Flavonoid derivatives compounds, and their use as drugs

Abstract

The invention relates to compounds and their therapeutic use, said compounds having the formula (I) wherein, for example, wherein, for example, • α is 0, • δ is 0, • X is O, • V is H2C=O, • n is 1 and m is 0, • R1 is a hydrogen atom, • R2 is a methyl group and R3 is -OH, • Z is (A1).

Description

[0001] FLAVONOID DERIVATIVES COMPOUNDS, AND THEIR USE AS DRUGS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to flavonoid derivatives compounds and their uses as drugs.

[0004] PRIOR ART

[0005] The first theory of oncogenic energetic metabolic reprogramming describes a shift of ATP production from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis that may result in higher cellular proliferation rate of cancer cells. Nowadays, the definition of metabolic reprogramming has been broadened and a better knowledge of tumor biology and its interplay with micro-environment allowed the recognition of the deregulation of cellular metabolism of cancer cells as a new hallmark in cancer. Indeed, cancer cells take advantage of an intrinsic cellular metabolic flexibility to grow based on their ability to switch their use of energy substrates (e.g. glucose, amino acids, and fatty acids) and their metabolite production (e.g. ATP and building blocks for macromolecules). All these changes reveal specific metabolic vulnerabilities and addictions of cancer cells compared to normal cells. At the level of patients, hypermetabolism, which can be detected by PET-scan, is usually associated with a poorer survival and can lead to cachexia.

[0006] Oncogenic metabolism relies on a bi-directional inter-organelle dialogue between mitochondria and nucleus to allow cellular adaptation at the transcriptomic, metabolomic and proteomic levels favoring cancer progression. As almost all mitochondrial proteins (1.500 proteins approximately) are nuclear- encoded and imported into the organelle, multiple specialized protein import machineries have evolved to adapt the mitochondrial proteome to meet the cancer cell needs. One of these import machineries, the so-called apoptosis-inducing factor (AIF) / coiled-coil-helix domain-containing protein 4 (CHCHD4) complex controls the redox-regulated import of a family of small cysteine motif-carrying proteins that are pivotal for cell survival and adaptation to stress. Once imported within the intermembrane space and correctly folded, this set of proteins participates to mitochondrial bioenergetics, redox regulation, lipid homeostasis, calcium accumulation and / or mitochondrial ultrastructure to maintain normal cell homeostasis. In a pathophysiological context, some of these proteins could play a key role in cancer development. This is the case of AIF, the genetic modulation of which has been demonstrated in vitro and in vivo to control mitochondrial biology and the cell fate via the biogenesis of respiratory chain and the import of nuclear-encoded mitochondrial proteins such as TRIAPI . Moreover, the importance of the AIF role has been demonstrated in a mouse model of human KRAS mutated lung cancer and proposed to serve as a prognostic factor for lung cancer. Similarly, CHCHD4 confers a metabolic vulnerability to cancer cells and regulates tumour proliferation and epithelio-mesenchymal transition (EMT)-related phenotypes, through respiratory chain-mediated metabolism.

[0007] Flavonoids are a group of naturally occurring plant compounds, classified as secondary metabolites, known for their diverse health benefits and bioactive properties. They are abundant in various fruits, vegetables, grains, and herbal medicines, playing key roles as pigments and contributing to the fragrance and taste of fruits, flowers, and seeds. More than 9,000 types of flavonoids have been identified, with their chemical structure first characterized in the early 20th century. These compounds act as antioxidants, anti -cancer anti-inflammatory, antibacterial, and cardioprotective effects. They also help plants by protecting them from UV radiation, supporting physiological functions, and regulating cellular processes. For humans, flavonoids have significant pharmacological activities and are essential in promoting health. Widely consumed in foods like tea, red wine, apples, and onions, their daily intake varies between 20 mg and 500 mg. Given their multifaceted benefits, flavonoids remain a vital area of research in medical and pharmaceutical applications. Unfortunately, due to low solubility, poor absorption and rapid metabolism, use of flavonoids in cancer treatment is not satisfactory.

[0008] Therefore, new compounds that are able to bind to AIF and disrupt AIF / CHCHD4 complex, that are non-toxic and that are not rapidly metabolized are requested. SUMMARY OF THE INVENTION

[0009] An aim of the invention is to provide flavonoid derivatives that are not rapidly metabolized.

[0010] Another aim of the invention is to provide antitumoral drugs that are well tolerated by patients and can be used as part of chemotherapy.

[0011] Another aim of the invention is to provide the use of flavonoid derived compounds to treat cancers such as tongue cancer, non-small cell lung cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, leukemia, osteosarcoma or head and neck cancer.

[0012] Another aim of the invention is to provide drugs liable to treat pediatric cancers.

[0013] DETAILED DESCRIPTION

[0014] The invention relates to a compound of formula (I): wherein: a and are equal to 0 or 1, and cannot represent simultaneously 1,

[0015] 5 is equal to 0 or 1,

[0016] X is chosen among: CH2 or O,

[0017] V is a nitrogen atom or H2C=O, n and m are different and are equal to 0 or 1,

[0018] Ri is chosen among: hydrogen atom or a methyl group,

[0019] R2 is chosen among: a hydrogen atom, a methyl group, a cyclopentyl group or a CH2 and

[0020] R3 is chosen among: a -OH, an -O-CH3 group, an -O-CH2-cyclopropyl group , an -O-octyl group, an -O-cyclopentyl group, group or an oxygen atom, optionally R2 and R3 are linked to form a methylenedioxy group, • R.4 and R5 are chosen among: a hydrogen atom or a methyl group,

[0021] • Z represents a group chosen among: and when a = 1 and P = 0

[0022] ■ X is CH2 and V is a nitrogen atom

[0023] ■ n = 0 and m = 1 or when a = 0 and = 1

[0024] ■ X is O and V is H2C=0

[0025] ■ n = 1 and m = 0, or when a = 0 and P = 0

[0026] ■ X is O and V is H2C=O

[0027] ■ n = 1 and m = 0, and when 5 = 0,

[0028] ■ R2 is chosen among: a hydrogen atom, a methyl group or a cyclopentyl group and Rs is chosen among: a OH, an O-CHs group, an -O-CHs-cyclopropyl group, an -O-octyl group, an -O-cyclopentyl group, group, or when 5 = 1,

[0029] ■ R2 is CH2 and Rs is an oxygen atom and R2 and Rs are linked to form a methylenedioxy group, for its use in the treatment of cancer, in particular tongue cancer, non-small cell lung cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, leukemia, osteosarcoma or head and neck cancer.

[0030] It has been unexpectedly found that compounds of formula (I) are able to bind on AIF, to disrupt enough amount of AIF / CHCHD4 complexes, in order to treat cancer.

[0031] The invention relates to compounds for their use of formula (I), under the form of pharmaceutically acceptable salts or isomers, in particular under pure form, or enantiomers or a mixture of said isomers and enantiomers.

[0032] The expression “pharmaceutically acceptable salts” means all pharmaceutically or physiologically acceptable salt forms of the compounds of formula (I) which may be formed, by protonation of a nitrogen of an amino group, with an inorganic or organic acid, or as a salt that is formed in the presence of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Examples of acid addition salts comprise, mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2- hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2- naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts.

[0033] The expression “enantiomers” means two compounds of the same molecular formulae but having a different stereochemical configuration.

[0034] In the formula (I), the dotted bond between R2 and R3 is present when 5 is 1. It means that R2 is CH2, Rs is an oxygen atom and R2 and Rs are linked to form a methylenedioxy group. The compound of formula (I) has thus the following structure:

[0035] wherein Z, Ri, R4, R5, X, V, a and P have the meaning defined above.

[0036] When a = 1 and P = 0, the compound of formula (I) has thus the following structure: wherein Z, Ri, R4 and Rshave the meaning defined above.

[0037] When a = 0 and = 1, the compound of formula (I) has thus the following structure: wherein Z, Ri, R2, and R3 have the meaning defined above.

[0038] When a = 0 and P = 0, the compound of formula (I) has thus the following structure: wherein Z, Ri, R2 and Rshave the meaning defined above.

[0039] According to another embodiment, the invention relates to a compound as defined above of the formula (I), for its use in the treatment of osteosarcoma. According to another embodiment, the invention relates to a compound as defined above of the formula (I), for its use in the treatment of lung cancer.

[0040] According to another embodiment, the invention relates to a compound as defined above of the formula (I), for its use in the treatment of head and neck cancer.

[0041] According to another embodiment, the invention relates to a compound for its use as defined above, of

[0042] In this embodiment, the compound of formula (II) has thus the following structure:

[0043] According to another embodiment, the invention relates to a compound for its use as defined above, of

[0044] • is equal to 0 or 1,

[0045] • n = 1,

[0046] • Z represents a group chosen among:

[0047] • 5, Ri, R2, Rs, and V have the meaning defined above.

[0048] According to another embodiment, the invention relates to a compound for its use as defined above, of formula (III): wherein:

[0049] • a = 0

[0050] • P = o,

[0051] • n = 1,

[0052] • Z represents a group chosen among:

[0053] • 5, Ri, R2, Rs, X and V have the meaning defined above.

[0054] In this embodiment, the compound of formula (III) has thus the following structure: wherein Z, Ri, R2 and Rs have the meaning defined above.

[0055] According to another embodiment, the invention relates to a compound for its use as defined above, of formula (III):

[0056]

[0057] • Z represents a group chosen among:

[0058] • 5, Ri, R2, Rs, X and V have the meaning defined above.

[0059] In this embodiment, the compound of formula (I) has thus the following structure: wherein Z, Ri, R2, and Rs have the meaning defined above. In this embodiment, Z comprises an ether function.

[0060] According to another embodiment, the invention relates to a compound for its use as defined above, having a formula chosen among the following ones:

[0061] LF18 LF19 LF20

[0062] According to another embodiment, the invention relates to a compound for its use as defined above, of formula:

[0063] According to another embodiment, the invention relates to a compound for its use as defined above, in the treatment of pediatric cancer.

[0064] The expression “pediatric cancer” means cancers that occur between birth and 14 years of age. Childhood cancers are very rare and may differ from adult cancers in the way they grow and spread, how they are treated, and how they respond to treatment.

[0065] According to another embodiment, the invention relates to a compound for its use as defined above, formulated for an administration of active substance at a range of 10 mg / kg to 100 mg / kg of body weight.

[0066] According to another embodiment, the invention relates to a compound for its use as defined above, in a unitary form comprising from 70 mg to 700 mg of active substance (for a human being weighing 70 kg).

[0067] According to another embodiment, the invention relates to a compound for its use as defined above, said compound maintaining the Mitochondrial Membrane Potential (MMP or A m). measured using fluorescent probe Tetramethylrhodamine Methyl Ester (TMRM) in flow cytometry.

[0068] The expression “maintain the Mitochondrial Membrane Potential (MMP or A m)" means that said potential remains unchanged at a fixed value with a variation of 20% (positive or negative variation), compared to a control condition of no treatment, without applying any stress.

[0069] The compounds of the present invention maintain the Mitochondrial Membrane Potential whereas natural flavonoids like eupatilin and diosmetin lower it (cf example 35).

[0070] The invention relates to a compound for its use as defined above, of formula (IA): e the meaning defined above.

[0071] According to another embodiment, the invention relates to a compound for its use as defined above, of formula (IIA): (IIA) wherein:

[0072] • a = 1 and P = 0,

[0073] • m = 1,

[0074] • Z, X, V, Ri, R4 and R5 have the meaning defined above.

[0075] The invention also relates to a compound for its use as defined above, of formula (10): he meaning defined above.

[0076] Some of the compounds of formula (I) are new.

[0077] The invention relates to a new compound of formula (III): wherein:

[0078] • a and are equal to 0 or 1, and cannot represent simultaneously 1,

[0079] • 5 is equal to 0 or 1,

[0080] • X is O,

[0081] • V is H2C=O,

[0082] • n is 1,

[0083] • Ri is a hydrogen atom,

[0084] • R2is chosen among: a hydrogen atom, a methyl group, a cyclopentyl group or a CH2and R3 is chosen among: a -OH, an -O-CH3 group, an -O-CH2-cyclopropyl group , an -O-octyl group, an -O-cyclopentyl group, group or an oxygen atom, optionally R2 and R3 are linked to form a methylenedioxy group,

[0085] • Z represents a group chosen among: and when 5 = 0,

[0086] ■ R2 is chosen among: a hydrogen atom, a methyl group or a cyclopentyl group and

[0087] ■ R3 is chosen among: a OH, an O-CH3 group, an -O-CH2-cyclopropyl group, an -O-octyl group, an -O-cyclopentyl group, or when 5 = 1,

[0088] ■ R2 is CH2 and R3 is an oxygen atom and R2 and R3 are linked to form a methylenedioxy group.

[0089] The invention relates to new compounds of formula (III), under the form of pharmaceutically acceptable salts, or isomers, in particular under pure form, or enantiomers or a mixture of said isomers and enantiomers.

[0090] The expression “pharmaceutically acceptable salts” means all pharmaceutically or physiologically acceptable salt forms of the compounds of formula (I) which may be formed, by protonation of a nitrogen of an amino group, with an inorganic or organic acid, or as a salt that is formed in the presence of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Examples of acid addition salts comprise, mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2- hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2- naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts.

[0091] According to another embodiment, the invention relates to a new compound, of formula (III) as defined

[0092] • Z represents a group chosen among:

[0093] In this embodiment, Z comprises an ether function.

[0094] According to another embodiment, the invention relates to the news compounds having a formula chosen among the following ones:

[0095] LFO2 LFO3

[0096]

[0097] According to another embodiment, the invention relates to a new compound, of formula:

[0098] M30-E05.

[0099] According to another embodiment, the invention relates to a compound as defined above, said compound enabling to maintain the Mitochondrial Membrane Potential (MMP or A m). measured using fluorescent probe Tetramethylrhodamine Methyl Ester (TMRM) in flow cytometry.

[0100] Unlike the compounds of the present invention, natural flavonoids like eupatilin and diosmetin decrease the Mitochondrial Membrane Potential (cf. example 35).

[0101] The invention is also related to the use of the new compounds as defined above as drugs.

[0102] The invention relates to a compound as defined above, for its use as a drug.

[0103] The invention also relates to a process of preparation of M30-E05 comprising the step of: a) a reduction step of 7-(benzyloxy)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-5-hydroxy-4H- chromen-4-one with palladium on carbon and hydrogen gas, to obtain 2-[3-(cyclopentyloxy)-4- methoxyphenyl] -5 ,7-dihydroxy-2,3 -dihydro-4H-chromen-4-one, b) a Williamson coupling reaction step of the above mentioned 2-[3-(cyclopentyloxy)-4- methoxyphenyl] -5 ,7-dihydroxy-2,3 -dihydro-4H-chromen-4-one with 1 - [3 , 4 - bis(benzyloxy)phenyl]-2-iodoethanone and potassium carbonate, to obtain 7-{2-[3,4- bis(benzyloxy)phenyl]-2-oxoethoxy}-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-5-hydroxy- 4H-chromen-4-one, c) a reduction step of the above mentioned 7-{2-[3,4-bis(benzyloxy)phenyl]-2-oxoethoxy}-2-[3- (cyclopentyloxy)-4-methoxyphenyl]-5-hydroxy-4H-chromen-4-one, with hydrogen gas and palladium hydroxide, to obtain M30-E05,

[0104] According to another embodiment, the invention relates to a process as defined above, further comprising the prior steps of preparation of l-[3,4-bis(benzyloxy)phenyl]-2 -iodoethanone: al) a double Williamson coupling reaction step with 3,4-dihydroxyacetophenone, benzyl chloride and potassium carbonate, to obtain l-[3,4-bis(benzyloxy)phenyl]ethenone, a2) an iodation step of the above mentioned l-[3,4-bis(benzyloxy)phenyl]ethenone with copper(II) oxide and iodine, to obtain l-[3,4-bis(benzyloxy)phenyl]-2-iodoethanone In this embodiment, the process of preparation of M30-E05 is a 5 -step process starting from 7- (benzyloxy)-2-[3 -(cyclopentyloxy)-4-methoxyphenyl] -5 -hydroxy-4H-chromen-4-one and 3,4- dihydroxyacetophenone .

[0105] According to another embodiment, the invention relates to a product containing a compound of formula(I) as defined above and an immunotherapeutic agent or an anti-cancer agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer, in particular tongue cancer, non-small cell lung cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, leukemia, osteosarcoma or head and neck cancer.

[0106] According to another embodiment, the invention relates to a product for it use as defined above, said immunotherapeutic agent being chosen from:

[0107] - an anti-immuno-oncologic agent (anti I-O) such as an anti-macrophage antibody (i.e. immunogen being spleen cells)

[0108] - an inhibitor of PD 1 such as nivolumab and pembrolizumab

[0109] - an inhibitor of PD-L1 such as atezolizumab and durvalumab,

[0110] - an inhibitor of CTLA-4 such as ipilimumab

[0111] - an antibody selected from the group consisting of siglec 15 antibody, anti- phosphatidylserine, anti- 0X40, anti-CD73, anti-TIM3, anti-CD24, anti-CD47, anti-PDl, anti- PDL1, anti-CTLA4, anti-GITR, anti-CD27, anti-CD28, anti-CD 122, anti-TIGIT, anti- VISTA, anti-ICOS, and anti-LAG3, or -vaccines derived from viral, bacterial, and phagic components and their adjuvants.

[0112] According to another embodiment, the invention relates to a product for it use as defined above, said anti -cancer agent being chosen from:

[0113] - alkylating agents such as alkylsulfonates in particular busulfan, dacarbazine, procarbazine, cloretazine, nitrogen mustards such as chlormethine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, nitrosoureas such as carmustine, lomustine, semustine, streptozocin, altretamine, fotemustine;

[0114] - antineoplastic alkaloids such as vincristine, vinblastine, vinorelbine, vindesine;

[0115] - taxanes such as paclitaxel or taxotere;

[0116] - antineoplastic antibiotics such as actinomycin, bleomycin;

[0117] - intercalating agents such as mitoxantrone, etoposide, bleomycin, actinomycin D, amsacrine, alliptinium;

[0118] - antineoplastic antimetabolites: folate antagonists, methotrexate; inhibitors of purine synthesis; analogues such as mercaptopurine, 6-thioguanine; inhibitors of pyrimidine synthesis, aromatase inhibitors, capecitabine, pyrimidine analogs such as fluorouracil, gemcitabine, cytarabine and cytosine arabinoside; brequinar, nelarabine;

[0119] - group I and II topoisomerase inhibitors such as irinotecan, exatecan, topotecan, teniposide, camptothecin or etoposide;

[0120] - anticancer hormone agonists and antagonists including tamoxifen;

[0121] - kinase inhibitors, such as imatinib, nilotinib and dasatinib, midaustorin, sorafenib, lestaurtinib, tandutinib, sirolimus, everolimus or tensirolimus;

[0122] - growth factor inhibitors;

[0123] - anti-inflammatories such as pentosan polysulfate, corticosteroids, prednisone, dexamethasone;

[0124] - ceplene (histamine dihydrochloride);

[0125] - antracyclines such as daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, aclarubicin, annamycin, doxorubicin, mitomycin and methramycin;

[0126] - anticancer metal complexes, platinum derivatives such as cisplatin, carboplatin, oxaliplatin, satraplatin;

[0127] - alpha interferon;

[0128] - triphenylthiophosphoramide;

[0129] - antiangiogenic agents;

[0130] - thalidomide;

[0131] - inhibitors of famesyl-tranferase such as tipifamib;

[0132] - inhibitors of DNA methyltransferase such as MG98;

[0133] - immunotherapy adjuvants such as gemtuzumab ozogamicin, HuM 195;

[0134] - biotherapeutic agents such as CT388-I L3; or - antisense such as GTI-2040.

[0135] According to another embodiment, the invention relates to a product for it use as defined above, said anti -cancer is a chemotherapy agent.

[0136] According to another embodiment, the invention relates to a product for it use as defined above, said anti -cancer is a chemotherapy agent chosen from:

[0137] - alkylating agents such as cyclophosphamide, ifosfamide and busulfan,

[0138] - platinum-based agents such as cisplatine, carboplatin and oxaliplatin

[0139] - antimetabolites such as methotrexate, 5 -fluorouracil,

[0140] - anthracyclines such as doxorubicin, epirubicin, daunorubicin,

[0141] - topoisomerase inhibitors such as irinotecan, etoposide, topotecan;

[0142] - mitotic inhibitors such as paclitaxel, docetaxel, vincristine

[0143] - corticosteroids such as prednisone, dexamethasone

[0144] - targeted therapies such as imatinib, trastuzumab, erlotinib; or

[0145] - monoclonal antibodies such as rituximab, bevacizumab.

[0146] According to another embodiment, the invention relates to a product for it use as defined above, said anti -cancer is a chemotherapy agent chosen from:

[0147] Altretamine; Bendamustine; Busulfan; Carboplatin; Carmustine; Chlorambucil; Cisplatin; Cyclophosphamide; Dacarbazine; Ifosfamide; Lomustine; Mechlorethamine; Chlormethine; Melphalan; Oxaliplatin; Temozolomide; Thiotepa; Trabectedin; Carmustine; Lomustine; Streptozocin; Azacitidine; 5 -fluorouracil (5-FU); 6-mercaptopurine (6-MP); Capecitabine (Xeloda); Cladribine; Clofarabine; Cytarabine (Ara-C); Decitabine; Floxuridine; Fludarabine; Gemcitabine (Gemzar); Hydroxyurea; Methotrexate; Nelarabine; Pemetrexed (Alimta); Pentostatin; Pralatrexate; Thioguanine; Trifluridine / tipiracil combination; Daunorubicin; Doxorubicin; Epirubicin; Idarubicin; Valrubicin; Bleomycin; Dactinomycin; Mitomycin-C; Mitoxantrone; Irinotecan; Topotecan; Etoposide (VP- 16); Mitoxantrone; Teniposide; Cabazitaxel; Docetaxel; Nab-paclitaxel; Paclitaxel; Vinblastine; Vincristine; Vinorelbine; Prednisone; Methylprednisolone; Dexamethasone; Arsenic trioxide; Asparaginase; Eribulin; Hydroxyurea; Ixabepilone; Mitotane; Omacetaxine; Pegaspargase; Procarbazine; Romidepsin; Vorinostat; or Raltitrexed.

[0148] According to another embodiment, the invention relates to a product for it use as defined above, said anti -cancer is a chemotherapy agent chosen from cisplatin (CIS), doxorubicine (DOXO) or methotrexate (MTX).

[0149] LIST OF FIGURES

[0150] Figure 1. Correlation between the AutoDock Vina binding free energies of the 7 Pretswick compounds (black circle) on AIF monomer (1M6I) and dimer (4BUR) and their log(IC5o) values measured experimentally. Grey squares that are positioned on the correlation line indicates the binding free energies of the 5 compounds from the French Chemical National Library.

[0151] Figure 2. Contact frequency of AIF monomer residues with PW02, PW04, A002, A004, LF08 and LF12 binding modes (graphs) and localization of the most frequently contacted ones on AIF surface (white areas indicate residues that are contacted more than 20% of the ligand binding modes).

[0152] Figure 3. Contact frequency of AIF dimer residues with PW02, PW04, A002, A004, LF08 and LF12 (in top and bottom graphs for chain A and B, respectively) and localization of the most frequently contacted ones on AIF surface (white areas indicate residues in chain A (top) and B (bottom) that are contacted more than 10% of the ligand binding modes).

[0153] Figure 4. Cytotoxicity of a panel of 8 OS cell lines were determined, using colorimetric assay after 72 h of treatment with a range of different M30-E05 concentrations diluted in appropriate culture medium without antibiotics. Lysis solution, reagent solution and stop solution were added serially, following the manufacture's protocol. Therefore, the half maximal inhibitory concentration (IC50) for M30-E05 was determined using a nonlinear regression with log(Agonist) vs. response - Variable slope equation [Y=100 / (l+10A((LogEC50-X)*HillSlope)) ] in Prism 8.4.2 (GraphPad). Figure 5. Effects of M30-E05 on clonogenic formation of HOS, HOS-R / DOXO, HOS-R / MTX cells were evaluated by a clonogenic assay, using the approximate IC50 (lOpM of M30- E05). Clonogenic assay was conducted 10 days after treatment.

[0154] Figure 6. Representative Western-blotting showing AIF, GAPDH expression are significantly downregulated in parental HOS after 24 h, 48 h, 72 h and 96 h of 5mM of siRNA treatment with respective target. Each gene expression was detected by Western blotting with specific antibody. Vinculin was used as a loading control to normalize the signals. Therefore, 48 h was determined as a good time point for siAIF treatment. A previous procedure was repeated to determine the IC50 of each condition (including HOS + siAIF and HEK293 as a control for non-cancerous cell line)

[0155] Figure 7. Effect of M30-E05 on peripheral blood mononuclear cells (PBMCs) cell viability. PBMCs were isolated by Ficoll and seeded at 20.000 cells and treated with DMSO diluted in RPMI + 10%FBS as a control or M30-E05 diluted in RPMI and 10%FBS for 72 h.

[0156] Figure 8. Effect of M30-F05 on peripheral blood mononuclear cells (PBMCs) cell viability. PBMCs were isolated by Ficoll and seeded at 20.000 cells and treated with DMSO diluted in RPMI (Roswell Park Memorial Institute medium) + 10%FBS as a control or M30-E05 diluted in RPMI and 10%FBS for 72 h.

[0157] Figure 9. Cellular effects of M30-E05 treatment on different adult cancer cell lines. IC50 was determined as previously described protocol. IC50 was determined using a nonlinear regression with log(Agonist) vs. response - Variable slope equation [Y=100 / (l+10A((LogEC50-X)*HillSlope)) ] in Prism 8.4.2 (GraphPad)

[0158] Figure 10. Alterations in mitochondrial proteins import were detected by non-denaturing electrophoresis gel and western-blot. Monomer to dimer AIF oligomerisation is modified by a 24 h M30-E05 cell treatment by heat (60°C, 30 minutes), ionic strength (IM NaCl, 30 minutes), IC50 Mitoxantrone (MitoX) and IC5o M3O-EO5 and 2xICso M30-E05.

[0159] Figure 11. Mitochondrial respiration (OCR) was measured by Seahorse analysis and total ATP content detection by luminescence.

[0160] Figure 12. Protein expression respectively of AIF4, CHCHD4, MICU1 and Cox 17 in presence of M30- E50 at its IC50 over time expressed in hours. Panel (A) represents AIF, panel (B) represents CHCHD4, panel (C) represents MICU1 and panel (D) represents Coxl7

[0161] Figure 13. Cell death was evaluated by Annexin V / PI analysis at different time points to evaluate apoptosis induction. Statistical analysis, ns, non significant * P<0.05, **P<0.0I, *** P0.001, **** PO.OOOl. ICsoof mitoxantrone (MitoX) = 0.2 mM; ICso of M30-E05 = 8.3 mM.

[0162] Figure 14. Total ATP measured by luminescence (ATPlite assay). Statistical analysis, * P<0.05; **P<0.0I.

[0163] Figure 15. Apoptosis measured by AnnexinV / PI by flow cytometry. Right panel shows early and late apoptosis analysis. Statistical analysis, ns, non significant; * P<0.05; **P<0.0I.

[0164] Figure 16. Analysis of differentially expressed metabolites identified in Human osteosarcoma (HOS) cell lines treated with corresponding IC50 of M30-E05 during 72 h

[0165] Figure 17. Analysis of differentially expressed metabolites identified in HOS -doxorubicin resistant (HOS-R / DOXO) cell lines treated with corresponding IC50 of M30-E05 during 72 h

[0166] Figure 18. Analysis of differentially expressed metabolites identified in HOS -methotrexate resistant (HOS-R / MTX) cell lines treated with corresponding IC50 of M30-E05 during 72 h

[0167] Figure 19. Analysis of differentially expressed metabolites identified in HOS cell lines treated with corresponding IC50 of M30-E05 during 72 h. Different metabolites (the top 16) identified by the PLS- DA according to the VIP score on the x-axis. Colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group. In general, all the mentioned metabolites are up-regulated in M30-E05 -treated condition, except down-regulation was found in Glycerophosphocholine (HMDB00086).

[0168] Figure 20. Analysis of differentially expressed metabolites identified in HOS-R / DOXO cell lines treated with corresponding IC50 of M30-E05 during 72 h. Different metabolites (the top 16) identified by the PLS-DA according to the VIP score on the x-axis. Colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group. In general, all the mentioned metabolites are up-regulated in M30-E05 -treated condition, except down-regulation was found in Glycerophosphocholine (HMDB00086).

[0169] Figure 21. Analysis of differentially expressed metabolites identified in HOS-R / MTX cell lines treated with corresponding IC50 of M30-E05 during 72 h. Different metabolites (the top 16) identified by the PLS-DA according to the VIP score on the x-axis. Colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group. In general, all the mentioned metabolites are up-regulated in M30-E05 -treated condition, except down-regulation was found in Glycerophosphocholine (HMDB00086).

[0170] Figure 22. Cytotoxic effect of E05 abolishes mitochondrial effect on PDX models, M30-E05 potently decreases the viability of different OS PDX ex vivo models. Cytotoxicity of M30-E05 against a panel of cultured subcutaneous.

[0171] Figure 23. Cytotoxic effect of E05 abolishes mitochondrial effect on PDX models, M30-E05 potently decreases the viability of different OS PDX ex vivo models. Cytotoxicity of M30-E05 against a panel of orthotopic paratibial

[0172] Figure 24. PDX ex vivo models were cultured in DMEM + 20% FBS and their mitochondrial activities were analyzed by Seahorse MitoStress Test after 24h treatment with lOpM of M30-E05.

[0173] Figure 25. Western blot analysis showing the M30-E05 treatment effect after 48 h and 72 h on AIF / CHCHD4 complex protein expression of different PDX models. Relative intensity of bands of treated PDX to untreated PDX normalized to 1 is indicated.

[0174] Figure 26. Apoptotic rates of secondary in vitro cultured GR-OS-18 (Gustave Roussy-Osteosarcoma) Ortho were determined by Annexin V / PI analysis at different time points to evaluate apoptosis induction (annexin V / PI, flow cytometry). Statistical analysis, ns, non significant * P<0.05, **P<0.01.

[0175] Figure 27. Apoptotic rates of GR-OS-15 Ortho were determined by Annexin V / PI analysis at different time points to evaluate apoptosis induction (annexin V / PI, flow cytometry). Statistical analysis, ns, non significant * P<0.05, **P<0.01.

[0176] Figure 28. Mice (5 mice per group) were administrated orally various quantity (0, 10, 25, 50, lOOmg / kg) of M30-E05 dissolved in 0.5% of methylcellulose 5 days per week for 4 weeks. The body weight was measured the day of the gavage and shows no loss in body weight. (A) is the control group. (B) is the group where lOmg / kg of M30-E05 were given. (C) is the group where 25mg / kg of M30-E05 were given. (D) is the group where 50mg / kg of M30-E05 were given. (E) is the group where lOOmg / kg of M30-E05 were given.

[0177] Figure 29. Representative HE staining photos of 6 organs (heart (A), lung (B), kidney (C), liver (D), brain (E) and spleen (F)) of lOOmg / kg M30-E05 -treated mice. No sign of toxicity was found in those mice.

[0178] Figure 30. GR-OS-18 SC was chosen to subcutaneously implant into NSG (NOD scid gamma) mice to construct xenograft growth models and were exposed to lOOmg / kg M30-E05 (n =18) during 2 weeks, compared to vehicle-only control group. Tumor volumes for each group are shown. Statistical analysis, ns, non significant * P<0.05, **P<0.01

[0179] Figure 31. Picture of the tumor of a mice of the control group of GR-OS-18 subcutaneously implanted (left) and the tumor of a mice that was exposed to lOOmg / kg M30-E05. Figure 32. Scatter plot exhibiting the most enriched GO biological process results from all differentially expressed genes.

[0180] Figure 33. Heat map of apoptosis-related genes that are differentially expressed based on z-score normalized pseudocounts.

[0181] Figure 34. Cell viability of 11 lung adenocarcinoma cell lines treated with M30-E05. Dose-response curves represent cell viability measured using a LDH assay at increasing concentrations of M30-E05 after 72 hours of treatment. Individual experiments are shown (n > 3). 0.002% DMSO was used as the solvent of M30-E05 and as a control for calculation of percentage of viability.

[0182] Figure 35. Cell viability of wild-type Mouse Embryonic Fibroblast (MEF WT) and AIFM1 KO MEF (MEF AIF KO) cells treated with M30-E05. Dose-response curves represent cell viability measured using a LDH assay at increasing concentrations of M30-E05 after 72 hours of treatment. Individual experiments are shown (n > 2). IC50, inhibitory concentration yielding to 50% cell viability. 0.002% DMSO was used as the solvent of M30-E05 and as a control for calculation of percentage of viability.

[0183] Figure 36. Western Blot analysis showing the basal expression levels of AIF, CHCHD4 and Coxl7 in different lung adenocarcinoma cell lines. Vinculin was used as loading controls to normalize. Normalized data were used to compared with corresponding protein expression in HOS cells. Data are presented as the mean ± SD from three independent experiments (n = 3). Statistical significance was assessed using ANOVA with Sidak’s correction for multiple comparisons to compare between cell lines versus HOS cells. Significance levels are indicated as follows: not significant (ns), p < 0.0332 (*), p < 0.0021 (**), p < 0.0002 (***), and p < 0.0001 (****).

[0184] Figure 37. Illustrative 3D synergy map of M30-E05 in combination with CIS, DOXO, MTX at depicted concentrations in HOS, HOS-R / DOXO, HOS-R / MTX cell lines obtained using SynergyFinder tool (https : / / synergyfinder .fimm .fi / ) .

[0185] Figure 38. Cell viability of HOS cells treated with different analogs of M30-E05. Dose-response curves represent cell viability measured using a LDH assay at increasing concentrations of each analog after 72 hours of treatment. Individual experiments are shown (n > 2). IC50, inhibitory concentration yielding to 50% cell viability. 0.002% DMSO was used as the solvent of M30-E05 and as a control for calculation of percentage of viability.

[0186] Figure 39. Volcano plot of differential protein expression for DMSO-treated versus M30-E05 -treated HOS cells. The plot displays log2 fold change (log2FC) on the x-axis and -loglO(adjusted p-value) on the y-axis, with proteins classified as up-regulated (red, log2FC > 0.5), down-regulated (blue, log2FC < -0.5), or not significant (grey) based on an adjusted p-value threshold of 0. 1. Dashed lines indicate the significance thresholds for log2FC (±0.5) and adjusted p-value (0.1), highlighting proteins with statistically significant differential expression. The analysis was performed using the limma package in R, with data from four replicates per condition, and the plot was generated using ggplot2.

[0187] Figure 40. KEGG pathway enrichment analysis of differentially expressed proteins. Significantly enriched KEGG pathways were identified using clusterProfiler with all detected proteins as background. The dot plot displays the top enriched pathways ranked by adjusted p-value. The x-axis represents the GeneRatio (proportion of differentially expressed proteins mapping to a pathway relative to all proteins in that pathway).

[0188] Figure 41. Mitochondrial membrane potential assessed by flow cytometry using lOOnM TMRM. fluorescent probe labelling for 20 min. HOS cells were analyzed at different time points following M30- E05 treatment. Oligomycin and CCCP were used as positive controls for membrane potential modulation, and unstained cells served as negative controls for gating. Three independent experiments were performed. Data represent the mean±SD. Statistical significance was assessed using ANOVA with Sidak’s multiple comparison test correction. EXAMPLES

[0189] EXAMPLE 1 - Materials and Methods

[0190] Cell cultures and patient-derived xenografts (PDX). Human osteosarcoma cell lines HOS Parental, HOS -doxorubicin resistant Dox-R / DOXO, and HOS -methotrexate resistant HOS-R / MTX were generously provided by Dr N. Gaspar (Marques da Costa et al., 2019). Adult cancer cell lines and HEK293 were provided by Dr J. Wiels (CNRS UMR 9018, Gustave Roussy, Villejuif). All the cell lines were routinely cultured in Dulbecco’s modified Eagle medium (DMEM, high glucose, GlutaMAX Supplement, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma- Aldrich, Paraguay Origin), 1% Penicillin-Streptomycin (Gibco) at 37°C in humidified atmosphere (5% CO2 and 95% air), under mycoplasma free conditions. The 7 PDXs GR-OS-9, GR-OS-10, GR-OS-11, GR-OS-12, GR-OS- 15, GR-OS-18, GR-OS-20 were established from relapsed or refractory osteosarcoma within the MAPPYACTS PDX project as published previously (Marques Da Costa et al., 2023). Of note, FBS concentration for in vitro PDX secondary culture was 20%.

[0191] Human blood samples were obtained from the ‘Etablissement Francais du Sang’, Hopital Saint-Louis, Paris, France and peripheral blood mononuclear cells (PBMCs) were freshly prepared by density gradient centrifugation. siRNA transfection. To knock down AIF expression for evaluating the specificity of M30-E05, 12.500, 18.750, 25.000, 31.250 (HOS cells / cm3were transfected with ON-TARGETplus Human AIFM1 siRNA (Ref. L-011912-00-0005, Dharmacon) or ON-TARGETplus GAPDH Control Pool (Ref.D-001830-10-05, Dharmacon) for 96h, 72h, 48h, 24h, using 0.2pL, 0.16pL, 0.2pL and 0.08pL of DharmaFECT 1 Transfection Reagent (Ref. T-2001-03, Dharmacon), respectively according to the manufacturer’s instructions to determine the best time point for subsequent experiments. 48h was chosen to conduct further experiments. In details, siRNA pools targeting AIF, containing 4 selected siRNA duplexes each with a modification pattern that addresses off-target effects caused by both strands (SMART pool).

[0192] Transcriptomic analysis. Approval by the steering committee of the MAPPYACTS trial (NCT02613962, Dr B. Geoerger) for 42 refractory or relapsed OS patient RNA-sequencing data and UNICANCER transbone scientific committee of the OS2006 trial (NCT00470223 - Dr N Gaspar) for 82 diagnostic OS patient RNA-sequencing data. Expression of each component of the AIF / CHCHD4- Ca2+axis was analyzed in the 2 databases using Mann-Whitney test to observe expression changes between non-resistant vs. resistant OS tumors.

[0193] Gene set enrichment analysis (GSEA). Cell line RNA-sequencing results were obtained by Dr N. Gaspar and A. Marchais’s team. GSEA was performed using the gseGO function in the clusterProfiler package, applying the KEGG pathway enrichment to reveal the most significant dysregulated pathways.

[0194] Metabolomics a, Sample preparation cells and supernatant. 300.000 cells were cultured in 6 well-plates and treated with corresponding drugs during 72h. After a soft rinse with cold PBS, cells were then lysed adding 500 pL of cold methanol / water (9 / 1, v / v, -20°C) with internal standards (ISTD). After a quick scrap, supernatants were pooled, with two wells by condition in a single microtube. Supernatants in microtubes were vortexed for 800 g for 5 minutes, and centrifuged at 15000 g for 10 min at +4 °C. Supernatants were split in two parts: 150 pL were used for GC-MS experiment in injection vial and 300 pL were used for UHPLC (ultra-high performance liquid chromatography)-MS experimentation. Regarding the LC- MS aliquots, aliquots were evaporated and dried extracts were solubilized with 150 pL of MilliQ water. Biological samples were kept at -80°C until injection or transferred in vials for direct analysis by UHPLC / MS.

[0195] After evaporation, dry GC-MS aliquot was spiked with 50 pL of methoxyamine (20 mg / mL in pyridine) and stored at room temperature in the dark, overnight. Then, 80 pL of MSTFA (N-Methyl-N- trimethylsilyl -trifluoroacetamide) was added and final derivatization occurred at 40°C during 30 minutes. Samples were directly injected into GC-MS. b, Targeted analysis of nucleotides and cofactors by ion pairing ultra-high performance liquid chromatography (UHPLC) coupled to a Triple Quadrupole (QQQ) mass spectrometer. Targeted analysis was performed on a RRLC 1290 system (Agilent Technologies, Waldbronn, Germany) coupled to a Triple Quadrupole 6470 (Agilent Technologies) equipped with an electrospray source. 10 pL of sample were injected on a Column Zorbax Eclipse XDB-C18 (100 mm x 2.1 mm particle size 1.8 pm) from Agilent technologies. Gradient mobile phase consisted of water with 2mM of dibutylamine acetate concentrate (DBAA) (phase A) and acetonitrile (phase B). Flow rate was set to 0.4 mL / min, and gradient as follow: initial condition was 90% phase A and 20% phase B, maintained during 3 min. Molecules were then eluted using a gradient from 10% to 95% phase B over 1 min. Column was washed using 95% mobile phase B for 2 minutes and equilibrated using 10% mobile phase B for 1 min. Scan mode used was the MRM (multiple reaction monitoring) for biological samples. Peak detection and integration of the analytes were performed using the Agilent Mass Hunter quantitative software (B. 10. 1). c, Widely-targeted analysis of intracellular metabolites gas chromatography (GC) coupled to a triple quadrupole (QQQ) mass spectrometer. GC-MS / MS method was performed on a coupling gas chromatography / triple quadrupole 7890B / 7000C (Agilent Technologies, Waldbronn, Germany). The scan mode used was the MRM for biological samples. Peak detection and integration of analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01), exported as tables and processed with R software (version 4.0.3) and the GRMeta package (Github / kroemerlab).

[0196] Western blot. Immunoblotting was performed on cell lysates in NETN buffer supplemented with antiprotease cocktail (ThermoFisher, #A32963). Briefly, the cell lysates were separated on 10- 15% Tris-glycine SDS-PAGE gel (Invitrogen Ref. NP0321; NP0322; NP0326) and transferred to PVDF membrane. Membranes were blocked in 5% milk in TBS with 0.1% TWEEN-20 (TBST) for 1 hour at room temperature followed by overnight incubation with indicated primary antibodies (MICU1 (Ref. HPA037479) (ATLAS Antibodies, 1: 1,000), MICU2 (Ref.HPA045511) (ATLAS Antibodies, 1: 1,000), MCU (Ref.AMAB91189) (ATLAS Antibodies , 1: 1000), AIF (Ref. 5318S) (Cell Signaling, 1: 1000), CHCHD4 (Ref.21090-l-AP) (Proteintech 1: 1000), Vinculine (Ref. ab219649) (Abeam 1: 1000), GAPDH (Ref.2118) (Cell Signaling, 1: 1000) in TBST with 5% BSA. Membranes were washed and incubated with secondary antibody (Horseradish peroxidase (HRP)-labeled rabbit anti-mouse (Cat#315-035-003, Jackson ImmunoResearch, 1:5000) and goat anti -rabbit antibodies (Cat#l 11-035- 144, Jackson ImmunoResearch, 1:5000)) for 2 hours at room temperature, followed by visualization with the Substrat HRP Immobilon Western (Ref. WBKLS0500, Millipore) and imaged with Amersham™ ImageQuant™ 800. Equal loading was verified by immunoblotting with Vinculin, GAPDH, then normalization of results was performed using free Image J software.

[0197] Lactate Dehysdrogenase Assay (LDH). The half maximal inhibitory concentration (IC50) at 72 h of each compound for all cell line was determined by CytoTox 96® Non-Radioactive Cytotoxicity Assay according to manufacturer instructions (Ref.G1781 Promega). This assay measures the conversion of a tetrazolium (INT) salt to a formazan product which presents a reddish color. The intensity of color produced is proportional to the number of cells lysed. Absorbance is measured using a 96-well microplate reader at 490nm wavelength (TECAN Infinite M200). The half-maxial inhibitory concentration (IC50) was determines using log(agonist) vs. response - Variable slope equation (Y=Bottom + (Top-Bottom) / (l+10A((LogEC50-X)*HillSlope))) by GraphPad Prism5 software (GraphPad Software Inc., California, USA).

[0198] Cell death analysis. Cells were seeded in 6-well plates at 60.000 cells / well for HOS, and 150.000 cells / wells for all in vitro PDX secondary cultures (except 200.000 cells / wells for GR-OS-lOsc and GR-OS-15sc) and treated with M30-E05 at IC50 for 24, 48, 72 h. Culture medium and cells were collected and centrifuged. Supernatant was discarded and cells were stained with APC Annexin V at 12 pg / mL and propidium iodide (PI) at 0.5 mg / mL (BioLegend, Cat#640932). Data acquisition was performed using BD Accuri™ C6 Plus Flow Cytometer. At least 10.000 events were acquired for each sample, and all data were analyzed using FlowJo™ Software (BD Life Sciences). Clonogenic assay. HOS cells were seeded at a density of 500 cells / well in 6-well plates. After 24 h of incubation, cells were treated with lOpM of M30-E05 and kept in the incubator for 10 days under standard conditions. At day 10 after the treatment, colonies on the plate were washed twice with PBS and incubated with Crystal Violet (REF#HT90132, Sigma-Aldrich) for 30 minutes. Photo was taken using Amersham™ ImageQuant™ 800 imaging system.

[0199] ATP Assay. ATPlite 1 step Luminescence Assay system from PerkinElmer (Ref.6016736) was used to evaluate the total ATP contents that reflect the catabolic / anabolic status of the cells by measuring total ATP concentration based on the reaction between ATP with added luciferase and D-Luciferine to produce Oxiluciferine and detectable light. Cells were cultured on white 96- well microplates, provided with the kit (PerkinElmer) and total ATP content was measured 24 h after with TECAN Infinite 200 Pro microplate reader.

[0200] Bioenergetic profiling using Seahorse XFe96 Assay. To determine the metabolic changes in ATP production, parameters such as the rate of cellular mitochondrial respiration and the rate of extracellular acidification, represented respectively by Oxygen consumption rate (OCR) and Glycolytic efflux rate (GlycoPER), were measured using the Seahorse XFe96 Analyzer (Agilent) in real-time in 96-well plates. In the untreated conditions, 2.000 cells / well, 4.000 cells / well and 3.000 cells / well were plated 72 h before assay for parental HOS, well for HOS-R / DOXO for HOS-R / MTX respectively. In the treated conditions, 10.000 cells / well, 20.000 cells / well and 15.000 cells / well were treated during 72 h before assay for parental HOS, well for HOS-R / DOXO for HOS-R / MTX respectively. Three different protocols were used to evaluate together or separately OCR and glycoPER values by injecting different treatments, following manufacturer protocols [Seahorse XF Real-Time ATP Rate Assay (Oligomycin, Rot / AA) (Cat 103592-100); Seahorse XF Cell Mito Stress Test (Cat 103015-100); Seahorse XF Glycolytic Rate Test (Cat 103344-100)].

[0201] Cell counting and normalized analysis. After Seahorse assay, cells were incubated with 2.5pg / ml of Hoechst in 150pL of PBS solution for 15 minutes at 37 °C and observed under the Cytation 1 and counted using Gen5 software (Agilent). Images are captured by DAPI overlay channels or phasecontrast. Raw data analysis and normalization were made using Seahorse analytics.

[0202] Transmission electron microscopy. For ultrastructural studies, cells were fixed in 2% glutaraldehyde in 0.1 M Sorensen phosphate buffer (pH 7.3) for 1 h at 4°C, post-fixed with 2% osmium tetroxide for 1 h at room temperature and stained en bloc in 2% uranyl acetate in 30% methanol for 1 h. Following dehydration through a graded ethanol series, cells were embedded in Epon™ 812. Polymerization was complete after 48 h at 60 °C. Ultrathin sections were stained with standard uranyl acetate and lead citrate and observed with FEI Tecnai 12 electron microscope. Digital images were taken with a SIS MegaviewIII CCD camera.

[0203] Chemicals. All compounds were solubilized in dimethyl sulfoxide (DMSO) at 25mM stock solutions, and stored at -20°C. For in vivo use, the compound was washed in Chloroform: Methanol 1: 1 and then, air-dried by speedvac over-night and then, dissolved in 0.5% methylcellulose (Merck, M7027), freshly before use. Methylcellulose at 0.5% was used as vehicle for in vivo tests. A001, A003 and A004 were provided from the French National Chemical Library.

[0204] Mice studies. Experiments were validated by the CEEA26 Ethic Committee (approval number: APAFIS #37810-2022062308201583) aggreed by the French Ministry of Research in application of articles R.214-87 a R.214-126. For all in vivo experiments, animals were purchased at Gustave Roussy (Villejuif, France) and conducted in the respective animal facilities following standard animal regulations, health and care, and ethical controls. a, In vivo dose escalation. For safety investigation, the 25 female NOD.Cg-PrkdcscidIL2rgtmlWjl / SzJ (NSG) mice were divided into 5 different groups, each receiving different oral doses of the M30-E05 compound: Control (Omg / kg, n=5); lOmg / kg (n=5), 25mg / kg (n=5), 50mg / kg (n=5) and lOOmg / kg (n=5)). M30-E05 was freshly prepared and dissolved in 0.5% methyl cellulose prior to each treatment day. The mice were treated during 4 weeks (5 days / weeks). Behavior, physical appearance, and body weight were monitored and measured to assess any visible potential adverse effects of M30-E05. At the end of the experiment, blood and different organs (e.g., liver, lung, kidney, spleen, brain and heart) as well as bone leg were collected at the experimental endpoint, fixed in 4% PFA (paraformaldehyde), and processed for histological analysis. Blood was used for plasma collection. b, Subcutaneous patient-derived xenografts (PDX). 2-5mm soft frozen tumor fragments from GROS- 18 PDX were xenotransplanted for amplification and posteriorly transplanted subcutaneously into immunocompromised NOD. 35 mice (NSG) (21 females and 14 males) under anesthesia (3% induction, 2% maintenance isoflurane, and 1.5 L / min air). 17 mice were assigned to the control group, receiving the vehicle only for 5 days per week for two weeks. 18 mice were assigned to the treatment group, receiving 100 mg / kg M30-E05. Mice were treated for 5 days per week for 2 weeks. Clinical observations included monitoring skin changes, behavior, posture, response to handling, and abnormal movements. Body weight and tumor volume were measured twice weekly. Tumor volume was calculated according to the equation: V (mm3)=width2 (mm2) x length (mm) / 2. The experiments lasted until tumors reached specific endpoints detailed in the ethical projects. At mice sacrifice, blood, tumor and various organs (bone, liver, lung, kidney, spleen, and brain) were collected for mass spectrometry and western-blot (snap-frozen by immersing the cryovials in liquid nitrogen) and histology. Tissues fixed in 4% PFA and included in FFPE (Formalin-Fixed Paraffin-Embedded) blocs were sectioned and stained with hematoxylin and eosin (H&E). An experienced histopathologist at the Gustave Roussy Preclinical Evaluation Department assessed all sections.

[0205] ELISA. Blood from 25 treated mice and vehicle-treated mice used for the in vivo efficiency were centrifuged at 2000 rpm, 5 minutes at 4°C for plasma collection. The mouse cardiac troponin-I (TNNI3) levels in plasma were measured using an ELISA Kit (REF#EEL112, Invitrogen), following the manufacturer’s instructions.

[0206] Computational study. Both AIF monomer (co-crystalized with one FAD (Flavin adenine dinucleotide), PDB (protein database) ID: 1M6I [Ye et al. Nat Struct Biol 2002; 9: 680-684]) and dimer (co-crystalized with one FAD and two NAD, PDB ID: 4BUR [Ferreira et al. Biochemistry 2014; 53: 4204-15]) were considered in this investigation. Missing residues of each structure (except those at terminal ends) were first added using SWISS-MODEL webserver [Waterhouse et al. Nucleic Acids Res 2018; 46: W296-W303], Then each initial structure was submitted to a 500 ns (AIF monomer) or 1 ps (AIF dimer) molecular dynamics (MD) simulation in explicit solvent, by using AMBER99SB-ILDN [Lindorff-Larsen et al. Proteins 2010; 78: 1950-8] and TIP3P [Jorgensen et al. J Chem Phys 1983; 79: 926-35] all-atom force field and GROMACS software [Abraham et al. SoftwareX 2015; 1-2: 19-25], Finally, the 5 most populated clusters of each protein conformational ensemble were extracted from their MD trajectory and used as receptors in subsequent docking calculations.

[0207] Each studied ligand was blindly docked 100 times on the whole surface of each of the AIF clusters (without specifying any preferred binding site), by using AutoDock Vina software [Trott & Olson. J Comput Chem 2010; 31: 455-6], Each docking calculation generating 10 binding modes, atotal of 5.000 binding modes of each ligand was obtained for each of the AIF monomer and dimer.

[0208] Cell lines (lung cancer). A panel of lung adenocarcinoma cell lines HOP62, A437, H522, H1650, H1793, H1838, H1975, H2030, H2343 were generously provided by Dr Luc Friboulet (INSERM UMR981, Gustave Roussy). All the cell lines were routinely cultured in Dulbecco’s modified Eagle medium (DMEM, high glucose, GlutaMAX Supplement, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma- Aldrich, Paraguay Origin) and 1% Penicillin-Streptomycin (Gibco) at 37°C in humidified atmosphere (5% CO2 and 95% air), under mycoplasma free conditions.

[0209] Transcriptomic analysis. Total RNAs were isolated using the NucleoSpin® RNA (Macherey- Nagel, 740955) according to the manufacturer’s instruction, stored at -80°C. RNA quality control were performed on NanoDrop™ 2000. RNA-sequencing results were obtained by Novogene. Differentially expressed genes (DEGs) between untreated HOS cells and the M30-E05 IC50 treated HOS cells were identified using DESeq2 package (absolute log2 fold change > 1 and adjusted p-value < 0.05). Therefore, the DEGs were uploaded into DAVID bioinformatics tool (Database for Annotation, Visualization and Integrated Discovery) and analyzed for enriched GO terms in the biological process category. 1

[0210] SynergyFinder. Evaluation of drug interactions between M30-E05 and conventional chemotherapeutic agents Cisplatin (CIS), Doxorubicin (DOXO), and Methotrexate (MTX) was conducted on parental (HOS) and DOXO- or MTX-resistant (HOS-R / DOXO, HOS-R / MTX) models. HOS (2000 cells / well), HOS-R / DOXO (7000 cells / well) and HOS-R / MTX (5000 cells / well) were seeded in 96-well plates and treated with a combination of M30-E05 and one chemotherapy, in the form of a matrix, for 72 hours. Cell viability was measured using LDH assay, and data was uploaded on the SynergyFinder platform (https: / / synergyfmder.fimm.fi / ). Using the zero interaction potency (ZIP) model, an overall synergy score was calculated for each matrix, with values below 10 indicating antagonism, in between -10 and 10 as additivity, and above 10 as synergism. 2D and 3D interaction maps were also provided, in which the colors green, white, and red demonstrate antagonism, additivity, and synergism, respectively. Experiments were repeated at least 3 times.

[0211] LC-MS / MS analyses. Proteins from DMSO-treated and M30-E05 -treated were extracted in 30 mM Tris buffer (pH 7.4) with 8 M urea, 4% CHAPS, and protease inhibitors by sonication and centrifugation, as described by our collaborator. After centrifugation (15,000 x g for 15 min at 4 °C) to remove cellular debris and ultracentrifugation at 105,000 x g for I h at 4 °C to remove subcellular particles, cytosoluble proteins were stored at -80 °C until further analysis. Total protein concentration in each sample was determined using the Pierce™ BCA Protein Assay according to the manufacturer's instructions (Thermo Scientific). Samples were sent to proteomic platform to acquire data by nano liquid chromatography tandem mass spectrometry (MS / MS). Briefly, the resulting peptide mixtures were separated on a 75 pm x 250 mm C18 lonOpticks Aurora 2 column (Ion Opticks Pty Ltd., Bundoora, Australia) with a NanoElute HPLC system (Bruker Daltonik GmbH) at a flow rate of 400 nL / min at 50°C. The separation was performed with a buffer gradient (buffer A: 0.1% formic acid, 98% H2O MilliQ, 2% acetonitrile; buffer B: 0.1% formic acid, 100% acetonitrile) for 120 min (2%— 15% buffer B for 60 min; up to 25% at 90 min; up to 37% at 100 min; up to 95% at 110 min and finally 95% for 10 min to wash the column). The column was coupled in-line to a timsTOF Pro (Bruker Daltonik GmbH) with a CaptiveSpray ion source (Bruker Daltonik GmbH). LC-MS / MS data were acquired by the PASEF method66 with a total cycle time of 1.31 s, comprising 1 TIMS MS scan and 10 PASEF MS / MS scans. The 10 PASEF scans (100 ms each) contained, on average, 12 MS / MS scans per PASEF scan.

[0212] MS data were processed with the Data Analysis 5.1 software to produce the peak list of MS and MS / MS spectra. Peptide and protein identification was then performed using the Mascot database search engine (http: / / www.matrixscience.com) using its automatic decoy database search to calculate a false discovery rate (FDR) as previously described. MS / MS spectra were simultaneously compared with the UniProt KB UP000005640 human proteome database restricted to one protein sequence per gene. The mass tolerance for MS and MS / MS was set at 15 pmm and 0.05 Da. The enzyme was set to full trypsin with allowed miscleavage. The modifications are fixed carbamidomethylation of cysteines, variable oxidation of methionine, variable acetylation of lysine and N-terminal proteins, and variable deamidation of asparagine and glutamine. Identification results from Mascot (.dat files) were imported into the Proline Studio software v2.1 .2.66 This software was then used to validate the identification of proteins with a peptide rank = 1, an FDR of 1% on the peptide spectra match score and peptides with a minimum score of 30 (-10*LOG10(P), where P is the absolute probability).

[0213] Proteomics data were analyzed in R using the limma package (Bioconductor) for differential expression and ggplot2 for visualization. Raw protein intensity values from DMSO-treated and IC50 M30-E05- treated HOS cells conditions (n = 4 replicates per group) were used to perform the analysis. Proteins with fewer than three non-missing values per condition were excluded. Data were log2-transformed (with a pseudocount of 1) and normalized by quantile normalization. A design matrix was constructed to model group effects, and a linear model was fitted to the normalized intensities using ImFit. Differential expression was estimated with contrasts (DMSO-treated and IC50 M30-E05 -treated HOS cells), followed by empirical Bayes moderation (eBayes). Significance thresholds were set at adjusted p < 0.1 (Benjamini-Hochberg correction) and |log2 fold change] > 0.5. Results were visualized as volcano plots, with proteins classified as up- or down-regulated according to these criteria.

[0214] Functional enrichment analyses were performed in R using the clusterProfiler package with annotation from org.Hs.eg.db. Differentially expressed proteins (DEPs) identified from the DMSO-treated and IC50 M30-E05 -treated HOS cells comparison were used as the test set, and all detected proteins in the proteomics dataset served as the background. Over-representation analyses were conducted for Gene Ontology (GO) terms (Biological Process, Molecular Function, and Cellular Component) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Enrichment significance was assessed by hypergeometric testing with Benjamini-Hochberg correction for multiple comparisons, applying a false discovery rate (FDR) threshold of 0.05 for GO terms and 0.1 for KEGG pathways. Results were visualized as dot plots and bar plots showing the top enriched categories. Additionally, pathway-specific protein lists were generated by mapping DEPs to enriched KEGG terms for downstream interpretation.

[0215] Mitochondrial membrane potential. To analyze the mitochondrial membrane potential, HOS cells were seeded at 110.000 cells / 3.5 cm2and allowed to adhere for 24h, before treatment of M30-E05 at ICso for 24h. Next, cells were trypsinized and incubated with lOOnM Tetramethylrhodamine methyl ester (TMRM) for 10 minutes at 37°C, in the dark. Data acquisition was performed using BD Accuri™ C6 Plus Flow Cytometer. At least 10.000 events were acquired for each sample, and all data were analyzed using FlowJo™ Software (BD Life Sciences).

[0216] Statistics. Statistical analysis (Mann-Whitney test) was performed using GraphPad Prism for scatter plot graphs in RNA-Seq analysis. ***P<0.001; **P<0.002, *P<0.033. Statistical analysis is performed using one-way ANOVA test. * P<0.05, **P<0.01, *** P0.001, **** P0.0001 with GraphPad Prism software.

[0217] EXAMPLE 2 - Synthesis of M30-E05

[0218] M30-E05 (2 - [3 -(cyclopentyloxy)-4-methoxyphenyl] -7-[2-(3 ,4-dihydroxyphenyl)ethoxy] -5 -hydroxy- 4H-chromen-4-one) was synthesized in 5 steps starting from dihydroxyacetophenone and 3’-O- cyclopentyl-5-desoxydiosmetine (Patent G. Lewin ; EP 0832 886 Al)

[0219] To a solution of 3,4-Dihydroxyacetophenone (3.70 g, 24.32 mmol, 1.0 eq) in DMF (120 mL) were successively added K2CO3 (16.80 g, 121.60 mmol, 5 eq) and benzylchloride (6.72 mL, 58.36 mmol, 2.4 eq) under N2 The resulting mixture was stirred at 120 °C overnight. The mixture was diluted with DCM (dichloromethane) (50 mL), filtered on celite and washed with H2O (3x50 mL). The organic layers were dried over anhydrous MgSCL and concentrated in vacuo to give the product as white solid which was used without further purification, (6.85 g, 85%).

[0220] ’H NMR (300 MHz, Chloroform-d) 5 7.61 (d, J = 2.1 Hz, 1H), 7.53 (dd, J = 8.4, 2.1 Hz, 1H), 7.49 - 7.28 (m, 10H), 6.94 (d, J = 8.4 Hz, 1H), 5.24 (s, 2H), 5.20 (s, 2H), 2.51 (s, 3H).

[0221] 13C NMR (75 MHZ, Chloroform-d) 5 196.8, 153.4, 148.8, 136.9, 136.6, 130.9, 128.7, 128.7, 128.2, 128.1, 127.5, 127.2, 123.6, 114.0, 113.2, 71.4, 71.0, 26.3

[0222] OD026 A mixture of OD003 (9.82 g, 30.0 mmol, 1 eq), CuO (3.29 g, 41.36 mmol, 1.4 eq) and iodide (10.50 g, 41.36 mmol, 1.4 eq) in MeOH (100 mL) was heated at 70 °C until no starting material is detected by TLC after 4h. The mixture was filtered on celite and the solvent was removed. The residue obtained was diluted in DCM (50 mL) washed with Na2S2C>3 (3x50 mL), and dried over anhydrous MgSCL. Evaporation of the solvent gave the product OD026, which was used in the next step without further purification, as a colorless solid (11.05 g 82%).

[0223] ’H NMR (300 MHz, Chloroform-d) 5 7.62 (d, J = 2. 1 Hz, 1H), 7.57 (dd, J = 8.4, 2.1 Hz, 1H), 7.50 - 7.29 (m, 10H), 6.95 (d, J = 8.4 Hz, 1H), 5.25 (s, 2H), 5.21 (s, 2H), 4.26 (s, 2H).

[0224] 13C NMR (75 MHz, Chloroform-d) 5 191.5, 153.9, 149.0, 136.8, 136.4, 128.8, 128.7, 128.2, 128.1, 127.5, 127.2, 127.0, 124.3, 114.8, 113.1, 71.4, 71.0, 1.5.

[0225] HRMS-ESI (m / z) : calcd for [M+H]+C22H19IO3, 459.0457; found : 459.0464

[0226] 00001

[0227] 7-O-Benzyl-3’-O-cyclopentyl-diosmetine was added (5 g, 11.00 mmol, 1 eq) in a mixture of MeOH / EtOAc (80 / 20 mL). A suspension of Pd / C (600 mg, 10 % w / w) was introduced to the mixture. The mixture was stirred overnight under atmospheric H2 pressure and at room temperature (20-25 °C). After removal of Pd / C by filtration, the solution was evaporated to afford the product as yellow solid. (3.68g, 92%)

[0228] ’H NMR (300 MHz, DMSO-de) 5 12.91 (s, 1H), 10.78 (br, 1H), 7.63 (dd, J = 8.5, 2.2 Hz, 1H), 7.51 (d, J = 2.2 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 6.89 (s, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.20 (d, J = 2.1 Hz, 1H), 5.05 - 4.86 (m, 1H), 3.84 (s, 3H), 2.02 - 1.84 (m, 2H), 1.81 - 1.66 (m, 4H), 1.63 - 1.50 (m, 2H).

[0229] 13C NMR (75 MHz, DMSO) 5 181.7, 164.2, 163.3, 161.4, 157.3, 153.0, 147.2, 122.8, 119.9, 112.2, 112.1, 103.7, 98.8, 94.0, 79.8, 55.7, 32.1, 23.6.

[0230] HRMS-ESI (m / z) : calcd for [M+H]+C2IH2IO6, 369.1338 ; found : 369.1334.

[0231] 00023

[0232] OD026 (1.64 g, 3.58 mmol, 1.1 eq) and K2CO3 (675 mg, 4.89 mmol, 1.5 eq) were added to a solution of OD001 (1.20 g, 3.26 mmol, 1.0 eq) in CH3CN (30 mL). The resulting mixture was stirred overnight at 90 °C, and then filtered on celite. After removal of the solvent by vacuum evaporation, the obtained residue was diluted with CH2CI2 (50 mL), washed with water (2x30 mL) and dried over anhydrous MgSCEto afford the product as a brownish solid (1.87 g, 82%).

[0233] ’H NMR (300 MHz, Chloroform-d) 5 12.82 (s, 1H), 7.62 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.52 - 7.28 (m, 13H), 6.97 (t, J = 7.9 Hz, 2H), 6.56 (s, 1H), 6.51 (s, 1H), 6.34 (s, 1H), 5.31 - 5.13 (m, 6H), 4.85 (s, 1H), 3.92 (s, 3H), 1.91 (m, 6H), 1.64 (m, 2H).

[0234] 13C NMR (75 MHz, Chloroform-d) 5 191.6, 182.5, 164.5, 163.9, 162.5, 157.7, 154.2, 153.7, 149.1, 148.1, 136.7, 136.4, 128.8, 128.7, 128.3, 128.2, 127.7, 127.5, 127.2, 123.7, 123.2, 120.2, 114.0, 113.3, 112.7, 111.9, 106.2, 104.7, 98.5, 93.8, 81.1, 71.4, 71.0, 70.6, 56.2, 32.9, 24.2.

[0235] HRMS-ESI (m / z) : calcd for [M+H]+C43H39O9, 699.2594 ; found : 699.25.89.

[0236] Activated 20% Pd / C (480 mg) was added to a suspension of OD023 (1.38 g, 2.0 mmol, 1.0 eq) in THF (20 mL), and the mixture was stirred under an atmospheric H2 pressure for 5 days. Resulting mixture was fdtered through a short path of celite, eluted with dichloromethane. After removal of the solvent, the crude residue was purified by preparative liquid chromatography to afford M30-E05 as a yellowish solid (270 mg, 27%).

[0237] ’H NMR (300 MHz, DMSO-de) 5 12.91 (s, 1H), 8.72 (s, 1H), 8.67 (s, 1H), 7.70 (dd, J = 8.6, 2.2 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.12 (d, J = 8.6 Hz, 1H), 6.99 (s, 1H), 6.82 (d, J = 2.2 Hz, 1H), 6.71 (d, J = 2.0 Hz, 1H), 6.67 (d, J = 7.9 Hz, 1H), 6.57 (dd, J = 7.9, 2.0 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 5.01 (t, J = 5.7 Hz, 1H), 4.24 (t, J = 6.9 Hz, 2H), 3.85 (s, 3H), 2.89 (t, J = 6.8 Hz, 2H), 2.02 - 1.86 (m, 2H), 1.80 - 1.53 (m, 6H).

[0238] 13C NMR (75 MHz, DMSO) 5 181.9, 164.4, 163.6, 161.1, 157.2, 153.2, 147.2, 145.0, 143.8, 128.5, 122.7, 120.1, 119.6, 116.3, 115.4, 112.3, 112.1, 104.7, 103.9, 98.3, 93.2, 79.8, 69.4, 55.8, 34.0, 32.1, 23.6.

[0239] HRMS-ESI (m / z) : calcd for [M+H]+C29H29O8, 505.1862 ; found : 505.1865.

[0240] IR (u.cm-1) 2966, 2866, 1654, 1608, 1500, 1442, 1257, 1166, 1064, 908, 642.

[0241] EXAMPLE 2b - Synthesis of M30-F05

[0242] Activated 20% Pd / C (480 mg) was added to a suspension of OD023 (1.38 g, 2.0 mmol, 1.0 eq) in THF (20 mL), and the mixture was stirred under an atmospheric H2pressure for 7 days. Resulting mixture was filtered through a short path of celite, eluted with dichloromethane. After removal of the solvent, the crude residue was purified by preparative liquid chromatography to afford M30-F05 as a yellowish solid (170 mg, 20%).

[0243] EXAMPLE 3 - Synthesis of LF02 EXAMPLE 5 - Synthesis of LF12

[0244] EXAMPLE 6 - Synthesis of LF12bis

[0245] EXAMPLE 7 - Synthesis of LF21

[0246] EXAMPLE 8 - Synthesis of LF21bis

[0247] Molecular Weight: 472,5370

[0248] OD245-Red

[0249] The compound was prepared according to the following scheme: , , , , , , , , , , , J= 8.3 Hz, 1H), 6.85 (d, J= 2.1 Hz, 1H), 6.78 (d, J= 8.0 Hz, 1H), 6.67 (dd, 7= 8.2, 2.1 Hz, 1H), 6.64 (d,7 = 2.2 Hz, 1H), 6.58 (s, 1H), 6.30 (d,7 = 2.2 Hz, 1H), 4.26 (t,7 = 6.9 Hz, 2H), 2.97 (t,7 = 6.92H).13C NMR (75 MHz, Acetone-7) 8183.1, 165.8, 165.4, 163.1, 158.7, 150.2, 146.6, 145.9, 144.6, 130.6, 123.8, 121.2, 120.2, 117.0, 116.7, 116.2, 116.1, 114.3, 104.3, 99.2, 93.7, 70.5, 35.5.

[0250] EXAMPLE 11 - Synthesis of OD263

[0251] 00263

[0252] The compound was prepared according to the following scheme:

[0253] OD263

[0254] 'H NMR (300 MHz, Chloroform-^ 87.45 (dd, J= 8.6, 2.1 Hz, 1H), 7.31 (d, J= 2.2 Hz, 1H), 6.93 (d, J= 8.6 Hz, 1H), 6.85-6.83 (m, 3H), 6.56 (d, J = 2.2 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 4.87-4.83 (m, 1H), 4.25 (t, J= 7.0 Hz, 2H), 3.94 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.87 (s, 3H), 2.03 - 1.81 (m, 6H), 1.72 - 1.60 (m, 2H).13C NMR (75 MHz, Chloroform-^ 8 177.7, 163.2, 161.1, 160.9, 159.9, 153.0, 149.2, 148.2, 148.1, 130.3, 124.1, 121.1, 119.5, 112.6, 112.5, 111.8, 111.7, 108.0, 96.4, 93.8, 80.9, 69.6, 56.6, 56.2, 56.1, 35.3, 33.0, 24.2.

[0255] EXAMPLE 12 - Synthesis of OD265

[0256] The compound was prepared according to the following scheme:

[0257] 00265

[0258] 'H NMR (300 MHz, Chloroform-^ 8 12.76 (s, 1H), 7.45 (dd, J= 8.5, 2.2 Hz, 1H), 7.32 (d, J= 2.2 Hz, 1H), 6.98 - 6.86 (m, 4H), 6.54 (s, 1H), 6.47 (d, J= 2.3 Hz, 1H), 6.36 (d, J= 2.3 Hz, 1H), 4.84 (tt, J= 5.8, 2.9 Hz, 1H), 4.54 (dd, J= 7.7, 3.7 Hz, 1H), 4.22 (dd, J= 10.1, 7.7 Hz, 1H), 4.08 (d, J= 3.7 Hz, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.35 (s, 3H), 2.01 - 1.80 (m, 6H), 1.72 - 1.57 (m, 2H).13C NMR (75 MHz, Chloroform-J) 8 182.3, 164.5, 164.1, 162.2, 157.6, 153.4, 149.4, 149.2, 147.9, 130.5, 123.6, 119.9, 119.6, 112.5, 111.7, 111.3, 109.8, 105.7, 104.5, 98.7, 93.3, 81.62, 80.9, 72.6, 57.1, 56.1, 56.0, 32.8, 24.1.

[0259] EXAMPLE 13 - Synthesis of OD169

[0260] Molecular Weight: 518,5180

[0261] 00163

[0262] The compound was prepared according to the following scheme:

[0263] 'H NMR (300 MHz, DMSO-^e) <5 12.98 (s, 1H), 8.96 (s, 1H), 8.88 (s, 1H), 7.14 - 7.06 (m, 3H), 6.78 (d, J= 2.1 Hz, 1H), 6.72 (d, J= 8.0 Hz, 1H), 6.66 - 6.62 (m, 1H), 6.60 (d, J= 2.1 Hz, 1H), 3.84 (s, 3H), 1.79 - 1.52 (m, 8H).13C NMR (75 MHz, DMSO-fifc) 8 182.5, 169.8, 164.4, 160.8, 156.2, 155.8, 153.4, 147.2, 145.2, 144.5, 124.9, 124.0, 122.4, 120.4, 120.3, 116.8, 115.6, 112.1, 108.1, 105.1, 104.3, 101.6, 79.7, 55.8, 32.2, 30.4, 23.7.

[0264] EXAMPLE 14 - Synthesis of OD240

[0265] The compound was prepared according to the following scheme: 'H NMR ' H NMR (300 MHz, THF-t / s) 5 12.96 (s, 1H), 7.62 (dd, J = 8.5, 2.2 Hz, 1H), 7.51 (d, J= 2.2 Hz, 1H), 7.17 (s, 2H), 7.05 (d, J= 8.6 Hz, 1H), 7.01 (d, J= 2.0 Hz, 1H), 6.77 (s, 1H), 6.63 (d, J= 2.0 Hz, 1H), 4.94 (tt, J = 5.6, 2.8 Hz, 1H), 3.90 - 3.82 (m, 3H), 1.95 - 1.79 (m, 6H), 1.68 - 1.57 (m, 2H).13C NMR (75 MHz, THF) 5 183.6, 165.8, 164.6, 163.1, 158.0, 157.8, 155.3, 149.3, 146.7, 140.4, 126.1, 124.4, 121.2, 120.1, 113.9, 113.0, 110.5, 109.3, 106.1, 105.3, 101.9, 81.6, 56.4, 33.7, 30.9.

[0266] EXAMPLE 15 - Synthesis of OD164

[0267] Molecular Weight 504,4910

[0268] 00164

[0269] The compound was prepared according to the following scheme:

[0270] 'H NMR (300 MHz, DMSO-t / e) 8 12.99 (s, 1H), 7.71 (dd, J= 8.6, 2.1 Hz, 1H), 7.58 (d, J= 2.2 Hz, 1H), 7.54- 7.50 (m, 2H), 7.24 (d, J = 2.1 Hz, 1H), 7.16 - 7.09 (m, 2H), 6.91 (d, J = 8.7 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 5.02-4.98 (m, 1H), 3.84 (s, 3H), 1.79 - 1.53 (m, 8H).13C NMR (75 MHz, DMSO-rfc) 8 182.5, 164.3, 163.7, 160.8, 156.2, 153.3, 151.7, 147.3, 145.4, 139.2, 124.9, 123.0, 122.4, 120.4, 118.9, 116.9, 115.6, 112.1, 108.0, 105.4, 104.3, 101.9, 79.7, 55.8, 32.2, 23.7.

[0271] EXAMPLE 16 - Synthesis of OD235-Ter

[0272] Molecular Weight: 490,5080

[0273] OD235-Ter

[0274] The compound was prepared according to the following scheme:

[0275] 'H NMR (500 MHz, DMSO-r / e) 512.90 (s, 1H), 8.72 (s, 2H), 7.69 (dd, J= 8.6, 2.2 Hz, 1H), 7.53 (d, J= 2.2 Hz, 1H), 7.11 (d,J=8.6 Hz, 1H), 6.98 (s, 1H), 6.80 (d,J=2.2 Hz, 1H), 6.71 (d,J=2.1Hz, 1H), 6.67 (d, 7=8.0 Hz, 1H), 6.57 (dd, J= 8.1, 2.1 Hz, 1H), 6.35 (d, J= 2.2 Hz, 1H), 4.23 (t, J= 6.9 Hz, 2H), 3.93 (d, J= 7.0 Hz, 2H), 3.87 (s, 3H), 2.89 (t, J= 7.0 Hz, 2H), 1.01 (t, J= 12 Hz, 1H), 0.63 - 0.53 (m, 2H), 0.37 - 0.29 (m, 2H).13C NMR (126 MHz, DMSO) 5 181.9, 164.4, 163.6, 161.1, 157.2, 152.4, 148.3, 145.1, 143.8, 128.5, 122.7, 120.1, 119.6, 116.4, 115.5, 111.8, 110.5, 104.7, 103.9, 98.3, 93.2, 73.1, 69.4, 55.7, 34.0, 10.1, 3.1.

[0276] EXAMPLE 18 - In silico characterization of compounds of the Invention on binding energy and binding site on AIF

[0277] The docking procedure described above was first used on 7 compounds from the Pretswick chemical library for which an experimental IC50 of the AIF / CHCHD4 interactions could be measured by the Alphascreen technology. In a second step, A0001-A0005 which could also inhibit the AIF / CHCHD4 complex, but the IC50 of which could not be determined accurately, were docked on AIF monomer and dimer, using the same protocol as described above. (Table 1). As show in Figure 1, ligand average binding energies yielded by AutoDock Vina on both AIF monomer and dimer could be fairly correlated with the experimental log(IC5o), indicating that the computational method is a helpful tool for identifying potential efficient inhibitors of AIF / CHCHD4 interactions.

[0278] The calculations show that these 5 compounds have overall better binding energies than the previous 7 Pretswick compounds and indicate that the 3 molecules A002, A004, and A005 are potentially better inhibitors of AIF / CHCHD4 interactions than the best molecule from Pretswick (Figure 1).

[0279] Next, the docking approach was applied on a series of 35 small molecules derived from A002, A004, and A005 compounds to guide the synthesis and test of more diverse promising inhibitors. Among them, 14 molecules were found with better binding energies than that of A005 on either AIF monomer or dimer (Table 2).

[0280] Table 2. Chemical structure and code of A002, A004, and A005 derivatives designed to inhibit AIF / CHCHD4 interactions. The two right columns indicate their binding energies on AIF monomer (M16I) and dimer (4BUR) yielded by AutoDock Vina docking calculations.

[0281] To gain insight into the mechanism of action of these AIF / CHCHD4 inhibitors, the preferred binding site of the best molecules from the Prestwick, French National Chemical Library, was further investigated, and derivative series on AIF monomer and dimer were designed. Strikingly, these molecules preferentially bind the same surface area on AIF protein which correspond to NAD binding pocket. This suggests that these molecules might competitively inhibit NAD binding, which in turn would prevent AIF dimerization. In AIF dimer which has NAD in its pocket, the preferred binding sites of the ligands are rather localized on chain A near its junction with chain B (Figure 2 and Figure 3). EXAMPLE 19 - Cytotoxicity study in cancer and normal cell line panels for osteosarcoma

[0282] Various osteosarcoma cell lines (HOS, HOS-R / DOXO, HOS-R / MTX, MG63, SAOS2, SAOS-R / MTX, 143B and U2OS) were treated with a dose-response of M30-E05 (8 doses, 1-lOOmM) for 72 h. Then, cell viability was evaluated by lactate deshydrogenase assay (LDH) assay, and an IC50 was calculated using PRISM software about lOmM for all cell lines, independently of doxorubicin and methotrexate drug resistance of some cell lines (Figure 4). To evaluate long term effects, M30-E05 was used at 10 mM in clonogenic assay in the 3 HOS cell lines, showing its ability to eliminate all cells in 14 days (Figure 5). In contrast, M30-E05 was less efficient on normal cells and on cells treated by siRNA against AIF to downregulate its expression. Thus, M30-E05 does not kill more than 50% of HEK293 cells for a lOOmM (IC50 = 62mM approx.) and its IC50 increased to 16.52mM when cells when AIF was knocked- down by siRNA (Figure 6). Then, the effect of M30-E05 (Figure 7). and with M30-F05 (Figure 8) was evaluated on freshly-isolated human peripheral blood mononuclear cells (PBMCs) and showed no toxicity until 100 pM.

[0283] To evaluate the effects of M30-E05 on adult cancer cells, cell lines from various tumoral origins were selected (Figure 9). M30-E05 displayed cytotoxicity on other type of cancer including head and neck cancers, ovarian cancer (both parental and resistant cell lines), lung, pancreatic cancer and leukemia. Of note, M30-E05 was able to display IC50 at 2-3 mM on ovarian cancers and some leukemia cell lines. This result shows that the interruption of AIF / CHCHD4 complex by M30-E05 has a lethal effect in a wide range of cell lines with different tumoral origins. IC50 of the different cell lines are reported on Table 3.

[0284] Table 3. ICso of the different cell lines

[0285] IC50 of M30-E05 was compared to other various anticancer compounds after 72h with various cancer cell lines. The results are in Table 4.

[0286] Table 4. Comparison of various IC50 on different cancer cell lines EXAMPLE 20 - Kinetic analysis of M30-E05 cellular effects for osteaosarcoma

[0287] HOS cells were treated with 2 sublethal doses of the compound (IC5o= 8 and IC25= 16 mM) and AIF dimerisation by non-denaturing electrophoresis and Western-blot was analyzed (Figure 11). The result shows that the compound interrupts the complex in cellulo at 24 h.

[0288] As a functional consequence of the disruption of the AIF dimer, a decrease in AIF, CHCHD4 and two nuclear-encoded substrates of the AIF / CHCHD4 pathway, namely MICU1 and Cox 17 was detected (Figure 10, Figure 11 and Figure 12). This protein import defect was not due to a change in mitochondrial mass, because the expression level of VDAC, an outer membrane protein, remained unchanged.

[0289] As expected from a defect in mitochondrio-nuclear communication leading to a perturbation of mitochondrial protein import, M30-E05 decreased mitochondrial oxygen consumption rate (OCR) (Figure 13) and increased total ATP due to an increase in glycolysis (Figure 14). This metabolic reprogrammation of energetic metabolism was accompanied by a loss of cell viability and cell death induction. Indeed, the compound induced apoptosis, as shown by the measurement of the permabilisation of the plasma cell membrane and the exposure of phosphatidylserine by Annexin V / PI labeling by flow cytometry at 24 h, 48 h and 72 h (Figure 15). When HOS cells were treated at IC50 dose of M30-E05 for 72h00, 50% loss of cell viability (Figure 15) and 15% cell death were detected (Figure 15).

[0290] EXAMPLE 21 - Effect of M30-E05 on lipid and aminoacid metabolism

[0291] Pathway analysis of metabolic signaling in HOS cell lines treated by M30-E05 were based on enrichment analysis procedures, thus identifying the most relevant metabolic pathways via pathway impact and adjusted p-value. The figures were drawn via MetaboAnalyst software v 4.0. MetaboAnalyst that was developed by Dr. Jianguo Xia of the Institute of Parasitology at McGill University. It is a free software for metabolic analysis, (http: / / www.metaboanalyst.ca / feces / home.xhtml)

[0292] The effect of M30-E05 on the metabolome of 3 HOS cell lines was then studied by ms / ms analysis. The perturbation induced by cell treatment by M30-E05 affected mainly mitochondrial b-oxidation of short and long chain fatty acids (Figure 16, Figure 17 and Figure 18). Moreover, some activation of amino acid metabolic pathways, such as arginine and proline metabolism, have been also detected in the enrichment analysis (Figure 19, Figure 20 and Figure 21).

[0293] The perturbation induced by cell treatment by M30-E05 affected mainly mitochondrial -oxidation of short and long chain fatty acids. Moreover, some activation of amino acid metabolic pathways, such as arginine and proline metabolism, have been also detected in the enrichment analysis (Figure 19, Figure 20 and Figure 21).

[0294] EXAMPLE 22 - Efficacy in ex vivo cultured PDX in osteaosarcoma

[0295] To investigate cancer models closer to patients, some effects of M30-E05 were analyzed in ex vivo cultured PDX. These models are particularly versatile and fragile (Marques Da Costa, 2023). Thus, not all PDX grow well ex vivo and they cannot be cultured more than 3 passages without losing their fitness. Thus, presented data are those who have been confirmed at least three times for PDX that grew.

[0296] First, M30-E05 ICsos was determined and it was oberved that even the PDX are multiresistant to chemotherapeutic agents (i.e. cisplatin, etoposide, doxorubicin... etc), the toxicity of M30-E05 appeared in a comparable range (IC50 =10-20mM approx.) to osteosarcoma cell lines excepted for GR-OS-l lsc (Figure 22, Figure 23).

[0297] The IC50 values for the subcutaneous PDX of various cell lines can be found on Table 5.

[0298] Table 5. IC50 values for the subcutaneous PDX 0 ’ various cell lines

[0299] The IC50 values for the orthotopic PDX of various cell lines can be found on Table 6.

[0300] Table 6. IC50 values for the orthotopic PDX of various cell lines

[0301] Second, the data show that M30-E05 decreased basal respiration to a minimal level, excepted for GR- OS-15sc (Figure 24).

[0302] Third, surprisingly, the mitochondrial protein import of AIF, CHCHD4 and MICU 1 appeared modulated in variable ways by M30-E05, some PDX being inhibited whereas other being stimulated depending on the duration of treatment (Figure 25). Explanation of these findings is still elusive, but so far, indicates that M30-E05 affects the mitochondrial import function of AIF / CHCD4 complex in the 5 PDX tested. Finally, in at least two PDX, M30-E05 treatment leads to apoptotic cell death induction as shown by Annexin V / PI labelling (Figure 26 and Figure 27).

[0303] EXAMPLE 23 - Toxicity and antitumor activity of M30-E05 in subcutaneous OS PDX

[0304] As a preclinical safety assessment, a dose-escalation study was performed in NSG mice to determine the maximal tolerated dose. Ms / ms analysis of plasma and bone showed the accumulation of M30-E05 in bone of all 5 mice in lOOmg / kg M30-E05 treatment group but no M30-E05 was found in plasma extracted from the same mice. This result indicates the rapid metabolization of M30-E05 in animal body and M30-E05 could be delivered to the leg bone. No statistical difference in body weight was observed in comparison with the control group who received only the vehicle (Figure 28). The behavior and the fur aspects remained unaffected during the experiment. Moreover, the HE staining (ematoxylin and Eosin staining) of all lOOmg / kg M30-E05 -treated mice organs (heart, lung, liver, kidney, spleen, brain) showed no morphological alterations while compared with control group (Figure 29). Finally, the mouse cardiac troponin-I (TNNI3) levels remained undetectable in plasma that were collected at the sacrifice (not shown) suggesting no acute cardiotoxicity.

[0305] The GR-OS-18 PDX model was evaluated at advanced stage, i.e. with established and growing tumors of 80-150 mm3volumes at 14 days after tumor transplantation. Mice were randomly assigned to M30- E05 treatment at 100 mg / kg day, administered via oral gavage during 14 days. Control animals were treated adequately with 0.5% methyl cellulose as the vehicle solution. M30-E05 effectively inhibited subcutaneous osteosarcoma PDX in vivo growth with a tumor growth inhibition of 41% after 2 weeks of treatment at 100 mg / kg (753+ / - 479.5 mm3), compared to the empty vector group (1370.85 + / - 741.1 mm3). In contrast, the tumor in the control group grew extremely fast, the tumor volume is doubled every 3 days. As showed by dose escalation experiment, no observable toxicity was recorded during treatment session at 100 mg / kg (Figure 30 and Figure 31).

[0306] EXAMPLE 24 - Cytotoxicity study in cancer and normal cell line panels for lung cancer

[0307] This example is carried out according to the conditions of Example 19. The cell lines chosen are reported on Table 7.

[0308] Table 7. Cell lines of lung cancer for the cytotoxicity study

[0309] EXAMPLE 25 - Kinetic analyses of M30-E05 cellular effects for lung cancer This example is carried out according to the conditions of Example 20. The cell lines chosen are the 5 cell lines from table 7 of example 16 that show the lowest IC50 for M30-E05.

[0310] EXAMPLE 26 - Cytotoxicity study in cancer and normal cell line panels for head and neck cancer

[0311] This example is carried out according to the conditions of Example 19. The cell lines chosen are reported on Table 8. Table 8. Cell lines of head and neck cancer for the cytotoxicity study

[0312] EXAMPLE 27 - Kinetic analyses of M30-E05 cellular effects for head and neck cancer

[0313] This example is carried out according to the conditions of Example 20. The cell lines chosen are the 5 cell lines from table 8 of example 26 that show the lowest IC50 for M30-E05.

[0314] EXAMPLE 28 - Impact of M30-E05 on the transcriptome.

[0315] To investigate in depth the impact of M30-E05 on the transcriptome, bulk RNA-sequencing analysis of M30-E05 -treated HOS cells at 24h and the corresponding untreated condition were performed.

[0316] Gene Ontology (GO) analysis of M30-E05 -treated HOS cells revealed different enriched pathways, among which apoptosis was significantly represented (p-value = 0.0372) (Figure 32).

[0317] In addition, detailed heatmap of deregulated genes involved in the apoptosis gene set showed increases in some pro-apoptotic genes including tumor suppressor TP53, PARP4 and ITPR1, as well as decreases in some anti-apoptotic genes such as BCL1L1 (BCL-XL), MCL1. (Figure 33).

[0318] EXAMPLE 29 - Cytotoxicity of M30-E05 in cell line panels for lung cancer

[0319] To assess the cytotoxicity of M30-E05, a panel of lung adenocarcinoma cell lines, including HOP62, A427, H522, A549, H1650, H1793, H1838, H1975, H2030, H2342 was treated with M30-E05 (ranging from 1 to 100 pM dissolved in 0.002% DMSO) for 72h. Cell viability (Figure 34) was then evaluated using a lactate dehydrogenase (LDH) assay, and the IC50 of M30-E05 was determined using GraphPad Prism software. IC50 of the different cell lines are reported on Table 9.

[0320] Across all tested cell lines, the lowest IC50 was 3.85pM for A427 cells, while the highest IC50 was 62.8 IpM for H2030 cells.

[0321] Table 9. ICso of the different cell lines

[0322] EXAMPLE 30 - Cytotoxicity of M30-E05 in wild-type mouse embryonic fibroblast.

[0323] The same cell viability assay on wild-type (WT) mouse embryonic fibroblast (MEF) (MEF WT) and AIFM1 knocked-out MEF (MEF AIF KO) was performed (Figure 35, table 10). The compound showed three times less efficacy in MEF AIF KO (IC50 = 13.77 pM), compared to its WT cells (IC50 = 4.37 pM). This result suggests the on-target effect of M30-E05.

[0324] Table 10. ICso of the different cell lines

[0325] EXAMPLE 31 - Protein expression

[0326] The protein expression of AIF, CHCHD4 and two substrates of the AIF / CHCHD4 pathway (MICU1 and COX 17) were examined in order to evaluate their relationship with the sensitivity of M30-E05 (Figure 36).

[0327] Interestingly, A427 cells display significantly higher levels of AIF, MICU1, and COX 17 compared to HOS cells. Although not statistically significant, the lowest expressions of AIF and CHCHD4 were observed in A2342, HOP62, and H1650 cells, whereas the lowest levels of C0X17 and MICU1 were detected in HOP62 and H2030 cells. Coherent with the IC50 results, A427 was the most sensitive cell line (IC50 = 3.85pM), exhibiting the highest expression of AIF, CHCHD4, MICU1 and Coxl7.

[0328] In constrast, the IC50 values of M30-E05 for HOP62, H1650, H2030, A2342 were 31.31pM, 7.86pM, 62.8 IpM, and 13.06pM, respectively. Notably, among them, H2030 is the most resistant cell line, while HOP62 ranked as the third most resistant cell line. Future studies should focus on the genetic backgrounds and phenotypic characteristics of this panel of lung adenocarcinoma cell lines to better understand the mechanisms underlying these differences in M30-E05 sensitivity and the expression of AIF, CHCHD4 as well as their substrates.

[0329] EXAMPLE 32 - Potential of M30-E05 in overcoming conventional chemotherapeutic resistance

[0330] To assess the potential of M30-E05 in overcoming conventional chemotherapeutic resistance, its interaction with three standard osteosarcoma drugs (cisplatin (CIS), doxorubicin (DOXO), and methotrexate (MTX)) was evaluated using the ZIP model in SynergyFinder (Figure 37). Synergy scores (Table 11) were defined as antagonistic (< -10), additive (-10 to 10), or synergistic (> 10). In the parental model HOS, while combining M30-E05 with CIS, DOXO, MTX, HOS cells demonstrated the additivity effect. Interestingly, M30-E05 showed the highest ZIP synergy score (50.488) with DOXO when treated in HOS-R / DOX, which indicates that our molecule could resensitize HOS-R / DOXO to DOXO. In addition, synergism was found between M30-E05 and MTX in HOS-R / DOXO (17.697), and between M30-E05 and DOXO in HOS-R / MTX (11.903). These results highlight that M30-E05 could be a valuable candidate for incorporation into the treatment regimen for resistant OS, showing synergy with chemotherapies.

[0331] Table 11. Synergy scores

[0332] EXAMPLE 33 - Impact of structural change A small library of M30-E05 analogs was synthesis to systematically evaluate how structural changes affect the efficacy of engineered flavonoids. Six compounds, namely OD235-Ter, OD164, OD265, OD240, OD169 and OD263, were selected based on their solubility in DMSO.

[0333] Cell viability (Figure 38) was then assessed using a lactate dehydrogenase (LDH) assay, and the ICso of each analog was calculated using GraphPad Prism software (Table 12).

[0334] Table 12. ICso of the different compounds

[0335] Dose-response curves revealed heterogeneous sensitivities across HOS cells. ICso values ranged from 9.69 pM (OD 240, most sensitive) to 51.67 pM (OD 169) with OD 265 showing insensitivity until the concentration of 200 pM. Noteworthy, as determined, ICso of M30-E05 in HOS cells is 8.3 pM and none of the synthesized analogs demonstrated superior efficacy, indicating that M30-E05 remains the most effective compound in-cellulo.

[0336] These findings suggest that while the current set of analogs provides valuable insights into the structureactivity relationship, additional rounds of chemical optimization will be required to further enhance efficacy.

[0337] EXAMPLE 34

[0338] Proteomic profiling of HOS cells treated with M30-E05 at its ICso for 48 h revealed alterations in protein expression, as illustrated by the volcano plot (Figure 39).

[0339] KEGG pathway enrichment analysis identified significant changes in pathways associated with neurodegeneration, including Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease, as well as oxidative phosphorylation and proteasome function (Figure 40).

[0340] Closer analysing of the Alzheimer’s disease pathway protein set (Table 13) highlighted the deregulation of multiple mitochondrial respiratory chain subunits (COX7A2L, MT-CO2, NDUFA2, NDUFA4, NDUFA1, NDUFS3, NDUFS7) as well as proteasome components (PSMA7, PSMD14) and signaling proteins involved in Wnt and NF-KB pathways.

[0341] These data suggest that M30-E05 strongly impacts mitochondrial oxidative phosphorylation, consistent with its proposed mechanism of action, while also influencing proteostasis and survival signaling cascades.

[0342]

[0343] Table 13. Analysis of Alzheimer’s disease pathway protein set

[0344] EXAMPLE 35 - Effect of M30-E05 on mitochondrial membrane potential.

[0345] Results

[0346] Strikingly, tetramethylrhodamine, methyl ester (TMRM) measurements of mitochondrial membrane potential (A m) remained unchanged after M30-E05 cell exposure, indicating that the observed respiratory defects observed by Seahorse assay and structural defects observed by fluorescence microscopy are independent of A m (Figure 41).

[0347] Discussion

[0348] Mechanistically, some flavonoids such as eupatilin and diosmetin promote apoptosis primarily through redox imbalance, mitochondrial dysfunction, and suppression of survival signaling pathways, but their mechanism of action differ between them and are distinct from the mechanism of M30-E05. Eupatilin induces apoptosis by decreasing A m. suppressing anti-apoptotic BCL-xL, increasing pro-apoptotic BAK, inducing CytC release, and activating caspase-3 cleavage. These effects are accompanied by excessive ROS production and suppression of the PI3K / AKT axis, together with activation of MAPK stress signaling (Zhong et al., 2016) (Wu et al., 2020) (Lee et al., 2021) (Rosa et al., 2024). Diosmetin similarly triggers cell death across diverse cancers by decreasing anti-apoptotic proteins (Bcl-2, XIAP), oncogenic c-Myc, while increasing pro-apoptotic regulators (Bax, Bak) and cleaved caspase-3, cleaved caspase-8, cleaved PARP, p53, p21, and FOXO3a. Excessive ROS production was also observed via inhibition of Nrf2, thus limiting invasion through downregulation of MMP-2 / 9 via PKC / MAPK signaling (Liu et al., 2016, p. 9) (Oak et al., 2018) (Wang et al., 2019) (Ma and Zhang, 2020) (Zhao et al., 2021). Taken together, both compounds kill tumor cells both in vitro and in vivo models by inducing mitochondrial apoptosis through loss of A m. ROS overproduction and inhibition of pro-survival signaling. In contrast, M30-E05, though belonging to the same flavonoid family (i.e, similar pharmacophore), exerts its activity through disruption of the mitochondrial AIF / CHCHD4 proteinprotein interaction, inducing metabolic reprogramming rather than primarily acting on oxidative stress and inhibiting pro-survival signaling pathways.

[0349] Thus, in HOS cells a marked decrease in OCR while A m remains statistically unchanged (Figure 41) has been observed, suggesting another mechanism of action on mitochondria beyond A m collapse, potentially linked to mitochondrial proteome alteration via altered import / assembly of respiratory substrates and downstream metabolic reprogramming. However, an effect on oxidative stress as a part of the M30-E05 mechanism of action cannot be excluded, and this would deserve future investigation by measuring ROS levels and enzyme activities such as SOD, catalase, and NADPH oxidase. Together, this comparison highlights that the chemical modifications introduced in M30-E05 can profoundly affect its cellular target, mechanisms of action, and consequences, underscoring the originality of M30-E05 compared to other anti -cancerous natural flavonoids.

[0350] References

[0351] Lee, J.-Y., Bae, H., Yang, C., Park, S., Youn, B.-S., Kim, H.-S., Song, G., Lim, W., 2020. Eupatilin Promotes Cell Death by Calcium Influx through ER-Mitochondria Axis with SERPINB 11 Inhibition in Epithelial Ovarian Cancer. Cancers (Basel) 12, 1459. https: / / doi.org / 10.3390 / cancersl2061459

[0352] Lee, M., Yang, C., Song, G., Lim, W., 2021. Eupatilin Impacts on the Progression of Colon Cancer by Mitochondria Dysfunction and Oxidative Stress. Antioxidants (Basel) 10, 957. https: / / doi.org / 10.3390 / antioxl0060957

[0353] LI, Y.-Y., WU, H„ DONG, Y.-G., LIN, B„ XU, G„ MA, Y.-B., 2015. Application of eupatilin in the treatment of osteosarcoma. Oncol Lett 10, 2505-2510. https: / / doi.org / 10.3892 / ol.2015.3563

[0354] Liu, J., Wen, X., Liu, B., Zhang, Q., Zhang, J., Miao, H., Zhu, R., 2016. Diosmetin inhibits the metastasis of hepatocellular carcinoma cells by downregulating the expression levels of MMP-2 and MMP-9. Mol Med Rep 13, 2401-2408. https: / / doi.org / 10.3892 / mmr.2016.4872

[0355] Ma, A., Zhang, R., 2020. Diosmetin Inhibits Cell Proliferation, Induces Cell Apoptosis and Cell Cycle Arrest in Liver Cancer. Cancer Manag Res 12, 3537-3546. https: / / doi.org / 10.2147 / CMAR.S240064

[0356] Oak, C., Khalifa, A.O., Isali, I., Bhaskaran, N., Walker, E., Shukla, S., 2018. Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest. Int J Oncol 53, 835-843. https: / / doi.org / 10.3892 / ijo.2018.4407

[0357] Rosa, A., Piras, L., Pollastro, L., Sogos, V., Appendino, G., Nieddu, M., 2024. Comparative Evaluation of Anticancer Activity of Natural Methoxylated Llavones Xanthomicrol and Eupatilin in A375 Skin Melanoma Cells. Life 14, 304. https: / / doi.org / 10.3390 / lifel4030304

[0358] Shen, Z., Shao, J., Dai, J., Lin, Y., Yang, X., Ma, J., He, Q., Yang, B., Yao, K., Luo, P., 2015. Diosmetin protects against retinal injury via reduction of DNA damage and oxidative stress. Toxicol Rep 3, 78-86. https: / / doi.Org / 10.1016 / j.toxrep.2015.12.004

[0359] Wang, C., Li, S., Ren, H., Sheng, Y., Wang, T., Li, M., Zhou, Q., He, H., Liu, C., 2019. AntiProliferation and Pro-Apoptotic Effects of Diosmetin via Modulating Cell Cycle Arrest and Mitochondria-Mediated Intrinsic Apoptotic Pathway in MDA-MB-231 Cells. Med Sci Monit 25, 4639-4647. https: / / doi.org / 10.12659 / MSM.914058

[0360] Wu, Z., Zou, B., Zhang, X., Peng, X., 2020. Eupatilin regulates proliferation and cell cycle of cervical cancer by regulating hedgehog signalling pathway. Cell Biochem Lunct 38, 428-435. https: / / doi.org / 10. 1002 / cbf.3493

[0361] Zhao, L., Hong, X., Li, D., Wei, Z., Ci, X., Zhang, S., 2021. Diosmetin induces apoptosis in ovarian cancer cells by activating reactive oxygen species and inhibiting the Nrf2 pathway. Med Oncol 38, 54. https: / / doi.org / 10.1007 / sl2032-021-01501-l

[0362] Zhong, W.-F., Wang, X.-H., Pan, B., Li, F., Kuang, L., Su, Z.-X., 2016. Eupatilin induces human renal cancer cell apoptosis via ROS-mediated MAPK and PI3K / AKT signaling pathways. Oncol Lett 12, 2894-2899. https: / / doi.org / 10.3892 / ol.2016.4989

Claims

CLAIMSwherein: a and are equal to 0 or 1, and cannot represent simultaneously 1,5 is equal to 0 or 1,X is chosen among: CH2 or O,V is a nitrogen atom or H2C=O, n and m are different and are equal to 0 or 1,Ri is chosen among: hydrogen atom or a methyl group,R2 is chosen among: a hydrogen atom, a methyl group, a cyclopentyl group or a CH2 andRs is chosen among: a -OH, an -O-CH3 group, an -O-CH2-cyclopropyl group , an -O-octyl group, an -O-cyclopentyl group,group or an oxygen atom, optionally R2 and R3 are linked to form a methylenedioxy group,• R4 and R5 are chosen among: a hydrogen atom or a methyl group,• Z represents a group chosen among:and when a = 1 and P = 0■ X is CH2 and V is a nitrogen atom■ n = 0 and m = 1 or when a = 0 and = 1■ X is O and V is H2C=0■ n = 1 and m = 0, or when a = 0 and P = 0■ X is O and V is H2C=O■ n = 1 and m = 0, and when 5 = 0,R2 is chosen among: a hydrogen atom, a methyl group or a cyclopentyl group and Rs is chosen among: a OH, an O-CH3 group, an -O-CH2-cyclopropyl group, an -O-octyl group, an -O-cyclopentyl group,group, or when 5 = 1,■ R2 is CH2 and R3 is an oxygen atom and R2 and R3 are linked to form a methylenedioxy group, for its use in the treatment of cancer, in particular tongue cancer, non-small cell lung cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, leukemia, osteosarcoma or head and neck cancer.

2. A compound for its use, according to claim 1, of formula (II):1.

3. A compound for its use, according to claim 1, of formula (III):• is equal to 0 or 1,• n = 1,• Z represents a group chosen among:

4. A compound for its use, according to claim 1, having a formula chosen among the following ones:LF18 LF19 LF205. A compound for its use, according to any one of claims 1 or 3 to 4, of formula:

6. A compound for its use, according to any one of claims 1 to 5, in the treatment of pediatric cancer.wherein:• a and are equal to 0 or 1, and cannot represent simultaneously 1,• 5 is equal to 0 or 1,• X is O,• V is H2C=O,• n is 1,• Ri is a hydrogen atom,• R2is chosen among: a hydrogen atom, a methyl group, a cyclopentyl group or a CH2andRs is chosen among: a -OH, an -O-CHs group, an -O-CH2-cyclopropyl group , an -O-octyl group, an -O-cyclopentyl group,group or an oxygen atom, optionally R2and Rs are linked to form a methylenedioxy group,• Z represents a group chosen among:and when 5 = 0,■ R2 is chosen among: a hydrogen atom, a methyl group or a cyclopentyl group and■ R3 is chosen among: a OH, an O-CH3 group, an -O-CH2-cyclopropyl group, an -O-octyl group, an -O-cyclopentyl group,or when 5 = 1,■ R2 is CH2 and R3 is an oxygen atom and R2 and R3 are linked to form a methylenedioxy group.

8. A compound, according to claim 7, of formula (III):wherein:• Z represents a group chosen among:

9. A compound, according to claim 7, having a formula chosen among the following ones:

10. A compound, according to any one of claims 7 to 9, of formula:M30-E05.

11. A compound, according to any one of claims 7 to 10, for its use as a drug.

12. Product containing a compound of formula(I) according to claim 1 and an immunotherapeutic agent or an anti-cancer agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer, in particular tongue cancer, non-small cell lung cancer, ovarian carcinoma, ovarian cancer, pancreatic adenocarcinoma, pancreatic cancer, leukemia, osteosarcoma or head and neck cancer.

13. Product for it use according to claim 12, said immunotherapeutic agent being chosen from:- an anti-immuno-oncologic agent (anti I-O) such as an anti-macrophage antibody (i.e. immunogen being spleen cells)- an inhibitor of PD 1 such as nivolumab and pembrolizumab- an inhibitor of PD-L1 such as atezolizumab and durvalumab,- an inhibitor of CTLA-4 such as ipilimumab- an antibody selected from the group consisting of siglec 15 antibody, anti- phosphatidylserine, anti- 0X40, anti-CD73, anti-TIM3, anti-CD24, anti-CD47, anti-PDl, anti- PDL1, anti-CTLA4, anti-GITR, anti-CD27, anti-CD28, anti-CD 122, anti-TIGIT, anti- VISTA, anti-ICOS, and anti-LAG3, or -vaccines derived from viral, bacterial, and phagic components and their adjuvants.

14. Product for it use according to claim 12, said anti -cancer agent being chosen from- alkylating agents such as alkylsulfonates in particular busulfan, dacarbazine, procarbazine, cloretazine, nitrogen mustards such as chlormethine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, nitrosoureas such as carmustine, lomustine, semustine, streptozocin, altretamine, fotemustine;- antineoplastic alkaloids such as vincristine, vinblastine, vinorelbine, vindesine;- taxanes such as paclitaxel or taxotere;- antineoplastic antibiotics such as actinomycin, bleomycin;- intercalating agents such as mitoxantrone, etoposide, bleomycin, actinomycin D, amsacrine, alliptinium;- antineoplastic antimetabolites: folate antagonists, methotrexate; inhibitors of purine synthesis; analogues such as mercaptopurine, 6-thioguanine; inhibitors of pyrimidine synthesis, aromatase inhibitors, capecitabine, pyrimidine analogs such as fluorouracil, gemcitabine, cytarabine and cytosine arabinoside; brequinar, nelarabine;- group I and II topoisomerase inhibitors such as irinotecan, exatecan, topotecan, teniposide, camptothecin or etoposide;- anticancer hormone agonists and antagonists including tamoxifen;- kinase inhibitors, such as imatinib, nilotinib and dasatinib, midaustorin, sorafenib, lestaurtinib, tandutinib, sirolimus, everolimus or tensirolimus;- growth factor inhibitors;- anti-inflammatories such as pentosan polysulfate, corticosteroids, prednisone, dexamethasone;- ceplene (histamine dihydrochloride);- antracyclines such as daunorubicin, epirubicin, pirarubicin, idarubicin, zorubicin, aclarubicin, annamycin, doxorubicin, mitomycin and methramycin;- anticancer metal complexes, platinum derivatives such as cisplatin, carboplatin, oxaliplatin, satraplatin;- alpha interferon;- triphenylthiophosphoramide;- antiangiogenic agents;- thalidomide;- inhibitors of famesyl-tranferase such as tipifamib;- inhibitors of DNA methyltransferase such as MG98;- immunotherapy adjuvants such as gemtuzumab ozogamicin, HuM 195;- biotherapeutic agents such as CT388-I L3; or- antisense such as GTI-2040.

15. Product for it use according to claim 12, said anti-cancer is a chemotherapy agent, preferably being chosen from:- alkylating agents such as cyclophosphamide, ifosfamide and busulfan,- platinum-based agents such as cisplatine, carboplatin and oxaliplatin- antimetabolites such as methotrexate, 5 -fluorouracil,- anthracy clines such as doxorubicin, epirubicin, daunorubicin,- topoisomerase inhibitors such as irinotecan, etoposide, topotecan;- mitotic inhibitors such as paclitaxel, docetaxel, vincristine- corticosteroids such as prednisone, dexamethasone- targeted therapies such as imatinib, trastuzumab, erlotinib, or- monoclonal antibodies such as rituximab, bevacizumab, more preferably said chemotherapy agent is chosen from Altretamine; Bendamustine; Busulfan; Carboplatin; Carmustine; Chlorambucil; Cisplatin; Cyclophosphamide; Dacarbazine; Ifosfamide; Lomustine; Mechlorethamine; Chlormethine; Melphalan; Oxaliplatin; Temozolomide; Thiotepa; Trabectedin; Carmustine; Lomustine; Streptozocin; Azacitidine; 5 -fluorouracil (5-FU); 6- mercaptopurine (6-MP); Capecitabine (Xeloda); Cladribine; Clofarabine; Cytarabine (Ara-C); Decitabine; Floxuridine; Fludarabine; Gemcitabine (Gemzar); Hydroxyurea; Methotrexate; Nelarabine; Pemetrexed (Alimta); Pentostatin; Pralatrexate; Thioguanine; Trifluridine / tipiracil combination; Daunorubicin; Doxorubicin; Epirubicin; Idarubicin; Valrubicin; Bleomycin; Dactinomycin; Mitomycin-C; Mitoxantrone; Irinotecan; Topotecan; Etoposide (VP- 16); Mitoxantrone; Teniposide; Cabazitaxel; Docetaxel; Nab-paclitaxel; Paclitaxel; Vinblastine; Vincristine; Vinorelbine; Prednisone; Methylprednisolone; Dexamethasone; Arsenic trioxide; Asparaginase; Eribulin; Hydroxyurea; Ixabepilone; Mitotane; Omacetaxine; Pegaspargase; Procarbazine; Romidepsin; Vorinostat; or Raltitrexed, in particular said chemotherapy agent is chosen from cisplatin (CIS), doxorubicine (DOXO) or methotrexate (MTX).

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