB Life, 2008, 58(372-373)] and > 3 pmol/L for MDA using thiobarbituric acid method [Banjare et al., J. Sci. Soc., 2017; 44(137-9)]).

Of note, the upregulation of ACSL4 enzyme level in damaged organ tissues was also reported as putative biomarker of ferroptosis (ACSL4 expression can be monitored by transcriptomic and proteomic approaches).

Ferroptosis plays important roles in multiple system diseases, including but not limited to nervous system diseases, heart diseases, liver diseases, gastrointestinal diseases, lung diseases, kidney diseases, pancreatic diseases, ... (Li et al., 2020, cell death and disease). Pathologies associated with ferroptosis affecting the heart include myocardial ischemiareperfusion injury, notably occurring after artery ligation, cardiomyopathy, notably doxorubicin-induced cardiomyopathy [Li et al., 2020], and cardiovascular disease, notably aortic dissection [Chen et al., Pharmacol. Res., 2022, 177, 106122], among others [Li et al., Free Radio. Biol. Med., 2020, 160, 303-318; Qin et al., Biomed. Pharmacot her, 2021 , 141 , 111872],

Pathologies associated with ferroptosis affecting the central nervous system include strokes, notably ischemic stroke [Li et al., 2020] or hemorrhagic stroke [Li et al., JCI Insight, 2017, 2(7):e90777], traumatic brain injury [Xie et al., CNS Neurosci Ther, 2019, 25:465-475], contusion spinal cord injury [Zhang et al., Neural Regen. Res., 2019, 14(3):532], epilepsy, including mitochondrial disease-related epilepsy and intractable epilepsy [Kahn-Kirby et al., PloS One., 2019, 14(3)], and neurodegenerative disorders, in particular chronic neurodegenerative disorders, more particularly Alzheimer’s disease [Li et al., 2020], Huntington’s disease [Mi et al., Neuromolecular Med., 2019, 21 , 110-119], Parkinson’s disease [Do Van et al., Neurobiol Dis., 2016, 94: 169-78], amyotrophic lateral sclerosis (Charcot’s disease) [Li et al., 2020], multiple sclerosis [Luoqian et al., Cell Mol Immunol., 2022, 19(8), 913-924], Friedreich’s ataxia [Cotticelli et al., J Pharmacol Exp Ther, 2019, 369(1): 47-54], periventriculor leukomalacia [Skouta et al., J. Am. Chem. Soc., 2014, 136, 4551-4556] and dementia, which may be linked to one or several of the previous pathologies. Pathologies associated with ferroptosis affecting the eyes include vision loss, in particular due to cataract [Wei et al., Free Radio Biol Med., 2021 , 167, 94-108], and retinal disorders, notably Stargardt disease and age-related macular degeneration (AMD), in particular dry AMD [Sun et al., Invest Ophth Vis Sci., 2018, 59(9), 2482; Chen et al., J. Biol. Chem., 2021 , 296, 100187],

Pathologies associated with ferroptosis affecting the liver include chronic liver diseases as well as acute liver injury and acute liver failure. Among chronic liver diseases, mention should be made of non-alcoholic steatohepatitis (NASH) [Qi et al., Am J Pathol., 2020, 190(1)], chronic infections such as hepatitis B and C [Cappelletti et al., Int J Mol Sci., 2020, 21(14)] and alcoholic cirrhosis [Zhou et al., Hepatol Commun., 2019, 3(5)]. Acute liver failure may notably result from a drug-induced liver injury (DILI), such as acetaminophen (APAP)- induced liver injury [Yamada et al., Cell Death Dis., 2020, 11(2)], from an ischemiareperfusion injury induced by a septic or hemorrhagic shock [Friedmann Angeli et al., Nat Cell Biol., 2014, 16(12):1180-91], from fulminant viral hepatitis, from auto-immune origin or from alcohol intake.

Pathologies associated with ferroptosis affecting the skin include skin inflammatory diseases, such as psoriasis [Li et al., Cell Death Dis., 2020, 11(88)], and toxic epidermal necrolysis (Lyell syndrome) [Zhang et al., J Invest Dermatol., 2020, 140(7), S79],

Pathologies associated with ferroptosis affecting the kidneys include acute kidney injury (AKI) (also known as acute renal failure), such as crystal (oxalate)-, folic acid (FA)-induced AKI [Martin-Sanchez et al., 2017] and cisplatin-induced AKI [Deng et al., J Clin Invest., 2019, 129(11); Mishima et al., J Am Soc Nephrol., 2020, 31(2); Hu et al., Cell Death Dis., 2020, 11(1)], renal ischemia-reperfusion injury [Li et al., 2020], and acute tubular necrosis [Friedmann Angeli et al., 2014],

Pathologies associated with ferroptosis affecting the lungs include chronic obstructive pulmonary disease (COPD) [Yoshida etal., Nat Commun., 2019, 10, 3145], bronchial asthma [Tao et al., Oxid Med Cell Longev., 2020], lung injury caused by a bacterial infection, notably by Pseudomonas aeruginosa [Dar et al., J Clin Invest., 2018, 128(10), 4639-4653] or Mycobacterium tuberculosis [Amaral et al., J Exp Med., 2019, 216(3): 556-570] and pulmonary fibrosis, such as radiation induced-lung fibrosis (RILF) [Li et al., J Inflamm., 2019, 16:11] and paraquat-induced pulmonary damage [Rashidipour et al., Toxicology, 2020, 433- 434:152407],

Pathologies associated with ferroptosis affecting the gut include necrotizing enterocolitis [Subramanian et al., Acta Physiologica Sinica, 2020, 72(3)] and inflammatory bowel diseases, such as Crohn’s disease [Mayr et al., Nat Commun., 2020,

ulcerative colitis.

Pathologies associated with ferroptosis affecting the whole body include haemochromatosis [Imoto et al., Transfus Apher Sci., 2018, 57(4), 524-531], p-thalassemia [Sposi, N. M., Oxidative Stress and Iron Overload in ^-Thalassemia: An Overview, 2019, DOI: 10.5772/intechopen.90492], hemolytic disorders [Youssef et al., 2019, Ferroptosis in Hemolytic Disorders. In: Tang D. (eds) Ferroptosis in Health and Disease. Springer, Cham.], cytokinic storm during a viral infection [Edeas et al., Int J Infect Dis., 2020, 97; Yang and Lai, Cell Death Discov., 2020, 6], radiation-induced necrosis [Wu et al., Front Oncol., 2020, 10], rheumatoid arthritis [Xie et al., Inflammation., 2020, doi: 10.1007/s10753-020-01338-2], type I diabetes [Bruni et al., Cell Transplant., 2018, 27(6)], insulin resistance related to obesity, and pathologies related to stress-induced premature tissue senescence, such as atherosclerosis [Bai et al., Free Radio Biol Med., 2020, 160], hypertension [Yang et al., Clin Exp Hypertens., 2020, 42(8)] and type II diabetes [Li et al., Nutrients., 2020, 12(10)].

In particular, the affection or disease is associated with ferroptosis and is preferably selected from the group consisting of: degenerative affection or disease such as neurodegenerative diseases or disorders and retinal degenerative diseases or disorders, stroke, traumatic brain injury, type I diabetes, liver injury, kidney injury, heart injury, liver fibrosis, cancers such as hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, lung cancer, adrenocortical carcinomas, clear cell renal cell carcinomas, diseases related to transplantation.

Preferably, the disorder associated with ferroptosis is selected from the group consisting of myocardial ischemia-reperfusion injury, notably occurring after artery ligation or myocardial necrosis in myocardial infarction; strokes, notably ischemic stroke or hemorrhagic stroke; traumatic brain injury; neurodegenerative disorders, in particular chronic neurodegenerative disorders, more particularly Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (Charcot’s disease) and multiple sclerosis; vision loss, in particular due to retinal detachment or cataract; retinal disorders, notably Stargardt disease or age-related macular degeneration (AMD), in particular dry AMD; chronic liver diseases, notably non-alcoholic steatohepatitis (NASH); acute liver injury and acute liver failure, notably resulting from a drug-induced liver injury (DILI), such as acetaminophen (APAP)-induced liver injury, or from an ischemia-reperfusion injury induced by a septic or hemorrhagic shock; and acute kidney injury (AKI) or acute renal failure, such as folic acid (FA)-induced AKI, cisplatin-induced AKI, renal ischemia-reperfusion injury and acute tubular necrosis.

In particular, the disorder associated with ferroptosis is selected from the group consisting of neurodegenerative disorders, in particular chronic neurodegenerative disorders, more particularly Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (Charcot’s disease) and multiple sclerosis; vision loss, in particular due to retinal detachment or cataract; retinal disorders, notably Stargardt disease or age-related macular degeneration (AMD), in particular dry AMD; acute liver injury and acute liver failure, notably resulting from a drug-induced liver injury (DILI), such as acetaminophen (APAP)- induced liver injury, or from an ischemia-reperfusion injury induced by a septic or hemorrhagic shock; and acute kidney injury (AKI) or acute renal failure, such as folic acid (FA)-induced AKI and cisplatin-induced AKI.

Necroptosis plays important role in the pathogenesis of various diseases across the body, including conditions of the neurologic, cardiovascular, pulmonary, and gastrointestinal systems. Necroptosis also plays a role in infectious and autoimmune diseases. Necroptosis was also reported to mediate organ rejection in particular in cardiac and renal allografts (Khoury et al. Am j pathology, 2020; Jouan-Lanhouet et al. semin. Cell dev. Biol. 2014).

In particular, the affection or disease is associated with ferroptosis (with or without necroptosis) and can be selected from the group consisting of: degenerative affection or disease such as neurodegenerative diseases or disorders, degenerative eye diseases or disorders such as retinal degenerative diseases or disorders, vision loss, stroke, traumatic brain injury, infectious diseases, autoimmune diseases such as psoriasis and rheumatoid arthritis, inflammatory diseases such as inflammatory bowel diseases, type I diabetes, liver injury such as acute liver failure, kidney injury, heart injury, myocardial infarction, aortic aneurysm, atherosclerosis, chronic obstructive pulmonary disease, acute respiratory distress disorder, liver fibrosis, pancreatitis, diseases related to transplantation.

The disorder associated with both ferroptosis and necroptosis may be acute liver injury and acute liver failure, acute renal failure or acute kidney injury (AKI), acute tubular necrosis, radiation-induced necrosis, ischemic disorders affecting the heart, brain, or kidneys, rheumatoid arthritis, psoriasis, neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s diseases, lateral amyotrophic sclerosis (Charcot’s disease) and dementia, dry (atrophic) age-related macular degeneration (AMD), haemochromatosis, necrotizing enterocolitis, non-alcoholic steatohepatitis (NASH), Friedreich’s ataxia, inflammatory bowel diseases such as Crohn’s disease, toxic epidermal necrolysis (Lyell’s syndrome), type I diabetes, and diseases related to stress-induced premature tissue senescence, including age-related disorders such as atherosclerosis, hypertension and type II diabetes.

In some embodiments, the affection or disease is a degenerative affection or disease associated with ferroptosis, notably with both ferroptosis and necroptosis. In particular, the degenerative affection or disease is selected from the group consisting of neurodegenerative diseases or disorders and degenerative eye diseases or disorders, more particularly from the group consisting of neurodegenerative diseases or disorders and retinal degenerative diseases or disorders.

Neurodegenerative diseases or disorders, which are primarily characterized by neuron loss include Alzheimer’s disease, Parkinson’s disease, prion disease, Amyotrophic lateral sclerosis, Friedreich ataxia, motor neuron disease, Huntington’s disease, spinal muscular atrophy, and spinocerebellar ataxia.

Degenerative retinal diseases or disorders, in particular characterized by neuron loss, include glaucoma, macular degeneration (MD) (including age-related macular degeneration), retinitis pigmentosa (RP), retinoblastoma, diabetic retinopathy (DR).

When used in therapeutic applications, the regulated necrotic cell death inhibitor may be used in the form of a pharmaceutical composition.

As used herein, the term “pharmaceutical composition” refers to a composition having preventive and curative properties towards a degenerative affection or disease as defined herein.

The pharmaceutical compositions can be intended to oral, sublingual, ocular, nasal, subcutaneous, intramuscular, intravenous, transdermal/topical, local or rectal administration. The active ingredient can be administered in unit forms for administration, mixed with conventional pharmaceutical carriers, to animals or to humans. Suitable unit forms for administration comprise the forms for oral administration, such as tablets, gelatin capsules, powders, granules and oral solutions or suspensions, the forms for sublingual and buccal administration, the forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and the forms for rectal administration.

When a solid composition is prepared in the form of tablets, the main active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like before being compressed. The tablets may be coated with sucrose or with other suitable materials, or they may be treated in such a way that they have a prolonged or delayed activity and they continuously release a predetermined amount of active principle.

A preparation in gelatin capsules is obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard gelatin capsules. A preparation in the form of a syrup or an elixir may contain the active ingredient together with a sweetener, an antiseptic, or also a taste enhancer or a suitable coloring agent.

The water-dispersible powders or granules may contain the active ingredient mixed with dispersing agents or wetting agents, or suspending agents, and with flavor correctors or sweeteners.

For rectal administration, suppositories are used which are prepared with binders which melt at rectal temperature, for example cocoa butter or polyethylene glycols.

For parenteral, intranasal or intraoccular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which contain pharmacologically compatible dispersing agents and/or wetting agents are used.

The active principle may also be formulated in the form of microcapsules, optionally with one or more carrier additives.

The regulated necrotic cell death inhibitor can be used in a pharmaceutical composition at a dose ranging from 0.01 mg to 1000 mg a day, administered in only one dose once a day or in several doses along the day, for example twice a day. The daily administered dose advantageously varies from 5 mg to 500 mg, and more advantageously from 10 mg to 200 mg. However, it can be necessary to use doses out of these ranges, which could be noticed by the person skilled in the art.

The pharmaceutical composition of the invention may further comprise another active compound, useful in particular as regulated necrotic cell death inhibitor, such as N- acetylcysteine (NAC) or a pharmaceutical salt or a derivative thereof.

Pharmaceutically acceptable salts of NAC include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, sulfuric, acetic, oxalic, tartaric acids, and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-(ethylamino)ethanol, histidine, procaine and the like.

In the framework of the present invention, the term “derivative of NAC” is intended to mean any compound which is structurally related to NAC and which possesses a similar pharmacological activity. For instance, the following compound, known as N-acetylcysteine amide (NACA) [Sunitha et al., Clin Pharmacol Then, 2013, 47(5), 884-890] is a derivative of NAC:

NACA IV. Regulated necrotic cell death inhibitor

For the purpose of the invention, the expression “regulated necrotic cell death inhibitor” (hereinafter “the inhibitor”) refers to a compound which allows to preserve at least partially cells from a regulated necrotic death. In other words, a regulated necrotic cell death inhibitor is a compound comprising means for inhibiting regulated necrotic cell death (in particular ferroptosis), and/or a compound comprising preserving at least partially cells from a regulated necrotic death (in particular ferroptosis). In particular, when cells are exposed to a regulated necrotic cell death inducer, treatment with the inhibitor allows to improve cell viability, which can be easily verified by the skilled person using methods well known in the art, such as those described in the examples of the present description.

The inhibitor of regulated necrotic cell death is advantageously a ferroptosis and/or a necroptosis inhibitor. Preferably, the inhibitor of regulated necrotic cell death is a ferroptosis inhibitor, and optionally a necroptosis inhibitor.

Tests for identifying ferroptosis inhibitors are for instance disclosed in WO2022157392, from pages 69 to 81 (experimental part, section “II. Biological Activity of the compounds of the invention). A preferred model is SH-SY5Y human neuroblastoma cell line treated with ferroptosis inducer RSL3. A ferroptosis inhibitor is characterized by its ability to protect cells from cell death in a dose-dependent manner and to decrease lipid peroxidation induced by a ferroptosis inducer such as RSL3 in the model SH-SY5Y.

Tests for identifying necroptosis inhibitors are for instance disclosed in page 29 of EP3362450B1 , specially in paragraph [178], A necroptosis inhibitor is characterized by its ability to protect cells from cell death induced by a necroptosis inducer, in a dose-dependent manner in this test, for example with an ECso of 25 pMor less (ECso is the half maximal effective concentration of the drug).

The regulated necrotic cell death inhibitor inhibitor used in the invention may be in particular sibiriline, nigratine or a derivative thereof.

Examples of known ferroptosis and/or necroptosis inhibitor are Resveratrol (CAS number 501-36-0, see for instance doi.org/10.1016/j.fct.2022.113586, doi.org/10.1002/jemt.24335 or J Cell Mol Med. 2023; 27(20): 3075-3089), Ferrostatin 1 (CAS Number: 347174-05-4 see for instance Cell 2012; 149(5): 1060-72 or Trends Pharmacol Sci. 2023;44(12):902-916) or Liproxstatin (CAS Number 950455-15-9, see for instance Biochem Biophys Res Commun. 2019; 520(3): 606-611 or Neural Regen Res. 2021 ; 16(3): 561-566), and NEC1 F (described in particular in (Tonnus et al. Nat Commun 12, 4402 (2021), DOI: 10.1038/s41467-021- 24712-6).

The Inventors have previously disclosed two new classes of regulated necrotic cell death inhibitors, the first one consisting of sibiriline derivatives (WO 2017/064217, WO 2017/064216, and WO 2022/157392), and the second one consisting of nigratine derivatives (WO 2018/073321).

Sibiriline (S1) Nigratine (N1)

IV.1. Sibiriline and derivatives thereof

In a particular embodiment, the inhibitor of a pharmaceutical composition according to the invention is sibiriline (S1) or a derivative thereof. Said inhibitor may thus correspond to any compound disclosed in WO 2017/064217, WO 2017/064216 or WO 2022/157392.

In this particular embodiment, the inhibitor is preferably a compound of general formula (I)

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

(i) when Y^^X is Y'^^^X _I

X is N, Y is N(R₂) and Z is C(H);

(ii) when Y^’X is Y^^x ,

- X is N(Ri), and

- Y is N or N⁺(O’) and Z is C(R₃), or

Y is CH and Z is N, or

Y and Z are CH; and wherein:

■ Ri and R₂ represent, independently of each other, a hydrogen atom, CN, NO₂, OR?, SRs, NR9R10, C(O)Rii, CO₂Ri₂, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRIGRI7, S(O)RS, SO₂RS’, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl, a (Ci-C6)alkyl-[O-(Ci-C6)alkyl]_m-NRNiRN2 group with m ranging from 1 to 6, an aryl, an aryl-(Ci-Ce)alkyl, a heterocyclyl, or a heterocyclyl-(Ci- Ce)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO₂, ORis, SR19, NR20R21, C(O)R22, CO2R23, OC(O)R₂4, NR₂₅C(O)R26, C(O)NR₂₇R28, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ R3, R4, R4b and R5 represent, independently of each other, a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, C(O)Rss, CO2R34, OC(O)Rs5, NR3sC(O)R37, C(O)NR3SR39, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR40, SR41 and NR42R43, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl- (Ci-Ce)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NR₅IC(O)R₅2, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ Re represents a hydrogen atom, a (Ci-Ce)alkyl, an aryl-(Ci-Ce)alkyl, a heterocyclyl-(Ci- Ce)alkyl, a (Ci-C6)alkyl-[O-(Ci-C6)alkyl]_m-NRN’iRN’2 group with m’ ranging from 1 to 6, or a (Ci-Ce)alkylcarbonyl group, said (Ci-Ce)alkyl, aryl-(Ci-Ce)alkyl, and (C1- C6)alkylcarbonyl group being optionally substituted with one or more substituents selected from the group consisting of OH, SH, NH2, a (Ci-Ce)alkoxy, a (Ci-Ce)thioalkoxy, a (Ci-Ce)alkylamino and a di((Ci-Ce)alkyl)amino group;

■ Rs and Rs’ represent, independently of each other a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group;

■ R7-R10, R12, R14 and R16-R17 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group;

■ R11 , Risand R15 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl, a (Ci-Ce)alkoxy, a (Ci-Ce)alkylamino or a di((Ci- Ce)alkyl)amino group;

■ Ris to R28 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group;

■ R29 to R39 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, heterocyclyl-(Ci-C6)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, said aryl group being optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, OR55, SR56, NR57R58, C(O)Rs9, CO2R60, OC(O)Rei, NR62C(O)R63, C(O)NR64R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ R40 to R43 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group;

■ R44 to R54 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group;

■ R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, aryl- (Ci-Ce)alkyl group or an aryl group; and RNI, RN-1, RN2 and RN’2 represent, independently of each other, a hydrogen atom, a (Ci- Ce)alkyl group, an aryl-(Ci-Ce)alkyl group or an aryl group.

For the purpose of the invention, the term “pharmaceutically acceptable” is intended to mean what is useful to the preparation of a pharmaceutical composition, and what is generally safe and non-toxic, for a pharmaceutical use.

The term “pharmaceutically acceptable salt or solvate” is intended to mean, in the framework of the present invention, a salt or solvate of a compound which is pharmaceutically acceptable, as defined above, and which possesses the pharmacological activity of the corresponding compound.

The pharmaceutically acceptable salts comprise:

(1) acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acid and the like; or formed with organic acids such as acetic, benzenesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxynaphtoic, 2-hydroxyethanesulfonic, lactic, maleic, malic, mandelic, methanesulfonic, muconic, 2-naphtalenesulfonic, propionic, succinic, dibenzoyl-L-tartaric, tartaric, p- toluenesulfonic, trimethylacetic, and trifluoroacetic acid and the like, and

(2) base addition salts formed when an acid proton present in the compound is either replaced by a metal ion, such as an alkali metal ion, an alkaline-earth metal ion, or an aluminium ion; or coordinated with an organic or inorganic base. Acceptable organic bases comprise diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

Acceptable solvates include conventional solvates such as those formed during the last step of the preparation of the compounds due to the presence of solvents.

The term “halogen”, as used in the present invention, refers to a fluorine, bromine, chlorine or iodine atom.

The terms “(Ci-C6)alkyl”, as used in the present invention, refers to a straight or branched saturated hydrocarbon chain containing from 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

The term “(Ci-C6)haloalkyl”, as used in the present invention, refers to a (Ci-Ce)alkyl group as defined above in which part or all of the hydrogen atoms is replaced with a halogen atom as defined above. This means that the (Ci-Ce)alkyl group is substituted by at least one halogen atom. It can be for example a trifluoromethyl group. The term “aryl”, as used in the present invention, refers to an aromatic hydrocarbon group comprising preferably 6 to 10 carbon atoms and comprising one or more, notably 1 or 2, fused rings, such as, for example, a phenyl or naphtyl group, advantageously a phenyl group. The term “heterocyclic” as used in the present invention refers to a saturated, unsaturated (i.e. not aromatic) or aromatic monocyclic or bicyclic group comprising two fused, bridged or spiro rings, preferably fused rings, advantageously comprising 5 to 10, notably 5 or 6, atoms in each ring, in which the atoms of the ring(s) comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms.

A saturated heterocyclic group is more particularly a 5- or 6-membered saturated monocyclic heterocyclic group such as a pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, thiazolidinyl, isothiazolidinyl, oxazolidinyl, isoxazolidinyl, imidazolidinyl, pyrazolidinyl, triazolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl group.

An unsaturated heterocyclic group is more particularly an unsaturated monocyclic or bicyclic heterocyclic group, each cycle comprising 5 or 6 members, such as a pyrrolinyl, dihydrofuranyl, dihydrothiophenyl, thiazolinyl, isothiazolinyl, oxazolinyl, isoxazolinyl, imidazolinyl, pyrazolinyl, triazolinyl, dihydropyridinyl, tetrahydropyridinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, dihydropyridazinyl, tetrahydropyridazinyl, dihydropyrazinyl, tetrahydropyrazinyl, dihydrotriazinyl, tetrahydrotriazinyl, indolinyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophenyl, 1 ,3-benzodioxolyl, 1 ,3-benzoxathiolyl, benzoxazolinyl, benzothiazolinyl, benzimidazolinyl, chromanyl or chromenyl group.

An aromatic heterocyclic group, also called heteroaryl group, is more particularly an aromatic monocyclic or bicyclic heterocyclic group, each cycle comprising 5 or 6 members, such as a pyrrolyl, furanyl, thiophenyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl (such as 1 , 3, 5-triazinyl), indolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, purinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl or quinoxalinyl group.

The term “aryl-(Ci-C6)alkyl”, as used in the present invention, refers to an aryl group as defined above bound to the molecule via a (Ci-Ce)alkyl group as defined above. In particular, the— (Ci-Ce)alkyl-aryl group is a benzyl group.

The term “heterocyclyl— (Ci-C6)alkyl”, as used in the present invention, refers to a heterocyclyl group as defined above bound to the molecule via a (Ci-Ce)alkyl group as defined above. In particular, the— (Ci-Ce)alkyl- heterocyclyl group is 5- or 6-membered saturated monocyclic heterocyclic group as defined above bound to the molecule via a (Ci- Ce)alkyl group as defined above. The term “(Ci-C6)alkylcarbonyl”, as used in the present invention, refers to a (Ci-Ce)alkyl group as defined above bound to the molecule via a -C(=O)- group, including, but not limited to, acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and the like.

The term “(Ci-C6)alkoxy”, as used in the present invention, refers to a (Ci-Ce)alkyl group as defined above bound to the molecule via an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, t-butoxy, n- pentoxy, n-hexoxy, and the like.

The term “(Ci-C6)thioalkoxy”, as used in the present invention, refers to a (Ci-Ce)alkyl group as defined above bound to the molecule via a sulfur atom, including, but not limited to, thiomethoxy, thioethoxy, n-thiopropoxy, iso-thiopropoxy, n-thiobutoxy, iso-thiobutoxy, secthiobutoxy, t-thiobutoxy, n-thiopentoxy, n-thiohexoxy, and the like.

The term “(Ci-C6)alkylamino”, as used in the present invention, refers to a

-NHAIk group with Aik representing a (Ci-Ce)alkyl group as defined above, including, but not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, n-butylamino, isobutylamino, sec-butylamino, t-butylamino, n-pentylamino, n-hexylamino, and the like.

The term “di(Ci-C6)alkylamino”, as used in the present invention, refers to a -nAlkiAlk2 group with Alki and Alk2 representing, independently of one another, a (Ci-Ce)alkyl group as defined above, including, but not limited to, dimethylamino, diethylamino, ethylmethylamino and the like.

In a particular embodiment, in the compound of the following general formula (I):

■ Ri and R2 represent, independently of each other, a hydrogen atom, CN, NO2, OR7, SRs, NR9R10, C(O)Rii, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRisRi7, S(O)RS, SO2RS’, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, ORis, SR19, NR20R21, C(O)R22, CO2R23, OC(O)R24, NR2sC(O)R26, C(O)NR27R28, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ R3, R4, R4b and R5 represent, independently of each other, a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, C(O)Rss, CO2R34, OC(O)Rs5, NR3sC(O)R37, C(O)NR3SR39, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR40, SR41 and NR42R43, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl- (Ci-Ce)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NR₅IC(O)R₅2, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group; ■ Rs represents a hydrogen atom, a (Ci-Ce)alkyl, an aryl-(Ci-Ce)alkyl or a -CH2-CH2-O- CH2-CH2-NH2 group, or a (Ci-C6)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, SH, NH2, a (Ci-Ce)alkoxy, a (Ci-Ce)thioalkoxy, a (Ci-C6)alkylamino and a di((Ci-C6)alkyl)amino group;

■ R11 , Risand R15 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl, a (Ci-Ce)alkoxy, a (Ci-C6)alkylamino or a di((Ci- Ce)alkyl)amino group;

■ R29 to R39 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, said aryl group being optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, OR55, SR₅₆, NR57R58, C(O)R₅₉, C0₂R₆O, OC(O)R₆I, NR₆₂C(O)R₆3, C(O)NR₆₄R₆5, a (Ci-C₆)alkyl and a (Ci-Ce)haloalkyl group;

■ R44 to R54 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group; and

R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group.

According to particular embodiments, R1 represents a hydrogen atom, CN, NO2, OR7, SRs, NR9R10, C(O)Rii, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRIGRI7, S(O)RS, SO2RS’, a (C1- Ce)alkyl, a (Ci-Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci- Ce)alkyl group, wherein said aryl or heterocyclyl group (which may be part of a aryl-(Ci- Ce)alkyl or heterocyclyl-(Ci-C6)alkyl group) is optionally substituted by one or more substituents, notably one susbtituent, selected from the group consisting of a halogen atom, CN, NO2, OR18, SR19, NR20R21, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl, notably a halogen atom, NO2, OR18, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular NO2 and OR18, and wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group.

According to other particular embodiments, R1 represents a hydrogen atom, CN, NO2, OR7, SRs, NR9R10, C(O)Rii, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRIGRI7, a (Ci-Ce)alkyl, a (Ci- 1

Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents as defined above.

According to still other particular embodiments, Ri represents a hydrogen atom, CN, OR?, C(O)Rii, CO2R12, OC(O)Ri3, SChRs’, a (Ci-Ce)alkyl, a heterocyclyl or a heterocyclyl-(Ci- Ce)alkyl group, wherein said heterocyclyl group (which may be part of a heterocyclyl-(Ci- Ce)alkyl group) is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, ORis, SR19, NR20R21, a (C1- Ce)alkyl and a (Ci-Ce)haloalkyl group, notably a halogen atom, NO2, ORis, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular NO2 and ORis, preferably said heterocyclyl group is optionally substituted by NO2, and wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group.

According to yet other particular embodiments, R1 represents a hydrogen atom, CN, OR7, C(O)Rii, CO2R12, OC(O)Ri3, a heterocyclyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said heterocyclyl group is optionally substituted by one or more substituents as defined above.

In a preferred embodiment, R1 represents a hydrogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom or a (Ci-C3)alkyl group, preferably a hydrogen atom.

In the above embodiments, the (Ci-Ce)alkyl group, which may be part of an aryl-(Ci-Ce)alkyl group or a heterocyclyl-(Ci-C6)alkyl group, is preferably a (Ci-C3)alkyl group.

In the above embodiments, the aryl group, which may be part of an aryl-(Ci-Ce)alkyl group, is preferably a phenyl group.

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably saturated or aromatic, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms, such as a morpholinyl, a pyridinyl or a piperazinyl, for instance a morpholinyl or pyridinyl group.

In the above embodiments, Rs and Rs’ represent, independently of each other a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, notably a (Ci-Ce)alkyl or an aryl group, in particular, an aryl group, such as a phenyl group.

In the above embodiments, R7-R10, R12, R-u and R16-R17 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, and Rn, Risand R15 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl- (Ci-Ce)alkyl, a (Ci-Ce)alkoxy, a (Ci-Ce)alkylamino or a di((Ci-Ce)alkyl)amino group, notably a hydrogen atom, a (Ci-Ce)alkyl, an aryl, a (Ci-Ce)alkylamino or a di((Ci-Ce)alkyl)amino group, in particular a (Ci-C3)alkyl, an aryl such as a phenyl, a (Ci-C3)alkylamino or a di((Ci- C3)alkyl)amino group. In particular, in the above embodiments, R? to R17 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, notably a hydrogen atom, a (Ci-Ce)alkyl or an aryl group, typically a hydrogen atom, a (Ci-C3)alkyl or an aryl group, wherein the aryl group, which may be part of an aryl-(Ci-Ce)alkyl group, is preferably a phenyl group.

According to particular embodiments, R2 represents a hydrogen atom, CN, NO2, OR?, SRs, NR9R10, C(O)Rii, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRIGRI7, S(O)RS, SO2RS’, a (C1- Ce)alkyl, a (Ci-Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci- Ce)alkyl group, wherein said aryl or heterocyclyl group (which may be part of a aryl-(Ci- Ce)alkyl or heterocyclyl-(Ci-C6)alkyl group) is optionally substituted by one or more substituents, notably by onesusbtituent, selected from the group consisting of a halogen atom, CN, NO2, ORis, SR19, NR20R21, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl, notably a halogen atom, NO2, ORis, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular NO2 and ORis, and wherein R18 to R21 represent, independently of each other, a hydrogen atom or a (C1- Ce)alkyl group.

According to other particular embodiments, R2 represents a hydrogen atom, CN, NO2, OR7, SRs, NR9R10, C(O)Rii, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRIGRI7, a (Ci-Ce)alkyl, a (C1- Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents as defined above.

According to still other particular embodiments, R2 represents C(0)Rn, CO2R12, C(O)NRi₆Ri7, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom ORis, SR19, NR20R21, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, and wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group.

According to yet other particular embodiments, R2 represents CO2R12, C(O)NRieRi7, a (C1- Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, notably CO2R12, C(O)NRieRi7 or an aryl-(Ci- Ce)alkyl group, wherein said aryl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, ORis, SR19 and NR20R21, notably ORis, and wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group. In the above embodiments, the (Ci-Ce)alkyl group, which may be part of an aryl-(Ci-Ce)alkyl group or a heterocyclyl-(Ci-C6)alkyl group, is preferably a (Ci-C3)alkyl group.

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably saturated, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms, such as a morpholinyl, a pyridinyl or a piperazinyl group, notably a piperazinyl group.

In the above embodiments, R7-R10, R12, R-u and R16-R17 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, and Rn, Risand R15 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl- (Ci-Ce)alkyl, a (Ci-Ce)alkoxy, a (Ci-Ce)alkylamino or a di((Ci-Ce)alkyl)amino group, notably a hydrogen atom, a (Ci-Ce)alkyl, an aryl, a (Ci-Ce)alkylamino or a di((Ci-Ce)alkyl)amino group, in particular a (Ci-C3)alkyl, an aryl such as a phenyl, a (Ci-C3)alkylamino or a di((Ci- C3)alkyl)amino group.

In particular, in the above embodiments, R7 to R17 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, notably a hydrogen atom, a (Ci-Ce)alkyl or an aryl-(Ci-Ce)alkyl group, typically a hydrogen atom, a (Ci-C3)alkyl or an aryl group, wherein the aryl group, which may be part of an aryl-(Ci-Ce)alkyl group, is preferably a phenyl group.

According to other particular embodiments, R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, OR29, SR30, NR31R32, C(O)Rss, CO2R34, OC(O)R35, NR3GC(O)R37, C(O)NR3SR39, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, and wherein R29 to R39 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, or an aryl-(Ci-Ce)alkyl group, notably a hydrogen atom or a (Ci-Ce)alkyl group, typically a hydrogen atom or a (Ci-C3)alkyl group. According to other particular embodiments, R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, OR29, SR30, NR31R32, C(O)Rss, CO2R34, OC(O)R35, NR3GC(O)R37 or C(O)NR3SR39, wherein R29 to R39 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, or an aryl-(Ci-Ce)alkyl group, notably a hydrogen atom or a (C1- Ce)alkyl group. According to still other particular embodiments, Rs represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, OR29, SR30, NR31R32, OC(O)R35, NR3GC(O)R37, a heterocyclyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47 and a (Ci-Ce)alkyl a group, preferably, R3 represents a hydrogen atom, a halogen atom, CN, NR31R32, OC(O)R35, a heterocyclyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a OR44, SR45 and NR46R47, notably OR44, and wherein R29 to R37 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, or an aryl- (Ci-Ce)alkyl group, notably a hydrogen atom or a (Ci-Ce)alkyl group, typically a hydrogen atom or a (Ci-C3)alkyl group.

In the above embodiments, R44 to R47 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, notably a hydrogen atom or a (Ci-C3)alkyl group.

According to yet other particular embodiments, R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, OR29, SR30, NR31R32, C(O)Rss, CO2R34, OC(O)R35, NR3GC(O)R37 or C(O)NR3SR39, preferably a hydrogen atom, a halogen atom, OR29, SR30, NR31R32, OC(O)RS5 or NR₃₆C(O)R₃7, more preferably a hydrogen atom or OC(O)R35, wherein R29 to R37 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, or an aryl-(Ci-Ce)alkyl group, notably a hydrogen atom or a (Ci-Ce)alkyl group, typically a hydrogen atom or a (Ci-C3)alkyl group.

In some preferred embodiments, R3 represents a hydrogen atom, a halogen atom or a (C1- Ce)alkyl group, in particular a hydrogen atom, a halogen atom or a (Ci-C3)alkyl group, preferably a hydrogen atom or a halogen atom .

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably aromatic, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms, such as a pyridinyl, a pyrimidinyl, a pyrazolyl, a piperazinyl or a piperidinyl group, for instance a a pyridinyl, a pyrimidinyl or a pyrazolyl group. In the above embodiments, the (Ci-Ce)alkyl group, which may be part of an aryl-(Ci-Ce)alkyl group or a heterocyclyl-(Ci-C6)alkyl group, is preferably a (Ci-C3)alkyl group.

According to a particular embodiment of the present invention, R4 represents a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl group, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group (which may be part of a aryl-(Ci-Ce)alkyl or heterocyclyl-(Ci- Ce)alkyl group) is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NR₅IC(O)R₅2, C(O)NR53RS4, a (Ci-Ce)alkyl and a (C1- Ce)haloalkyl group, notably a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)R48, CO2R49, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, preferably C(O)R48 and a (C1- Ce)alkyl group.

According to another particular embodiment of the present invention, R4 represents a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl group, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents as defined above.

According to still another particular embodiment of the present invention, R4 represents a hydrogen atom, a halogen atom, OR29, SR30, NR31R32, a (Ci-Ce)alkyl, an aryl, or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NR53Rs4, a (Ci-Ce)alkyl and a (C1- Ce)haloalkyl group, in particular C(O)R48, CO2R49, C(O)NR53Rs4, a (Ci-Ce)alkyl and a (C1- Ce)haloalkyl group, preferably C(O)R48 and a (Ci-Ce)alkyl group.

According to yet another particular embodiment of the present invention, R4 represents a hydrogen atom, a halogen atom, NR31R32, a (Ci-Ce)alkyl, an aryl or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of C(O)R48, CO2R49, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, preferably C(O)R48 and a (C1- Ce)alkyl group.

In a preferred embodiment, R4 represents a hydrogen atom, a halogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom or a (Ci-C3)alkyl group, preferably a hydrogen atom.

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably saturated, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms, such as a piperazinyl, a piperidinyl, a pyridinyl, a pyrimidinyl or a pyrazolyl group, for instance a piperazinyl or a piperidinyl group. In the above embodiments, R29 to R32 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, said aryl group, which may be part of an aryl-(Ci-Ce)alkyl group, being preferably a phenyl and being optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR55, SR56, NR57R58, C(O)Rs9, CO2R60, OC(O)Rei, NRs2C(O)R63, C(O)NR64R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, advantageously a halogen atom, OR55, SR56, NR57R58, C(O)RS9, CO2R60, OC(O)Rei, NRG2C(O)R63, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, notably C(O)Rs9, CO2R60, C(O)NR64R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)Rs9, wherein R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl or an aryl group, notably an aryl group, preferably a phenyl group.

In the above embodiments, R44 to R54 represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl or an aryl group, notably an aryl group, preferably a phenyl group.

In the above embodiments, the (Ci-Cs)alkyl group, which may be part of an aryl-(Ci-Cs)alkyl group or a heterocyclyl-(Ci-Cs)alkyl group, is preferably a (Ci-C3)alkyl group.

According to a particular embodiment of the present invention, R4b represents a hydrogen atom, a halogen atom, OR29, SR30, NR31R32, a (Ci-Cs)alkyl, a (Ci-Cs)haloalkyl group, an aryl, a heterocyclyl, an aryl-(Ci-Cs)alkyl or a heterocyclyl-(Ci-Cs)alkyl group, wherein said aryl or heterocyclyl group (which may be part of a aryl-(Ci-Cs)alkyl or heterocyclyl-(Ci-Cs)alkyl group) is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NRSIC(O)RS2, C(O)NRS3RS4, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, notably a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NRs3Rs4, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, in particular C(O)R48, CO2R49, C(O)NRs3Rs4, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, preferably C(O)R48 and a (Ci-Cs)alkyl group.

According to a particular embodiment of the present invention, R4b represents a hydrogen atom, a halogen atom, OR29, SR30, NR31R32, a (Ci-Cs)alkyl, an aryl, or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)R48, CO2R49, C(O)NR53Rs4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, preferably C(O)R48 and a (Ci-Ce)alkyl group.

According to yet another particular embodiment of the present invention, R4b represents a hydrogen atom, a halogen atom, NR31R32, a (Ci-Ce)alkyl, an aryl or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of C(O)R48, CO2R49, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Cs)haloalkyl group, preferably C(O)R48 and a (Ci- Ce)alkyl group.

According to still another particular embodiment of the present invention, R4b represents a hydrogen atom, a halogen atom, a (Ci-Cs)alkyl group, OR29 or NR31R32, preferably a hydrogen atom, a halogen atom, a (Ci-C3)alkyl group or OR29, more preferably a hydrogen atom.

In a preferred embodiment, R4b represents a hydrogen atom, a halogen atom or a (C1- Ce)alkyl group, in particular a hydrogen atom or a (Ci-C3)alkyl group, preferably a hydrogen atom.

In the above embodiments, the aryl group, which may be part of an aryl-(Ci-Cs)alkyl group, is preferably a phenyl group.

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably saturated, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms.

In the above embodiments, R29 to R32 represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl, an aryl or an aryl-(Ci-Cs)alkyl group, said aryl group, which may be part of an aryl-(Ci-Ce)alkyl group, being preferably a phenyl and being optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR55, SR56, NR57R58, C(O)Rs9, CO2R60, OC(O)Rei, NRG2C(O)R63, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Cs)haloalkyl group, advantageously a halogen atom, OR55, SR56, NR57R58, C(O)RS9, CO2R60, OC(O)Rei, NRG2C(O)R63, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, notably C(O)Rs9, CO2R60, C(O)NR64R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)Rs9, wherein R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl or an aryl group, notably an aryl group, preferably a phenyl group. Preferably, R29 to R32 represent, independently of each other, a hydrogen atom or a (Ci-Cs)alkyl group, notably a hydrogen atom.

In a particular embodiment of the present invention, R4 is as defined above and R4b represents a hydrogen atom, a halogen atom, OR29 or NR31R32, wherein R29 to R32 represent, independently of each other, a hydrogen atom or a (Ci-Cs)alkyl group, notably a hydrogen atom, preferably R4b represents a hydrogen atom, a halogen atom or OR29, more preferably a hydrogen atom. In a particular embodiment of the present invention, Rs represents a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, a (Ci-Cs)alkyl, a (Ci-Cs)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR40, SR41 and NR42R43, an aryl, a heterocyclyl, an aryl-(Ci-Cs)alkyl or a heterocyclyl-(Ci-Cs)alkyl group, wherein said aryl or heterocyclyl group (which may be part of a aryl-(Ci-Cs)alkyl or heterocyclyl-(Ci-Cs)alkyl group) is optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NRSIC(O)RS2, C(O)NRS3RS4, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, notably a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NRS3RS4, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, in particular a halogen atom, OR44, SR45, NR46R47, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, notably a halogen atom, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, preferably a (C1- Cs)alkyl group.

In the above embodiment, R29 to R32 represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl, an aryl or an aryl-(Ci-Cs)alkyl group, said aryl group, which may be part of an aryl-(Ci-Cs)alkyl group, being preferably a phenyl and being optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of a halogen atom, CN, NO2, OR55, SR56, NR57R58, C(O)Rsg, CO2R60, OC(O)Rei, NRs2C(O)Rs3, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Cs)haloalkyl group, advantageously a halogen atom, OR55, SR56, NR57R58, C(O)RS9, CO2R60, OC(O)Rei, NRG2C(O)R63, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, notably C(O)Rs9, CO2R60, C(O)NR64Re5, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)Rs9, wherein R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl or an aryl group, notably an aryl group, preferably a phenyl group. Preferably, R29 to R32 represent, independently of each other, independently of each other, a hydrogen atom or a (Ci-Cs)alkyl group.

In another particular embodiment of the present invention, Rs represents a hydrogen atom, a halogen atom, a (Ci-Cs)alkyl, a (Ci-Cs)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents, notably one substituent, selected from the group consisting of OR40, SR41 and NR42R43, an aryl-(Ci-Cs)alkyl or a heterocyclyl-(Ci- Cs)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, notably a halogen atom, a (Ci-Cs)alkyl and a (Ci-Cs)haloalkyl group, preferably a (Ci-Cs)alkyl group.

In yet another particular embodiment of the present invention, Rs represents a hydrogen atom, a halogen atom, a (Ci-Cs)alkyl, a (Ci-Cs)haloalkyl group or a heterocyclyl-(Ci-Cs)alkyl group, said alkyl or haloalkyl group being optionally substituted by OR40, and said heterocyclyl being optionally substituted by one or more (Ci-Ce)alkyl group.

In a preferred embodiment, R5 represents a hydrogen atom, a halogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom or a (Ci-C3)alkyl group.

Preferably, R5 represents a hydrogen atom.

In the above embodiments, the heterocyclyl group, which may be part of a heterocyclyl-(Ci- Ce)alkyl group, is in particular a 5- or 6-membered, saturated, unsaturated (i.e. not aromatic) or aromatic, notably saturated, monocyclic group, in which the atoms of the ring comprise one or more, advantageously 1 to 3, heteroatoms selected from O, S and N, preferably O and N, the remainder being carbon atoms, such as a piperazinyl group.

In the above embodiments, R40 to R43 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, notably a hydrogen atom.

In the above embodiments, R44 to R54 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group, notably an aryl group, preferably a phenyl group.

In a particular embodiment of the present invention, Rs represents a hydrogen atom, a (C1- C3)alkyl, an aryl-(Ci-C3)alkyl or a -CH2-CH2-O-CH2-CH2-NH2 group, or a (Ci-Ce)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, SH, NH2, a (Ci-C3)alkoxy, a (Ci-C3)thioalkoxy and a (Ci-C3)alkylamino group, preferably Rs represents a hydrogen atom, a methyl, an ethyl, a benzyl, a -CH2-CH2-O-CH2- CH2-NH2 or a (Ci-C6)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, NH2 and SH, in particular Rs represents a hydrogen atom, a -CH2-CH2-O-CH2-CH2-NH2 or an ethyl group, notably an ethyl group.

In a preferred embodiment, Rs represents a hydrogen atom, a (Ci-C3)alkyl or a -CH2-CH2-O- CH2-CH2-NH2 group, in particular a hydrogen atom or a -CH2-CH2-O-CH2-CH2-NH2 group.

In a first aspect of the present invention,

js Y^' , X is N, Y is N(R2) and Z is C(H), and the inhibitor of a pharmaceutical composition according to the invention is thus of the following general formula (l.i):

wherein R2, R4, R4b, Rs and Rs are as defined in any one of the above embodiments.

In particular, Rs represents a hydrogen atom or a (Ci-Ce)alkyl group, preferably a (Ci-C3)alkyl group, notably a methyl or an ethyl group, advantageously Rs represents an ethyl group.

In a second aspect of the present invention,

, X is N(Ri), Y is N and Z is C(Rs), and the inhibitor of a pharmaceutical composition according to the invention is thus of the following general formula (l.ii.a):

wherein R1, R3, R4, R4b, Rs and Rs are as defined in any one of the above embodiments.

In particular, Rs represents a hydrogen atom, a (Ci-C3)alkyl such as an ethyl, a -CH2-CH2-O- CH2-CH2-NH2 or a (Ci-Ce)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, NH2 and SH, advantageously Rs represents a hydrogen atom, a -CH2-CH2-O-CH2-CH2-NH2 or a (Ci-C3)alkyl such as an ethyl group.

In a third aspect of the present invention,

, X is N(Ri), Y is N⁺(O') and Z is C(Rs), and the inhibitor of a pharmaceutical composition according to the invention is thus of the following general formula (l.ii.b):

In a fourth aspect of the present invention,

, X is N(Ri), Y is CH and Z is N, and the inhibitor of a pharmaceutical composition according to the invention is thus of the following general formula (l.ii.c):

wherein R1, R4, R4b, Rs and Rs are as defined in any one of the above embodiments.

In a fifth aspect of the present invention,

, X is N(Ri) and Y and Z are CH, and the inhibitor of a pharmaceutical composition according to the invention is thus of the following general formula (l.ii.d):

wherein Ri, R4, R4b, Rs and Rs are as defined in any one of the above embodiments.

In particular, Rs represents a hydrogen atom, a (Ci-Ce)alkyl group or an aryl-(Ci-Ce)alkyl group, preferably a hydrogen atom, a (Ci-C3)alkyl group or an aryl-(Ci-C3)alkyl group, notably a hydrogen atom, a methyl, an ethyl or a benzyl group, advantageously Rs represents a hydrogen atom, a methyl or a benzyl group, typically a hydrogen atom.

In a preferred embodiment, the inhibitor is a compound of the following general formula (l.iii):

wherein R3, R4, R4b, Rs and Rs are as defined in any one of the above embodiments, and wherein Y’ is N or CH.

In particular:

R3, R4, R4b and R5 represent, independently of each other, a hydrogen atom, a halogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom, a halogen atom or a (C1- C3)alkyl group, preferably a hydrogen atom or a halogen atom, and

Rs represents a hydrogen atom, a (Ci-C3)alkyl or a -CH2-CH2-O-CH2-CH2-NH2 group, in particular a hydrogen atom or a -CH2-CH2-O-CH2-CH2-NH2 group.

The regulated necrotic cell death inhibitor may be selected from the group consisting of compounds S1 to S4, represented below, and the pharmaceutically acceptable salts and/or solvates thereof.

In particular, the inhibitor of a pharmaceutical composition according to the invention may be selected from the group consisting of compounds S1 to S4 and the pharmaceutically acceptable salts and/or solvates thereof.

IV.2. Nigratine and derivatives thereof

In a particular embodiment, the inhibitor of a pharmaceutical composition according to the invention is nigratine (N1) or a derivative thereof.

In this particular embodiment, the inhibitor is preferably a compound of the following general formula (II):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

■ Xi, X₂ and X3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl or an OH group, or a group selected from ORx, SRx, SO₂Rx and NRxRz, wherein at least one of Xi, X2 and X3 represents a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group, or a group selected from ORx, SRx, SChRx and NRxRz, wherein Rx a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, Rz is a hydrogen atom or a (Ci-Ce)alkyl group, and the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORee, -NRGTRGS, -SRGS, -S(0)R?O, -SO2R71, -OCOR72, -CO2R73, ■ CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN);

■ Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl or an OH group, or a group selected from ORY, SRY, SOZRY and NRYRZ, wherein at least one of Y1, Y2 and Y3 represents a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group, or a group selected from ORY, SRY, SOZRY and NRYR’Z, wherein RY is a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group,

R’z is a hydrogen atom or a (Ci-C6)alkyl group, and the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORse, -NRGYRGS, -SRGS, -S(0)R7O, -SO2R71, -OCOR72, -CO2R73, ■ CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN); and

■ Res to R77 are, independently of one another, a hydrogen atom or a (Ci-C6)alkyl group.

According to a particular embodiment of the present invention, Xi, X2 and X3 represent, independently of each other, a hydrogen atom, or a group selected from ORx, SRx, SOzRx and NRxRz, wherein at least one of Xi, X2 and X3 is not a hydrogen atom.

In another particular embodiment of the present invention, Xi, X2 and X3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl group, an OH or an ORx group, wherein at least one of Xi, X2 and X3 represents an ORx group.

In still another particular embodiment of the present invention, Xi, X2 and X3 represent, independently of each other, a hydrogen atom or an ORx group, wherein at least one of Xi, X2 and X3 represents an ORx group.

In the above embodiments, Rx is preferably a (Ci-C6)alkyl group, notably a (Ci-C3)alkyl group such as methyl, ethyl, n-propyl, more preferably methyl.

In the above embodiments, the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORee, -NRGYRBS, -SRe9, -S(0)R7O, -SO2R71, -OCOR72, -CO2R73, -CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN).

In another embodiment, Xi represents a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group, or a group selected from ORx, SRx, SOzRx and NRxRz, wherein Rx is selected from a (Ci- Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, Rx being preferably a (Ci-Ce)alkyl group, notably a (Ci-C3)alkyl group such as methyl, ethyl, n-propyl, more preferably methyl; and X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, preferably a hydrogen atom.

In still another embodiment, Xi represents a group selected from ORx, SRx, SChRx and NRxRz, wherein Rx is selected from a (Ci-Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, Rx being preferably a (Ci-Ce)alkyl group, notably a (Ci-C3)alkyl group such as methyl, ethyl, n- propyl, more preferably methyl; and X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, preferably a hydrogen atom.

In a preferred embodiment, Xi represents an ORx group, Rx being advantageously a (C1- Ce)alkyl group.

In another preferred embodiment, X2 and X3 each represent a hydrogen atom.

In a yet another preferred embodiment, Xi represents an ORx group, wherein Rx is selected from a (Ci-C6)alkyl, an aryl and an aryl-(Ci-C6)alkyl group, Rx being advantageously a (Ci- Ce)alkyl group, notably a (Ci-C3)alkyl group such as methyl, ethyl, n-propyl, more advantageously methyl; and X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-C6)alkyl group, advantageously a hydrogen atom.

In the above embodiments, the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORse, -NRGTRBS, -SRGS, -S(0)R?O, -SO2R71, -OCOR72, -CO2R73, -CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN).

According to a particular embodiment of the present invention, Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, or a group selected from ORY, SRY, SO2RY and NRYR’Z, wherein at least one of Y1, Y2 and Y3 is not a hydrogen atom.

In another particular embodiment of the present invention, Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl group, an OH, or an ORY group, wherein at least one of Y1, Y2 and Y3 represents an ORY group.

In still another particular embodiment of the present invention, Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom or an ORY group, wherein at least one of Y1, Y2 and Y3 represents an ORY group.

In the above embodiments, RY is preferably a -(Ci-C6)alkyl-aryl group, such as benzyl or - CHs-naphtyl, more preferably benzyl.

In another embodiment, Y1 represents a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group, or a group selected from ORY, SRY, SO2RY and NRYR’Z, wherein RY is selected from a (Ci- Ce)alkyl, an aryl and an aryl-(Ci-C6)alkyl group, RY being preferably a -(Ci-C6)alkyl-aryl group, such as benzyl or -CHs-naphtyl, more preferably benzyl; and Y2 and Y3 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, preferably a hydrogen atom.

In still another embodiment, Yi represents a group selected from ORY, SRY, SO2RY and NRYR’Z, wherein RY is selected from a (Ci-Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, RY being preferably -(Ci-Ce)alkyl-aryl group, such as benzyl or -CHs-naphtyl, more preferably benzyl, and Y2 and Y3 represent, independently of each other, a hydrogen atom or a (C1- Ce)alkyl group, preferably a hydrogen atom.

In a preferred embodiment, Y1 represents an ORY group, wherein RY is selected from a (C1- Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, RY being advantageously a -(Ci-C6)alkyl-aryl group, such as benzyl or -CHs-naphtyl, more preferably benzyl; and Y2 and Y3 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, advantageously a hydrogen atom.

According to a particular embodiment of the present invention:

■ Xi, X2 and X3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl or an ORx group, wherein at least one of Xi, X2 and X3 is not a hydrogen atom, and wherein Rx is selected from a (Ci-Ce)alkyl, an aryl and an aryl- (Ci-Ce)alkyl group; and

■ Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl group, an OH or an ORY group, wherein at least one of Y1, Y2 and Y3 represents a (Ci-C6)alkyl, an aryl, an aryl-(Ci-C6)alkyl group or an OR_y group, wherein RY is selected from a (Ci-C6)alkyl, an aryl and an aryl-(Ci-C6)alkyl group.

According to another particular embodiment:

■ Xi represents a (Ci-C6)alkyl, an aryl, an aryl-(Ci-C6)alkyl or an ORx group, wherein Rx is a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group;

■ X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-C6)alkyl group; and

■ Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-C6)alkyl, an aryl, an aryl-(Ci-C6)alkyl, an OH or an ORY group, wherein at least one of Y1, Y2 and Y3 represents a (Ci-C6)alkyl, an aryl, an aryl-(Ci-C6)alkyl or an ORY group, wherein RY is selected from a (Ci-C6)alkyl, an aryl and an aryl-(Ci-C6)alkyl group.

According to yet another particular embodiment: ■ Xi represents an ORx group, wherein Rx is selected from a (Ci-Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, Rx being advantageously a (Ci-Ce)alkyl group, notably a (Ci- C3)alkyl group such as methyl, ethyl, n-propyl, more advantageously methyl;

■ X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, advantageously a hydrogen atom;

■ Y1 represents an ORY group, wherein RY is selected from a (Ci-Ce)alkyl, an aryl and an aryl-(Ci-Ce)alkyl group, RY being advantageously a -(Ci-Ce)alkyl-aryl group, such as benzyl or -CHs-naphtyl, more preferably benzyl; and

■ Y2 and Y3 each represent, independently of each other, a hydrogen atom or a (C1- Ce)alkyl group, advantageously a hydrogen atom.

According to a preferred embodiment, the inhibitor of a pharmaceutical composition according to the invention is of the following formula (II. i):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

Rx represents a (Ci-Ce)alkyl group, notably a (Ci-C3)alkyl group such as methyl, ethyl, n- propyl, more advantageously methyl, and

RY represents an aryl-(Ci-Ce)alkyl group, such as benzyl or -CHs-naphtyl, more preferably benzyl.

The inhibitor of a pharmaceutical composition according to the invention may notably be selected from the group consisting of compounds N1 to N4, represented below, and the pharmaceutically acceptable salts and/or solvates thereof.

In particular, the inhibitor of a pharmaceutical composition according to the invention is compound N1 or a pharmaceutically acceptable salt and/or solvate thereof.

FIGURES

Figure 1 : Morphological parameter retinal pigment epithelia (ARPE-19) cell differentiated with compounds of formula (I) or (II) or known ferroptosis inhibitors and sodium iodate. Representative phase contrast images of proliferative ARPE-19 cells in culture medium to JO (A). Representative phase contrast images of proliferative ARPE-19 cells in culture medium for 5 days (B). Representative phase contrast images of human retinal pigment epithelia cell ARPE-19 cultured for 5 days with 10 mM sodium iodate and compound S1 (C), compound S3 (D), compound N1 (E), Resveratrol (F), Ferrostatin 1 (G). Scale bars represent 400pm for all images.

Figure 2: Histogram representing the length of the ARPE-19cell neurite not differentiated or differentiated by the compounds of formula (I) or (II) and sodium iodate after processing phase microscopy images by the neuronJ (Fiji) software. (A) compound S1- (B) compound S3 -(C) compound N1-(D) Resveratrol-(E) Ferrostatin— Bars represent the mean ± SEM (Standard error of the mean) using Student’s f-test . ****p < 0.0001 ***, p < 0.002

Figure 3: A - Representative confocal image of ARPE-19 undifferentiated (CTRL) and differentiated after three days of treatment with compound S1 and sodium iodate. After treatment, cells were fixed and immunostained with pill Tubulin antibody (green) and DAPI (blue). B - Histograms representing relative intensity of pill Tubulin with respect to the number of nuclei.

Figure 4: A - Representative confocal image of ARPE-19 undifferentiated (CTRL) and differentiated after six days treatment with compound S1 + sodium iodate treatment, compound N1+sodium iodate treatment or compound S3+ sodium iodate. Cells were also treated with a known nucleic acid (nuclear) staining Hoechst 33342, tri chlorhydrate, trihydrate (HOECHST) and a cell-permeable calcium ion indicator Fluo-4 acetoxymethyl ester. B - Histogram representing the relative fluorescence of Fluo-4 acetoxymethyl ester (a cell-permeable fluorescent calcium indicator, also known a fluo 4AM F14201 , commercially available from Invitrogen) of the ARPE-19 cell -Bars represent the mean ± SEM using Student’s t-test. ***, p < 0.002, **, p < 0.001

Figure 5: A - Representative confocal image of ARPE-19 undifferentiated (CTRL) and differentiated cells after six days treatment compound S1 and sodium iodate treatment, compound N1 + sodium iodate treatment or compound S3 and sodium iodate, after 0, 10s or 60s light exposure. B - Histogram representing the Integrated optical density (IOD) ratio (lODt/IODo) of fluo 4AM.

Figure 6: Morphological parameter of human neuroblastoma SH-SY5Y cells differentiated with compounds of formula (I) or (II) or known ferroptosis inhibitors and Erastin (ERA). Representative phase contrast images of proliferative SH-SY5Y cells in culture medium to JO (A). Representative phase contrast images of proliferative SH-SY5Y cells in culture medium for 2 days (B). Representative phase contrast images of human neuroblastoma cell line differentiated SH-SY5Y cultured for 5, 6 or 7days (F) with 10 pM erastin and compound S1 (C), compound S6 (D), S5 (E), compound S2 (F), compound S7 (G), compound S9 (H), compound S8 (I), NEC1 F (I), Ferrostatin 1 (K), Resveratrol (L), Liproxstatin (M). Scale bars represent 400pm for all images. Compounds S5 to S9 are inhibitors of formula (I)

Figure 7: Histogram representing the length of the shsy-5y cell neurite not differentiated or differentiated by the compounds of formula (I) or (II) and erastin (ERA) after processing phase microscopy images by the neuronJ (Fiji) software. (A) compound S1- (B) compound S6-(C) compound S5-(D) compound S2- (E) compound S9-(F) compound S7- (G) compound S8- (H) NEC1 F- (I) Resveratrol- (J) ferrostatin 1- (K) liproxstatin - Bars represent the mean ± SEM using Student’s t-test. ****p < 0.0001 ***, p < 0.002, **p < 0.01 , *p < 0.05.

Figure 8: Analysis of the gene expression of certain neuronal markers in differentiated cells after five days of treatment of SH-SY5Y cells with compound S1 and erastin. (A) Expression levels are shown as increases or decreases relative to undifferentiated cells. The fold change in gene expression relative to undifferentiated cells was calculated using B2M, PPI A, and GAPDH simultaneously as reference genes and using the delta-delta Ct method - Bars represent the mean ± SEM using Student’s t-test. ****p < 0.0001 ***, p < 0.002, **p < 0.01 , *p < 0.05. (B) Gene expression of neuronal markers at five days of differentiation. The circles at the center of each network represent a centered hierarchical structure visualizing the key processes in which the connected genes are involved. For example, SYP is a neuronal marker and more specifically involved in the synaptic secretion process. increased expression levels are shown in black and reduced expression levels in grey.

Figure 9: A - Representative confocal image of SH-SY5Y undifferentiated (CTRL) and differentiated after five days of treatment with compound S1 and erastin (ERA). After treatment, cells were fixed and immunostained with pill Tubulin antibody (green DNA stain) and DAPI (4',6-diamidino-2-phenylindole, blue-fluorescent DNA stain, commercially available from Invitrogen as Alexa Fluor 488, FITC, GFP). B - Histograms representing relative intensity of pill Tubulin with respect to the number of nuclei.

Figure 10: A - Representative confocal image of SH-SY5Y undifferentiated (CTRL) and differentiated after five days of treatment with compound S1and erastin. After treatment, cells were fixed and immunostained with Tyrosine hydroxylase (green) and DAPI (blue). B - Histograms representing relative intensity of tyrosine hydroxylase with respect to the number of nuclei.

Figure 11 : A - Representative confocal image of SH-SY5Y undifferentied (CTRL) and differentiated after six days of treatment with compound S1 + erastin (ERA), differentiated after seven days of treatment with compound S2 and erastin, differentiated after seven days of treatment with compound S9 and erastin. B - Histogram representing the relative fluorescence of fluo 4AM of the shsy-5y cell -Bars represent the mean ± SEM using Student’s t-test . **, p < 0.01.

Figure 12: Histogram representing cell death of the SH-SY5Y cells differentiated with compounds of erastin and S1 for 7 days and 24h of Parkinson treatment with rotenone, after processing phase microscopy images by the neuronJ (Fiji) software. (A) rotenone 0.05 pM - 0.5 pM supplemented or not of S1 at 20 pM - Bars represent the mean ± SEM using Student’s t-test. ****p < 0.0001 ***, p < 0.002, **p < 0.01 , *p < 0.05. ROT stands for rotenone. Figure 13: Morphological parameter of human neuroblastoma SH-SY5Y cells differentiated with compounds of erastin and S1 for 7 days and 24h of Parkinson treatment with rotenone. Representative phase contrast and IP stained images of human neuroblastoma cell line differentiated stained with propidium iodide, (a) CTRL - (b) S1 - (c) rotenone 0.05 pM - (d) rotenone 0.1 pM - (e) rotenone 0.2 pM - (f) rotenone 0.4 pM - (g) rotenone 0.5 pM - (h) rotenone 0.05 pM with S1 20pM - (i) rotenone 0.1 pM with S1 20 pM - (j) rotenone 0.2 pM with S1 20 pM - (k) rotenone 0.4 pM with S1 20 pM - (I) rotenone 0.5 pM with S1 20 pM. Scale bars represent 400 pm for all images. ROT stands for rotenone.

Figure 14: Histogram representing the length of the SH-SY5Y cells differentiated with compounds of erastin and S1 for 7 days and 24h of Parkinson treatment with rotenone, after processing phase microscopy images by the neuronJ (Fiji) software. (E) rotenone 0.05 pM - 0.5 pM supplemented or not of S1 at 20 pM - Bars represent the mean ± SEM using Student’s t-test. ****p < 0.0001 ***, p < 0.002, **p < 0.01 , *p < 0.05. ROT stands for rotenone. Figure 15: Morphological parameter of human neuroblastoma SH-SY5Y cells differentiated with compounds of erastin and S1 for 7 days and 24h of Parkinson treatment with rotenone. Representative phase contrast images of human neuroblastoma cell line differentiated. (A) CTRL - (B) S1 - (C) rotenone 0.05 pM - (D) rotenone 0.1 pM - (E) rotenone 0.2 pM - (F) rotenone 0.4 pM - (G) rotenone 0.5 pM - (H) rotenone 0.05 pM with S1 20 pM - (I) rotenone 0.1 pM with S1 20 pM - (J) rotenone 0.2 pM with S1 20 pM - (K) rotenone 0.4 pM with S1 20 pM - (L) rotenone 0.5 pM with S1 20 pM. Scale bars represent 400 pm for all images. ROT stands for rotenone.

16: Representative confocal image of SH-SY5Y undifferentiated (CTRL) and differentiated after 7 days of treatment with compound of erastin and S1. At 7 days of differentiation treatment the cells were treated for 24 hours with rotenone at 0.1 pM alone or co-treated with 20 pM of S1. After treatment, cells were fixed and immunostained with tyrosine hydroxylase (green) and DAPI (blue). (1) confocal image of tyrosine hydroxylase, (2) confocal image of DAPI, (3) confocal image of tyrosine hydroxylase and DAPI: MERGE. ROT stands for rotenone.

17: Histograms representing relative intensity of tyrosine hydroxylase with respect to the number of nuclei. ROT stands for rotenone.

EXAMPLES

The following examples are given for illustrative purposes only. They should not be construed as limiting the invention in any way.

The cell differentiation induced by regulated necrosis cell death inhibitors was evaluated in cellular models of pathologies associated with regulated necrotic cell death using different inducers.

Compounds S1 to S4 have already been described (see e.g. WO2022157392, EP3362450 or EP3362449). Compounds S5 to S9 are all compounds of formula (I). They could be prepared following the methods described in WO2022157392, EP3362450 or EP3362449.

Compound N1 has already been described (see e.g. WO 2018/073321 ; Delehouze et al. (Sci. Report, 2022).

Material and Methods

Cell culture o The SH-SY5Y neuronal cells (Human Neuroblastoma Cell line) were maintained in standard DMEM with GlutaMAX medium (GIBCO), supplemented with 10% fetal bovine serum (GIBCO), at 37 °C in presence of 5% CO2. o The ARPE-19 cells (Human Retinal pigment epithelial cell line) were cultured in standard DMEM/F12 medium (GIBCO), supplemented with 10% fetal bovine serum (GIBCO), at 37 °C in presence of 5% CO2.

Example 1

SH-SY5Y and ARPE-19 cells were seeded in 96-well plates at a density of 5000 cells per well, following overnight incubation. Cells were treated with 10 pM (SH-SY5Y cells) of erastin or 10 mM of sodium iodate (ARPE-19 cells) for 7 days.

Erastin was purchased from Selleck Chemical and sodium iodate from Sigma Aldrich. Cell viability was assessed by MTS assay (CellTiter 96® aQueous Non-Radioactive Cell Proliferation Assay; Promega, Fitchburg, Wl, USA) according to the manufacturer’s instructions. This assay is based on the reduction of the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) by viable cells to form a colored formazan product. After treatment, cells were incubated 3 h at 37 °C, 5% CO2 with MTS. The absorbance was measured using a microplate reader at 490 and 630 nm and the percentage of viability was calculated by dividing the absorbance of testing compound by the absorbance of DMSO treated cells (control).

Analysis of differentiation effects of molecules

Cells (SHSY-5Y and ARPE 19) were seeded in a 96-well plate or in a 6-well plate in their respective medium overnight. The next day they were treated with a regulated necrotic cell death inhibitor compound and a regulated necrotic cell death inducer. The analysis of cell morphology was carried out after processing phase microscopy (Incucyte) images by the Neurone J (Fiji) software. In a second stage, immunohistochemistry studies were carried out to highlight the expression of neural markers

• SH-SY5Y

Neuronal differentiation protocol developed for SH-SY5Y cell line requires only one work step and 3 to 5 days of incubation depending on inhibitor compound. After differentiation, cells present increased levels of beta III Tubuline (said protein is primarily expressed in neurons and may be involved in neurogenesis and axon guidance and maintenance) and tyrosine hydroxylase (said protein is a marker for dopamine neurons). The immortalized and proliferative cell line SH-SY5Y is one of the most commonly used cell line in neuroscience and neuroblastoma research. However, undifferentiated SH-SY5Y cells share few properties with mature neurons and are typically locked in an early neuronal differentiation stage, characterized biochemically by the low presence of neuronal markers (Biedler et al., 1978, Gilany et al., 2008). In spite of that, surprisingly the inventors have managed to differentiate human neuroblastoma SH-SY5Y cells to a neuronal-like phenotype. Indeed, by the induction of ferroptosis in vitro to mimic in vivo damaged in several neurodegenerative diseases, and its inhibition with regulated necrotic cell death inhibitors, the human neuroblastoma SH-SY5Y cells are able to acquire neuron-like phenotypes with neurite outgrowth and branches. The inventors have observed a complete decrease in cellular proliferation rate and the induction of extensive neurites outgrowth by the differentiation protocol of SH-SY5Y cells developed. This change in morphology was observed as early as 24 h of treatment. • ARPE19

The retinal pigment epithelium (RPE), located between the neurosensory retina and the choroid, is a monolayer of cells vital for the maintenance of photoreceptor cell function and integrity. The RPE also plays a critical part in the development of the retina and in some species participates in the regeneration of functional neural retinal tissue after injury through RPE cell dedifferentiation, proliferation, and redifferentiation into the cellular elements of the retina (a process termed transdifferentiation). To regenerate a complete retina, RPE cells must be able to transdifferentiate into several cell types including neurons and glial cells. These findings suggest that a mechanism to activate transdifferentiation toward neural cells might exist, at least in part, in human RPE cells and raises the possibility that RPE could, with the appropriate manipulation, be induced to produce new retinal cell types, replacing those lost as a result of degenerative disease. Following 3 days of treatment with 25pM of regulated cell death inhibitors, cells were morphologically similar to neurons, with an elongated cell body and the formation of neurite branching.

Regulated necrotic cell death inhibitors could provide a new and effective approach to the treatment of a number of currently poorly addressed degenerative conditions which affect, inter alia, for example the eyes, brain, heart, and liver.

• Statistical Analysis

Results were expressed as means ± SEM. Mean differences between two experimental groups were assessed using the non-parametric Student’s t-test. All statistical analyses were achieved with the GraphPad Prism 9.5 software. Calculated P values are integrated on histograms and graphs. Significance is shown as follows: * P < 0.05, ** P < 0.01 , *** P < 0.001 and **** P < 0.0001 (compare between control group and treated groups).

Results

2D cellular models of disease associated with regulated necrotic cell death

The cell differentiation induced by regulated necrosis inhibitors was evaluated in cellular models of pathologies associated with regulated necrotic cell death using different inducers. Regulated necrosis is involved in several pathologies or dysfunctions of the organism and notably in liver damage linked to drug toxicity. Regulated necrosis is also involved in the pathophysiology of degenerative diseases, such as retinal degeneration or neurodegenerative diseases such as Parkinson's disease, or in neurological disorders associated with excitotoxicity such as trauma or stroke.

Model of eye disease

Age-related macular degeneration (hereinafter AMD), is characterized by vision loss caused by degeneration of the central cells of the retina, called the macula. Oxidative stress has been shown to play an important role in retinal cell loss through the initiation of non-apoptotic cell death, including ferroptosis. One model used to study retinal cell death is that of human ARPE-19 cells, a retinal pigment epithelial cell line, in the presence of sodium iodate (NalCh, a regulated necrotic cell death inducer able to induce ferroptosis but also necroptosis).

FIGURES 1 and 2 specifically shows that the compounds of formula (I) or (II) as well as Resveratrol and Ferrostatin promote outgrowth neurite in ARPE-19 cells.

FIGURE 3 more specifically demonstrates that compounds of formula (I) or (II) increase the neuronal marker pill Tubulin.

FIGURE 4 shows that the compounds of formula (I) or (II) allow establishing highly specialized physiological traits specific to photoreceptors in ARPE-19 cells.

FIGURE 5 more specifically shows that compounds of formula (I) or (II) induce membrane hyperpolarization of photoreceptors in response to light in ARPE-19 cells.

To conclude, the treatment with Compound S1 , or compound S3 or compound N1 or Resveratrol or Ferrostatin 1 not only protected ARPE-19 cells from regulated necrotic cell death induced by sodium iodate but also induced differentiation of these cells towards a neuron-like phenotype.

Neurotoxicity and excitotoxicity models

The biological activity of interest was demonstrated in the SH-SY5Y neuronal cell line, a human neuroblastoma cell line. Regulated necrotic cell death was induced by erastin in this cell line. Erastin is a well-described ferroptosis inducer and also a molecular tool for studying neuronal pathologies.

Figures 6 and 7 shows that the compounds of formula (I) or (II) promote outgrowth neurite in SH-SY5Y cells.

Figure 8 shows that the compounds of formula (I) or (II) promote the expression of neuronal markers. Analysis of the gene expression of certain neuronal markers revealed an increase in these markers in differentiated cells after five days of treatment. A significant increase in Synaptophysin was observed, linked to the synaptic secretion system. There was also an increased expression of markers linked to dopaminergic function, such as KCNJ6, RET and NR4A2. On the contrary, a decrease of the expression of SOX2 was observed (SOX2 expression being known to be a marker of pluripotent neural stem cells).

Figure 9 shows that the compounds of formula (I) or (II) increase the neuronal marker pill Tubulin.

Figure 10 shows that the compounds of formula (I) or (II) increase the neuronal marker tyrosine hydroxylase.

Figure 11 demonstrates that the compounds of formula (I) or (II) allow the establishment of highly specialized physiological traits specific to neuron like in shsy-5y cells.

The results obtained thus clearly showed the effectiveness of compound S1 (ferroptosis and necroptosis inhibitor), compound S2 (ferroptosis inhibitor), compound S5 (ferroptosis inhibitor), compound S6 (ferroptosis inhibitor), compound S9 (ferroptosis inhibitor), compound S7 (ferroptosis and necroptosis inhibitor), compound S8 (ferroptosis inhibitor), NEC1 F (ferroptosis and necroptosis inhibitor), Resveratrol (ferroptosis inhibitor), Ferrostatin 1 (ferroptosis inhibitor), and Liproxstatin (ferroptosis inhibitor) on these neurotoxicity models as well as an induction of neuronal differentiation.

Conclusion

The results describe notably a new method and a new therapeutic approach using regulated necrotic cell death inhibitors, in particular the compounds of formula (I) or (II). The data showed that when the inhibitor is used to protect cells from regulated necrosis death such as necroptosis and/or ferroptosis it can induce the differentiation of the cells to specialized cells, in particular to neurons. The treatment of the cells by necrotic cell death inhibitors, in particular of formula (I) or (II) as defined herein, under a pathological -like context, produces cell protection effects and its differentiation.

The invention can be used in vitro to induce the differentiation of cells to specialized cells, in particular for inducing neuronal differentiation and improve the development of more relevant in vitro models or cell differentiation kits.

The invention can also be used as a therapeutic approach in particular for inducing differentiation of pre-necrotic cells, i.e. cells wherein regulated necrosis pathways have been activated, either naturally (for instance in the case of a disease or disorder associated with regulated necrotic cell death) or typically by addition of a regulated necrotic cell death inducer (for instance in the case of cancer therapy by targeting tumor cells with a regulated necrotic cell death inducer).

In particular, the invention can be used as a curative approach for the first time for the treatment of a disease or disorder associated with regulated necrotic cell death, in particular degenerative diseases and severe diseases for which cells are difficult to regenerate, in particular neurodegenerative diseases or disorders and retinal degenerative diseases or disorders. This therapeutic strategy is therefore a promising tool to improve the management of patients suffering from severe pathologies such as Alzheime”s disease, Parkinson's disease, prion disease, Amyotrophic lateral sclerosis, Friedreich ataxia, motor neuron disease, Huntington's disease, spinal muscular atrophy, and spinocerebellar ataxia, glaucoma, macular degeneration (MD) (including age-related macular degeneration), retinitis pigmentosa (RP), retinoblastoma, diabetic retinopathy (DR).

In particular, the invention can be used as a therapeutic approach in the treatment of cancer.

Example 2

Material and Methods

Cell culture o The SH-SY5Y neuronal cells (Human Neuroblastoma Cell line) were maintained in standard DMEM with GlutaMAX medium (GIBCO), supplemented with 10% fetal bovine serum (GIBCO), at 37 °C in presence of 5% CO2.

Propidium Iodide (PI) staining

SH-SY5Y cells were seeded in 96-well plates at a density of 5000 cells per well, following overnight incubation. Cells were treated with 10 pM (SH-SY5Y cells) of erastin for 7 days.

Erastin was purchased from Selleck Chemical from Sigma Aldrich. PI staining was assessed by Propidium Iodide (Invitrogen) at 0.5 pg/mL per well. PI staining is a method to assess cell viability and detect dead cells. PI is a small molecule that does not cross the cell membranes of living cells due to their impermeability to this compound. When cells are damaged or dead, their cell membrane becomes permeable, allowing PI to penetrate inside the cell, to interact specifically with nucleic acids. Once bound to DNA, PI emits an intense red fluorescence. After treatment, cells were incubated 15 min at 37°C, 5% CO2 with PI at 0.5 pg/mL per well. The fluorescence was measured by Incucyte S3 and the ratio of red to total cell confluence was calculated.

Cell-based assays for Parkinson's disease using differentiated SH-SY5Y

SH-SY5Y were incubated in a differentiation medium containing compounds S1 and Erastin for 7 days, and then differentiated cells were treated with Rotenone for 24 hours alone or rotenone with compounds S1. Cell death was analyzed after 24 hours of treatment using the propidium iodide immunofluorescence technique. The morphological state of the cells was observed by calculating the length of the neurites. Levels of specific marker protein in dAergic neurons (tyrosine hydoxylase) in cells were analyzed using the immunofluorescence technique.

Statistical Analysis

Results were expressed as means ± SEM (standard error of measurement). Mean differences between two experimental groups were assessed using the non-parametric Student’s t-test. All statistical analyses were achieved with the GraphPad Prism 9.5 software. Calculated P values are integrated on histograms and graphs. Significance is shown as follows: * P < 0.05, ** P < 0.01 , *** P < 0.001 and **** P < 0.0001 (compare between control group and treated groups).

Results Parkinson model

Figures 12 and 13 show that rotenone induces neuronal like cell death of human neuroblastoma SH-SY5Y cells differentiated with a compound of formula (I) and Erastin (ERA). The treatment with compound S1 protects differentiated cells from rotenone-induced death.

Figures 14 and 15 show that the different concentrations of rotenone on neuronal cells (differentiated from SH-SY5Y cells with a compound of formula (I) and Erastin (ERA)) cause a decrease in neurite size. The treatment with compound S1 allows a maintenance of the length of nerites in these differentiated cells.

Figures 16 and 17 show that rotenone at 0.1 pM induces a decrease in the expression of the dopaminergic neuronal marker tyrosine hydroxylase in neuron cells ((differentiated from SH- SY5Y cells with a compound of formula (I) and Erastin (ERA)). This is restored with the treatment with compound S1 .

Conclusion

The SH-SY5Y cells differentiated into a neuron like via erastin and compound S1 treatment were used as a model of Parkinson’s disease using the specific inhibitor of complex 1 of the mitochondrial respiratory chain, rotenone. In fact, in vitro rotenone is known to mimic the pathological effects of Parkinson’s disease and is therefore widely used to induce a parkinsonian model.

In this study, the inventors were able to show that the cells treated with erastin and compound S1 allowed the obtaining of a neuron-like model that is also a very good model for studying Parkinson’s disease. Indeed, the use of rotenone on neuron like causes cell death, a decrease in neurite size and expression of tyrosine hydroxylase.

In addition to having demonstrated here that this model is relevant for neurodegenerative diseases of such as Parkinson’s disease, the inventors also demonstrate the protective properties of the S1 compound.

Claims

1 . A method for inducing cell differentiation, comprising contacting in vitro or ex vivo at least one cell to be differentiated with an effective amount of a regulated necrotic cell death inducer and of a regulated necrotic cell death inhibitor, to obtain a differentiated cell, wherein the cell to be differentiated has not been obtained by a method involving the destruction of human embryos.

2. The method of claim 1 , wherein the at least one cell to be differentiated is a nonspecialized cell or a neuroblast or a retinal pigment epithelium cell.

3. The method of claim 1 or 2, wherein the cell to be differentiated is a mammal cell, preferably a human cell.

4. The method of any of claims 1 to 3, wherein the differentiated cell is a specialized cell, advantageously a neuron.

5. The method of any one of claims 1 to 4, for inducing neuronal differentiation.

6. Use of a kit for inducing cell differentiation in vitro or ex vivo, the kit comprising: a regulated necrotic cell death inducer, and a regulated necrotic cell death inhibitor.

7. The method of any of claims 1 to 5, or the use of claim 6, wherein the regulated necrotic cell death inducer is a ferroptosis inducer, such as sodium iodate, erastin, RSL3, ML162, ML210, 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP+), cisplatin, rotenone, FIN56, Sorafenib, buthionine sulfoximine, a salt or solvate thereof, or mixtures thereof.

8. A regulated necrotic cell death inhibitor for use as a medicament for inducing differentiation of pre-necrotic cells of a patient in need thereof.

9. The regulated necrotic cell death inhibitor for use according to claim 8, wherein said patient suffering from an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, more particularly a degenerative affection or disease.

10. The regulated necrotic cell death inhibitor for use according to claim 8, wherein the patient is pretreated or co-treated with a regulated necrotic cell death inducer, in particular the patient is suffering from cancer.

11 . The regulated necrotic cell death inhibitor for use according to claim 8, in combination with a regulated necrotic cell death inducer, in particular for a simultaneous, separate or sequential use.

12. A regulated necrotic cell death inhibitor for use for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, by inducing differentiation of pre-necrotic cells of a patient in need thereof, wherein the affection or disease is preferably selected from the group consisting of: degenerative affection or disease, vision loss, stroke, traumatic brain injury, infectious diseases, autoimmune diseases, inflammatory diseases, type I diabetes, liver injury, kidney injury, heart injury, myocardial infarction, aortic aneurysm, atherosclerosis, chronic obstructive pulmonary disease, acute respiratory distress disorder, liver fibrosis, pancreatitis, diseases related to transplantation, in particular from the group consisting of neurodegenerative diseases or disorders and retinal degenerative diseases or disorders.

13. A kit comprising: c) A first therapeutic agent, which is a regulated necrotic cell death inducer, and d) A second therapeutic agent, which is a regulated necrotic cell death inhibitor, for separate, simultaneous, or sequential use of the first and the second therapeutic agents, as medicament, in particular for inducing differentiation of pre-necrotic cells of a patient in need thereof or for treating an affection or disease associated with regulated necrotic cell death, in particular ferroptosis, advantageously by inducing differentiation of pre-necrotic cells.

14. The method of any of claims 1 to 5 and 7, or the use of claims 6 and 7, or the regulated necrotic cell death inhibitor for use of any of claims 8 to 12, or the kit for use of claim 13, wherein the regulated necrotic cell death inhibitor is: a) a compound of general formula (I)

(I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

(iii) when Y^^X is Y'^^^X _I

X is N, Y is N(R₂) and Z is C(H);

(iv) when Y^’X is Y^^x ,

- X is N(Ri), and

- Y is N or N⁺(O’) and Z is C(R₃), or

Y is CH and Z is N, or

Y and Z are CH; and wherein:

■ Ri and R₂ represent, independently of each other, a hydrogen atom, CN, NO₂, OR?, SRs, NR9R10, C(O)Rii, CO₂Ri₂, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRieRi7, S(O)RS, SO₂RS’, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl, a -(Ci-C6)alkyl-[O-(Ci-C6)alkyl]_m-NRNi N62 group with m ranging from 1 to 6, an aryl, an aryl-(Ci-Ce)alkyl, a heterocyclyl, or a heterocyclyl-(Ci- Ce)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO₂, ORis, SR19, NR₂OR₂I, C(O)R₂₂, CO₂R₂3, OC(O)R₂₄, NR₂SC(O)R₂6, C(O)NR₂?R₂8, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ R3, R₄, R₄b and R5 represent, independently of each other, a hydrogen atom, a halogen atom, CN, OR₂9, SR30, NR3iR₃₂, C(O)R₃3, CO₂Rs₄, OC(O)Rs5, NR3sC(O)R37, C(O)NR3SR39, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR₄O, SR₄I and NR₄₂R₄₃, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl- (Ci-Ce)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO₂, OR₄₄, SR₄5, NR₄SR₄7, C(O)R₄8, CO₂R₄9, OC(0)RSO, NR₅IC(O)R₅2, C(O)NR₅₃R₅₄, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group;

■ Re represents a hydrogen atom, a (Ci-Ce)alkyl, an aryl-(Ci-Ce)alkyl, a heterocyclyl-(Ci- Ce)alkyl, a -(Ci-C6)alkyl-[O-(Ci-C6)alkyl]_m-NRN’i N’2 group with m’ ranging from 1 to 6, or a (Ci-Ce)alkylcarbonyl group, said (Ci-Ce)alkyl, aryl-(Ci-Ce)alkyl, and (C1- C6)alkylcarbonyl group being optionally substituted with one or more substituents selected from the group consisting of OH, SH, NH₂, a (Ci-Ce)alkoxy, a (Ci-Ce)thioalkoxy, a (Ci-Ce)alkylamino and a di((Ci-Ce)alkyl)amino group; ■ Rs and Rs’ represent, independently of each other a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group;

■ R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, aryl- (Ci-Ce)alkyl group or an aryl group; and

■ RNI, RN’1 , RN2 and RN’2 represent, independently of each other, a hydrogen atom, a (Ci- Ce)alkyl, an aryl-(Ci-Ce)alkyl group or an aryl group; or

(b) a compound of the following general formula (II):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

■ Xi, X2 and X3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl or an OH group, or a group selected from ORx, SRx, SO₂Rx and NRxRz, wherein at least one of Xi, X2 and X3 represents a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group, or a group selected from ORx, SRx, SO₂Rx and NRxRz, wherein

Rx a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group, Rz is a hydrogen atom or a (Ci-Ce)alkyl group, and the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORee, -NRGTRGS, -SRGS, -S(0)R?O, -SO2R71, -OCOR72, -CO2R73, ■ CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN);

■ Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl, an aryl-(Ci-Ce)alkyl or an OH group, or a group selected from ORY, SRY, SO2RY and NRYR'Z, wherein at least one of Y1, Y2 and Y3 represents a (Ci-Ce)alkyl, an aryl or an aryl-(Ci- Ce)alkyl group, or a group selected from ORY, SRY, SO2RY and NRYR’Z, wherein RY is a (Ci-C6)alkyl, an aryl or an aryl-(Ci-C6)alkyl group, R’z is a hydrogen atom or a (Ci-C6)alkyl group, and the aryl groups are optionally substituted with one or several groups selected from a halogen atom, -ORse, -NRGYRGS, -SRGS, -S(0)R7O, -SO2R71, -OCOR72, -CO2R73, ■ CONR74R75, -CO2R76, nitro (-NO2) and cyano (-CN); and

15. The method or the use or the kit for use or the regulated necrotic cell death inhibitor for use of claim 14, wherein the regulated necrotic cell death inhibitor is a compound of formula (I), wherein:

■ R1 represents a hydrogen atom, CN, NO2, OR7, SRs, NR9R10, C(0)Rn, CO2R12, OC(O)Ri3, NRi₄C(O)Ri5, C(O)NRi₆Ri7, S(O)RS, SO₂RS’, a (Ci-C₆)alkyl, a (C1- Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one substituent selected from the group consisting of a halogen atom, CN, NO2, ORis, SR19, NR20R21, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group and Rs and Rs’ represent, independently of each other a (Ci-Ce)alkyl or an aryl group, preferably an aryl group, preferably, R1 represents a hydrogen atom, CN, OR7, C(0)Rn, CO2R12, OC(O)Ri3, SChRs’, a (Ci-Ce)alkyl, a heterocyclyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said heterocyclyl group is optionally substituted by NO2, more preferably, R1 represents a hydrogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom or a (Ci-C3)alkyl group, advantageously a hydrogen atom;

■ R2 represents C(0)Rn, CO2R12, C(O)NRisRi7, a (Ci-Ce)alkyl, a (Ci-Ce)haloalkyl, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom ORis, SR19, NR20R21, a (Ci-Ce)alkyl and a (Ci- Ce)haloalkyl group, wherein Ris to R21 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, preferably R2 represents CO2R12, C(O)NRisRi7 or an aryl-(Ci-Ce)alkyl group, wherein said aryl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR18, SR19 and NR20R21, notably ORis;

■ R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, OR29, SR30, NR31R32, C(O)RS3, CO2R34, OC(O)RS5, NR₃₆C(O)R37, C(O)NR₃₈R39, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, a (Ci-Ce)alkyl and a (C1- Ce)haloalkyl group, wherein R29 to R39 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, and R44 to R47 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, preferably R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, OR29, SR30, NR31R32, OC(O)RS5, NR₃₆C(O)R₃7, a heterocyclyl or a heterocyclyl-(Ci- Ce)alkyl group, wherein said heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47 and a (Ci-Ce)alkyl a group, more preferably R3 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, CN, NR31R32, OC(O)RS5, a heterocyclyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a OR44, SR45 and NR46R47, notably OR44, even more preferably R3 represents a hydrogen atom, a halogen atom or a (Ci-C3)alkyl group, advantageously a hydrogen atom or a halogen atom;

■ R4 represents a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, a (C1- Ce)alkyl, a (Ci-Ce)haloalkyl group, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, CN, NO2, OR44, SR45, NR46R47, C(O)R48, CO2R49, OC(0)Rso, NRSIC(O)R52, C(O)NR53RS4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, preferably R4 represents a hydrogen atom, a halogen atom, OR29, SR30, NR31R32, a (C1- Ce)alkyl, an aryl or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, C(O)R48, CO2R49, C(O)NR53Rs4, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, more preferably R4 represents a hydrogen atom, a halogen atom, NR31R32, a (C1- Ce)alkyl, an aryl or a heterocyclyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of C(O)R48 and a (Ci-Ce)alkyl group, even more preferably R4 represents a hydrogen atom or a (Ci-C3)alkyl group, advantageously a hydrogen atom,

R4b represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl group, OR29 or NR31R32, preferably a hydrogen atom, a halogen atom, a (Ci-C3)alkyl group or OR29, more preferably a hydrogen atom or a (Ci-C3)alkyl group, advantageously a hydrogen atom, wherein, in the above definitions of R4 and R4b, R29 to R32 represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl, an aryl or an aryl-(Ci-Ce)alkyl group, said aryl group being optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR55, SR56, NR57R58, C(O)Rs9, CO2R60, OC(O)Rei, NR₆₂C(O)R₆₃, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, notably C(O)RS9, CO2R60, C(O)NRG4R65, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, in particular C(O)RS9, R48 represents a hydrogen atom, a (Ci-Ce)alkyl or an aryl group, notably an aryl group, and R55 to Res represent, independently of each other, a hydrogen atom, a (Ci-Ce)alkyl or an aryl group, notably an aryl group;

■ R5 represents a hydrogen atom, a halogen atom, CN, OR29, SR30, NR31R32, a (C1- Ce)alkyl, a (Ci-Ce)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR40, SR41 and NR42R43, an aryl, a heterocyclyl, an aryl-(Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, OR44, SR45, NR46R47, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, wherein R29 to R32 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, and R40 to R43 represent, independently of each other, a hydrogen atom or a (Ci-Ce)alkyl group, notably a hydrogen atom, preferably R5 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl, a (C1- Ce)haloalkyl group, said alkyl or haloalkyl group being optionally substituted by one or more substituents selected from the group consisting of OR40, SR41 and NR42R43, an aryl- (Ci-Ce)alkyl or a heterocyclyl-(Ci-C6)alkyl group, wherein said aryl or heterocyclyl group is optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a (Ci-Ce)alkyl and a (Ci-Ce)haloalkyl group, more preferably, R5 represents a hydrogen atom, a halogen atom, a (Ci-Ce)alkyl, a (C1- Ce)haloalkyl group or a heterocyclyl-(Ci-C6)alkyl group, said alkyl or haloalkyl group being optionally substituted by OR40, and said heterocyclyl being optionally substituted by one or more (Ci-Ce)alkyl group, even more preferably, Rs represents a hydrogen atom or a (Ci-C3)alkyl group, advantageously a hydrogen atom; and

■ Rs represents a hydrogen atom, a (Ci-C3)alkyl, an aryl-(Ci-C3)alkyl or a -CH2-CH2-O- CH2-CH2-NH2 group, or a (Ci-Cs)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, SH, NH2, a (Ci-C3)alkoxy, a (Ci-C3)thioalkoxy and a (Ci-C3)alkylamino group, preferably Rs represents a hydrogen atom, a methyl, an ethyl, a benzyl, a -CH2-CH2-O- CH2-CH2-NH2 or a (Ci-Cs)alkylcarbonyl group optionally substituted with one or more substituents selected from the group consisting of OH, NH2 and a thiomethyl group, in particular Rs represents a hydrogen atom or a -CH2-CH2-O-CH2-CH2-NH2 group.

16. The method or the use or the kit for use or the regulated necrotic cell death inhibitor for use of claim 14, wherein the regulated necrotic cell death inhibitor is a compound of formula (II), wherein:

■ Xi represents a (Ci-Cs)alkyl, an aryl, an aryl-(Ci-Cs)alkyl or an ORx group, wherein Rx is a (Ci-Cs)alkyl, an aryl or an aryl-(Ci-Cs)alkyl group;

■ X2 and X3 represent, independently of each other, a hydrogen atom or a (Ci-Cs)alkyl group; and

■ Y1, Y2 and Y3 represent, independently of each other, a hydrogen atom, a (Ci-Cs)alkyl, an aryl, an aryl-(Ci-Cs)alkyl, an OH or an ORY group, wherein at least one of Y1, Y2 and Y3 represents a (Ci-Cs)alkyl, an aryl, an aryl-(Ci-Cs)alkyl or an ORY group, wherein RY is selected from a (Ci-Cs)alkyl, an aryl and an aryl-(Ci-Cs)alkyl group.

17. The method or the use or the kit for use or the regulated necrotic cell death inhibitor for use of claim 16 wherein:

■ Xi represents an ORx group, Rx being advantageously a (Ci-Cs)alkyl group;

■ Y1 represents an ORY group, RY being advantageously an aryl-(Ci-Cs)alkyl group, and Y2 and Y3 each represent a hydrogen atom.

18. The method or the use or the kit for use or the regulated necrotic cell death inhibitor for use of claim 14, wherein the regulated necrotic cell death inhibitor inhibitor is:

(a) a compound of the following general formula (l.iii):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein: Rs, R4, R^ and R5 represent, independently of each other, a hydrogen atom, a halogen atom or a (Ci-Ce)alkyl group, in particular a hydrogen atom, a halogen atom or a (C1- Cs)alkyl group, preferably a hydrogen atom or a halogen atom, and

Rs represents a hydrogen atom, a (Ci-Cs)alkyl or a -CH2-CH2-O-CH2-CH2-NH2 group, in particular a hydrogen atom or a -CH2-CH2-O-CH2-CH2-NH2 group; or

(b) a compound of the following general formula (II. i):

or a pharmaceutically acceptable salt and/or solvate thereof, wherein:

Rx represents a (Ci-Ce)alkyl group, notably a (Ci-Cs)alkyl group such as methyl, ethyl, n-propyl, more advantageously methyl, and

19. The method or the use or the kit for use or the regulated necrotic cell death inhibitor for use of claim 14, wherein the regulated necrotic cell death inhibitor is selected from the group consisting of: