FIELD OF THE INVENTION:

The present invention relates to compounds targeting the Bfl-1 anti-apoptotic protein and uses thereof for the treatment of cancer.

BACKGROUND OF THE INVENTION:

Many of the cellular events that initiate malignant transformation of a normal cell

(e.g., activation of oncogenes) also activate oncogenic stress pathways that usually cause the cell to enter apoptosis. In order for these cells to survive and cause cancer, they typically must have acquired changes in other cellular pathways to prevent apoptosis. Through their capacity to interact within each other, pro- and anti-apoptotic proteins of the Bcl-2 family are important regulators and executioners of the intrinsic apoptotic pathway. Expression of the Bcl-2 proteins is often deregulated in cancer and thus dictates whether or not many current cancer therapies are effective, because most of these treatments result in stress signals that ultimately must activate the apoptosis machinery of cancer cells. Increased expression of pro-survival members is recognized as a hallmark of many cancers, and drugs that can prevent their action would be very valuable. One approach currently being developed in anti-cancer drug discovery is to search for BH3 mimetics capable of occupying and blocking the hydrophobic pocket of anti- apoptotic Bcl-2 family members necessary for interacting with pro-apoptotic proteins. Illustration of such strategy is the identification of ABT-737, a small molecule with high affinity for Bcl-2, Bcl-xl and Bcl-w ¹ and more recently the development of ABT-199 a selective inhibitor of Bcl-2 ² . Navitoclax an orally bioavailable derivative of ABT-737 is currently evaluated in phase 2 clinical trials ^3"5 . Besides a dose- limiting toxicity towards platelets for navitoclax, a limitation for the use of ABT-199 and navitoclax is the expression by tumor cells of Mcl-1 or Bfl-1 (BCL2A1) anti-apoptotic proteins that was shown to confer resistance to ABT-737 ^6"8 , thus emphasizing the need for BH3 mimetics specific for Mcl-1 or Bfl-1.

Since its discovery in 1995 as a gene overexpressed in stomach cancer ⁹ , high expression of Bfl-1 has been documented in various types of cancers (for review see ⁸ ) and particularly in lymphoid malignancies, as a sub-group of diffused large B-cell lymphoma (DLBCL) ¹⁰ , mediastinal B cell lymphoma (MLBCL) ¹¹ and mantle cell lymphoma ¹² . Bfl-1 was also implicated in the emergence of resistance of B- CLL subjects to fludarabine treatment ¹³ ' ¹⁴ . Further studies using RNA interference strategies demonstrated that inhibition of Bfl-1 sensitize fresh B-CLL or malignant B-cell lymphoma cell lines to chemotherapeutic agents such as Cisplatin and Fludarabine, and therefore validated Bfl-1 as a therapeutic target in B cell malignancies ^13~15 . Peptide aptamers that specifically interact with the hydrophobic groove of Bfl-1 were recently identified. Said aptamers disrupt the interaction of Bfl-1 with its pro- apoptotic partners such as the pro-survival protein Bax. As for RNA interference strategy it was demonstrated that anti-Bfl-1 aptamers sensitize malignant B-cell lymphoma cell lines to chemotherapeutic agents ¹⁶ . Thus, disrupting interactions of Bfl-1 with pro-apoptotic partners appears to be an efficient strategy to overcome its pro-survival activity in malignant cells.

SUMMARY OF THE INVENTION:

The present invention relates to compounds targeting the Bfl-1 anti-apoptotic protein and uses thereof for the treatment of cancer. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Here the inventors describe the discovery of small molecules targeting Bfl-1 anti- apoptotic protein using high-throughput screening (FITS) approach of a chemical library followed by a ligand-based screening approach (similarity and substructure search). The inventors found two compounds (BDM 44931 , BDM 44898) that specifically interact with Bfl-1, inhibit Bfl-l protective activity and promote cell death of malignant B cells. Finally, the inventors selected BDM 44898 compound for in vivo assays, and demonstrated its ability to reduce tumor growth of lymphoid tumor xenograft established in immunocompromised mice.

Accordingly, the present invention relates to a method of the treatment of cancer in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one compound selected from the group consisting of BDM 44898, BDM 44931 and pharmaceutical acceptable salts thereof. As used herein the term "BDM 44931" refers to the compound having the general formula (I):

The compound was disclosed in DATABASE chemcats [Online] 3 July 2013 (2013-07-03), Database accession no. 0149027232.

As used herein the term "BDM 44898" refers to the compound having the general formula (II):

This compound was previously described in Christensen QH, Grove TL, Booker SJ, Greenberg EP. A high-throughput screen for quorum-sensing inhibitors that target acyl- homoserine lactone synthases. Proc Natl Acad Sci U S A. 2013 Aug 20;110(34): 13815-20. doi: 10.1073/pnas.1313098110. Epub 2013 Aug 7. This compound was also disclosed in DATABASE chemcats [Online] 3 July 2013 (2013-07-03), Database accession no. 0150673791.

Preparing the compounds of the invention is within the skill of the organic chemistry art. The compounds described herein can be conveniently prepared from commercially available starting materials, compounds known in the literature, or readily prepared intermediates, by employing standard synthetic methods and procedures known to those skilled in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the synthetic steps presented may be varied for the purpose of optimizing the formation of the compounds described herein. The preparation methods can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C), infrared spectroscopy (FT -IR), spectrophotometry (e.g., UV -visible), or mass spectrometry (MS), or by chromatography such as high performance liquid chromatograpy (HPLC) or thin layer chromatography (TLC). Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The reactions of the processes can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of solvents. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

Pharmaceutically acceptable salts of the compounds of formula (I) or (II) include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids, which form non-toxic salts. Examples include the acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate and trifluoroacetate and xinafoate salts. For a review on suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley- VCH, Weinheim, Germany, 2002). Typically the pharmaceutically acceptable salts of compounds of formula (I) or (II) may be prepared by one or more of three methods by reacting the compound of formula (I) or (II) with the desired acid or base. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non- ionized.

The compounds of the invention may also exist in both unsolvated and solvated forms. The term "solvate" is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term "hydrate" is employed when said solvent is water.

Hereinafter all references to compounds of formula (I) or (II) include references to salts, solvates and complexes thereof and to solvates and complexes of salts thereof. The compounds of the invention include compounds of formula (I) or (II) as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof whenever relevant. So-called "pro-drugs" of the compounds of formula (I) or (II) are also within the scope of the invention. Thus certain derivatives of compounds of formula (I) or (II) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) or (II) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as "prodrugs". Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) or (II) with certain moieties known to those skilled in the art as "pro-moieties" as described, for example, in "Design of Prodrugs" by H. Bundgaard (Elsevier, 1985).

In some embodiments, the subject suffers from a cancer selected from the group consisting of breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non- Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

In some embodiments, the subject suffers from a haemato logical malignancy selected from the group consisting of leukemia, lymphoma or myeloma.

In some embodiments, the lymphoma is a mature (peripheral) B-cell neoplasm. In specific embodiments, the mature B-cell neoplasm is selected from the group consisting of B- cell chronic lymphocytic leukemia/small lymphocytic lymphoma; B-cell prolymphocytic leukemia; Lymphoplasmacytic lymphoma; Marginal zone lymphoma, such as Splenic marginal zone B-cell lymphoma (+/-villous lymphocytes), Nodal marginal zone lymphoma (+/-monocytoid B-cells), and Extranodal marginal zone B-cell lymphoma of mucosa- associated lymphoid tissue (MALT) type; Hairy cell leukemia; Plasma cell myeloma/plasmacytoma; Follicular lymphoma, follicle center; Mantle cell lymphoma; Diffuse large cell B-cell lymphoma (including Mediastinal large B-cell lymphoma, Intravascular large B-cell lymphoma, and Primary effusion lymphoma); and Burkitt's lymphoma/Burkitt's cell leukemia.

In some embodiments, the lymphoma is selected from the group consisting of multiple myeloma (MM) and non Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia (WM) or B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL). In some embodiments, Non-Hodgkin's Lymphoma (NHL) falls into one of two categories, aggressive NHL or indolent NHL. Aggressive NHL is fast growing and may lead to a subject's death relatively quickly. Untreated survival may be measured in months or even weeks. Examples of aggressive NHL includes B-cell neoplasms, diffuse large B-cell lymphoma, T/NK cell neoplasms, anaplastic large cell lymphoma, peripheral T-cell lymphomas, precursor B-lymphoblastic leukemia/lymphoma, precursor T- lymphoblastic leukemia/lymphoma, Burkitt's lymphoma, Adult T-cell lymphoma/leukemia (HTLV1+), primary CNS lymphoma, mantle cell lymphoma, polymorphic posttransplantation lymphoproliferative disorder (PTLD), AIDS-related lymphoma, true histiocytic lymphoma, and blastic NK-cell lymphoma. The most common type of aggressive NHL is diffuse large cell lymphoma. Indolent NHL is slow growing and does not display obvious symptoms for most subjects until the disease has progressed to an advanced stage. Untreated survival of subjects with indolent NHL may be measured in years. Nonlimiting examples include follicular lymphoma, small lymphocytic lymphoma, marginal zone lymphoma (such as extranodal marginal zone lymphoma (also called mucosa associated lymphoid tissue— MALT lymphoma), nodal marginal zone B-cell lymphoma (monocytoid B- cell lymphoma), splenic marginal zone lymphoma), and lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemialn some cases, histologic transformation may occur, e.g., indolent NHL in subjects may convert to aggressive NHL. In some embodiments, the leukemia is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and small lymphocytic lymphoma (SLL). Acute lymphocytic leukemia is also known as acute lymphoblastic leukemia and may be used interchangeably herein. Both terms describe a type of cancer that starts from the white blood cells, lymphocytes, in the bone marrow.

In some embodiments, the compounds of the invention are used in combination with a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to 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, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; 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 CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. , calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, 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; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofuran; spiro germanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-1 1); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments, the compounds of the invention are administered to the subject in combination with a compound which targets a member of Bcl-2 family. Typically, said compound is selected from the group consisting of ABT-199, ABT-263 and ABT-737.

As used herein the term "ABT-737" has its general meaning in the art and refers to 4- {4-[(4 -Chlorobiphenyl-2-yl)methyl]piperazin- 1 -yl} -N-{[4-( {(1 R)-3-(dimethylamino)- 1 - [(phenylsulfanyl)methyl]propyl}amino)-3-nitrophenyl]sulfonyl }benzamide.

As used herein the term "ABT-199" has its general meaning in the art and refers to 4- [4-[[2-(4-Chlorophenyl)-4,4-dimethylcyclohex- 1 -en- 1 - yl]methyl]piperazin- 1 -yl]-N-[[3-nitro- 4- [ [(tetrahydro-2H- pyran-4-yl)methyl]amino ]phenyl] sulfonyl] -2- [( 1 H- pyrrolo [2,3 - b]pyridin-5-yl)oxy]benzamide.

As used herein, the term "ABT-263" or "Navitoclax" has its general meaning in the art and refers to R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[l,r-bip henyl]-2- yl)methyl)piperazin- 1 -yl)-N-((4-((4-morpholino- 1 -(phenylthio)butan-2-yl)amino)-3- ((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide.

In some embodiments, compounds of the invention are administered to a subject having a refractory cancer, i.e. a cancer that is resistant to chemotherapeutic agents. In particular the subject suffers from a cancer which is resistant to a fludarabine or cisplatine treatment. In some embodiments, the subject suffers from a cancer that is a resistant cancer to a BCL-2 inhibitor (e.g. ABT-737, ABT-199 or ABT-263).

By a "therapeutically effective amount" is meant a sufficient amount of the compound to treat cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The compounds of the invention are administered as a formulation in association with one or more pharmaceutically acceptable excipients to form pharmaceutical composition.

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 administered 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, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle (i.e. a compound of the invention), alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. In particular, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular 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 which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Compounds of the invention can be formulated into a composition in a neutral or salt form as above described. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Compounds of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1. Screening to identify molecules targeting Bfl-1 and displaying cytotoxic properties. 346 compounds were screened at 30 μΜ to find inhibitors of Bfl-1 (30 nM) binding to Bim BH3 (15 nM). (A) Screening flow chart. On the basis of the results of the screening algorithm, BDM 44931 and BDM 44898 were selected and tested in apoptotic cellular assays and for mechanism of action studies. DRC: Dose-Response Curve; QC: quality control of the library samples. (B) Chemical structure of BDM 44931 and BDM 44898. (C) Cytotoxic activity of BDM 44931 and BDM 44898. BP3 and IM9 cells were incubated 24 h with compounds at 25 μΜ. Cell viability was evaluated using a metabolic assay (ATPlite™) and data are presented as percent of viable cell compared to untreated control. Data are presented as mean ± SEM of 3 independent experiments.

Figure 2. Inhibitory effect of BDM 44931 and BDM 44898 in FP assays against apoptotic and anti-apoptotic proteins of the Bcl-2 family. BDM 44931 (A) and BDM 44898 (B) effect on Bfl-1 binding to Bim BH3 or to other pro-apoptotic peptides (Bax and Bak). BDM 44931 (C) and BDM 44898 (D) effect on Bfl-1 or other anti-apoptotic proteins (Bcl-2, Bcl-xl and Mcl-1) binding to Bim BH3. Increasing concentrations of compounds were pre-incubated for 30 min with anti-apoptotic proteins before BH3 peptide addition in PBS buffer, pH 7.4, 1% DMSO, 0.01% Triton X-100™. The concentrations of anti-apoptotic proteins used in the different protein/protein interactions are the folio wings: Bfl-1 (30 nM) / Bim (15 nM), Bfl-1 (300 nM) / Bax (10 nM), Bfl-1 (400 nM) / Bak (10 nM), Bcl-2 (15 nM) / Bim (15 nM), Bcl-xl (10 nM) / Bim (15 nM), Mcl-1 (10 nM) / Bim (15 nM). FP was measured after 15 min. Data are mean of 2 incubates.

Figure 3. Pro-apoptotic effect of BDM 44931 and BDM 44898 compounds towards lymphoma cell lines. BP3 and IM9 cells were treated 24 h with increasing concentrations of BDM 44931 (A) and BDM 44898 (B) molecules. Percent of viable cells was evaluated by propidium iodide / Annexin V double staining and flow cytometry analysis. Data are presented as mean ± SEM of 3 independent experiments. Figure 4. Involvement of apoptotic mitochondrial pathway. MEF WT or Bax/Bak

DKO were treated 24 h with indicated molecule concentrations of BDM 44931 (A) and BDM 44898 (B). Percent of viable cells was evaluated by propidium iodide staining and flow cytometry analysis. Data are presented as mean ± SEM of 3 independent experiments. Figure 5. BDM 44898 delays BP3 tumor growth in vivo. (A) BP3 or IM9 cells

(5xl0 ⁶ ) were inoculated subcutaneously into the right flank of 5-week-old female SCID/Beige mice. Calipers were used to measure the length and the width of each tumor 2-3 times per week. Tumor volume was estimated by applying the following equation: volume = length x width2 x 0.52. (B) BP3 cells (5xl0 ⁶ ) were inoculated subcutaneously into the right flank of 5- week-old female SCID/Beige mice, when reach approximately 150 mm3, mice were divided into 3 groups and injected intra peritonealy on day 10, 11, 12, 15, 16 and d 17 with either PBS, or vehicle (20mL/kg cyclodextrine Crysmed 100 mM) or BDM 44898 (20mg/kg). Tumor growth was measured and results were compared using a two-tailed paired t-test. A p value < 0.05 x was considered as statistically significant. * indicated statistical significance between vehicle- and BDM_44898-treated mice or between PBS- and BDM_44898-treated mice. EXAMPLE:

1- Bfl-1 library Within a first high-throughput screening (HTS) through APTASCREEN® technology using a 61,200 compound library, 70 molecules were selected for their ability to disrupt the interaction between Bfi-1 with one of the functionally Bfi-1 binding aptamers (Brien G et al, Biochemistry 2011) previously identified and/or with one of Bfl-1 's natural ligand: the pro- apoptotic protein Bim. Those 70 compounds were further analyzed. Medicinal chemistry (synthetic feasibility and potentials for optimizations) and chemo informatics analysis (fingerprint and clustering computations) of the molecules together with the pharmacological results (inhibition of the Bfi-l/Bim interaction in a dose-dependent manner and initial selectivity profiling), patent search and in silico ADME/Tox profiling of the 70 hits led to the selection of 12 compounds.

We then used a ligand-based screening approach (similarity and substructure search) starting from these 12 initial hits to generate a short list of about 400 compounds for screening through fluorescence polarization (FP) assay-based HTS and further characterization in both in vitro and in vivo models. The goal here was to obtain compounds with increased potency (better than ΙμΜ) as compared to the 12 initial compounds and to obtain a preliminary structure activity relationship (SAR). The best compounds, as estimated experimentally and displaying a favorable in silico ADME/Tox profile (computed with our package FAF-Drugs 3, and Pipeline Pilot) and good ligand-efficiency were then docked into the 3D structures of the Bel- 2 family such as to attempt to predict the most likely binding pose and assist further rational optimization of the molecules. The docking was carried out with Surflex, Dock, Autodock Vina and LigandFit using pre-generated multi- conformer structures of the receptors using a recent protocol that we have developed. Such in silico approach led to the selection of 346 compounds, collectively named "Bfl-1 library".

2- Bfl-1 library screen

HTS to identify Bfl-1 -targeting compounds displaying cytotoxic properties

A FP assay was set up to follow the interaction of the recombinant human GST-Bfi-1- ACterm protein with FITC-Bim BH3. FP assays were performed in 10 mM PBS pH 7.4, 100 mM NaCl, 2.7 mM KC1, 0.01% Triton X-100™ and 1% DMSO in black 96-well microplates .Twenty five Bfl-1 protein (30nM final) were incubated with 25 test compound at room temperature for 30 minutes. Then, 25 FITC-Bim peptide (15nM final) were added. After an incubation of 15 to 45 min at room temperature, direct fluorescence and FP were measured using a Victor3TM VI 420 Perkin Elmer plate reader (excitation filter 485 nm and emission filter 535 nm). The 346 compounds of the "Bfl-1 library" were screened at the concentration of 30 μΜ for their capacity to disrupt Bfl-l/Bim interaction (Fig. 1A). Compounds that were found to inhibit significantly the Bfl-l/Bim interaction were defined as primary positives and advanced in confirmatory sequential steps including 1) repeat single- dose testing in triplicate, 2) dose-responsive competitive binding and 3) confirmation of identity and purity of the library samples. This process yielded 16 positives with 5μΜ< IC ₅ o < 30 μΜ. After activity analysis (sigmoidal aspect of the DRC, maximal inhibition effect ...) and taking account of chemical information we selected 2 compounds BDM 44931 and BDM 44898 that were also active in a secondary TR-FRET assay (data not shown).

BDM 44931 and BDM 44898 were then evaluated for their cytotoxic activity towards B cell lines by using ATP lite™ metabolic assay. We used the diffused large B cell lymphoma (DLBCL) cell line BP3 and the B lymphoblastoid cell line IM9 that highly express Bfl-1 mRNA and protein and that are sensitive to Bfl-1 down regulation (Brien G et al, Oncogene 2007). These cell lines also express other anti apoptotic Bcl-2 protein family members (Bcl-xL and Mcl-l for BP3 and Bcl2 and Bcl-xL for IM9) (Brien G et al, Oncogene 2007). Treatment with 12.5 μΜ of BDM 44931 or BDM 44898 for 24h efficiently induced cell death of the B lymphoblastoid cell lines BP3 and IM9. BDM 44898 demonstrated the more potent cytotoxic activity with less than 10% treated cells that are still viable following treatment (Fig. 1C). Characterization of compound selectivity for Bfl-1 anti- apoptotic protein

It is known that Bfl-l interacts with multiple pro-apoptotic proteins, potentially through the same BH3 domain binding site. Thus, to characterize compound selectivity for Bfl-l/Bim interaction we developed a panel of FP assays dedicated to Bcl-2 proteins. The concentrations of anti-apoptotic and pro-apoptotic partners used in each complex were as followed: Bfl-l (300 nM) / Bax (10 nM), Bfl-l (400 nM) / Bak (10 nM), Bcl-2 (15 nM) / Bim (15 nM), Bcl-xl (10 nM) / Bim (15 nM), Mcl-l (10 nM) / Bim (15 nM). All FP assays were validated using gambogic acid as a positive reference compound (data not shown).

We first observed that BDM_ 44931 and BDM_ 44898 displaced Bfl-1 from its interaction with either FITC-Bax or FITC-Bak BH3 peptides as efficiently as from its interaction with FITC-Bim BH3 peptides (Fig. 2A and 2B). Estimated IC50 values are in the same range for both BDM 44931 (1.3 μΜ, 3.2 μΜ and 4 μΜ, for Bfl-l/Bim, Bfl-l/Bax and Bfl-l/Bak, respectively) and BDM 44898 (1.6 μΜ for Bfl-l/Bim and 3.2 μΜ for both Bfl- l/Bax and Bfl-l/Bak) (Table 1).

Table 1 : Summary of BDM 44931 and BDM 44898 properties.

BDM 44931 BDM_44898

Protein protein interaction inhibition (IC ₅₀ ,

μΜ)

Bim/Bf -1 1.3 1.6

Bax/Bfl-1 3.2 3.2

Bak/Bfl-1 4.0 3.2

Bim/bcl-2 >100 >100

Bim/Bclxl >100 >100

Bim/Mcll >100 >100

Proapoptotic effect (EC50, μΜ)

BP3 5.2 1.6

IM9 1.6 0.8

RS4.11 2.4 1.1

SudHL4 8.1 > 25

MEF viability

DKO/WT fold 1.8 3

Cytotoxicity (EC50, μΜ)

PBMC 5.8 28.1

In vitro ADME

Gluthathione reactivity (3 heavy ; 0 nul) 0 0

Mouse microsomal stability (Clint 534 667

Solubility (μΜ) 3.5 0.6

Pharmacokinetics, mouse (20 mg/kg IP)

AUC (min.ng/mL) 47106 75 677

Cmax (ng/mL) 251 472 We next evaluated BDM 44931 and BDM 44898 for their capacity to inhibit FITC- Bim BH3 binding to Bcl-2, Bcl-xl and Mcl-l proteins (Fig. 2C and 2D). BDM 44931, showed no significant inhibition of Bim binding to Bcl-xl and Mcl-l proteins and a weak inhibition of Bcl-2/Bim interaction, compared to Bfl-l/Bim interaction. Estimated IC ₅₀ were > 100 μΜ for Bcl-xl/Bim, Mcl-l/Bim and Bcl-2/Bim vs an estimated IC ₅₀ of 1.3 μΜ for Bfl- l/Bim (Fig. 2C and Table 1). BDM 44898, showed no significant inhibition of Bim binding to Bcl-2, Bcl-xl and Mcl-l proteins, but efficiently inhibited Bim/Bfl-1 interaction with an estimated IC ₅₀ of 1.6 μΜ (Fig. 2D and Table 1).

3- Characterization of BDM 44931 and BDM 44898 for their in vitro biological activities

Biological activity of selected compounds towards B cell lymphoma.

In order to further characterized their biological activity we tested the capacity of compounds BDM 44931 and BDM 44898 to induce apoptosis of B lymhoma cell lines. For this purpose, BP3 and IM9 cells were cultured for 24h in the presence of increasing concentrations of compounds BDM 44931 or BDM 44898 and apoptosis was assessed by propidium iodide/ Annexin V double staining and flow cytometry analysis. We observed that both compounds induced an efficient apoptosis of both cell lines. Estimated EC50 values for molecule BDM 44931 are of 5.2 μΜ for BP3 and 1.6 μΜ for IM9. Estimated values for BDM 44898 are of 1.6 μΜ for BP3 and 0.8 μΜ for IM9 (Fig. 3 and Table 1).

Anti-apoptotic Bcl-2 members suppress apoptosis by inhibiting Bax and Bak pro- apoptotic Bcl-2 members, and cells lacking both Bax and Bak proteins are resistant to apoptotic stimuli that act through Bax/Bak-dependent disruption of mitochondrial function. To assess the specificity of compounds, for this pathway we used murine embryonic fibroblasts (MEF) either WT or Bax/Bak DKO. We observed that WT MEFs are 1.8 and 3 fold more sensitive to cell death induced by BDM 44931 and BDM 44898 respectively than DKO MEFs (Fig. 4 and Table 1).

Interestingly we observed that PBMC are less sensitive to cell death induced by

BDM 44898 than the BP3 and IM9 B lymphoblastoid cell lines. Estimated EC50 values are of 28.1 μΜ for PBMC vs 1.6 and 0.8 for BP3 and IM9 respectively (Table 1).

4- Preliminary characterization of BDM 44931 and BDM 44898 in in vivo assays Compounds BDM 44931 and BDM 44898 from the "Bfl-1 library", demonstrated physicochemical properties and metabolic stability suitable for a proof-of-concept in the animal, their pharmacokinetics in vivo were thus studied. After a single administration of 20mg/kg intraperitonealy, we measured compound concentrations in plasma during 4 hours. We observed that mice treated with BDM 44898 were 1.6 fold more exposed to the compound with an AUC of 75 677 min.ng/mL and a Cmax of 472 ng/mL, compared to BDM 44931 (AUC of 47 106 min.ng/mL and a Cmax of 251 ng/mL. After 4 hours, both compounds were always present in plasma at concentrations higher than 200 ng/mL.

Based on criteria cited just above we selected BDM 44898 compound for in vivo assays, and evaluated its ability to reduce tumor growth of lymphoid tumor xenograft established in immunocompromised mice.

In a first set of experiments we evaluated tumor growth of the B lymphoblastoid cell line IM9 and the DLBCL cell line BP3 in SCID/Beige immunodeficient mice. Both cell lines expressed high levels of Bfl-1 and are sensitive BDM 44898 in vitro. BP3 or IM9 cells (5xl0 ⁶ ) were implanted subcutaneously into the right flank of 5-week-old SCID/Beige mice. A rapid tumor growth was observed in mice inoculated with BP3 cells, tumors being detected at day 8 and reaching the maximal 2500 mm ³ volume around day 20 (Fig 5 A). Following IM9 inoculation tumors became detectable around day 20 and reached 1500 mm ³ volume at day 45 (Fig 5A).

We thus decided to evaluate anti-tumor activity within the BP3 xenograft tumor model. Mice were implanted subcutaneously, on day 10 after inoculation mice with equivalent tumor size (150 mm ³ ) were divided into 3 groups and injected intraperitonealy on day 10, 1 1, 12, 15, 16 and d 17 with either PBS, or vehicle (20mL/kg cyclodextrine Crysmed 100 mM) or BDM_44898 (20mg/kg). Mice treated with BDM_44898 showed a statistically significant decrease of tumor growth compared with PBS treated animals (p<0.005 at dl5 and p<0.001 at day 16) or vehicle treated animals (p<0.05 at day 17) (Fig 5B).

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