BE1028754B1 - 5α-Hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol ANALOGS AND PHARMACEUTICAL COMPOSITIONS COMPRISING THEM FOR USE IN THE TREATMENT OF CANCER - Google Patents

Description

5a-Hydroxy-6B-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3B-ol ANALOGS AND COMPOSITIONS

PHARMACEUTICALS COMPRISING IT FOR

USE IN THE TREATMENT OF CANCER Technical area

[1] The invention relates to the field of sterol compounds and more particularly to analogues of the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol and compositions pharmaceuticals comprising it for use in the treatment of cancer. Technology background

[2] The term "cancer" or "cancerous tumor" encompasses a group of diseases characterized by the uncontrolled multiplication and spread of abnormal cells. If the cancer cells are not eliminated, the evolution of the disease will more or less quickly lead to the death of the affected person.

[3] Cancer management involves surgery, radiotherapy and chemotherapy, which can be used alone or in combination, simultaneously or sequentially. Chemotherapy uses antineoplastic agents which are drugs that prevent or inhibit the maturation and proliferation of neoplasms. Antineoplastic agents work by effectively targeting rapidly dividing cells. Because antineoplastic agents affect cell division, tumors with a high growth rate (such as acute myeloid leukemia and aggressive lymphomas, including Hodgkin's disease) are more susceptible to chemotherapy because a greater proportion targeted cells undergo cell division at any time. Malignant tumors with slower growth rates, such as indolent lymphomas, tend to respond much more modestly to chemotherapy. However, the development of drug resistance is a persistent problem during chemotherapy treatment. For example, conventional treatment for acute myeloid leukemia (AML) includes the combined administration of cytarabine with an anthracycline, such as daunorubicin. The overall 5-year survival rate is 40% in young adults and about 10% in elderly patients. Response rates vary considerably with aging, from 40% to 55% in patients over 60 and from 24% to 33% in patients over 70. This is even worse for the elderly with unfavorable cytogenetic profiles and death within 30 days of treatment ranges from 10% to 50% with age and worsening. Moreover, the restriction of the use of these molecules is also due to side effects, and in particular to the emergence of chronic cardiac toxicity (linked to anthracyclines). The toxic mortality rate linked to intensive chemotherapy is 10 to 20% in patients over 60 years of age.

[4] With this risk-benefit profile of the conventional diet, only 30% of older people with newly diagnosed AML receive antineoplastic chemotherapy.

[5] In recent decades, there has been only a modest improvement in outcomes for younger AML patients, but none for adults over 60 (most AML patients) .

[6] There is therefore a real need to develop molecules useful in the treatment of these cancerous tumors which present problems of chemoresistance and intrinsic toxicity of antineoplastic drugs. The aforementioned data underline the need to find new approaches which combine both a reduction in the dosage regimens of antineoplastic agents for the treatment of chemosensitive tumors with a reduction in the resistance of chemoresistant tumors to the antineoplastic agent.

[7] Document EP3272350B1 discloses the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol, known as Dendrogenin A, useful for the treatment chemoresistant tumours. Dendrogenin A is able to restore the sensitivity of chemoresistant tumors to an antineoplastic agent or to increase the effects of antineoplastic agents on tumors, which in turn makes it possible to reduce the effective cytotoxic dose of antineoplastic agents against chemosensitive tumors.

[8] The object of the present invention is to provide new compounds and analogues of the compound Dendrogenin A, useful for treating cancerous tumours, in particular chemosensitive and/or chemoresistant tumours.

[9] Surprisingly, the inventors have discovered that specific analogues of the compound Dendrogenin A exhibit pharmacological activity comparable to Dendrogenin A.

Summary

[10] The first subject of the invention is a compound of formula (I); x N AU ss N 3 0 De ge Me) 7 À î î = $3 © | À = A 8 + gn, TT N * $LS Ÿ

SRE U N N ST N. | 8 SS

NN or a pharmaceutically acceptable salt of such a compound, in which: Rı is chosen from F, N(R4)2, SR4, S(Ra)2, SOR4, SO2R4 or OH with R4 chosen from: H; a C1 to C20 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl and aryl groups, said aryl group optionally containing heteroatoms chosen from N, O, and S, Ra equals Rs are chosen from CH; or CF: ; and when Ry is OH, Ra and Rs are CF3, for its use as a medicament for regressing a mammalian cancerous tumor.

[11] A second subject of the invention is a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one compound of formula (I) for its use in causing a cancerous mammalian tumor to regress.

Definitions

[12] In this description, unless otherwise specified, it is understood that when an interval is given, it includes the upper and lower bounds of said interval.

[13] In the present invention, throughout this description and the appended claims, the following terms, unless otherwise indicated, shall be understood to have the following meaning:

[14] The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and containing stoichiometric or sub-

stoichiometric values of one or more pharmaceutically acceptable solvent molecules such as ethanol. The term “hydrate” refers when said solvent is water.

[15] The term "human" refers to a subject of either sex and at any stage of development (i.e. newborn, infant, juvenile, adolescent, adult).

[16] The term "patient" refers to a warm-blooded animal, more preferably a human, who is awaiting reception or receiving medical treatment and/or who will be the subject of a medical procedure.

[17] By “pharmaceutically acceptable” it is understood that the ingredients of a pharmaceutically acceptable product are compatible with each other and are not harmful to the patient of this product.

[18] The term “pharmaceutical vehicle” as used in this text means an inert carrier or medium used as a solvent or diluent in which the pharmaceutically active agent is formulated and/or administered. Non-limiting examples of pharmaceutical carriers include creams, gels, lotions, solutions and liposomes.

[19] The term "administration" means to deliver, the active agent or active ingredient (e.g. the compound of formula (I), in a pharmaceutically acceptable composition, to the patient in which a condition, symptom and/or a disease must be treated.

[20] The terms "treat" and "treatment" as used herein include alleviating, alleviating, stopping, curing a condition, symptom and/or disease.

[21] The term “analogue” as used herein means a compound having a similar chemical structure to another compound, but differing from it in some component.

It may differ by one or more atoms, functional groups, substructures, which are replaced by other atoms, functional groups or substructures. Analogs may have different physical, chemical, biochemical or pharmacological properties. In the present invention, the analogs have identical or similar pharmacological properties with respect to the reference compound.

[22] By "chemoresistant cancer" we mean a cancer in a patient where the proliferation of cancerous cells cannot be prevented or inhibited by means of an antineoplastic agent or a combination of antineoplastic agents usually used to treat this cancer , at a dose acceptable to the patient. Tumors may be inherently resistant prior to chemotherapy, or resistance may be acquired during treatment by tumors initially responsive to chemotherapy.

[23] By "chemosensitive cancer" is meant a cancer in a patient which responds to the effects of an antineoplastic agent, i.e. where the proliferation of cancer cells can be prevented by means of said antineoplastic agent at a acceptable dose for the patient.

[24] The compound of formula (I) belongs to the group of steroids. The numbering of the carbon atoms of the compound of formula (I) therefore follows the nomenclature defined by PIUPAC in Pure & Appl. Chem., Vol.61, No.10, pp.1783-1822,1989. The numbering of the carbon atoms of a compound belonging to the group of steroids according to IUPAC is illustrated below: 24°; 24 at 20 22 26 2 8 23 29 Tos it CS N 27 ‚ 1° ° >> 8 2 84 5

GIS 4 8

[25] In the present invention, the following abbreviations mean: AML: acute myeloid leukemia; Dendrogenin A: 5α-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol; MCF-7: Michigan Cancer Foundation-7; DMEM: Dulbecco's Modified Eagle Medium; FCS: fetal calf serum; ChEH: Cholesterol Epoxide Hydrolase Neuro2a: murine neuroblastoma; CTL: Control; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS: phosphate buffered saline; DMSO: dimethyl sulfoxide; OD: optical density or absorbance; CT: cholestane-3B,5a,6B-triol; OCDO: 6-oxo-cholestan-3B,5a-diol; 5,80-EC: 5,60-epoxycholesterol;

_ BE2020/5764 Tam: Tamoxifen; TLC: thin layer chromatography. Description of embodiments

[26] The invention relates firstly to a compound of formula (I); $ j x os, $ y SAS EN a TS ] en ses RR 3: Ÿ N $ 7 © 3 À a. d | $ = 5 N = => Nd > Ne ae > $ î

Ÿ Ÿ oe Hs > N ST bu | HA

MM N X ST NN ga” Le l X a N 3 $

K.A.

IVES or a pharmaceutically acceptable salt of such a compound, in which: Ry is chosen from F, N(R4)2, SRa4, S(R4)2, SOR4, SOzR4 or OH with R4 chosen from: H; a C1 to C20 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl and aryl groups, said aryl group optionally containing heteroatoms chosen from N, O, and S, Ra equals Rs are chosen from CH; or CF: ; and when Ry is OH, Ra and Rs are CF3, for its use as a medicine.

[27] According to one embodiment, the invention relates to a compound of formula (I); AL Ne N 3 > <L- a sc su = Ÿ î

Ÿ Ÿ ee TS en Ng ge” EN or a pharmaceutically acceptable salt of such a compound, in which: Ry is chosen from F, N(R4)2, SR4, S(R4)2, SOR4, SOzR4 or OH with R4 chosen from: H; a C1 to C20 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl and aryl groups, said aryl group optionally containing heteroatoms chosen from N, O, and S, Ra equals Rs are chosen from CH; or CF: ; and when Ry is OH, Ra and Rs are CF3, for its use as a medicament for regressing a mammalian cancerous tumor.

[28] Unless otherwise indicated, in the compound of formula (|), the chemical groups R+, Ra, Rs and Ra independently have the definitions indicated. In the case where several R4 groups are present in a formula (I), they may be identical or different.

[29] In the present invention: - the carbonyl group is chosen from aldehydes, ketones, carboxylic acid, esters, amides and/or anhydride. - the term "aryl" as used herein means a polyunsaturated aromatic hydrocarbyl group having a single ring optionally comprising a heteroatom (such as phenyl, furan, pyridine, triazole, primidine or pyrrole) or several aromatic rings fused together (eg naphthyl) or covalently bonded, typically containing 5 to 12 atoms; preferably 6 to 10, in which at least one ring is aromatic. The aromatic ring may optionally include one or two additional rings (cycloalkyl, heterocyclyl or heteroaryl) fused thereto. The term "aryl" is also intended to include partially hydrogenated derivatives of carbocyclic systems, in which at least one ring is aromatic. Examples of such partially hydrogenated derivatives include 1,2,3,4-tetrahydronaphthyl, fluorenyl and 1,4-dihydronaphthyl. - the term “allyl” refers to an alkene functional group of semi-developed formula H:C=CH-CHz-. - the term sulphide is a chemical compound where sulfur is combined with another chemical element or one of its radicals. - the radical of carbon 3 of the compound of formula (I) can be in position a or B. Position B being preferred.

[30] According to one embodiment, when Rı is N(R4)2, SR4, S(Ra4)2, SOR4 or SO:R4; then R2 and Rs are CHz.

[31] According to one embodiment, when R: is F, then Ra and Rs are CH> or Ra and R3 are CF:. These are the compounds:

[32]-5a-Hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro; -5a-Hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro-26-27-di-trifluoro-methyl.

[33] According to one embodiment, when Rı is OH, then Ra and Rs are CFs, it is the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan -3-ol-26-27-di-trifluoro-methyl.

[34] According to a preferred embodiment according to the invention, the radicals of the compound of formula (I) are Rı=F; Ra and R3 = Chas. The corresponding compound is 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-fluoro, for example 5a-hydroxy-6B-[2-(1H-imidazol- 4-yl)-ethylamino]-cholestan-3α-fluoro or 5α-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-fluoro. More preferably, the compound is 5α-hydroxy-6B8-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3α-fluoro.

[35] According to a preferred embodiment of the compound of formula (I), when R: is chosen from N(R4)2, SR4, S(R4)2, SOR4 or SOzR4; then Ra and Rs are CHs, the compound of formula (I) is chosen from: - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-amino; - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methyl! sulfide; - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methylsulfonyl.

[36] According to one embodiment, in the compound of formula (I), Rı is a sulphide radical.

[37] According to one embodiment of the compound of formula (I), R1=SR4; Ra and Rs= CHa. This is the compound of formula: I _ LE me NB Pr Kg a LS H

ln; BE2020/5764

[38] According to one embodiment of the compound of formula (I), Rı=SOR4; Ra and R3 = Cha. This is the compound of formula: >, ax, j A ee, N Fan 5 N + Ra JL A7 % > de a ve *

5. 5 A da ol Ks HLN a fi > U >

HH

[39] According to one embodiment of the compound of formula (I), R1=SOzR4; R2 and R3 = CH. This is the compound of formula: ae N i Ë en ee oe

EN Ná Hi SS N R 4, The RS

[40] According to one embodiment, the compound of formula (I) is intended for use in the treatment of: breast, prostate, colorectal, lung, bladder, skin, uterus, cervix, mouth, brain, stomach, liver, throat, larynx, esophagus, bone, ovary, pancreas, kidney, retina, sinus, nasal cavity, testicle, thyroid, vulva, lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, acute myeloid leukemia or acute lymphocytic leukemia, multiple myeloma , Merkel cell carcinoma or mesothelioma.

[41] According to one embodiment, the cancer is acinar adenocarcinoma, acinar carcinoma, acrolentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, rest tumor adrenal, adrenocortical carcinoma, aldosterone-secreting carcinoma, alveolar soft tissue sarcoma, ameloblastic thyroid carcinoma, angiosarcoma, apocrine carcinoma, Askin tumor, astrocytoma, basal cell carcinoma, basaloid carcinoma, basosquamous carcinoma, bile duct cancer , bone marrow cancer, botryoid sarcoma, bronchioalveolar carcinoma, bronchogenic adenocarcinoma, bronchogenic carcinoma, carcinoma ex pleomorphic adenoma, chloroma, cholangiocellular carcinoma, chondrosarcoma, choriocarcinoma, choroid plexus carcinoma, clear cell adenocarcinoma, colon cancer, comedocarcinoma, carcinoma cortisol producer, columnar cell carcinoma, liposa differentiated cancer, ductal adenocarcinoma of the prostate, ductal carcinoma, ductal carcinoma in situ, duodenal cancer, eccrine carcinoma, embryonic carcinoma, endometrial carcinoma, endometrial stromal carcinoma, epithelioid sarcoma, Ewing's sarcoma, exophytic carcinoma, fibroblastic sarcoma, fibrocarcinoma, fibrolamellar carcinoma, fibrosarcoma, thyroid follicular carcinoma, gallbladder cancer, gastric adenocarcinoma, giant cell carcinoma, giant cell sarcoma, giant cell bone tumor, glioma, glioblastoma multiforme, cell carcinoma granulosa, head and neck cancer, hemangioma, hemangiosarcoma, hepatoblastoma, hepatocellular carcinoma, Hürthle cell carcinoma, ileal cancer, invasive lobular carcinoma, inflammatory breast carcinoma, intraductal carcinoma, intraepidermal carcinoma, jejunal cancer, sarcoma Kaposi tumor, Krukenberg tumor, Kulchitsky cell carcinoma, sarcoma Kupffer cell carcinoma, large cell carcinoma, laryngeal cancer, lentigo maligna melanoma, liposarcoma, lobular carcinoma, lobular carcinoma in situ, lymphoepithelioma, lymphosarcoma, malignant melanoma, medullary carcinoma, medullary thyroid carcinoma, medulloblastoma, meningeal carcinoma, micropapillary carcinoma, mixed cell sarcoma, mucinous carcinoma, mucoepidermoid carcinoma, mucosal melanoma, myxoid iposarcoma, myxosarcoma, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, nodular melanoma, non-clear cell kidney cancer, non-small cell lung cancer, oat cell carcinoma, ocular melanoma, oral cancer, osteoid carcinoma, osteosarcoma, ovarian cancer, Paget's carcinoma, pancreatoblastoma, papillary adenocarcinoma, papillary carcinoma, papillary thyroid carcinoma, pelvic cancer, periampullary carcinoma, phyllodes tumor, cancer pituitary gland, pleomorphic liposarcoma, pleuropulmonary blastoma, carcinoma primary intraosseous oma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, round cell liposarcoma, scar cancer, schistosomal bladder cancer, schneiderian carcinoma, sebaceous carcinoma, ring cell carcinoma, skin cancer, small cell lung cancer, small cell osteosarcoma, soft tissue sarcoma, spindle cell sarcoma, squamous cell carcinoma, stomach cancer, superficial spreading melanoma, synovial sarcoma, telangiectatic sarcoma, terminal canal carcinoma, testicular cancer , thyroid cancer, transient cell carcinoma, tubular carcinoma, tumorigenic melanoma, undifferentiated carcinoma, urethral adenocarcinoma, bladder cancer, cancer of the uterus, carcinoma of the uterine body, melanoma of the uterus, cancer of the vagina, verrucous carcinoma, villous carcinoma, well-differentiated liposarcoma, Wilms tumor or germ cell tumors.

[42] In a preferred embodiment, the compound of formula (II) is for use in the treatment of breast cancer in mammals.

[43] According to one embodiment, the compound is for use in the treatment of a chemosensitive cancer.

[44] According to a particularly preferred embodiment, the compound of formula (I) is intended for use in the treatment of a chemoresistant cancer.

[45] According to one embodiment, the chemoresistant cancer is a hematological or blood cancer, such as leukemia, in particular acute myeloid leukemia or acute lymphocytic leukemia, lymphoma, in particular non-Hodgkin's lymphoma and myeloma multiple.

[46] In one embodiment, the cancer is chemoresistant to daunorubicin, cytarabine, fluorouracil, cisplatin, all-trans-retinoic acid, arsenic trioxide, bortezomib, or one of of their combinations.

[47] All references to compounds of formula (I) include references to salts, multicomponent complexes and their liquid crystals. All references to compounds of formula (I) also include references to polymorphs and their usual crystals.

[48] The compound according to the invention may be in the form of pharmaceutically acceptable salts. A pharmaceutically acceptable salt of the compound of formula (I) comprises the acid addition thereof.

[49] Suitable acid salts are formed from acids that form non-toxic salts. For example chosen from: acetate, adipate, benzoate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, furamate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, chloride hydrochloride, hydrobromide, bromide, hydroiodide, iodide, isethionate, lactate, malate, maleate, malonate mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccarate, sterate, succinate, tannate, tartrate salts, tosylate, trifluoroacetate and xinofoate. Preferably, the pharmaceutically acceptable salt of the compound of formula (|) is formed from lactate.

[50] Pharmaceutically acceptable salts of compounds of formula (I) may be prepared by one or more of the following three methods: (i) by reacting the compound of formula (I) with the desired acid; (ii) by removing an acid or base labile protecting group from a suitable precursor of the compound of formula (I) or by opening the cycle of a suitable cyclic precursor, for example a lactone or a lactam, using the desired acid or base; or ill) by conversion of a salt of the compound of formula (I) into another by reaction with an appropriate acid or base or by means of an appropriate ion exchange column.

[51] These three reactions are usually carried out in solution. The salt obtained can precipitate and be collected by filtration or can be recovered by evaporation of the solvent.

The degree of ionization of the salt obtained can vary from completely ionized to almost non-ionized.

[52] The second subject of the invention relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one compound according to the invention, as described above for its use in regressing a cancerous tumor in a mammal.

[53] According to one embodiment, the pharmaceutical composition further comprises at least one other therapeutic agent.

[54] According to a preferred embodiment, this other therapeutic agent is an antineoplastic agent.

[55] In one embodiment, the antineoplastic agent is a DNA damaging agent such as camptothecin, inotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, chlorambucil, chlormethine, busulfan, treosulfan or thiotepa, an antitumor antibiotic such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, actinomycin D, mitomycin, bleomycin or plicamycin, an antimetabolite such as 5-fluorouracil, cytarabine, fludarabine or methotrexate, an antimitotic such as paclitaxel, docetaxel, vinblastine, vincristine, vindesine or vinorelbine, or various antineoplastic agents such as bortezomib, all-trans-retinoic acid, arsenic trioxide, or one of their combination products.

[56] According to one embodiment, the pharmaceutical composition is used in the treatment of cancer in a patient suffering from a tumor which is chemoresistant to said antineoplastic agent when it is not administered in combination with a compound according to the invention .

[57] According to one embodiment, the pharmaceutical composition is used in the treatment of cancer in a patient suffering from a tumor which is chemosensitive to said antineoplastic agent, and the dose of the antineoplastic agent administered to said patient in combination with a compound according to the invention or one of its pharmaceutically acceptable salts is lower than the dose of the antineoplastic agent when it is not administered in combination with a compound according to the invention. In particular, the dose of the antineoplastic agent administered to said patient in combination with a compound according to the invention or one of its pharmaceutically acceptable salts is lower than the dose of the antineoplastic agent administered alone, without any other active principle.

[58] The pharmaceutical composition according to the invention may also further comprise other active therapeutic compounds commonly used in the treatment of the pathology set out above.

[59] According to one embodiment, the pharmaceutical composition of the invention can be administered by any route, in particular by route: intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal ), nasal spray inhalation, using tablet, capsule, solution, powder, gel, particle formulation; and contained in a syringe, an implanted device, an osmotic pump, a cartridge, a micropump; or any other means appreciated by the skilled artisan well known in the art. Site-specific administration can be performed, for example, intratumoral, intra-articular, intrabronchial, intra-abdominal, intracapsular, intra-cartilaginous, intracavitary, intracerebellar, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac , intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal or transdermal in a suitable dosage comprising the usual non-toxic and pharmaceutically acceptable vehicles. Preferably, the pharmaceutical composition is in a form suitable for being administered intravenously, subcutaneously, intraperitoneally or orally. The oral route is particularly preferred.

[60] Besides warm-blooded animals such as mice, rats, dogs, cats, sheep, horses, cows and monkeys, the compound of the invention is also effective on humans.

[61] According to one embodiment, pharmaceutical compositions for administration of 976% of the compounds of this invention may be presented in unit dose form and may be prepared by any of the methods well known in the state of the art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, pharmaceutical compositions are prepared by bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition, the active object compound is included in an amount sufficient to produce the desired effect on the disease process or state. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, capsules, syrups, elixirs, solutions, mouth patches, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets. The pharmaceutical compositions containing the active principle can be in the form of an aqueous or oily suspension.

[62] According to one embodiment, the aqueous suspensions contain the active materials mixed with excipients suitable for the manufacture of aqueous suspensions. These excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; the dispersing or wetting agents may be a natural phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of alkylene oxide ethylene with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, such as polyoxyethylene monooleate sorbitol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitol monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[63] According to one embodiment, the oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, for example peanut, olive, sesame or coconut oil, or in a mineral such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those mentioned above and flavoring agents can be added to obtain a palatable oral preparation. These compositions can be preserved by adding an antioxidant such as ascorbic acid. Dispersible powders and granules which are suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives.

[64] Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. These formulations may also contain emollient, preservative, flavoring and coloring agents.

[65] The pharmaceutical compositions may be in the form of an aqueous or oleaginous suspension which can be injected in a sterile manner. This suspension can be formulated according to the known art using the appropriate dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable non-toxic diluent or solvent, for example a solution in 1,3-butane diol. Acceptable vehicles and solvents that can be used include; water, Ringer's fluid and isotonic sodium chloride solution. Additionally, sterile fixed oils are traditionally used as a solvent or suspending medium. For this purpose, any fixed oil can be used, including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid are used in the preparation of injectables.

[66] The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and which will therefore melt in the rectum to release the drug. These materials include cocoa butter and polyethylene glycols.

[87] In addition, the pharmaceutical compositions can be administered ocularly by means of solutions or ointments. Additionally, transdermal delivery of the compounds in question can be achieved by means of iontophoretic and other patches. For topical use, creams, ointments, jellies, solutions or suspensions are used.

[68] In the treatment of a mammal suffering from or at risk of developing cancer, a suitable dosage of the pharmaceutical composition of this invention may generally be from about 0.1 to 50,000 micrograms (ug) per kg body weight of the patient per day, which can be administered in single or multiple doses. The dosage level will preferably be from about 1000 to about 40,000 µg/kg per day, depending on many factors such as the severity of the cancer to be treated, the age and relative health of the subject, the route and the form of administration. For oral administration, this composition may be supplied in the form of tablets containing 1000 to 100000 micrograms of each of the active ingredients, in particular 1000, 5000, 10000, 15000, 20000, 25000, 50000, 75000, 100000 micrograms of each active ingredient . This composition can be administered on a 1 to 4 times daily schedule, for example once or twice daily. The dosage regimen may be adjusted to provide an optimal therapeutic response.

The invention also discloses a process for the manufacture of the compound of formula (1).

[69] According to one embodiment, the C3 fluorination process includes a dendrogenin A fluorination step, carried out using a fluorination reagent, for example Diethylaminosulfur trifluoride (DAST) or tetrafluoroborate. The fluorination reaction with DAST is described in the literature “Tetrahedron letters 1979, 20, 1823-1826, A new method for fluorination of sterols” (https:/doi.org/10.1016/S0040-4039(01)86228-6 ). The fluorination reaction with tetrafluoroborate is described in the literature “Org. Left, Vol.11, No. 21, 2009, 5050-5053, Aminodifluorosulfinium Tetrafluoroborate Salts as Stable and Crystalline Deoxofluorinating Reagents”.

[70] According to one embodiment, the process for the synthesis of 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluorodilactate comprises the following steps: - dissolving the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro in anhydrous ethanol then add lactic acid thereto; - stirring the mixture at room temperature for 3 h; - evaporation of the organic solvent.

The white powder obtained is the compound 5da-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluorodilactate.

[71] According to one embodiment of the method, the ambient temperature is between 15 and 40°C, for example 25 or 37, preferably 20°C.

; a / BE2020/5764 Brief description of figures

[72] The invention will be better understood, and other objects, details, characteristics and advantages thereof will appear more clearly during the following description of several particular embodiments of the invention, given for illustrative purposes only. and non-limiting, with reference to the accompanying drawings.

[73] [fig.1] Figure 1 shows the results of a cytotoxicity study of 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro on Neuro2a cells via trypan blue assay.

[74] [fig.2] Figure 2 illustrates the results of an MTT cell viability test performed on MCF-7 mammary tumor cells in the presence of the compound 5a-hydroxy-6B-[2-(1H-imidazol-4 -yl)-ethylamino]-cholestan-3a-fluoro.

[75] [fig. 3] Figure 3 illustrates the results of Cholesterol Epoxide Hydrolase (ChEH) activity in MCF-7 cells in the presence of the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino ]-cholestan-3a-fluoro.

EXAMPLES

[76] Various experiments were carried out in order to evaluate the characteristics of the compounds of formula (|).

[77] Example 1: Synthesis of the compound 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro: The first step is a synthesis of the compound 3a-fluorocholestane comprising the next steps: AN 9 d BF4

FF ONT C3HF ON, DCM - HO T.A, 24 h F Cholesterol 3a-fluorocholestane C27H480 C27HasF MW: 386.66 MW: 390.36

2.06 grams (g) of cholesterol was dissolved in 100 milliliters (mL) of anhydrous dichloromethane.

1.95 g of boron (diethylamine)difluorosulfonium tetrafluoride was added in several portions and 1.30 mL of trimethylamine trihydrofluoride was added thereto. The mixture thus obtained was left at room temperature for a period of 24 hours.

At the end of this time, the reaction was neutralized by adding 100 mL of a 5% Na-CO+ solution.

The mixture was transferred to a separating funnel and the aqueous phase was extracted twice with dichloromethane. The organic phases thus obtained were combined and washed with saturated NaCl.

The organic phase was dried over MgSO4 and then evaporated to obtain an oil.

The oil obtained was purified by column chromatography on silica gel with an eluent comprising hexane/Et:O in a 95/5 ratio. A white powder of 0.24 g of 3a-fluorocholestane was thus obtained. The final yield of the reaction is 12%.

The second step consists in synthesizing from 3a-fluorocholestane the compound 3a-fluoro-5,6-Epoxycholestane as follows: mCPBA ee a | DCM LJ F RT, 3h F E 15 3a-fluorocholestane 3a-fluro-5,6x-Epoxycholestane C27HasF C27H45FO MW: 390.36 MW: 402.66

0.54 g of meta-chloro-peroxybenzoic acid at 2.41 millimole (mmol) was dissolved in mL of methylene chloride and added dropwise to a mixture of 0.72 g of 20 3a-fluorocholestane at 1.85 mmol dissolved in 20 mL of chloride of methylene. The mixture thus obtained was stirred and maintained at ambient temperature for 3 hours. The mixture obtained was washed with an aqueous solution of sodium sulphite at 10% by weight, a saturated solution of sodium hydrogencarbonate and a saturated solution of sodium chloride. The organic phase was dried over anhydrous magnesium sulphate. Vacuum evaporation of the organic solvent was carried out and yielded 0.75 g of a white powder of 3a-fluoro-5,60-Epoxycholestane.

3a-Fluoro-5,6'-Epoxycholestane was used without further purification.

The third step is to synthesize 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro as follows: BuOH La 3a-fluroe-5 6a-lpoxycholestane 13070 , #3 h TR Osta MF KEA 402 66 Schrei BaN TOT SEHE CHE TAN Ser Oase FR

SAN ES RC

0.42 g of histamine in its basic form at 3.68 mmol was added to a 15 mL butanol solution comprising 0.75 g of the compound 3a-fluoro-5,6œ-Epoxycholestan-3p-ol at 1.84 mmol with stirring. The mixture was kept under stirring under reflux heating at a temperature of 130°C for 48 hours. The progress of the reaction can be monitored by thin layer chromatography (TLC) to follow the conversion of 3a-fluoro-5,60-Epoxycholestane.

After cooling, the mixture was diluted in 15 mL of methyl tert-butyl ether. The organic phase was washed with 4 times 15 ml of water and then once with a saturated solution of sodium chloride.

The organic phase was dried over anhydrous magnesium sulphate. The mixture was purified by column chromatography on silica gel on a purification machine comprising a prepacked 24 g column eluting with ethyl acetate/methanol in a 9/1 ratio. A white powder of 0.27 g of 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro was obtained. The final yield of the reaction is 29% with a purity greater than 95% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.

[78] Example 2: Preparation of a dilactate salt of the compound 5a-hydroxy-6B8-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro:

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[79] A dilactate salt of the compound 5da-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro was prepared as follows:; } to Au 5 DN: ì ì; $ Ï 3 3 pas PT gg rt od et HR win Ss "N ; pt NS EC ae x es SR a ST SR Uÿ € PS sie # FAR, a TR BE SS X EX

HRS ON HE Silk Rai SÖHNE bedstee aso anal

108.3 mg of lactic acid was added to a solution of 0.31g of 50-hydroxy-86-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro in 15 mL of anhydrous ethanol under agitation. Stirring was maintained at ambient temperature for 3 hours. Vacuum evaporation of the organic solvent yields a white powder of 0.42 g of 5a-hydroxy-6B8-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluorodilactate.

[80] Example 3: preparation of 3a amino and 3a sulfide derivatives of formula (|):

[81] The steps are as follows: uf FON DNG Fat _ Li en N! { + A Rs we . t 3 To. $ Ra = La dp Sr SEE ge v> we ni wo” SE Wes Ne en eran) Nes = 2555 qu IRAN PRE | PEA 5, 4h SORT 3 En DE Ke HA td Ra Ms” Da N ERS N nr, > He SUR I ee > ve Ba RD Nu = (Ras) el in the RS la Ru = RAU RES or INALN reaction gives products PRE ASK IS YOUR? secondaries like the ROSS and R,0:5 with R2 and R3 are CHz. Stir cholesterol in THF in the presence of NaH for five minutes at 70°C, add paratoluenesulfonic chloride (p-TsCl) and stir the mixture for 4 hours at 70°C. Add water, filter the reaction and evaporate the organic solvents. 299764 Extract the reaction product with DCM/H2O then dry over MgSOa4. Remove organic solvents by evaporation under vacuum. The product obtained is used as it is for the next step. Dissolve the product obtained in THF with stirring for 12 h with 1.1 equivalent of NuH (Nucleophile-hydrogen). NuH corresponds to R4SH or NH(R4)2 at 70°C. The reaction is stopped by adding water and the products were extracted by an ACOEYUH:20O system. The organic phase was dried over MgSO4 and the organic solvents evaporated under vacuum. The cholestan-3-sulphide and cholestan-3-amino derivative products are purified either by column chromatography or by recrystallization. The reaction path to obtain Dendrogenin A analogs are the same steps developed for the synthesis of Dendrogenin A.

[82] The products R4OS and R40:S will be obtained by oxidation of R4S with oxidizing agents such as m-CPBA or HO”.

[83] Example 4: preparation of 3B amino and 3B sulfide derivatives of formula (I): The steps are presented below: port a. king ord ge + Ba Ba Dissolve cholesterol, add NEt3 in DCM and add MsCl dropwise in DCM solution at room temperature for 1 h. Stir the reaction for 12 h, then evaporate the organic solvent and crystallize the product with MeOH. The product obtained is a white solid. The product obtained is used to obtain the 3B-sulfide and 3B-azide derivatives. The product obtained is dissolved in DCM then TMS-SR4 for the 3B-sulfide derivative or TMS-N3 for the 38-azide derivative is added to the solution. An addition of BF3*Et:O is carried out at room temperature. Then, it is stirred for 3 h.

[84] 3B-azide is reduced to 3B-amino by the action of LiAlH4 to Et:O and transformed into the products of formula (I) with reaction of R4X (X = Br, Cl or |) to Et:O (or pyridine) as a solvent. The reaction path to obtain the analogs of Dendrogenin A are the same steps developed for the synthesis of Dendrogenin A. The sulfur derivatives R40S and R40:S will be obtained by oxidation of R4S by usual oxidizing agents. This method is detailed in the literature “ORGANIC LETTERS; 2009, 11, 3, 567-570, Practical Synthesis of 38-Amino-5-cholestene and Related 36-Halides Involving i-Steroid and Retro-i-Steroid Rearrangements” (nttps://doi.org/10.1021/01802343z) .

[85] Example 5: Study of the cytotoxicity of 5a-hydroxy-6B8-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro:

[86] For this experiment, a cell culture medium was prepared. The culture medium consists of Dulbecco's Modified Eagle Medium (DMEM, marketed by Westburg under the reference LO BE12-604F), comprising 4.5 g/L Glucose with L-Glutamine, to which 10% serum is added. of fetal calf (SVF). Neuro2a cells (murine neuroblastoma) are introduced into this culture medium.

[87] 24-well dishes were seeded with 10,000 Neuro2a cells per well. After 72 hours (h) of culture under normal conditions, i.e. in an incubator at a temperature of 37°C at 5% Oz, we treat the Neuro2a cells for 48 hours with 5a-hydroxy-6B -[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro and 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B- ol at 100 nM, 1 MU and 10 MU. A control (CTL) is also carried out using the protocol described previously without the treatment with 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro and 5a- hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol. Cell survival is quantified by a trypan blue test with automatic counting by the Biorad TC20 device (TC20TM Automated Cell Counter). The trypan blue test is based on the integrity of cell membranes, which is broken in dead cells. Trypan blue stains dead cells blue. The Biorad TC20 cell counting device counts the proportion of blue and non-blue cells, and reports the percentage of cells. The results are shown in Figure 1. Figure 1 illustrates the ordinate of the percentage of cell survival compared to the control group.

[88] 1 is illustrated in Figure 1 that for 100 nM of treatment with 5a-hydroxy-6f-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro the percentage of living cells remains unchanged compared to the control group (CTL). Furthermore, for concentrations of 1 μm and 10 μm, the cell survival percentage is 75% and 30%. A similar activity is also observed between the two compounds tested.

In conclusion, cytotoxic activity of the compound 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro of formula (I) is observed towards Neuro2a tumor cells for concentrations in 1 µm and 10 µm 5α-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3α-fluoro.

[89] Example 6: The effect of 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro on MCF-7 cell viability:

[90] A cell viability test was performed on MCF-7 breast tumor cells (Michigan Cancer Foundation—7) overexpressing HER2 (ER(+) cells).

The MCF-7 cells are in a cell culture medium identical to Example 5 and are seeded in 12-well plates at 50,000 cells per well. 24 hours after seeding, the cells are treated with the vehicle solvate comprising water and ethanol with a 1% ethanol ratio, 5a-hydroxy-6B-[2-(1H-imidazol-4-yl )-ethylamino]-cholestan-3α-fluoro and 5α-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol at 1, 2.5 or 5 µM. The cells are observed under an inverted microscope and photographed via the microscope camera at 24 h and 48 h. Morphological changes of cells at 1 µM are very small. Only a few white vesicles are observed, reflecting the start of the autophagy phenomenon, causing cell death after 24 hours of treatment with 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan -3a-fluoro and 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol. The effects are more marked at 2.5 µM and 5 µM with an increase in the number of dead cells. Indeed, many white vesicles and collapsing cells. After 24 h of treatment with 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro at 5 μM, 99% of the cells observed are supernatants, indicating cell death and 1% of the cells are adherent and present white vesicles. After 48 h of treatment with 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro at 2.5 UM, a greater cytostatic effect is observed than at 24 h and more cells rounding up, indicating cell death. The cytostatic effect is illustrated by an inhibition of cell proliferation. After 48 h of treatment with 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3α-fluoro at 5 μM, all the cells are supernatants. Compared to treatment with 5a-hydroxy-6B6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol, treatment with 5a-hydroxy-6f-[2-(1 H-imidazol-4-yl)-ethylamino]-cholestan-3α-fluoro shows greater or equal effect after 24 h observation and similar after 48 h observation.

[91] Cell viability is measured by MTT labeling at 48 hours. This test is based on the use of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Tetrazolium is reduced by active living cell mitochondrial succinate dehydrogenase to formazan, a violet-colored precipitate. The quantity of precipitate formed is proportional to the quantity of living cells but also to the metabolic activity of each cell. Thus, a simple assay of the optical density at 540 nm by spectroscopy makes it possible to know the relative quantity of living and metabolically active cells. After 48 hours, the medium is aspirated, the cells washed with phosphate buffered saline (PBS) then incubated with MTT (0.5 mg/ml in PBS) for approximately 2 hours. The MTT solution is aspirated and then the purple crystals are solubilized in dimethylsulphoxide (DMSO). The OD (optical density) is measured at 540 nm.

[92] The results of this test are presented in figure 2. Figure 2 illustrates the ordinate of the percentage of cell viability compared to the control group. The control group is made in a similar way to the groups studied without the addition of the molecules studied in this text. In comparison to the control, a dose-dependent decrease in cell viability in MTT is measured for 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro and 5a -hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol. For a concentration of 5 µM, the viability is close to 0%. This reflects a capacity for destruction of mammary tumor cells by the compound of formula (I). These results are consistent with the observations made at 24 h and 48 h mentioned above.

[93] Example 7: Effect of 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro on Cholesterol Epoxide Hydrolase (ChEH) activity in cells MCF-7:

[94] The compounds 5,6a-epoxycholesterol (5,6a-EC) and 5,6B-epoxycholesterol (5,6B-EC) are oxysterols implicated in the anticancer pharmacology of tamoxifen, a widely used antitumor drug. They are both metabolized to cholestane-3B,5a,6B-triol (CT) by the enzyme cholesterol-5,6-epoxide hydrolase (ChEH), and CT is metabolized by the enzyme HSD11B2 (11B-Hydroxysteroid dehydrogenase 2 ) to 6-oxo-cholestan-3B,5a-diol (OCDO), a tumor-promoting oncosterone. The following experiment aims to demonstrate the ability of 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro to block ChEH and therefore limit the metabolism of oncosterone, a tumor promoting metabolite.

[95] MCF-7 cells are in a cell culture medium identical to Example 5 and are seeded in 6-well plates at 150,000 cells per well with 3 wells per treatment condition. 24 h after seeding, the MCF-7 cells are treated with ['“C]5,6a-EC (stock solution 1000X: 0.6 mM; 20 uCi/umol; final concentration 0.6 HM) alone or in combination with Tamoxifen ( tom). Tamoxifen is used as a positive control for 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3«a-fluoro and 5a-hydroxy-6B8-[2- (1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol (1uM for all molecules).

[96] After 24 hours of treatment, the media are collected and lipid extracts are prepared from the cell pellets by extraction with 100 μL of chloroform, 400 μL of methanol and 300 μL of water. Lipid extracts are analyzed by thin layer chromatography (TLC) using ethyl acetate (EtOAc) as eluent. The analysis is carried out using a plate reader and then by autoradiography. The results are presented in figure 3. It is observed the almost total metabolism of the epoxide in CT and OCDO (wells 2 and 4) and a total inhibition of ChEH activity by Tamoxifen and almost total (trace of CT) by 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro. Similar results are observed with 5α-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3B-01. In conclusion, 5a-hydroxy-68-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro possesses ChEH inhibitory activity similar to 5a-hydroxy-6B-[2-( 1H-imidazol-4-yl)-ethylamino]-cholestan-3B-ol.

[97] Although the invention has been described in connection with several particular embodiments, it is quite obvious that it is in no way limited thereto and that it includes all the technical equivalents of the means described as well as their combinations if these fall within the scope of the invention.

[98] The use of the verb "to comprise", "to understand" or "to include" and of its conjugated forms does not exclude the presence of other elements or other steps than those set out in a claim.

[99] In the claims, any reference sign in parentheses cannot be interpreted as a limitation of the claim.

Claims (1)

Claims [Claim 1] Compound of formula (I); Se > Ni” Ns HO ui N NH or a pharmaceutically acceptable salt of such a compound, wherein: Ry is selected from F, N(R4)2, SR4, S(R4)2, SOR4, SOzR4 or OH with R4 selected from: H; a C1 to C20 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl and aryl groups, said aryl group optionally containing heteroatoms chosen from N, O, and S, Ra equals Rs are chosen from CH; or CF: ; and when Ry is OH, Ra and R3 are CF3, for its use as a medicament for regressing a mammalian cancerous tumor. [Claim 2] A compound for use according to claim 1, wherein when R: is selected from N(R4)2, SRa4, S(R4)2, SOR4 and SOzR4, then Ra and R3 = CH:. [Claim 3] A compound for use according to claim 1 wherein when R: is F then Ra and R3 = CH: or Ra and Rs = CFs. [Claim 4] A compound for its use according to claim 1, wherein when Rı is OH, Ra and Rs are CF3, the compound of formula (I) is 5a-hydroxy-6B-[2-(1H-imidazol-4- yl)-ethylamino]-cholestan-3-ol-26-27-di-trifluoro-methyl. [Claim 5] A compound for its use according to claim 3, wherein the compound of formula (1) is 5da-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro . [Claim 6] A compound for its use according to claim 2, in which the compound of formula (I) is chosen from: - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-amino; - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methyl! sulfide; - 5a-hydroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methylsulfonyl; and Os, KO 8 to AN 4% [Claim 7] A compound for its use according to one of claims 1 to 6, in which the cancerous tumor is a chemosensitive cancer. [Claim 8] A compound for its use according to one of claims 1 to 6, in which the cancerous tumor is a chemoresistant cancer. [Claim 9] A compound for its use according to claim 8 wherein the chemoresistant cancer is a hematological or blood cancer, such as leukemia, in particular acute myeloid leukemia or acute lymphocytic leukemia, lymphoma, in particular lymphoma not -Hodgkin syndrome and multiple myeloma. [Claim 10] A compound for its use according to one of Claims 8 and 9, in which the said cancer is chemoresistant to daunorubicin, to cytarabine, to fluorouracil, to cisplatin, to all-trans-retinoic acid, to trioxide of arsenic, bortezomib or a combination thereof. [Claim 11] Pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one compound according to one of Claims 1 to 10 for its use in causing a cancerous mammalian tumor to regress. [Claim 12] Pharmaceutical composition for its use according to claim 11, further comprising at least one other therapeutic agent. [Claim 13] A pharmaceutical composition for use according to claim 12, wherein the other therapeutic agent is an antineoplastic agent. [Claim 14] A pharmaceutical composition for its use according to claim 13 for its use in the treatment of cancer in a patient suffering from a tumor which is chemoresistant to said antineoplastic agent when it is not administered in combination with a compound according to claim 14. one of claims 1 to 10. [Claim 15] A pharmaceutical composition for use according to claim 13 for use in the treatment of cancer in a patient suffering from a tumor which is chemosensitive to said antineoplastic agent, wherein the dose of the antineoplastic agent administered to said patient in combination with a compound according to one of claims 1 to 10 or one of its pharmaceutically acceptable salts is lower than the dose of the antineoplastic agent when it is not administered in combination with a compound according to one of claims 1 to 10. [Claim 16] Pharmaceutical composition according to one of Claims 11 to 15, characterized in that it is in a form suitable for being administered by any route, preferably intravenously, subcutaneously, intraperitoneally or oral.