Anti-proliferative imidazo[1,2-a]quinoxalines

Abstract

The present invention relates to the treatment of cancer. Here, the inventors evaluated EAPB02303 effects in PDAC cell lines and mouse models and investigated its mechanism of action. They showed a potent cytotoxic activity of EAPB02303 in combination with paclitaxel in vitro and in mice. Using a combination of transcriptomic, proteomic, biochemical and cellular assay, they demonstrated that EAPB02303 mechanism of action relies on its bioactivation by catechol-O-methyltransferase (COMT), resulting in the production of a methylated compound, EAPB04303, that effectively inhibits microtubule polymerization. Moreover, in a second study, the inventors established the first validation of a COMT-driven metabolism of EAPB02303 into a methylated compound, EAPB04303, in AML. Importantly, they demonstrated that the metabolite EAPB04303 induces growth arrest in AML cells, at earlier timepoints and lower concentrations than EAPB02303. Thus the present invention relates to an aromatic heterocyclic compound of general formula (I) and used thereof.

Description

[0001] NEW COMPOUNDS AND USE THEREOF

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine.

[0004] BACKGROUND OF THE INVENTION:

[0005] The Imiqualine family includes heterocyclic molecules based on scaffolds diversely substituted, and is divided into five series: imidazo[l,2-a]quinoxaline, imidazo[l,2-a]pyrazine, imidazo[l,5-a]quinoxaline, [l,2,4]triazolo[4,3-a]quinoxaline and pyrazolo[l,5-a]quinoxaline scaffolds. Among these compounds, first-generation hits, such as EAPB0503 and EAPB0203 belonging to the imidazo[l,2-a]quinoxaline series, showed cytotoxic activities at submicromolar concentrations in different cancer cell lines that were higher than those of reference molecules (vemurafenib, fotemustine, dacarbazine) in melanoma cell lines [1-2], These compounds inhibit microtubule polymerization by binding to the colchicine binding site in P-tubulin [3], Additional drug design studies led to the development of second-generation Imiqualine compounds with significantly higher cytotoxic activities. The most potent molecule, EAPB02303, demonstrates remarkable activity at nanomolar concentrations in different cancer cell lines, including Pancreatic Ductal Adenocarcinoma (PDAC) cell lines [4], In vivo, EAPB02303 reduced tumor size in mice xenografted with A375 human melanoma cells in a dose-dependent manner. Moreover, its half-life following intraperitoneal administration in mice was 6 hours using a dosage method validation according to the FDA and EMA guidelines [4-5], Comparison of the transcriptomic profiles induced by EAPB02303 and twelve well-known anti-cancer agents (e.g. microtubule disruptors, alkylating agents, anti-metabolic agents, targeted therapies) was performed and highlighted significant differences between EAPB02303 and these known anti-cancer agents. Furthermore, EAPB02303 failed to inhibit microtubule polymerization when added to purified tubulin using a standard polymerization protocol [4], Since those results, the mechanism of action underpinning EAPB02303 cytotoxic activity has been elucidated for its optimal pre-clinical development.

[0006] By 2030, pancreatic cancer could rise to second place among the causes of cancer mortality, making it one of the major public health problems. PDAC has an extremely poor prognosis, because of late diagnosis and broad primary resistance to therapies [6], Surgery is the only potentially curative treatment when PDAC is diagnosed at very early stage of development. The current standard of care includes gemcitabine alone or combined with thenanoparticle albumin-bound form of the microtubule-stabilizing agent paclitaxel (i.e. nab-paclitaxel) and FOLFIRINOX (5-fluorouracil, leucovorin, irinotecan and oxaliplatin) (Von Hoff et al. 2013a). These chemotherapy combinations increase patient survival, but the response is relatively poor with significant toxicity [7], Furthermore, acquired and innate resistance are usual in PDAC, and the development of new active compounds remains a challenge that requires extensive research.

[0007] In parallel, members of Imiqualine family exhibited an anticancer activity against solid tumors and hematological malignancies including acute myeloid leukemia (AML) [9-14], AML is a heterogenous, and complex blood malignancy characterized by the aberrant growth of undifferentiated myeloid progenitors. Despite the current advances in the clinical and therapeutic management of AML

[0015] , some karyotypes are still associated with adverse prognosis [16-23],

[0008] The inventors recently showed that the anti-AML potency of the hit molecule EAPB02303 from the second-generation series exceeded that of the first-generation Imiqualine EAPB0503 [10, 24, 25], Accordingly, EAPB02303 induced, at very low concentrations reaching the nanomolar range, growth arrest and apoptosis of AML cell lines and blasts derived from AML patients with diverse mutational signatures and karyotypes. At the molecular level, EAPB02303 inhibited the PI3K / AKT / mTOR signaling pathway, an essential regulator of AML cell growth and survival, constitutively upregulated in 60% of AML cases [26-29], The substantiation of these molecular targets was further reinforced in the multicellular organism Caenorhabditis elegans, where a role of EAPB02303 in reducing the activity of the PI3K / AKT and RAS / MAPK signaling was depicted

[0016] , Nevertheless, AML cells and patient-derived blasts expressing mutant nucleophosmin 1 (NPMlc), a mutagenetic pattern reported in 30% of AML patients [30, 31], were more sensitive to treatment with EAPB02303. Indeed, EAPB02303 induced NPMlc degradation in vitro and exclusively prolonged the survival of NPMlc AML xenograft mice, despite the high in vitro potency of this compound against AML cells with different karyotypes. To unveil the discrepancy between the broad in vitro activity of EAPB02303 against all tested AML karyotypes and its limited effect to confer survival advantage selectively in NPMlc AML, the potential metabolism of this molecule in AML cells was investigated.

[0009] SUMMARY OF THE INVENTION:

[0010] In a first study, the inventors evaluated EAPB02303 effects in PDAC cell lines and mouse models and investigated its mechanism of action. First, they measured the cytotoxicactivity of EAPB02303 alone or combined with standard chemotherapy drugs in 2D and 3D (spheroid) cultures of PDAC cell lines. Then, they evaluated its effect in patient-derived xenograft (PDX) mouse models. They showed a potent cytotoxic activity of EAPB02303 in combination with paclitaxel in vitro and in mice. Using a combination of transcriptomic, proteomic, biochemical and cellular assays, they demonstrated that EAPB02303 mechanism of action relies on its bioactivation by catechol-O-methyltransferase (COMT), resulting in the production of a methylated compound, notably EAP04303, that effectively inhibits microtubule polymerization. Analysis of The Cancer Genome Atlas (TCGA) data indicated that COMT is upregulated in PDAC compared with healthy tissue samples and this is associated with poor prognosis.

[0011] Moreover, in a second study, the inventors established the validation of a COMT-driven metabolism of EAPB02303 into a methylated compound in AML, as the addition of COMT inhibitors suppressed its antileukemic activity in vitro. Importantly, they demonstrated that the metabolite induces growth arrest in AML cells, at earlier timepoints and lower concentrations than EAPB02303. A striking G2 / M cell cycle arrest and apoptosis were observed in all tested AML cells, independently from the NPM1 mutagenic background. In accordance with identified molecular targets of EAPB02303, the derivative compound, EAPB04303, inhibits the PI3K / AKT / mTOR and RAS / MAPK pathways in all tested AML cells and induces the degradation of NPMlc in AML cell lines expressing this mutant protein. In vivo, the metabolite drastically reduced the leukemic burden in the bone marrow, spleen and liver of all treated AML xenograft mice. Finally, while EAPB02303 exclusively prolonged the survival of NPMlc AML xenograft mice, the metabolite EAPB04303 was more potent than EAPB02303 and significantly prolonged the survival of AML xenograft mice, independently from NPMlc. Collectively, those data shed light on the metabolism of EAPB02303 in AML and positions its metabolite, through its enhanced antileukemic performance as a potential therapeutic option against this complex blood malignancy.

[0012] Thus, the present invention relates to an aromatic heterocyclic compound of general formula (I) and use thereof.

[0013] Particularly, the invention is defined by its claims.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] Compounds of the invention

[0016] A first object to the invention relates to an aromatic heterocyclic compound of general formula (I):

[0017]

[0018] or a pharmaceutically acceptable salt thereof,

[0019] wherein n is between 0 and 2 and wherein if n=0:

[0020] - Rl, R2, R3, R4, R5 independently of each other represent a hydrogen atom, a hydroxy, methoxy, hydroxymethyl, dihydrogenated phosphate, dihydrogenated oxymethylphosphate, amino or methylamino group and

[0021] - R6 represents an amino or methylamino group,

[0022] wherein that at least 2 and / or at most 3 of the residues Rl, R2, R3, R4, R5 represent a hydrogen atom,

[0023] wherein that at least two successive residues Rl, R2, R3, R4, R5 are different from said hydrogen atoms, and

[0024] wherein if n=l or 2:

[0025] - Rl, R4, R5 independently represent a hydrogen atom, a hydroxy, methoxy, amino or methylamino group,

[0026] - R2 and R3 jointly represent two oxygen atoms or two nitrogen atoms,

[0027] - R6 represents an amino or methylamino group.

[0028] As used herein, the term “aromatic heterocycle” denotes the imidazofl ,2-a]quinoxaline structure.

[0029] As used herein, the term "amino" denotes a primary amine and the term “methylamino” denotes a secondary amine.

[0030] R2 and R3 are not connected when n=0. In a particular embodiment, the term “at least 2 and at most 3 of the residues” means 2 or 3 residues.

[0031] As used herein, the term "pharmaceutically acceptable salt" denotes, according to the invention, a pharmaceutically acceptable acid salt, that is to say with any non-toxic acid, including organic and inorganic acids. Such acids include acetic, benzenesulfonic, benzoic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, lactic, maleic,malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric and paratoluenesulfonic acid.

[0032] In a particular embodiment, the aromatic heterocyclic compound of the invention is characterized in that it corresponds to the formula (I):

[0033]

[0034] or a pharmaceutically acceptable salt thereof,

[0035] wherein n is between 0 and 2 and wherein if n=0:

[0036] - Rl, R4, R5 independently represent a hydrogen atom, a hydroxy, methoxy, amino, or methylamino group.

[0037] - R2 and R3 independently represent a hydroxy, methoxy, hydroxymethyl, dihydrogenated phosphate, dihydrogenated oxymethylphosphate, amino, or methylamino group.

[0038] - R6 represents an amino or methylamino group.

[0039] provided that at least 2 and / or at most 3 of the residues Rl, R4, R5 represent a hydrogen atom, and

[0040] wherein if n=l or 2:

[0041] - Rl, R4, R5 independently represent a hydrogen atom, a hydroxy, methoxy, amino, or methylamino group. - R2 and R3 together represent two oxygen atoms or two nitrogen atoms.

[0042] - R6 represents an amino or methylamino group.

[0043] In a particular embodiment, the aromatic heterocyclic compound of the invention is characterized in that it corresponds to the formula (I):

[0044]

[0045] (I)

[0046] or a pharmaceutically acceptable salt thereof,

[0047] wherein n is between 0 and 2 and wherein if n=0:

[0048] - Rl, R4, R5 independently represent a hydrogen atom, a hydroxyl group, or a methoxy group.

[0049] - R2 and R3 independently represent a hydroxyl group, a methoxyl group, or a dihydrogenated oxymethylphosphate group.

[0050] - R6 represents an amino or methylamino group.

[0051] provided that at least 2 or a maximum of 3 of the residues Rl, R4, and R5 represent a hydrogen atom, and

[0052] wherein if n=l or 2:

[0053] - Rl, R4, and R5 independently represent a hydrogen atom, a hydroxyl group, or a methoxy group.

[0054] - R2 and R3 together represent two oxygen atoms or two nitrogen atoms.

[0055] - R6 represents an amino or methylamino group.

[0056] In a particular embodiment, the aromatic heterocyclic compound of the invention is characterized in that it corresponds to the formula (I):

[0057]

[0058] or a pharmaceutically acceptable salt thereof,

[0059] wherein n is between 0 and 2 and wherein if n=0:

[0060] - Rl, R4, R5 independently represent a hydrogen atom

[0061] - R2 and R3 independently represent a hydroxy or methoxy group

[0062] - R6 represents an amino or methylamino group, and

[0063] wherein if n=l or 2:

[0064] - Rl, R4, R5 independently represent a hydrogen atom

[0065] - R2 and R3 together represent two oxygen atoms or two nitrogen atoms

[0066] - R6 represents an amino or methylamino group.In a particular embodiment, the aromatic heterocyclic compound of the invention is characterized in that it corresponds to the formula (I):

[0067]

[0068] (I)

[0069] or a pharmaceutically acceptable salt thereof,

[0070] wherein if n=0:

[0071] - Rl, R4, R5 independently represent a hydrogen atom

[0072] - R2 and R3 independently represent a hydroxyl or methoxy group

[0073] - R6 represents an amino or methylamino group.

[0074] In a particular embodiment, the compound of the invention is :

[0075] The 2-methoxy-5-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenol, the 3-methoxy-6-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)benzene- 1 ,2-diol, the 2 -m ethoxy - 5-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)benzene- 1 ,4-diol, the 2-methoxy-5-(4- (methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,3-diol, 2.3-dimethoxy-5-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 2.6-dimethoxy-3-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 2.4-dimethoxy-5-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 2-methoxy-4-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 4-methoxy-6-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,3-diol, 2-methoxy-4-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,3-diol, 3-methoxy-5-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)benzene- 1 ,2-diol, 2.6-dimethoxy-4-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 2.5-dimethoxy-4-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 2,3-dimethoxy-4-(4- (methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)phenol, 1 -(benzofd] [ 1 ,3 ]dioxol-5-yl)-N-methyli mi dazo[l,2-a]qui noxal in-4-amine, l-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-N-methylimidazof 1 ,2-a]qui noxal i n-4-ami ne, ((4-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)-l,2-phenylene)bis(oxy))bis(methylene) bis(dihydrogen phosphate), 4-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)-l,2-phenylene bis(dihydrogen phosphate), (2-methoxy-5-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenyl)methanol, 5-(4- (methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,2,3-triol, 4-(4- (methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,2,3-triol, 5-(4- (methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yl)benzene- 1 ,2,4-triol, 6-(4- (methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,2,4-triol, 3-(4- (methylamino)imidazo[ 1 ,2-a]quinoxalin- 1 -yl)benzene- 1 ,2,4-triol, 2-(4- (methylamino)imidazo[ 1 ,2-a]quinoxalin- 1 -yl)benzene- 1 ,3 ,5-triol, 5-(4-aminoimidazo[ 1 ,2-r / ]qiii noxal in-l-yl)-2-m ethoxyphenol, 3-(4-aminoimidazo[l,2-a]quinoxalin-l-yl)-6-m ethoxybenzene- 1 ,2-diol, 2-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-5-methoxybenzene- 1 ,4-diol, 5-(4-aminoimidazo[l,2-a]quinoxalin-l-yl)-2-methoxybenzene-l,3-diol, 5-(4-aminoimidazof 1 ,2-a]quinoxalin- 1 -yl)-2, 3 -dimethoxyphenol, 3 -(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2,6-dimethoxyphenol, 5-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2,4-dimethoxyphenol, 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2-methoxyphenol, 4-(4-aminoimidazof 1 ,2-a]quinoxalin- 1 -yl)-6-m ethoxybenzene- 1 ,3 -diol, 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2-m ethoxybenzene- 1 ,3 -diol, 5-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -y l)-3 -methoxybenzene- 1 ,2-diol, 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2,6-dimethoxyphenol, 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2,5-dimethoxyphenol, 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)-2, 3 -dimethoxyphenol, 1 -(benzofd] [ 1 ,3 ]dioxol-5-yl)imidazo[ 1 ,2-a]quinoxalin-4-amine, 1 -(2,3 -dihydrobenzofb] [ 1 ,4]dioxin-6-yl)imidazo[ 1 ,2-a]quinoxalin-4-amine, ((4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)- 1 ,2-phenylene)bis(oxy))bis(m ethylene) bis(dihydrogen phosphate), 4-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)- 1 ,2-phenylene bis(dihydrogen phosphate), (5-(4-aminoimidazo[l,2-a]quinoxalin-l-yl)-2-methoxyphenyl)methanol, 5-(4-aminoimidazo[l,2-a]quinoxalin-l-yl)benzene-l,2,3-triol, 4-(4-aminoimidazof 1 ,2-a]quinoxalin- 1 -yl)benzene- 1 ,2,3 -tri ol, 5-(4-aminoimidazo[ 1,2-a]qui noxal i n- 1 -yl)benzene- 1 ,2,4-triol, 6-(4-aminoimidazo[ 1 ,2-a]quinoxalin- 1 -yl)benzene- 1,2,4-triol, 3-(4-aminoimidazo[l,2-a]quinoxalin-l-yl)benzene-l,2,4-triol and 2-(4-aminoimidazof 1 ,2-a]quinoxalin- 1 -yl)benzene- 1 ,3 , 5-triol .

[0076] In a particular embodiment, the compound of the invention is a derivatives of the EAPB02303 (4-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)benzene-l,2-diol).

[0077] As used herein, the term ‘EAPB02303 derivatives” denotes to compounds EAPB04303 (2-methoxy-5-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenol) and EAPB04403 (2-methoxy-4-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenol) obtained by the metabolization of EAPB02303 by COMT.EAPB02303 is described in the formula II:

[0078]

[0079] EAPB04303 is described in the formula III:

[0080]

[0081] EAPB04403 is described in the formula IV:

[0082]

[0083] (IV)

[0084] As used herein, the term “COMT” for “catechol-O-methyltransferase“ denotes one of several enzymes that degrade catecholamines (neurotransmitters such as dopamine, epinephrine, and norepinephrine), catecholestrogens, and various drugs and substances displayind a catechol moiety. In humans, catechol-O-methyltransferase protein is encoded by the COMT gene (Entrez reference number: 1312). Two isoforms of COMT are produced: the soluble short form (S-COMT) and the membrane bound long form (MB-COMT).In a particular embodiment, the invention relates to an aromatic heterocyclic compound of general formula (I):

[0085]

[0086] or a pharmaceutically acceptable salt thereof,

[0087] wherein n is between 0 and 2 and wherein if n=0:

[0088] - Rl, R2, R3, R4, R5 independently of each other represent a hydrogen atom, a hydroxy, methoxy, hydroxymethyl, dihydrogenated phosphate, dihydrogenated oxymethylphosphate, amino or methylamino group and

[0089] - R6 represents an amino or methylamino group,

[0090] wherein that at least 2 and / or at most 3 of the residues Rl, R2, R3, R4, R5 represent a hydrogen atom,

[0091] wherein that at least two successive residues Rl, R2, R3, R4, R5 are different from said hydrogen atoms, and

[0092] wherein if n=l or 2:

[0093] - Rl, R4, R5 independently represent a hydrogen atom, a hydroxy, methoxy, amino or methylamino group,

[0094] - R2 and R3 jointly represent two oxygen atoms or two nitrogen atoms,

[0095] - R6 represents an amino or methylamino group and,

[0096] wherein the aromatic heterocyclic compound is not the compound EAPB02303 (4-(4-(methylamino)imidazo[ 1 ,2-a]qui noxal i n- 1 -yljbenzene- 1 ,2-diol).

[0097] Therapeutic uses

[0098] First, the inventors showed that EAPB02303, alone or combined with standard chemotherapy drugs, has a cytotoxic activity in several PDAC cell lines and confirmed these results in patient-derived xenograft mouse models. Moreover, they showed that EAPB02303 has an important in vitro and in vivo activity against AML.Thus, a second object of the invention relates to the aromatic heterocyclic compound of general formula (I) of the invention for use in the treatment of cancer in a subject in need thereof.

[0099] Particularly, the invention relates to a method for treating cancer in a subject in need thereof comprising administering a therapeutically effective amount of the aromatic heterocyclic compound of general formula (I) according to the invention.

[0100] Particularly, the invention relates to the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound for use in the treatment of cancer.

[0101] As used herein, the term “patient” or “subject” or “individual” refers to a subject to be treated by the compounds of the invention. In particular, the patient suffers from a Cancer. In one embodiment, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes and humans), rabbits, dogs, horses, cats, livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In one embodiment, the mammal is a human.

[0102] In a preferred embodiment, the “subject” is a human with a cancer, in particular a pancreatic cancer or an acute myeloid leukemia.

[0103] As used herein, the term “cancer” refers to liquid or a solid cancer. Cancer may be a cancer selected from the group consisting in adrenal cortical cancer, anal cancer, bile duct cancer (e.g. perihilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellularcarcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma,), vaginal cancer, vulvar cancer, uterine cancer (e.g. uterine leiomyosarcoma), leukemia (like acute myeloid leukemia, acute lymphoid leukemia, chronic myelomonocytic leukemia (CMML)...), lymphoma and myelodysplastic syndrome (MDS).

[0104] In one embodiment, the compounds and methods of the present invention are suitable for treating pancreatic cancer and particularly Pancreatic Ductal Adeno Carcinoma (PDAC) and resistant pancreatic cancer or leukemia and particularly acute myeloid leukemia.

[0105] In one embodiment, the compounds and methods of the present invention are suitable for treating cancer with COMT expression and particularly PDAC with COMT expression.

[0106] Pharmaceutical composition

[0107] A third object of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the aromatic heterocyclic compound of general formula (I) of the invention according to the invention.

[0108] Particularly, the invention relates to a pharmaceutical composition for use in the treatment of cancer in a subject in need thereof comprising a therapeutically effective amount of the aromatic heterocyclic compound of general formula (I) of the invention according to the invention.

[0109] Particularly, the compound is the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound.

[0110] The composition of the present invention may e.g. be formulated for any mode of administration suitable for the treatment of cancer. The form of the composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc. Then, the uses are adjusted to provide the optimum desired response (e.g., a therapeutic response).The pharmaceutical compositions may contain vehicles which are pharmaceutically acceptable for a formulation capable of treating cancer.

[0111] As used herein, the term “pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administrated to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluents, encapsulating material or formulation auxiliary of any type. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0112] “Carriers” or “vehicles” include any such material known in the art and may be any liquid, gel, solvent, liquid diluent, solubilizer, or like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroetheral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0113] In one embodiment, the use of liposomes and / or nanoparticles is contemplated for the vectorization of the compounds of the invention into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0114] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Lipidic nanocapsules are contemplated for use in the present invention, as they have been used with the first generation of Imiqualine compounds, notably EAPB0503 and EAPB0203.

[0115] Nanoparticles with alkyl backbone are a mode of administration of compounds described in the present invention. Organosilica nanoparticles, and more especially mesoporous and periodic mesoporous organosilica nanoparticles, combine the qualities of high-performance cargos, in terms of easy synthesis, versatile design, biodegradability through endogenous stimuli produced in cancerous environment, biocompatibility, high loading capacities, decorate surfaces to make them furtive and adapt vectorization patterns, such as sugars moieties, all or part of antibodies or peptidic sequences. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet the requirements are contemplated for use in the present invention, and such particles may be easily made.Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0116] In a particular embodiment, the pharmaceutic composition is a therapeutic composition.

[0117] According to the invention, the compounds of the invention or the pharmaceutical composition of the invention are administrated in a therapeutically effective amount.

[0118] Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutic compositions.

[0119] As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the compounds or of the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compounds or of the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the compounds or of the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician or a biologist having ordinary skill in the art may readily determine and prescribe the effective amount of the compounds or of the composition of the invention required. For example, the physician or the biologist could start doses of the compounds or of the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the compounds or of the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, and for example, the ability of a compound to inhibitinflammatory disorders and pain disorders may, for example, be evaluated in an animal model system predictive of efficacy in human. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg.

[0120] Administration may be topical, oral, intranasal, parenteral, intravenous, intrathecal, epidural, intraocular, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of the agent of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0121] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.

[0122] Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions. In particular, these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.

[0123] In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

[0124] Combination and kit of part

[0125] The fourth object of the invention relates to the aromatic heterocyclic compound of general formula (I) of the invention for use in the treatment of cancer in a subj ect in need thereof in combination with a further therapeutic active agent.

[0126] Particularly, the invention relates to the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound according to the invention for used in combination with further therapeutic active agent. In each of the embodiments of the therapeutical uses described in the present invention, the compound according to the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought (particularly cancer).

[0127] In one embodiment, the invention relates to a pharmaceutical composition according to the invention and a further therapeutic active agent for use in the treatment of cancer in a subj ect in need thereof.

[0128] In one embodiment, the cancer is a pancreatic cancer or an acute myeloid leukemia.As used herein, the term “therapeutic active agent”, “therapeutic agent” or “active agent” or “active substance” or “active principle” or “active ingredient” relates to a substance (particularly a chemical substance) inducing an effect such as a therapeutic or a preventive effect. It may be a bioactive chemical compound from a drug or the drug itself. Active agent can be a single molecule or a mixture of several substances.

[0129] Particularly, the therapeutic active agent may be conventional cancer therapies such as, e.g., radiotherapy, chemotherapy (or combinations thereof). Surgery can also be combined with the compounds of the invention.

[0130] In one embodiment, the therapeutic active agents used in combination with the aromatic heterocyclic compound of general formula (I) of the invention is anti-cancer antibodies, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anti-cancer immunogens, cell cycle control / apoptosis regulating agents, hormonal regulating agents, and other agents described below.

[0131] In some embodiments, the compounds of the present invention are used in combination with a chemotherapeutic agent. The term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g, Angew Chem Inti. Ed. Engl. 33:183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluorom ethylornithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.In particular embodiment, the compound EAPB02303 or its derivatives like the EAPB04303 and the EAPB04403 according to the invention is used in combination with the paclitaxel. Particularly, the EAPB02303 and the paclitaxel combination had a synergistic effect.

[0132] In some embodiments, the aromatic heterocyclic compound of general formula (I) of the invention is used in combination with a targeted cancer therapy. Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs," "molecularly targeted therapies," "precision medicines," or similar names. In some embodiments, the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor. The term “tyrosine kinase inhibitor” refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and / or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007 / 0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to, dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), sunitinib (Sutent; SU11248), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4-f][l,6]naphthyridin-3(2H)-one hydrochloride) derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described in, for example, U.S Patent Publication 2007 / 0254295, U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In some embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinicaltrial, more preferably at least one Phase II clinical, even more preferably at least one Phase III clinical trial, and most preferably approved by the FDA for at least one hematological or oncological indication. Examples of such inhibitors include, but are not limited to, Gefitinib, Erlotinib, Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP-547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS-032, PD-0332991, MKC-I (Ro-317453; R-440), Sorafenib, ABT-869, Brivanib (BMS-582664), SU-14813, Telatinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, andPD-0325901.

[0133] In some embodiments, the aromatic heterocyclic compound of general formula (I) of the invention is used in combination with an immunotherapeutic agent. The term "immunotherapeutic agent," as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and / or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and / or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and / or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves. Immunotherapy regimens may combine the use of nonspecific and specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on keyimmune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony-stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), IFN-beta (IFN-P) and IFN-gamma (IFN-y). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behavior and / or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and / or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSFs) contemplated by the present invention include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim) and erythropoietin (epoetin alfa, darbepoietin). Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSFs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. Various-recombinant colony stimulating factors are available commercially, for example, Neupogen® (G-CSF; Amgen), Neulasta (pelfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), Arnesp (erytropoietin). Combination compositions and combination administration methods of the present invention may also involve "whole cell" and "adoptive" immunotherapy methods. For instance, such methods may comprise infusion or re-infusion of immune system cells (for instance tumor-infiltrating lymphocytes (TILs), such as CC2+ and / or CD8+ T cells (for instance T cells expanded with tumor-specific antigens and / or genetic enhancements), antibody-expressing B cells or other antibody-producing or -presenting cells, dendritic cells (e.g., dendritic cells cultured with a DC-expanding agent such as GM-CSF and / or Flt3-L, and / or tumor-associated antigen-loaded dendritic cells), anti-tumor NK cells, so-called hybrid cells, or combinations thereof. Cell lysates may also be useful in such methods and compositions. Cellular "vaccines" in clinical trials that may be useful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96 (Antigenics), and Melacine® cell lysates. Antigens shed from cancer cells, and mixtures thereof (see for instance Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally admixed with adjuvants such as alum, may also be components in such methods and combination compositions.

[0134] In some embodiments, the aromatic heterocyclic compound of general formula (I) of the invention is used in combination with an antibody that is specific for a costimulatory molecule. Examples of antibodies that are specific to a costimulatory molecule include but are not limited to anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies.

[0135] In some embodiments, the second agent is an agent that induces, via ADCC, the death of a cell expressing an antigen to which the second agent binds. In some embodiments, the agent is an antibody (e.g. of IgGl or IgG3 isotype) whose mode of action involves induction of ADCC toward a cell to which the antibody binds. NK cells have an important role in inducing ADCC and increased reactivity of NK cells can be directed to target cells through use of such a second agent. In some embodiments, the second agent is an antibody specific to cell surface antigens, e.g., membrane antigens. In some embodiments, the second antibody is specific for a tumor antigen as described above (e.g., molecules specifically expressed by tumor cells), such as CD20, CD52, ErbB2 (or HER2 / Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, etc., particularly lymphoma antigens (e.g., CD20). Accordingly, the present invention also provides methods to enhance the anti-tumor effect of monoclonal antibodies directed against tumor antigen(s). In the methods of the invention, ADCC function is specifically augmented, which in turn enhances target cell killing, by sequential administration of an antibody directed against one or more tumor antigens, and an antibody of the present invention.

[0136] In some embodiments, the aromatic heterocyclic compound of general formula (I) of the invention is used in combination with radiotherapy. Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient. The source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium- 137,iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-ill.

[0137] Another object of the invention relates to a kit of part comprising the aromatic heterocyclic compound of general formula (I) of the invention or the pharmaceutical composition of the invention and at least one therapeutic active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

[0138] As used herein, the term “simultaneous use” denotes the use of the antibody and at least one active agent occurring at the same time.

[0139] As used herein, the term “separate use” denotes the use of the antibody and at least one active agent not occurring at the same time.

[0140] As used herein, the term “sequential use” denotes the use of the antibody and at least one active agent occurring by following an order.

[0141] Antibody-drug conjugates and use thereof

[0142] In a fifth object of the invention, the invention relates to an antibody conjugated to the aromatic heterocyclic compound of general formula (I) of the invention.

[0143] Particularly, the invention relates to an antibody conjugated to the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound according to the invention.

[0144] The conjugation of the compounds of the invention to an antibody will generate an "antibody-drug conjugates" or "ADCs".

[0145] As used herein the term “Antibody-drug conjugates” or “ADCs” are a class of pharmaceuticals designed to deliver highly toxic cargoes (payloads) to clinical targets. In ADCs, payloads (here the compounds of the invention) are the pharmaceutically active components.

[0146] In a particular embodiment, the DAR (drug-to-antibody ratio) of an antibody with one of the compounds (like the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound) of the invention can be 1, 2, 3, 4, 6, 7, 8, 9 or 10.

[0147] As used herein, the term “DAR” denotes the average number of drug molecules conjugated to an antibody.In a particular embodiment, the invention relates to an antibody conjugated to the aromatic heterocyclic compound of general formula (I) of the invention for use in the treatment of cancer in a subject in need thereof.

[0148] Particularly, the invention relates to an antibody conjugated to the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound according to the invention for use in the treatment of cancer in a subject in need thereof.

[0149] Typically, the antibody-drug conjugate compounds comprise a linker unit between the drug unit (here the compounds of the invention) and the antibody unit.

[0150] In one embodiment, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0151] In one embodiment, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, e.g. , a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide derivatives resulting in the release of active drug inside target cells (see, e.g, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).

[0152] Most typical are peptidyl linkers that are cleavable by enzymes that are present in 191P4D12-expressing cells. Examples of such linkers are described, e.g, in U.S. Pat. No.

[0153] 6,214,345, incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with the Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.

[0154] In one embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values.

[0155] Typically, the pH-sensitive linker hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used. (See,e.g, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0156] In one embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935).

[0157] In one embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg. Med. Chem.

[0158] 3(10): 1299-1304), or a 3-N-amide analog (Lau et al., 1995, Bioorg. Med. Chem. 3(10): 1305-12).

[0159] In one embodiment, the linker unit is not cleavable and the drug (here the compounds of the invention) is released by antibody degradation.

[0160] Typically, the linker is not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment,” in the context of a linker, means that no more than about 20 %, typically no more than about 15 %, more typically no more than about 10 %, and even more typically no more than about 5 %, no more than about 3 %, or no more than about 1 % of the linkers, in a sample of antibody-drug conjugate compound, are cleaved when the antibody-drug conjugate compound is present in an extracellular environment (e.g., in plasma). Whether a linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating with plasma the antibody-drug conjugate compound for a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the amount of free drug present in the plasma.

[0161] Techniques for conjugating molecules to antibodies, are well-known in the art (See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al.eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); “Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89 / 12624.) Typically, the nucleic acid molecule is covalently attached to lysines or cysteines on the antibody, through N-hydroxysuccinimide ester or maleimide functionality respectively.

[0162] Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve the homogeneity of the conjugate (Axup, J.Y., Bajjuri, K.M., Ritland, M., Hutchins, B.M., Kim, C.H., Kazane, S.A., Halder, R., Forsyth, J.S., Santidrian, A.F., Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, J.R., Flagella, K.M., Graham, R.A., Parsons, K.L., Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, D.L., Li, G., et al. (2010). Engineered thio-trastuzumab-DMl conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769-4778.). Junutula et al. (2008) developed cysteine-based site-specific conjugation called “THIOMABs” (TDCs) that are claimed to display an improved therapeutic index as compared to conventional conjugation methods. Conjugation to unnatural amino acids that have been incorporated into the antibody is also being explored for ADCs; however, the generality of this approach is yet to be established (Axup et al., 2012). In particular the one skilled in the art can also envisage Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag (e.g., Gin-containing peptide tags or Q- tags) or an endogenous glutamine that are made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Then a transglutaminase, can covalently crosslink with an amine donor agent (e.g., a small molecule comprising or attached to a reactive amine) to form a stable and homogenous population of an engineered Fc-containing polypeptide conjugate with the amine donor agent being site- specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine- containing tag or the accessible / exposed / reactive endogenous glutamine (WO 2012059882).

[0163] Diagnostic use

[0164] Thus, a sixth object of the invention also related to a method for predicting the survival time and / or the disease progression in a subject suffering from a pancreatic cancer i)determining in a sample obtained from the patient the expression level of COMT, ii) comparing said expression level determined at step i) with a predetermined reference value and iii) concluding that the method provides a good prognostic when the level of gene expression is lower than the predetermined reference value, or provides a bad prognostic when the level of gene expression is higher than the predetermined reference value.

[0165] As used herein, the term “sample” relates to sample obtained from the subject. The sample can be blood, peripheral-blood, serum, plasma, circulating cells, sample obtained from biopsy. In particular, the sample can be pancreatic cells or blood cells. As used herein, the term “normal sample” or “healthy sample” or “wild-type sample” refers to a sample from healthy tissue comprising.

[0166] As used herein, the term “predicting” relates to anticipating the presence and / or the progress of the disease as well as the survival time and / or the survival rate of the subject.

[0167] As used herein, the term “good prognostic” predictive of a subject without a progress of the disease and / or with a good survival time and / or with a good survival rate. In the same way, “bad prognostic” predictive of a subject with a constant progress of the disease and / or with an increased progress of the disease and / or a with bad survival time and / or with a bad survival rate.

[0168] As used herein, the term “survival time” refers to the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as the disease according to the invention. The survival time rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment. As used herein and according to the invention, the term “survival time” can regroup the term OS.

[0169] As used herein, the term “Overall survival (OS)” refers to the time from diagnosis of a disease such as the disease according to the invention until death from any cause. The overall survival rate is often stated as a two-year survival rate, which is the percentage of people in a study or treatment group who are alive two years after their diagnosis or the start of treatment.

[0170] In one embodiment, the method according to the invention is an in vivo method.

[0171] In one embodiment, the method according to the invention is an in vitro method.

[0172] As used herein, the term “reference value” or “predetermined reference value” refers to a number or value derived from population studies, including without limitation, patients of the same or similar age range, patients in the same or similar ethnic group, and patients having the same severity of disease. Such predetermined reference values can be derived from statisticalanalyses and / or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease. In the present invention, “reference value” refers to a value known compared with the value of the gene expression of the present invention. Typically, the reference is a threshold value or cut-off value.

[0173] As used herein, the term “threshold value” or “cut-off value” refers to a value which can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and / or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical consequences of false positive and false negative). In routine work, the reference value (cut-off value) may be used in the present method to discriminate samples of interest for the studied disease and therefore the corresponding patients.

[0174] Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be usedto draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. For example, the level of marker of interest has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the best expression level and sample 100 has the worst expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding ill patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also, for each of the 99 groups, the p value between both subsets was calculated. The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels. Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

[0175] The man skilled in the art also understands that the same technique of assessment of the expression level of a protein should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a protein of a patient subjected to the method of the invention. Such predetermined reference values of expression level may be determined for any genes of the invention defined above.

[0176] The result of which based on statistics, owing to its very good reliability, provides a very helpful information for the physician for making his clinical diagnosis and, where appropriate, prescribing steps to be taken for the management of the disease. It can help early identification of asymptomatic patients with high risk of disease progression. More generally, it advantageously allows a better monitoring and global disease management.

[0177] "Risk" relates to the probability that an event will occur over a specific time period, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of asubject compared either to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.

[0178] "Risk evaluation" or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of the disease, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population.

[0179] FIGURES:

[0180] Figure 1: Compound 1 was synthetized as described in Patinote and al.2021. A) Synthesis of EAPB04303: 2-methoxy-5-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenol. C18H16N4O2, Mw: 320.35 g / mol.

[0181] To a mixture of 1 -bromo-7V-methyl-imidazo[l ,2-a]quinoxalin-4-amine (1) (0.36 mmol) in DME / H2O (v / v, 1 / 1, 10 mL) were added 3-hydroxy-4-methoxyphenylboronic acid (0.43 mmol), tetrakis(triphenylphosphine)palladium (0.019 mmol) and sodium carbonate (0.89 mmol) in a microwave-adapted vial and sealed. The reaction was submitted to microwave irradiations during 20 min at 140 °C and then filtered on a Celite pad. The filtrate was concentrated under reduced pressure and purified by flash chromatography eluted with cyclohexane / ethyl acetate to ethyl acetate / methanol to afford EAPB04303 as solid in 43% yield with 98% purity.

[0182] 'H-RMN (ppm, 500 MHz, DMSO d6): 87.715 (d, 1H), 7.61 (q, 1H), 7.43 (s, 1H), 7.33 (m, 2H), 7.20 (d, 1H), 6.96 (m, 3H), 3,88 (s, 3H), 3,055 (d, 3H). / 13C NMR (ppm, 101 MHz, DMSO): 5 149.11, 148.55, 147.13, 138.29, 133.41, 132.17, 130.75, 127.03, 126.45, 125.73, 122.94, 122.14, 121.82, 117.61, 115.84, 112.77, 56.12, 40.62, 40.41, 40.20, 39.99, 39.79, 39.58, 39.37, 27.78. / MS (ESI +, QTof, m / z): 321.2 [M+H]+ / HRMS calculated for C18H17N4O2 321.1346, found 321.1347.

[0183] B) Synthesis of EAPB04403: 2-methoxy-4-(4-(methylamino)imidazo[l,2-a]quinoxalin-l-yl)phenol. C18H16N4O2, Mw: 320.35 g / mol.

[0184] To a mixture of l-bromo-7V-methyl-imidazo[l,2-a]quinoxalin-4-amine (1) (0.36 mmol) in DME / H2O (v / v, 1 / 1, 10 mL) were added 4-hydroxy-3-methoxyphenylboronic acid (0.43 mmol), tetrakis(triphenylphosphine)palladium (0.019 mmol) and sodium carbonate (0.89 mmol) in a microwave-adapted vial and sealed. The reaction was submitted to microwaveirradiations during 30 min at 150 °C and then filtered on a Celite pad. The filtrate was concentrated under reduced pressure and purified by flash chromatography eluted with cyclohexane / ethyl acetate to ethyl acetate / methanol to afford EAPB04403 as solid in 43% yield with 97% purity.

[0185] 'H-R.MN (ppm, 500 MHz, DMSO d6): 8 9.54 (s, 1H), 7.75 (q, 1H), 7.64 (d, 1H), 7.55 (s, 1H), 7.49 (m, 2H), 7.35 (s, 1H), 7.01 (m, 3H), 3.81 (s, 3H), 3.10 (m, 3H). / 13C NMR (ppm, 101 MHz, DMSO): 6 148.57, 148.39, 148.25, 148.13, 138.33, 133.36, 132.21, 131.08, 127.03, 126.43, 125.84, 123.63, 122.09, 121.30, 116.23, 115.90, 114.79, 56.27, 40.65, 40.44, 40.23, 40.02, 39.81, 39.60, 39.39, 27.78, 26.82, 0.65.

[0186] MS (ESI +, QTof, m / z): 321.2 [M+H]+ / HRMS calculated for C18H17N4O2 321.1346, found 321.1346.

[0187] Figure 2: EAPB02303 alone affects pancreatic cancer cell viability in vitro and reduces tumor growth in vivo. EAPB02303 effect on cell viability was assessed using the SRB assay in the indicated FOLFIRINOX-resistant (FR) and gemcitabine-resistant (GR) cell lines (A). Nude mice were xenografted with Pancpec (B) or P4604 (C) cells (n=10 mice / group) and treated with EAPB02303 (3mg / kg, 10 mg / kg, or 30 mg / kg) or vehicle (80% water, 0.9% NaCl, 10% DMSO, 10% Tween 20) 5 days / week for 30 days. Mouse weight and tumor size were measured throughout the experiment and Kaplan Meyer curves were computed.

[0188] Figure 3: EAPB02303 synergizes with paclitaxel in vitro and in vivo. A) Cell survival (SRB assay) was assessed in Pancpec and P4604 cells after co-incubation with the indicated drugs at increasing concentrations. The left matrices show the cell viability, expressed as percentage of survival, and the right matrices show the synergy, calculated as described in Materials and Methods. White: antagonism, black: additivity, grey: synergy (right panel). Nude mice were xenografted (subcutaneous injection) with Pancpec (B, C) or P4604 (D) cells and treated with EAP02303 (30 mg / kg 5 days / week) or / and paclitaxel (10 mg / kg 2 days / week), combination, or vehicle (80% water, 0.9% NaCl, 10% DMSO, 10%Tween 20) (n = 10 mice / group). Tumor size was measured throughout the experiment and Kaplan Meyer curves were computed.

[0189] Figure 4: EAPB02303 is bioactivated by catechol-O-methyltransferase (COMT) to elicit its activity at nanomolar concentrations. A) The hypothetical bioactivation of EAPB02303 by COMT would result in methylation at the 3’ or 4’ position of the catechol moiety, yielding EAPB04303 or EAPB04403. B) EAPB04303 and EAPB04403 were synthetized and their cytotoxicity in Pancpec and CFPAC-1 cells was assessed with the SRBassay and compared with that of EAPB02303. C) EAPB04303 was the most potent molecule with the lowest IC50, in line with the hypothesis that it is the active metabolite of EAPB02303.

[0190] Figure 5: (A) Patients’ survival curves in function of COMT expression level (high / low) in pancreatic cancer samples. RNA-seq and survival data were from the TCGA database and were downloaded from the Human Protein Atlas. (B) COMT expression levels in normal pancreas and primary pancreatic cancer tissue samples from the TCGA and GTEx databases.

[0191] Figure 6: COMT inhibitors suppress the anti-cancer activity of EAPB02303 but not that of EAPB04303. (A) AML cell lines (wt-NPMl OCLAML2, NPMlc OCLAML3) were treated with increasing concentrations of EAPB04303 for 24, 48 and 72 hours. (B) OCL AML2 or OCLAML3 were treated with single agents entacapone 6.25 pM (black line, square) or tolcapone 3.125 pM (black line, triangle) or EAPB02303 5 nM (grey line, circle), or double agents entacapone / EAPB02303 (red line, square) or tolcapone / EAPB02303 (red line, triangle).

[0192] (C) OCLAML2 or OCLAML3 were treated with EAPB04303 5 nM (circle), or double agents entacapone / EAPB04303 (square) or tolcapone / EAPB04303 (triangle). Cell proliferation was assessed using the trypan blue exclusion assay. The results shown represent the average of at least 3 independent experiments ± SD. Two-way ANOVA was performed to validate significance as compared to the untreated control.

[0193] Figure 7. EAPB04303 induces apoptotic cell death in AML cells. (A) Percent Cycle cells from Cell cycle analysis, (B) SubGO population from Cell cycle analysis (left panel) and Annexin-V cells (right panel) of OCI-AML2 and OCI-AML3 following 24h or 48h treatment with 5 nM EAPB04303. Histograms represent the average of 3 independent experiments ± SD.

[0194] (C) Western blot analysis of P53, P-P53, P21, PARP-1, caspase 3 and H3 in OCI-AML2 and OCI-AML3 cells treated with 5 nM EAPB04303 for 6, 24 and 48 hours. Histograms represent the average densitometries of P-P53 / P53, P21 / H3, PARP-1 / H3 and pro-caspase 3 / H3 ± SD. Student’s t-test was performed to validate significance as compared to the untreated control as follows: * -value < 0.05, ** / ?-value < 0.01 and *** / ?-value < 0.001.

[0195] Figure 8: EAPB04303 exerts potent in vivo efficacy against wt-NPMl and NPMlc AML xenografts. (A) Kaplan-Meier survival of untreated NSG mice injected with OCI-AML2 (n = 7 mice) or OCI-AML3 (n = 5 mice) or treated with EAPB04303 (n = 7 mice per condition). Day 0 represents the day of AML cancer cell injections (B) Graph showing the percentage of hCD45 in the BM of AML xenograft mice at the end of week 3 : OCLAML2 untreated control (n = 7 mice), OCI-AML2 group treated with EAPB04303 (n = 5 mice), OCI-AML3 untreatedcontrol (n = 4 mice), OCI-AML3 group treated with EAPB04303 (n = 5 mice). Statistical significance: * p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001.

[0196] EXAMPLES:

[0197] EXAMPLE 1: RESULTS ON PANCREATIC CANCER

[0198] Material & Methods

[0199] Cell lines and reagents.

[0200] All human PDAC cell lines were from ATCC (Rockville, MD, USA) and were cultured following the ATCC recommendations. All cell lines were authenticated (Eurofins Genomics) and routinely tested for mycoplasma contamination using the MycoAlert™ Mycoplasma Detection Kit (LT07-318, Lonza). The Pancpec and P4604 cell lines were derived from PDX of a primary tumor and peritoneum metastases of human pancreatic tumor specimens, respectively (PDX Platform, Institut de Recherche en Cancerologie de Montpellier). Gemcitabine-resistant and FOLFIRINOX-resistant PDAC cell lines were generated as previously described (Rabia et al, Mabs 2021). Primary cancer-associated fibroblasts (CAFs) were isolated from human PDAC samples using the outgrowth method, as described previously (Ogier C, Cancer letters 2018).

[0201] Chemicals, drugs and reagents.

[0202] Gemcitabine, paclitaxel, oxaliplatin, 5 -fluorouracil (5-FU) and colchicine were from the Montpellier Cancer Institute pharmacy. SN38 was purchased from Tocris (#2684). The drug concentrations in the FOLFIRINOX combination (oxaliplatin: 5 mM; 5-FU: 12 mM; SN38: 0.1 mM defined as dilution 1) were adapted from the concentrations detected in the blood of patients after treatment. Entacapone and tolcapone were purchased from Sigma-Aldrich (SML0654, SML0150).

[0203] EAPB02303 has been prepared as described in Patinote et al. 2021. EAPB04303 et EAPB04403 have been synthetized starting from l-bromo-N-methyl-imidazo[l,2-a]quinoxalin-4-amine according to the Suzuki Miyaura cross-coupling protocol described in Patinote et al. 2021. Syntheses for EAPB04303 and EAPB04403 are available in Figure 1A-B.

[0204] Starting materials and solvents for Imiqualines preparation were obtained from Sigma-Aldrich (Saint-Louis, MO, USA). Ultrapure water (18.2 MQ / cm) was prepared with a Milli-Q Plus 185 system (Millipore Corporation).

[0205] 2D cell growth assay.

[0206] Cell growth was evaluated using the SulfoRhodamine B (SRB) assay, as described by Combes et al (Combes CR 2019). Briefly, cells were seeded in 96-well plates and 24 hourslater, drugs were added in serial dilutions. Cells were incubated for 96 hours and the percentage of viable cells was calculated relative to the untreated controls and plotted as a function of the drug concentrations using the Prism software to determine the ICso (concentration that inhibits cell growth by 50%). The synergistic potential of the combinations was evaluated as described previously (Tosi BMC Cancer 2018).

[0207] 3D cell growth assay.

[0208] Spheroids (3D cultures) were generated by seeding cell suspensions (100 pL / well) at optimized densities (50-2500 cells / well) in ultra-low attachment 96-well round-bottom plates (Fisher Scientific). For spheroids with CAFs, a ratio of 1 tumor cell to 50 CAFs was used. After 2 days, spheroids were incubated with drugs and cell viability was assessed at day 8 using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), according to the manufacturer's instructions. Luminescence was measured using a 1450 MicroBeta TriLux Luminescence Microplate Reader (Perkin Elmer).

[0209] Flow cytometry.

[0210] For cell cycle analyses, cells were plated and after 24 hours they were incubated with drugs. At the selected time points, cells were detached and washed in ice-cold PBS, fixed in 75% ethanol, and stained in 100 pL of anti-phosphorylated histone H3 (PHH3) antibody at room temperature for 20 minutes. Then, cells were diluted in 500 pL of PBS to have a concentration of 10 pg / mL Propidium Iodide (PI) and 100 pg / mL RNAse A. Cell-cycle distribution and PHH3 signal were determined with a Gallios Cytometer (Beckman Coulter) and quantified using the Kaluza software (Beckman Coulter).

[0211] For apoptosis quantification, cells were seeded in 6-well plates and incubated with the indicated drugs for at least 24 hours. Cells were stained with FITC-labeled Annexin V and / or 7 -Amino- Actinomycin D (7-AAD; Annexin V / 7-AAD kit Beckman Coulter) and apoptosis was evaluated using a Gallios Cytometer and the Kaluza software (Beckman Coulter).

[0212] Whole-cell microtubule analysis.

[0213] Whole-cell microtubule polymerization assays were carried out by adapting the protocol described by Morisson and Hergenrother in 2012 (Morrison et Hergenrother 2012). Cells were plated in 6-well plates for 24 hours and incubated with drugs for 18 hours. Then, cells were detached, pelleted and 1 mL of permeabilization buffer (80 mM PIPES pH 6.8, 1 mM MgCh, 5 mM EDTA, 0.1% Triton X-100) at room temperature was added for 10 minutes. After centrifugation at 1500 rpm for 5 minutes, cells were incubated with microtubule stabilizing buffer (80 mM PIPES pH 6.8, 1 mM MgCh, 5 mM EDTA, and 0.5% Triton X-100) for 10 minutes. Glutaraldehyde auto- fluorescence was quenched by adding 0.7 mL of 1 mg / mLNaBH4 in PBS. Cells were pelleted, and the supernatant was removed by gentle aspiration. After a PBS wash, cells were incubated in 25 pL antibody diluting solution (PBS pH 7.4, 0.2% Triton X-100, 2% bovine serum albumin [BSA]) at 4°C for 1 hour, and then 0.5 pL of an anti-tubulin-FITC antibody was added to achieve the concentration of 1:50. Cells were incubated at 4°C in the dark for 3 hours, and then diluted in 200 pL PBS before transfer into flow cytometry tubes and analysis with a Gallios Cytometer (Beckman Coulter). The geometrical mean of the FITC channel was used to assess tubulin level that was normalized to the untreated condition (set to 100).

[0214] Western blot analysis.

[0215] Cells were lysed and blotted as previously described (Ogier C, Cancer letters 2019). Immunoreactions were revealed using fluorescent-labeled secondary antibodies and were visualized with the Odyssey XF Imaging System (LI-COR). Bands were quantified with the Image Studio Lite software and data were relative to the total protein amount measured using the Revert™ 700 Total Protein Stain for Western Blot Normalization. The effects of drugs on protein expression were expressed as fold change relative to the expression level in the untreated condition (set to 1).

[0216] Cellular thermal shift assay (CETSA),

[0217] CETSAs were carried out by adapting the protocols described by Jafari et al (Jafari et al. 2014) and Langeback et al (Langeback et al. 2019). For the protein melting curve analysis, cells were harvested, washed, and then, 500,000 cells were transferred into PCR tubes and pelleted. 100 pL of a 20 pM drug solution in PBS was added and cells were incubated at 37°C for 3 hours. Then, cells were heated using a Biometra Tri Analytik Jena thermocycler to the selected temperatures for 3 minutes. After heating, cells were immediately snap frozen in liquid nitrogen and stored at -80°C. Cells were then lysed by three cycles of freeze-thawing using liquid nitrogen and a thermocycler set at 20°C, before centrifugation at 15,000 g for 30 minutes.

[0218] 80 pL of supernatant was collected and diluted in 4x Laemmli buffer, vortexed, heated at 95°C for 5 minutes and loaded in 10% acrylamide gels for protein separation by electrophoresis.

[0219] For the IsoThermal Dose Response (ITDR-CETSA), cells were harvested, washed, and incubated in PCR tubes at the concentration of 10 million cells / mL in 50pL PBS. 50 pL of drugs was then added (2X concentration in PBS) and cells were heated, using a Biometra Tri Analytik Jena thermocycler, to 64°C for 3 minutes, snap frozen in liquid nitrogen and processed as described for the melting curves.RNA sequencing (RNA-seq) and data analysis.

[0220] Total RNA from CFPAC-1 and Pancpec cells incubated or not with EAPB02303 for 6 or 24 hours, in triplicate, was extracted using the Quick-RNA Miniprep Kit (ZymoResearch #R1055). RNA quality (RIN) was assessed using a 2100-Bioanalyzer (Agilent Technologies) by the NGS Core Facility Platform (IRMB, Montpellier). RNA samples with a RIN >7 were sequenced by the Brain Institute Genotyping and Sequencing Core Facility (iGenSeq, Paris). mRNA libraries were prepared and sequenced as already described (Fauvre et al. 2024). The raw data quality was evaluated with FastQC (https: / / www.bioinformatics.babraham.ac.uk / publications.html). Poor quality sequences and adapters were trimmed or removed with the software fastp (Chen et al. 2018), using default parameters, to retain only good quality paired reads. The Illumina DRAGEN bio-IT Platform (v3.8.4) was used for mapping to the reference human genome hg38 and for quantification with the Gencode v37 annotation gtf file. Library orientation, library composition and transcript coverage were checked with the Picard tools. The following analyses were done with the R software. Data were normalized with the DESeq2 (Love, Huber, et Anders 2014) packages before differential analysis using the glm framework likelihood ratio test from the DESeq2 workflow. Adjusted p-values for multiple hypotheses were calculated with the Benjamini-Hochberg procedure to control the false discovery rate (FDR). The enrichment analysis was performed with the clusterProfiler R package (Yu et al. 2012) and Gene Set Enrichment Analysis (GSEA) of the GO Biological Process and KEGG gene set collection (Liberzon et al.

[0221] 2015).

[0222] Reverse-phase protein array (RPPA),

[0223] RPPA analysis was carried out by MD Anderson Cancer Center. CFPAC-1 and Pancpec cells were incubated or not with EAPB02303 for 6 hours, in triplicate. After serial dilutions, protein extracts were spotted onto nitrocellulose-coated slides plus replicate controls. Specific antibodies were used to amplify the signal via a tyramide signal amplification system and were visualized by DAB (3,3’ Diaminobenzidine) colorimetric reaction to detect the proteins of interest. Each slide was probed with one antibody. The detection system used was a GenPoint™ staining kit from Agilent. Digital images of slides were obtained by scanning them on a Huron TissueScope scanner that produced 16-bit TIFF files. Spot intensities from the TIFF files were determined with the Array-Pro Analyzer software. Differentially expressed proteins were identified based on the normalized data between control and treated samples using the limma package in R; a FDR < 0.01 and logFC > 1 were used as cutoff.CRISPR-Cas9-mediated knock-out of COMT,

[0224] CFPAC-1 and Pancpec cells in which COMT was knocked out were generated using the COMT sgRNA CRISPR / Cas9 All-in-One Lentivector set (Human) (166181110595). CPF AC- 1 and Pancpec cells were infected with retroviral particles and after 96 hours, transduced cells were selected by adding 2 pg / mL puromycin for 6 days. Cells were amplified and cloned in 96-well plates. After 10 days, positive clones were re-seeded and screened by western blotting for COMT knock-out.

[0225] Metabolite dosage.

[0226] In vitro Pancpec cell sample preparation.

[0227] Pancpec and PancpecCOMT- / - cells were used to study EAPB02303 metabolism and particularly its biotransformation into EAPB04303 after addition or not of the COMT inhibitor entacapone at 6.25 pM. Cells were seeded for 24 hours and incubated with 2 mL of culture medium containing 100 nM of EAPB02303 for 15 and 60 minutes. Cells were detached using a scraper in ultrapure water to obtain a suspension of partially lysed cells. Cell suspensions were sonicated to complete cell lysis, followed by centrifugation at 14,000g for 15 minutes. Supernatants were frozen in liquid nitrogen before analysis by ultraperformance liquid chromatography -tandem mass spectrometry (UPLC-MS / MS). For each condition, five technically independent experiments were carried out.

[0228] Quantification of EAPB02303 and EAPB04303 by LC-MS / MS analyses.

[0229] Stock solutions, working solutions, calibration curves and sample preparation were prepared as already described (Chouchou A, JPBA 2018). All measurements were performed on an Acquity UPLC I-Class Plus System (Waters, Milford, USA) linked with a Waters Xevo TQD tandem triple quadrupole mass spectrometer. The chromatographic separation was conducted by injecting 5 pL of each calibration standard solution or in vitro sample on an Acquity UPLC BEH C18 column (1.7 pm, 2.1 mm x 50 mm) from Waters at 40°C. A mixture of acetonitrile (eluent A) and ultrapure water (eluent B) (50 / 50, v / v) with 0.1% formic acid was used as mobile phase at a flow rate of 0.5 mL / min without split. All solvents were filtered through a 0.20 pm millipore filter (Molsheim, France) before use and degassed 15 min in an ultrasonic bath. The autosampler was set at 4°C. The MS / MS was operated in positive electrospray ionization (ESI+), with operating conditions as follows: capillary voltage at 3.0 kV, desolvation temperature at 500 °C, source temperature at 150 °C, desolvation gas flow at 500 L / h, and cone gas flow at 150 L / h. Detection of EAPB02303 and EAPB04303 was performed using Multiple Reaction Monitoring (MRM). The optimal MRM transitions for precursor ion [M+H]+to specific product ion were m / z 307.2 291.2 for EAPB02303 m / z321.2 306.0 for EAPB04303, and m / z 277.1 117.1 for the internal standard (IS) (Fig 6A, B, C). Optimal MS / MS parameters (cone voltage and collision energy) for MRM transitions of EAPB02303, EAPB04303 and IS are 60V / 50V, 60V / 40V and 70V / 60V respectively. The acquisition dwell time was 108 ms for all transitions. Mass spectra were collected in scan mode (m / z 50-1000). The LC-MS / MS system was controlled and the analytical data were collected and processed using MassLynx software version 4.2 (Waters).

[0230] Immunofluorescence analyses.

[0231] Cells were plated on coverslips in 24-well plates. After 48 hours, cells were incubated with drugs for 6, 12 or 24 hours. After fixation with 3.7% formaldehyde in PBS for 20 minutes, cells were permeabilized with PBS / 0.5% Triton X-100 at room temperature for 15 minutes. After washes, cells were incubated in PBS / 2% BSA for 1 hour, and then with primary antibodies (1 / 500) (Supplementary Table 1) at 37°C for 90 minutes. Cells were washed twice with PBS / 0.1% Tween-20 and were incubated with the goat anti-mouse IgG Alexa 488 (Cell Signaling #4408; 1 / 50) and goat anti-rabbit AF568 (Invitrogen Al 1011; 1 / 500) secondary antibodies at 37°C for 45 minutes. Then, coverslips were washed with PBS / 0.1% Tween-20 three times and with PBS three times, followed by mounting with Everbrithe® and DAPI and analysis using an epifluorescence Zeiss Axio Imager 2 microscope.

[0232] Immunohistochemical analyses.

[0233] Immunohistochemistry analyses were performed as described previously (Leconet et al.

[0234] 2017). Signals were quantified with QuPath.

[0235] In vivo studies.

[0236] All in vivo experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (Agreement No. C34- 172-27). P4604 (5 x 106) or Pancpec (5 x 106) PDX-derived cells were grafted subcutaneously in 6-week-old female athymic mice, purchased from Charles Rivers (Le Malcourlet, France). Tumor-bearing mice were randomized in the different treatment groups (10 animals / group) when tumors reached a minimum volume of 150 mm3. Tumor volumes were calculated with the formula: DI x D2 x D3 / 2. Mice were treated by intra-peritoneal injection of EAPB02303 (30 mg / kg in vehicle: 80% isotonic water, 10% DMSO, 10% Tween 80) daily, and / or of with paclitaxel (10 mg / kg) twice per week, or vehicle alone daily for 4 weeks. For survival comparison, mice were sacrificed when tumors reached a volume of 1500 mm3.

[0237] COMT expression analysis.

[0238] COMT expression data in tumor and normal pancreatic tissues were downloaded via the Xena platform from the TCGA and Genotype-Tissue Expression (GTEx) databases. Data werevisualized as boxplots and expression levels in normal and tumor tissues were compared with the unpaired t-test using Prism vl0.0.2.

[0239] Statistical analysis.

[0240] Data were expressed as the mean ± SEM from at least three experiments. Statistical difference was set at p <0.05. The relationship between tumor growth and treatment was analyzed using a linear mixed regression model. The fixed part of the model included the number of days post-graft and treatment group; interaction terms were also evaluated. Random intercepts and random slopes were included to take into account the time effect. The model coefficients were estimated by maximum likelihood. A survival analysis was performed and the considered event was a tumor volume of 1,500 mm3. Survival rates were estimated using the Kaplan Meier method and survival curves were compared with the log rank test. Statistical significance was set at p <0.05. Statistical analyses were done with STATA 16 (Stata Corporation, College Station, TX).

[0241] Results

[0242] EAPB02303 inhibits PDAC cell viability and tumor growth.

[0243] We first assessed the effect of EAPB02303 on the viability of various PDAC cell lines cultured alone (2D models) or with CAFs in 3D spheroid models (data not shown). EAPB02303 ICso ranged between 5 nM in CFPAC-1 and 78 nM in Capan-1 cells and was in the same range as that of gemcitabine in most of the tested cell lines (data not shown). Co-culture of Pancpec tumor cells and CAFs (1:50 ratio) did not affect EAPB02303 cytotoxic effect (data not shown). This is consistent with EAPB02303 ability to inhibit CAF viability data not shown). Moreover, EAPB02303 displayed significant cytotoxic effect in gemcitabine-resistant (GR) MIA PaCa-2, BxPC3 and Pancpec cells and also in FOLFIRINOX-resistant (FR) MIA PaCa-2 cells, as indicated by the IC50 values in the same range as those of the parental cell lines, except for BxPC3 cells (Figure 2A).

[0244] We then assessed EAPB02303 in athymic mice xenografted with Pancpec or P4604 cells (subcutaneous injection). When tumor volume reached at least 150 mm3, we treated mice with EAPB02303 (3, 10, or 30 mg / kg) or vehicle alone every day for 30 days. EAPB02303 at 30 mg / kg significantly (p < 0.0001 in Pancpec and p = 0.0032 in P4604), but modestly reduced tumor growth. This led to an increase in overall survival without significant toxicity in both models (Pancpec : p=0.012, P4604 p = 0.0064) (Figure 2B and 2C).

[0245] These results demonstrated that EAPB02303 exerts a potent cytotoxic activity in vitro in PDAC cell lines with different mutational status and reduces tumor growth in mice.Efficacy of the EAPB02303 and paclitaxel combination in preclinical PDAC models.

[0246] Then, we assessed the effect of EAPB02303 combined with clinically approved drugs for PDAC (Figure 3A). The association of EAPB02303 with gemcitabine (Figure 3A) and radiotherapy (data not shown) was additive (black) in PDAC cell lines. Surprisingly, the EAPB02303 and paclitaxel combination had a synergistic effect in Pancpec and P4604 PDX-derived cells (Figure 3 A). This effect was confirmed in vivo, in mice xenografted with Pancpec and P4604 cells. The combination of EAPB02303 (30 mg / kg) and paclitaxel significantly inhibited tumor growth compared with each drug alone and vehicle in both models (p = 0.001 in Pancpec and p = 0.0007 in P4604) (Figure 3B and D). Kaplan-Meier curves showed that survival was increased in mice treated with the combination compared with the vehicle or EAPB02303 alone in the Pancpec model (p= 0.008 and p-0,0224 respectively) (Figure 3C). We did not observe any significant weight loss in any group (data not shown). We then analyzed by immunohistochemistry the drug effects on the expression of PHH3 (a mitosis marker) and cleaved caspase 3 (an apoptosis marker) in tumors at mid-treatment (data not shown). The percentage of PHH3 -positive cells was significantly increased in tumors from mice treated with the combination compared with mice treated with each drug alone or with vehicle (untreated) (p<0.0001). The percentage of cleaved caspase 3-positive cells was increased in tumors treated with the combination compared with untreated tumors (p=0.0008). Moreover, the percentages of PHH3- and cleaved caspase 3-positive cells were higher in EAPB02303 -treated than untreated tumors.

[0247] The proteomic and transcriptomic profiles of EAPB 02303 -treated PDAC cells reveal interactions with mitosis regulation and microtubule dynamics.

[0248] To elucidate EAPB02303 mechanism of action, we used transcriptomic and proteomic profiling. First, we evaluated the basal expression and phosphorylation levels of hundreds of proteins using the antibody -based RPPA in CFPAC-1 and Pancpec cells incubated or not with EAPB02303 (5xICso for 24 hours) for 24 hours. We identified 22 proteins that were differentially expressed or phosphorylated in both cell lines after incubation with EAPB02303 compared with untreated cells (data not shown). Many of these proteins are implicated in cell cycle and cell division, especially in the control of mitosis: aurora A, B, C, PRC1, PLK1, JUN, histone H3, CDK1, WEE1 data not shown), suggesting a major effect of EAPB02303 on mitosis regulation.

[0249] We then carried out RNA-seq of Pancpec and CFPAC-1 cells incubated with EAPB02303 (5xICso) or not for 6 hours (data not shown) or 24 hours (data not shown) (threereplicates). Incubation with EAPB02303 for 24 hours massively altered the transcriptomic profiles of both cell lines, particularly apoptosis and stress-related genes. In cells incubated for 6 hours, 75 and 24 genes were downregulated and 91 and 50 were upregulated in CFPAC-1 and Pancpec cells, respectively (data not shown) (LogFC =1, p-value = 0.01). GSEA performed using the MSigDB subset GO: Biological process showed 229 and 63 significantly enriched gene sets in CFPAC-1 and Pancpec cells, respectively, among which 19 gene sets were shared by both cell lines (data not shown). These 19 common gene sets are mainly related to processes involving microtubules (for instance, the gene set “microtubule-based process”) (data not shown). Incubation with EAPB02303 for 6 hours led to the downregulation mainly of genes encoding tubulin isoforms or tubulin pseudogenes (data not shown). This suggested an autoregulation based on the quantity of free tubulin monomers that could increase following disruption of microtubule polymerization, as previously reported (Cleveland et al. 1981). Overall, these data suggest a major effect of EAPB02303 on microtubule dynamics and mitosis.

[0250] EAPB02303 induces mitotic arrest and apoptosis.

[0251] To experimentally confirm these findings, we performed a cell cycle analysis by propidium iodide staining and found that in PDAC cell cultures incubated with EAPB02303 (ICso and 5xICso), cells accumulated in the G2-M phase at 24 hours and 48 hours compared with untreated controls (data not shown). Incubation with EAPB02303 for 48 hours increased also the percentage of cells in the sub-Gl phase, suggesting that they cannot engage in the next cell cycle steps and that they will die.

[0252] Analysis of PHH3 (mitosis marker) showed an accumulation of Pancpec cells in mitosis only at 24 hours (data not shown) and of CFPAC-1 cells in mitosis at 24 hours and 48 hours, associated with a typical rounded cells phenotype data not shown). As the formation of a complex between CDC2 and cyclin Bl is an important event for mitosis entry, we assessed their expression in cells incubated or not with EAPB02303 (IC50 and 5xICso) by western blotting. At 24 hours, cyclin B 1 expression was increased, whereas the expression of CDC2 phosphorylated at the inhibitory site Tyrl5 was decreased (data not shown), suggesting activation of the cyclin B1-CDC2 complex, in line with the accumulation of mitotic cells.

[0253] As drug-induced mitotic delay can lead to cell death through apoptosis (Topham and Taylor 2013), we investigated EAPB02303 effect on apoptosis induction by Annexin V and 7-AAD staining. In both Pancpec and CFPAC-1 cells, EAPB02303 (IC50 and 5xICso) triggered early and late apoptosis already after 24 hours of incubation. Apoptosis (early and late) ratereached 50% after 48h hours in Pancpec cells and after 72 hours in CFPAC-1 cells (data not shown).

[0254] These findings showed that EAPB02303 cytotoxic effect is achieved by blocking cells in mitosis and inducing apoptosis.

[0255] EAPB02303 inhibits microtubule polymerization.

[0256] As mitosis blockage is a common feature of agents that targets microtubule dynamics [8], we analyzed the effects on microtubule networks and cell morphology by immunofluorescence analysis of P-tubulin expression after incubation of Pancpec and CFPAC-1 cells with EAPB02303, colchicine, or paclitaxel for 6, 12 or 24 hours. There are two major classes of microtubule-targeting agents (MT A) (Goodson and Jonasson 2018): i) microtubulestabilizing agents (e.g. taxanes and epothilones) bind to microtubule polymers and stabilize them against disassembly; ii) microtubule-destabilizing agents (e.g. colchicine and vinca alkaloids) bind to tubulin dimers and destabilize microtubule polymers (Steinmetz and Prota 2018). We quantified the percentages of normal and abnormal microtubules (data not shown). As expected, colchicine and paclitaxel induced abnormal mitotic spindles with typical morphological changes, evoking destabilization (colchicine) or stabilization (paclitaxel) (data not shown). EAPB02303 also disrupted mitotic spindle formation in a dose-dependent manner (data not shown), yielding morphological changes similar to those observed with colchicine. This suggested that EAPB02303 could function as an inhibitor of microtubule polymerization. To test this hypothesis, we quantified the whole cell microtubule biomass after incubation with EAPB02303, colchicine, or paclitaxel for 18 hours (data not shown). Like colchicine, EAPB02303 led to a significant decrease in the quantity of cellular microtubules, thus confirming its activity as microtubule polymerization inhibitor. Lastly, we used CETSA to determine whether this effect could be attributed to direct binding to tubulin. This assay relies on the principle of the thermal stabilization of the target (here, P-tubulin) upon drug binding directly in living cells. As expected, incubation with paclitaxel (a known tubulin binder) for 3 hours increased the thermal stability of tubulin, as indicated by the increased tubulin detection at higher temperatures compared with untreated CFPAC-1 cells (data not shown). We obtained similar results after incubation with EAPB02303 for 3 hours.

[0257] These results demonstrated that EAPB02303 inhibits microtubule polymerization, as previously described for imidazo[l,2-a]quinoxaline compounds.Metabolization of EAPB02303 by Catechol-O-Methyl transferase.

[0258] As EAPB02303 does not inhibit microtubule polymerization when added to purified tubulin (Patinote et al. 2021), we hypothesized that this discrepancy between purified tubulin and in-cell experiments might be explained by EAPB02303 bioactivation in cells. EAPB02303 has a catechol moiety that is an attractive candidate for bioactivation by COMT (Figure 4A) to produce a hydro-methoxyphenyl metabolite. The yielded molecules would include 3 -hydroxy -4-methoxyphenyl, a functional group also involved in combretastatin A4 interaction with the colchicine binding site on tubulin (McLoughlin et O’Boyle 2020). Two mono-methoxy compounds could be considered following EAPB02303 metabolization by COMT: EAPB04303 (3 -hydroxy -4-methoxyphenyl derivative)) and EAPB04403 (4-hydroxy-3-methoxyphenyl derivative).

[0259] Moreover, the GSEA analysis within the KEGG pathway collection of the RNA-seq data of Pancpec and CFPAC cells incubated with EAPB02303 for 6 hours identified six common gene sets among which Parkinson’s disease was negatively enriched (data not shown). Due to the major implication of catecholamine metabolism and COMT inhibitors in Parkinson’s disease management, these results prompted us to investigate EAPB02303 bioactivation by COMT. First, we synthetized EAPB04303 and EAPB04403 from EAPB2303 in one step using a Suzuki-Miyaura cross-coupling reaction with the corresponding boronic acid (data not shown). Then, we measured their cytotoxic activities (SRB assay) in CFPAC- 1 and Pancpec cells. One of the predicted metabolites, EAPB04303, had a lower ICso than EAPB02303, while EAPB04403 was less potent (Figure 4B).

[0260] Then, we determined whether blocking COMT activity using the COMT inhibitors tolcapone and entacapone affected EAPB02303 cytotoxic effect (Figure 4C). EAPB02303 cytotoxic activity was strongly reduced by COMT inhibition with these two inhibitors. Conversely, COMT inhibition did not affect colchicine cytotoxicity, underscoring the specificity of COMT dependency in EAPB02303 mechanism of action. To confirm these results and avoid any conclusions based on off-target effects of the pharmacological inhibitors, we generated Pancpec and CFPAC- 1 cell lines in which COMT was knocked out using the CRISPR / cas9 technique (data not shown). In the negative control (Pancpec and CFPAC- 1 cells transduced with CRISPR luc), COMT expression was similar to that of parental cells (data not shown). In line with our previous results, EAPB02303 cytotoxic effect was drastically reduced in cells lacking COMT (COMT- / -), as indicated by the 100 times higher IC50 (data not shown). Conversely, the predicted metabolite EAPB04303 displayed its potent cytotoxic effect in both COMT- / - and COMT+ / + cell lines (data not shown). To determine whether bioactivation byCOMT was required for EAPB02303 binding to tubulin in cells, we performed ITDR-CETSA in CFPAC-1 COMT- / - cells incubated with increasing concentrations of EAPB02303 or EAPB04303. By heating cells at 64°C, a temperature that induces significant tubulin denaturation, ITDR-CETSA allowed calculating the ECso value at which each molecule stabilized tubulin. Unlike in COMT+ / + cells, in COMT- / - cells EAPB02303 could not induce tubulin stabilization even at the highest concentration tested (100 pM). Conversely, EAPB04303 had an ECso of 3 pM ±2 (data not shown). This demonstrated that in cells, EAPB02303 engagement with tubulin is possible after its bioactivation by COMT, and suggests that in cells, EAPB02303 cellular activity on microtubule dynamics at nanomolar concentrations is due to binding of its active metabolite EAPB04303 to P-tubulin (data not shown).

[0261] Then, we measured the presence and concentration of EAPB02303 and its predicted active metabolite EAPB04303 by UPLC-MS / MS in parental and COMT- / - Pancpec cells incubated with 100 nM EAPB02303, with or without the COMT inhibitor entacapone (6.25 pM). After 15 and 60 minutes of incubation, we measured the drug concentration in cell extracts. MRM chromatograms of EAPB02303 is: 307.2— >291.2, of EAPB04303: 321.2— > 306.0, and of IS 277.1^117.1. In untreated parental Pancpec cells, EAPB02303 and EAPB04303 were undetectable (below the limit of quantification, LoQ, of 0.49 ng / mL) (data not shown). In parental Pancpec cells incubated with EAPB02303 for 60 minutes, the concentrations of EAPB02303 and EAPB04303 were 3.5 ± 0.7 ng / mL and 22.4 ± 1.7 ng / mL, respectively, confirming the production of the predicted metabolite (data not shown). After 60 minutes of incubation with EAPB02303 (100 nM) and entacapone (6.25 pM), the concentration of EAPB02303 in parental Pancpec cells was 6.6 ± 1.1 ng / mL, while EAPB04303 was undetectable (< LoQ) (data not shown). Similarly, in COMT -I- Pancpec cells incubated with 100 nM EAPB02303 for 60 minutes, EAPB02303 concentration was 9.4 ± 0.8 ng / mL and EAPB04303 was undetectable (data not shown).

[0262] Altogether, these data demonstrated that in PDAC cells, EAPB02303 is effectively and rapidly transformed into EAPB04303 by COMT, supporting the hypothesis that EAPB04303 is the actual microtubule-interacting agent responsible for EAPB02303 cytotoxic activity.

[0263] COMT expression in PDAC samples.

[0264] Analysis of data from the TCGA database on human PDAC samples (n=176) showed a significant survival increase of patients with PDAC with low COMT mRNA expression (p =0.0021) (Figure 5A). Moreover, comparison of data from the TCGA and GTEx databasesshowed higher COMT expression in PDAC than normal pancreas tissue samples (p<0.001) (Figure 5B). These findings indicate that COMT could be used a biomarker to identify patients with PDAC who may respond to EAPB02303.

[0265] EXAMPLE 2: RESULTS ON ACUTE MYELOID LEUKAEMIA

[0266] Material & Methods

[0267] Cell culture.

[0268] OCI-AML2 (wild-type NPM1; wt-NPMl, from Dr. C. Teyssier) and OCI-AML3 (A AfY-mutation; NPMlc, from Dr. D. Bouscary) cells were grown in minimum essential medium alpha (MEM-a) (Ref 32561-029, Gibco, ThermoFischer Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine serum (FBS) (F9665 Sigma-Aldrich, St. Louis, Missouri, USA) and 1% penicillin-streptomycin (L0022-100-MS01L91000 Biowest Nuaille, France). Cells were seeded at a density of 2 x 105 / mL and cell viability was assessed using the trypan blue exclusion dye assay (T8154 lot #RNBK8531 Sigma-Aldrich, St. Louis, Missouri, USA) at 24h, 48h and 72h.

[0269] Preparation of compounds.

[0270] The synthesis and purity analysis of Imiqualine derivatives were performed as previously described

[0017] , Entacapone (Sigma- Aldrich SML0654), Tolcapone (Sigma- Aldrich SML0150), EAPB02303 and EAPB04303 were dissolved in dimethylsulfoxide (DMSO) to prepare a stock concentration of 10'2M, aliquoted and stored at -20°C. The working concentration of EAPB02303 and EAPB04303 was set at 5 nM. The final concentrations of entacapone and tolcapone were 6.25 and 3.125 pM, respectively.

[0271] Cell cycle analysis.

[0272] OCI-AML2 and OCI-AML3 cells treated with EAPB04303 for 24h and 48h were collected, washed with PBS, fixed with 100% cold ethanol and stored at -20°C overnight. Cell pellets were treated for 45 min with 100 pL of 200 pg / ml DNase-free RNase A (EN0531 ThermoFischer Scientific, Waltham, MA, USA). Cells were stained with 30 pL of 1 mg / mL propidium iodide (PI) (CAS 25535-16-4 Sigma-Aldrich, St. Louis, Missouri, USA) to assess the different stages of the cell cycle. Cell cycle analyses were performed using a Guava Easycyte 8 flow cytometer.

[0273] Annexin V / PI staining.

[0274] To assess the mechanism of cell death, OCI-AML2 and OCI-AML3 cells treated with EAPB04303 for 24h and 48h were harvested, washed with PBS, then stained with annexin V-FLUOS staining kit (11988549001 Roche) according to the manufacturer’s instructions.Immunoblot analysis.

[0275] Protein extracts were probed with the following antibodies: p53 (DO-1) (sc-126, Santa Cruz Biotechnology, Dallas, TX, USA), Phospho-p53 (Seri 5) (#9284 Cell Signaling Technology, Danvers, MA, USA), p21 Wafl / Cipl (12D1) (#9247 Cell Signaling Technology, Danvers, MA, USA), PARP-1 (F-2) (sc-8007, Santa Cruz Biotechnology, Dallas, TX, USA), Caspase-3 (31A1067) (sc-56053, Santa Cruz Biotechnology, Dallas, TX, USA), Akt (pan) (C67E7) (#4691 Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt (Ser473) (#9271 Cell Signaling Technology, Danvers, MA, USA), mTOR (7C10) (#2983 Cell Signaling Technology, Danvers, MA, USA), Phospho-mTOR (Ser2448) (#2971 Cell Signaling Technology, Danvers, MA, USA), p44 / 42 MAPK (Erkl / 2) (137F5) (#4695 Cell Signaling Technology, Danvers, MA, USA), Phospho-p44 / 42 MAPK (Erkl / 2) (Thr202 / Tyr204) (#4370 Cell Signaling Technology, Danvers, MA, USA), Anti-Nucleophosmin antibody NPM1 [3A9F1] (ab86712, Abeam, Cambridge, UK), NPMl-c (mutant) (PAI-46356, Invitrogen, ThermoFischer Scientific, Waltham, MA, USA) before incubation with the monoclonal horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse anti-rabbit IgG-HRP (sc-2357 lot #12022 Santa Cruz Biotechnology, Dallas, TX, USA) or m-IgGK BP-HRP (sc-516102 lot #12120 Santa Cruz Biotechnology, Dallas, TX, USA)). Loading control was performed by probing with Histone (H3) (abl791, Abeam, Cambridge, UK). Immunoblots were detected using the luminol detection kit (BioRad) and images were captured using BioRad Chemi doc MP system.

[0276] Xenograft animal studies.

[0277] C)DI \\\-scid I 1 ,2r-gamma (NSG) male and female mice were obtained from Jackson Laboratories (USA). Protocols were approved by the Institutional Animal Care and Utilization Committee of the American University of Beirut. 2xl06OCI-AML2 or OCI-AML3 cells were injected into the tail vein of six- to eight- week-old mice. Mice were treated intraperitoneally with EAPB04303 every other day over 3 weeks and divided into two groups: one group was monitored for survival while the second group was sacrificed to assess leukemic burden and organ infiltration. Human CD45 staining was performed to assess leukemic burden in bone marrow samples. Spleen and liver infiltration by AML cells was assessed through gross pathology and histopathology as previously described [4],

[0278] Statistical analysis.

[0279] All the data described in this study were run in at least three independent experiments. Data were reported as the average ± standard deviations. Statistical analysis was performedusing two-way ANOVA or Student’s t test. / J- value < 0.05 was considered statistically significant.

[0280] Results

[0281] EAPB04303 displays potent anti -proliferative activity against AML cell lines.

[0282] We previously demonstrated that EAPB02303 potently inhibits the growth of AML cell lines and patient-derived blasts from AML patients with different karyotypes (Makhoul et al., under review). We first investigated the anti-leukemic activity of the EAPB02303 metabolite, EAPB04303, on cell proliferation. Whilst 1 nM EAPB02303 had no effect on cell proliferation, EAPB04303 at the same concentration significantly decreased OCI-AML2 (wt-NPMJ) and OCLAML3 (NPMlc) cell growth by 30%, starting 48h post-treatment (p- value < 0.001 and 0.01, respectively) (Figure 6A). We previously showed that 5 nM EAPB02303 led to a median decrease in cell growth of OCI-AML2 and OCI-AML3, 24h post-treatment and completely abolished cell growth at 72h (Makhoul et al., under review). Comparing the anti -proliferative activity of the metabolite to its parental molecule, we show that at the low dose of 2.5 nM, EAPB04303 gradually reduced OCI-AML2 and OCI-AML3 cell viability by ~50%, as early as 24h, to completely abolish cell growth at 72h ( / ?-value < 0.001) (Figure 6A). Thus, the methylated derivative EAPB04303 proved more potent than EAPB02303 at lower concentrations against both NPMlc and vrt.-NPMl AML cell lines. For the remainder of the study, EAPB04303 was used at the same working concentration as EAPB02303 (5 nM) to allow an appropriate comparison between the activity of both derivatives.

[0283] COMT inhibitors suppress the anti-proliferative activity of EAPB02303 but not that of EAPB04303.

[0284] Given the chemical structure of EAPB02303 and its synthesized methoxylated metabolite EAPB04303, resulting from the methylation of one of the hydroxyl groups of EAPB02303 attached to the phenyl ring, one of the suggested enzymatic reactions involved in the biochemical activation of EAPB02303 is identical to the well-established methylation of catechol substrates by COMT enzymes

[0032] , To verify if COMT enzymes are implicated in the anti-proliferative activity EAPB02303, we used entacapone and tolcapone, two known inhibitors of COMT enzymes [33, 34], While these two COMT inhibitors alone did not show any anti-proliferative activity against AML cell lines (Figure 6B, 6C), entacapone or tolcapone completely reversed the antileukemic activity of EAPB02303 (Figure 6B). Conversely, the metabolite EAPB04303, as single agent or combined with entacapone or tolcapone significantlyinduced growth arrest in AML cells lines ( - value < 0.001) (Figure 6C). These results strongly suggest that the anti -leukemic potency of EAPB02303 depends on its methylation through the enzymatic activity of COMT in AML.

[0285] EAPB04303 induces G2 / M cell cycle arrest and massive apoptosis in AML cells. We previously demonstrated that EAPB02303 induces a significant accumulation of AML cell lines in the pre-GO state without affecting the cycling cells (Makhoul et al., under review). By contrast, EAPB04303 resulted in significant G2 / M cell cycle arrest 24h posttreatment (data not shown). Furthermore, whilst EAPB02303 induces OCI-AML2 and OCI-AML3 cell death at 48h, treatment with EAPB04303 resulted in significant AML cell death 24h earlier, with a significant increase in the percentage of late apoptotic / necrotic cells (p- value < 0.01) (Figure 7A,B,C). The EAPB04303 -driven apoptotic events started as early as 6h in both OCI-AML2 and OCI-AML3 with a significant increase in the levels of expression of the pro-apoptotic proteins P53, its active phosphorylated form Phospho-P53 and the downstream effector P21 (data not shown), as compared to EAPB02303 -mediated apoptosis which occurred between 24-48h of treatment (Makhoul et al., under review). Furthermore, EAPB04303 resulted in PARP-1 and caspase-3 cleavage starting 24h whilst the same events were observed only 48h post-treatment with EAPB02303 (data not shown) (Makhoul et al., under review). Taken altogether, these findings show that the second generation Imiqualine s-mediated apoptotic cell death is conserved between the two tested molecules, yet the activity of EAPB04303 is more pronounced and begins earlier than that EAPB02303. In addition, the effect of these molecules on the cell cycle phases differs between EAPB02303 and its metabolite EAPB04303.

[0286] The anti-leukemic activity of EAPB04303 implicates the same molecular players of EAPB02303 through the inhibition of the PI3K / AKT / mTOR and RAS / MAPK pathways.

[0287] Our recent reports demonstrated that the molecular mechanisms of EAPB02303 potency involves the inhibition of the activity of the PI3K / AKT / mTOR and RAS / MAPK signaling networks (

[0024] Makhoul et al., under review). We investigated whether these molecular cascades are involved in the mechanism of action of EAPB04303. Similar to EAPB02303 (Makhoul et al., under review), the treatment of OCI-AML2 and OCI-AML3 with EAPB04303 led to a gradual decrease in the levels of AKT, mTOR, and ERK between 24-48h, paralleled with a downregulation of their respective phosphorylated active forms (data not shown). These results demonstrate a parallelism in the mechanism of action of EAPB02303 and its metabolite, through the inhibition of the PI3K / AKT / mTOR and RAS / MAPK signaling pathways.Consistent with EAPB02303 (Makhoul et al., under review), OCI-AML3 cell lines expressing NPMlc were more sensitive to treatment with EAPB04303 and in a comparable downregulation of NPMlc in OCI-AML3 cell line was observed as early as 6h post-treatment (data not shown).

[0288] Overall, our results delineate preserved molecular targets of EAPB02303 and EAPB04303 with a similar mechanism of action underlying their anti-leukemic effects on AML.

[0289] EAPB04303 surpasses EAPB02303 anti-AML potency in vivo.

[0290] We recently demonstrated the potent in vivo activity of EAPB02303 (Makhoul et al., under review). Alas, despite the sharp decrease of leukemic burden in the bone marrow of treated xenograft mice, the favorable survival outcomes were exclusively seen in the NPMlc OCLAML3 xenograft cohort (Makhoul et al., under review). Thus, to explore the in vivo efficacy of EAPB04303, six- to eight- week-old female or male mice were intravenously injected with v -NPMl OCLAML2 or NPMlc OCI-AML3. Treatment with EAPB04303 was administered as described (data not shown). While all untreated AML xenograft mice succumbed around day 50 (n = 7 OCI-AML2 xenografts and n = 5 OCI-AML3 xenografts), EAPB04303 showed a striking and significant prolonged survival of treated OCI-AML2 and OCI-AML3 xenografts for over 120 or 160 days, respectively (4 out of 7 OCI-AML2 xenografts and 5 out of 7 OCI-AML3 xenograft mice treated are still alive, at least 6 months since the beginning of the experiment) (Figure 8A). This is contrary to the previous results obtained with EAPB0503 and EAPB02303, which prolonged the survival of OCI-AML3 xenograft mice for up to 100 and 120 days, respectively (Makhoul et al., under review). In alignment with the prolonged survival, and similar to EAPB02303, EAPB04303 alleviated leukemic burden in the BM of all treated mice and entirely diminished the %hCD45 in both OCI-AML2 and OCL AML3 xenografts ( - value < 0.001) (Figure 8B). Concurrent with this enhanced in vivo efficacy, EAPB04303 potently reduced splenomegaly manifested by the significant decrease in spleen size and weights ( -value < 0.001) (data not shown). Comparable with EAPB02303, liver gross pathology in OCI-AML2 and OCI-AML3 xenograft mice following treatment with EAPB04303 showed a normal gross macroscopy (data not shown). Consistently, H&E stain showed a clear infiltration of the liver in untreated xenograft mice injected with OCI-AML2 or OCI-AML3, while treatment with EAPB04303 preserved the liver’s normal architecture (data not shown). Altogether, our data emphasizes the broader in vivo anti-leukemic activity of EAPB04303 against AML.REFERENCES:

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Claims

- 54 -CLAIMS:

1. An aromatic heterocyclic compound of general formula (I):(I)or a pharmaceutically acceptable salt thereof,wherein n is between 0 and 2 and wherein if n=0:- Rl, R2, R3, R4, R5 independently of each other represent a hydrogen atom, a hydroxy, methoxy, hydroxymethyl, dihydrogenated phosphate, dihydrogenated oxymethylphosphate, amino or methylamino group and- R6 represents an amino or methylamino group,wherein that at least 2 or at most 3 of the residues Rl, R2, R3, R4, R5 represent a hydrogen atom,wherein that at least two successive residues Rl, R2, R3, R4, R5 are different from said hydrogen atoms, andwherein if n=l or 2:- Rl, R4, R5 independently represent a hydrogen atom, a hydroxy, methoxy, amino or methylamino group,- R2 and R3 jointly represent two oxygen atoms or two nitrogen atoms,- R6 represents an amino or methylamino group.

2. The aromatic heterocyclic compound according to the claim 1- 55 -wherein if n=0:- Rl, R4, R5 independently represent a hydrogen atom- R2 and R3 independently represent a hydroxyl or methoxy group- R6 represents an amino or methylamino group.

3. The aromatic heterocyclic compound according to the claim 2 wherein the compound is the EAPB04303 (2-methoxy-5-(4-(methylamino)imidazo[l,2-a]quinoxalin-l- yl)phenol) or the EAPB04403 (2-methoxy-4-(4-(methylamino)imidazo[l,2- a]qui noxal i n- 1 -yl)phenol).

4. An aromatic heterocyclic compound according to any one of claims 1 to 3 for use in the treatment of cancer in a subject in need thereof.

5. The aromatic heterocyclic compound according to the claim 4 wherein the compound is the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound.

6. The aromatic heterocyclic compound according to any one of claims 4 or 5 wherein the cancer is the pancreatic cancer or the AML.

7. A pharmaceutical composition comprising a therapeutically effective amount of the aromatic heterocyclic compound of general formula (I) of the invention according to any one of claims 1 to 3.

8. An aromatic heterocyclic compound of general formula (I) according to any one of claims 1 to 3 for use in the treatment of cancer in a subject in need thereof in combination with a further therapeutic active agent.

9. The aromatic heterocyclic compound according to the claim 8 wherein the further therapeutic active agent is the paclitaxel.

10. The aromatic heterocyclic compound according to the claim 9 wherein the paclitaxel is combined with the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound.

11. An aromatic heterocyclic compound of general formula (I) according to any one of claims 1 to 3 or the pharmaceutical composition according to the claim 7 and at least one therapeutic active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer.

12. An antibody conjugated to the aromatic heterocyclic compound of general formula (I) according to any one of claims 1 to 3.

13. The antibody conjugated according to the claim 12 wherein the compound is the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound.

14. An antibody conjugated to the aromatic heterocyclic compound of general formula (I) according to any one of claims 1 to 3 for use in the treatment of cancer in a subject in need thereof.

15. The antibody conjugated to the aromatic heterocyclic compound of general formula (I) according to the claim 13 for use in the treatment of cancer in a subject in need thereof wherein the compound is the EAPB02303 compound, the EAPB04303 compound or the EAPB04403 compound.

16. A method of treating a cancer in a subject in need thereof comprising administering said subject with an aromatic heterocyclic compound according to any one of claims 1 to 3.

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