5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-cholestane-3β-ol analogs and pharmaceutical compositions containing the same for use in the treatment of cancer

Abstract

The present invention provides a compound of general formula (I): [Formula 1] JPEG2023551363000057.jpg4160 and / or pharmaceutically acceptable salts of such compounds, and pharmaceutical compositions comprising at least said compounds.

Description

[Technical Field]

[0001] The present invention relates to the field of sterol compounds, and more particularly to analogs of the compound 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol and pharmaceutical compositions containing the same for use in the treatment of cancer. [Background technology]

[0002] The terms "cancer" or "malignant tumor" encompass a group of diseases characterized by the uncontrolled proliferation and spread of abnormal cells. If cancerous cells are not removed, the disease progresses rapidly to varying degrees, eventually leading to the death of the affected person.

[0003] Cancer management includes 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 growth of neoplasms. Antineoplastic agents work by effectively targeting rapidly dividing cells. Because antineoplastic agents affect cell division, tumors with high growth rates (e.g., acute myeloid leukemia and aggressive lymphomas, including Hodgkin's disease) are more sensitive to chemotherapy, as a larger proportion of target cells undergo cell division at any given time. Malignant tumors with slow growth rates, such as low-grade lymphomas, tend to respond much more mildly to chemotherapy. However, the development of chemotherapy resistance is an ongoing problem during chemotherapy treatment. For example, conventional treatment for acute myeloid leukemia (AML) includes the combination of cytarabine and anthracyclines, such as daunorubicin. The 5-year overall survival rate is 40% in young adults and about 10% in elderly patients. The response rate varies significantly with age, ranging from 40% to 55% in patients over 60 years of age and 24% to 33% in patients over 70 years of age. This is even worse in elderly patients with unfavorable cytogenetic profiles, and 30-day mortality within the course of treatment varies from 10% to 50% with age and worsening conditions. Furthermore, the use of these molecules is also limited by the occurrence of side effects, particularly chronic cardiotoxicity (associated with anthracyclines). The toxic mortality rate associated with intensive chemotherapy is 10% to 20% in patients over 60 years of age.

[0004] Due to the benefit-risk profile of conventional regimens, only 30% of newly diagnosed elderly patients with AML receive antineoplastic chemotherapy.

[0005] Over the past few decades, there has been a slight improvement in outcomes for younger patients with AML, but no improvement for adults over 60 (the majority of AML patients).

[0006] Therefore, there is a real need to develop useful molecules in the treatment of these cancerous tumors that present the problems of chemoresistance and intrinsic toxicity of anti-neoplastic drugs. The above data emphasizes the need to find a new approach that combines both a reduction in the dosing regimen of anti-neoplastic agents for the treatment of chemosensitive tumors and a reduction in the resistance of tumors that are chemoresistant to anti-neoplastic agents.

[0007] European Patent No. 3272350 discloses the compound 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol, which is known as dendrogenin A useful for the treatment of chemoresistant tumors and hereinafter referred to as DX101. Dendrogenin A can restore the sensitivity of chemoresistant tumors to anti-neoplastic agents or enhance the effect of anti-neoplastic agents on tumors, thereby sequentially reducing the effective cytotoxic dose of anti-neoplastic agents for chemosensitive tumors.

[0008] The literature of De Medina et al. (Biochimie, 2021, 95(3), 482-488, XP 028982107, Technical note: Hapten synthesis, antibody production and development of enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid dendrogenin A) describes a dendrogenin A derivative in which the alcohol at the 3β position is functionalized for use as a hapten for antibody production.

[0009] The literature of De Medina et al. (J.Med.Chem.,2009,52(23),7765-77,XP9131948,Synthesis of new alkylaminooxysterols with potent cell differentiating activities:identification of leads for the treatment of cancer and neurodegenerative diseases) describes dendrogenin A derivatives in which the alcohol at the 3β position is optionally functionalized with a methoxide or propoxide radical for the treatment of cancer.

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide novel compounds and analogs of the compound dendrogenin A that are useful for treating cancerous tumors, particularly chemosensitive and / or chemoresistant tumors.

Means for Solving the Problems

[0011] Surprisingly, the present inventors have discovered that certain analogs of the compound dendrogenin A (also referred to as DX101) exhibit pharmacological activities comparable to those of dendrogenin A.

[0012] A first object of the present invention is a compound of formula (I) for use as a medicament, more specifically as a medicament for reducing a cancerous tumor in a mammal:

[0013]

Chemical formula

[0014] or a pharmaceutically acceptable salt of such a compound, [[ID=3�]] wherein, R1 is F, N3, OC n H 2n+1, selected from NR2R3, SR2, SO2R2 (n≦8), R2 and R3 are independently selected from H, saturated or unsaturated C1-C8 alkyl groups, and optionally include one or more substituents selected from allyl groups, carbonyl groups, arene groups, and heterocyclic groups.

[0015] A second object of the present invention is a pharmaceutical composition comprising at least one compound of formula (I) in a pharmaceutically acceptable vehicle for use in reducing cancerous tumors in mammals. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 shows the results of a cytotoxicity study of Neuro2a cells with 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX111) using the trypan blue assay. [Figure 2] Figure 2 shows the results of an MTT cell viability assay performed on MCF-7 mammary tumor cells in the presence of the compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane. [Figure 3] Figure 3 shows the results of cholesterol epoxide hydrolase (ChEH) activity in MCF-7 cells in the presence of the compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane. [Figure 4] Figure 4 shows the pharmacokinetic profile of compound 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX103) in comparison with compound dendrogenin A (DX101). [Figure 5] Figure 5 shows the pharmacokinetic profile of compound 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX105) in comparison with compound dendrogenin A (DX101). [Figure 6]Figure 6 shows the pharmacokinetic profile of compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX111) in comparison with compound dendrogenin A (DX101). [Figure 7A] Figure 7A shows the progression of tumor growth and survival rates in mice treated with DX111 and DX101. [Figure 7B] Figure 7B shows the progression of tumor growth and survival rates in mice treated with DX111 and DX101. [Figure 8] Figure 8 shows the pharmacokinetic profile of compound 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX123) in comparison with compound dendrogenin A (DX101). [Modes for carrying out the invention]

[0017] [Definition] In this specification, unless otherwise specified, if a range is given, it is understood to include the upper and lower limits of that range.

[0018] In the present invention, throughout this description and the appended claims, the following terms should be understood to have the following meanings unless otherwise indicated:

[0019] The term "solvate" is used herein to refer to a molecular complex comprising the compound of the present invention and containing one or more pharmaceutically acceptable solvents, such as ethanol, in stoichiometric or quasi-stoichiometric amounts. The term "hydrate" refers to the case where the solvent is water.

[0020] The term "human" refers to an individual of any sex and any stage of development (i.e., neonatal, infancy, childhood, adolescence, or adulthood).

[0021] The term “patient” refers to a warm-blooded animal, more preferably a human, that is awaiting or receiving medical treatment and / or will be subject to medical treatment.

[0022] The term "pharmaceutically acceptable" means that the components of a pharmaceutically acceptable product are compatible with each other and are not harmful to patients receiving the product.

[0023] As used herein, the term “pharmaceutical vehicle” means an inert support or medium used as a solvent or diluent in which a pharmacoactive agent is formulated and / or administered. Non-limiting examples of pharmaceutical vehicles include creams, gels, lotions, solutions, and liposomes.

[0024] The term "administration" means delivering an activator or active ingredient (e.g., a compound of formula (I)) in a pharmaceutically acceptable composition to a patient whose condition, symptoms and / or disease is being treated.

[0025] As used herein, the terms “to treat” and “to treat” include reducing, alleviating, stopping or caring for a condition, symptom and / or disease.

[0026] As used herein, the term “analog” means a compound that has a similar chemical structure to another reference compound but differs in certain components. One or more atoms, functional groups, or substructures may be replaced by other atoms, functional groups, or substructures, thus differentiating the compound. Analogues may have different physical, chemical, biochemical, or pharmacological properties. In this invention, analogues relate to the compound dendrogenin A. These analogues have the same or similar pharmacological properties compared to the reference compound.

[0027] The term "chemotherapy-resistant cancer" means a patient's cancer in which the proliferation of cancer cells cannot be prevented or inhibited by an antineoplastic agent or combination of antineoplastic agents commonly used to treat such cancer, at doses tolerable to the patient. Tumors may be inherently resistant prior to chemotherapy, or resistance may be acquired during treatment by tumors that are initially sensitive to chemotherapy.

[0028] The term "chemosensitive cancer" means cancer in a patient that responds to the effects of an antineoplastic agent, that is, cancer in a patient whose cancer cells can be prevented from growing by the antineoplastic agent in a dose that is tolerable to the patient.

[0029] The compound of formula (I) belongs to the steroid group. Therefore, the numbering of the carbon atoms in the compound of formula (I) follows the nomenclature specified by IUPAC in the 1989 edition of Pure & Appl. Chem., Vol. 61, No. 10, pp. 1783-1822. The IUPAC numbering of carbon atoms in compounds belonging to the steroid group is shown below:

[0030] [ka]

[0031] In this invention, the following abbreviations have the meanings set forth below. AML: Acute myeloid leukemia; Dendrogenin A: 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol; MCF-7: Michigan Cancer Foundation-7; DMEM: Dulbecco's modified Eagle medium; FCS: Fetal bovine serum; CHEH: Cholesterol epoxide hydrolase; Neuro2a: Mouse neuroblastoma; CTL: Contrast; MTT: 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; PBS: Phosphate-buffered saline; DMSO: Dimethyl sulfoxide; OD: Optical density or absorbance; CT: Cholestane-3β,5α,6β-triol; OCDO:6-oxocholestane-3β,5α-diol; 5,6α-EC: 5,6α-epoxycholesterol; Tam: Tamoxifen; TLC: Thin-layer chromatography; PO: Oral; LC / MS: Liquid Chromatography / Mass Spectrometry

[0032] The first object of the present invention is a compound of formula (I) for use as a pharmaceutical:

[0033] [ka]

[0034] or a pharmaceutically acceptable salt of such a compound, During the ceremony, R1 is F, N3, OC n H 2n+1 , selected from NR2R3, SR2, SO2R2 (n≦8), R2 and R3 are independently selected from H, saturated or unsaturated C1-C8 alkyl groups, and optionally include one or more substituents selected from allyl groups, carbonyl groups, arene groups, and heterocyclic groups. According to one embodiment, the present invention relates to a compound of formula (I) for use as a pharmaceutical agent for reducing cancerous tumors in mammals:

[0035] [ka]

[0036] Or relating to pharmaceutically acceptable salts of such compounds, During the ceremony, R1 is F, N3, OC n H 2n+1 , selected from NR2R3, SR2, SO2R2 (n≦8), R2 and R3 are independently selected from H, saturated or unsaturated C1-C8 alkyl groups, and optionally include one or more substituents selected from allyl groups, carbonyl groups, arene groups, and heterocyclic groups.

[0037] In the present invention, The term "carbonyl group" refers to all functional groups containing an oxo group (an oxygen atom double-bonded to a carbon atom (=O)), and can be selected from aldehydes, ketones, carboxylic acids, esters, amides, and / or anhydrides; The term "allyl" refers to the alkene functional group with the half-expanded structure H2C=CH-CH2-. The term "sulfonyl" refers to a chemical compound in which a sulfur atom is bonded to two double-bonded oxygen atoms (=O) and their radicals. The term "arene" refers to all monocyclic and polycyclic aromatic hydrocarbons. The term "heterocyclic" refers to monocyclic and polycyclic aromatic compounds that contain one or more heteroatoms from O, S, and / or N as ring members. In the definition of the compound of formula (I) according to the present invention, the carbon 3 radical may be at the α-position or the β-position, with the β-position being a preferred embodiment.

[0038] According to one embodiment, the compound of formula (I) is a radical R1 = NR2R3, where R2 is H or COC n H 2n+1 It is an O-amino analog with R3=H.

[0039] In this embodiment, the compound of formula (I) is more specifically 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-3β-acetamide (named DX127).

[0040] In this embodiment, the compound of formula (I) is more particularly 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]-3β-amine (named DX125).

[0041] In this embodiment, the compound of formula (I) is more particularly 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]-3β-azide (named DX123).

[0042] According to yet another embodiment, the compound of formula (I) is 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX111).

[0043] According to yet another embodiment, the compound of formula (I) is an O-alkyl analog, with radical R1 = OC n H 2n+1 (n ≦ 8), and is selected from 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX103), 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX105), 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX115).

[0044] Even more preferably, the compound of formula (I) is an O-alkyl analog, such as 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (DX103) and 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (DX105).

[0045] According to a further embodiment, the compound of formula (I) is a sulfur analog, having radical R1 = SO2R2, where R2 is H or OCn H 2n+1 (n ≤ 8)

[0046] In this embodiment, the compound of formula (I) is preferably 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX129).

[0047] According to one embodiment, the compound of formula (I) is intended for use in the treatment of breast cancer, prostate cancer, colorectal cancer, lung cancer, bladder cancer, skin cancer, uterine cancer, cervical cancer, oral cancer, brain cancer, stomach cancer, liver cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, kidney cancer, retinal cancer, paranasal sinus cancer, nasal cavity cancer, testicular cancer, thyroid cancer, vulvar cancer, lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, acute myeloid leukemia or acute lymphoblastic leukemia, multiple myeloma, Merkel cell carcinoma or mesothelioma.

[0048] According to one embodiment, cancer includes acinar adenocarcinoma, acinar carcinoma, acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, adrenocortical quiescent tumor, adrenocortical carcinoma, aldosterone-secreting carcinoma, alveolar soft tissue sarcoma, thyroid ameloblastoma, angiosarcoma, apocrine carcinoma, askine tumor, astrocytoma, basal cell carcinoma, basaloid carcinoma, basosquamous carcinoma, biliary tract carcinoma, bone marrow carcinoma, staphyloid sarcoma, bronchoalveolar carcinoma, bronchogenic adenocarcinoma, bronchogenic carcinoma, and pleomorphic adenoma (e.g., pleomorphicAdenoma, cholangiocarcinoma, chondrosarcoma, choroidal cell carcinoma, choroidal plexus carcinoma, clear cell adenocarcinoma, colon cancer, comedone carcinoma, cortisol-producing adenoma, columnar cell carcinoma, differentiated liposarcoma, ductal adenocarcinoma of the prostate, adenocarcinoma of the prostate, in situ adenocarcinoma, duodenal cancer, eccrine carcinoma, embryonic carcinoma, endometrial carcinoma, endometrial stromal carcinoma, epithelioid sarcoma, Ewing's sarcoma, extrinsic carcinoma, fibroblastic sarcoma, fibrocarcinoma, fibrous layer carcinoma, fibrosarcoma, follicular thyroid cancer, gallbladder cancer, gastric adenocarcinoma, giant cell carcinoma, giant cell Sarcoma, giant cell bone tumor, glioma, glioblastoma multiforme, granuloma cell carcinoma, head and neck cancer, hemangioma, angiosarcoma, hepatoblastoma, hepatocellular carcinoma, Hürtle cell carcinoma, ileal cancer, lobular invasive carcinoma, inflammatory breast cancer, intraductal carcinoma, intraepidermal carcinoma, jejunal carcinoma, Krukenberg tumor, Krukiski cell carcinoma, Kupffer cell sarcoma, large cell carcinoma, laryngeal cancer, lentigo malignant melanoma, liposarcoma, lobular carcinoma, in situ lobular carcinoma, lymphoepithelioma, lymphosarcoma, malignant melanoma, medullary carcinoma, thyroid medullary carcinoma, medulloblastoma, meningeal carcinoma Micropapillary carcinoma, mixed cell sarcoma, myxoid carcinoma, mucoepidermoid carcinoma, mucosal melanoma, myxoid liposarcoma, myxosarcoma, nasopharyngeal carcinoma, nephroblastoma, neuroblastoma, nodular melanoma, non-clear cell renal carcinoma, non-small cell lung cancer, ovarian carcinoma, ocular melanoma, oral cancer, osteoid carcinoma, osteosarcoma, ovarian cancer, Paget's carcinoma, pancreatic blastoma, papillary adenocarcinoma, papillary thyroid carcinoma, pelvic cancer, periampuloma, phyllodes tumor, pituitary cancer, pleomorphic liposarcoma, pleuroblastoma, primary intraosseous carcinoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, round cell liposarcoma These include scar carcinoma, schistosomiasis bladder cancer, serous carcinoma, sebaceous carcinoma, ring cell carcinoma, skin cancer, small cell lung cancer, small cell osteosarcoma, soft tissue sarcoma, spindle cell sarcoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, synovial sarcoma, telangiectatic sarcoma, terminal ductal carcinoma, testicular cancer, thyroid cancer, transitional cell carcinoma, tubular carcinoma, neoplastic melanoma, undifferentiated carcinoma, urethral adenocarcinoma, bladder cancer, uterine cancer, uterine carcinoma, uterine melanoma, vaginal cancer, serous carcinoma, choriocarcinoma, well-differentiated liposarcoma, Wilms' tumor, or germ cell tumor.

[0049] In a preferred embodiment, the compound of formula (I) is intended for use in the treatment of mammalian breast cancer.

[0050] According to one embodiment, the compound is intended for use in the treatment of chemosensitive cancer.

[0051] According to a particularly preferred embodiment, the compound of formula (I) is intended for use in the treatment of chemotherapy-resistant cancer.

[0052] According to one embodiment, chemotherapy-resistant cancers include hematological or blood cancers, such as leukemia, particularly acute myeloid leukemia or acute lymphoblastic leukemia, lymphoma, particularly non-Hodgkin lymphoma, and multiple myeloma.

[0053] According to one embodiment, cancer is resistant to chemotherapy with daunorubicin, cytarabine, fluorouracil, cisplatin, all-trans retinoic acid, arsenic trioxide, bortezomib, or any combination thereof.

[0054] All references to the compound of formula (I) include references to its salts, multicomponent complexes, and liquid crystals. All references to the compound of formula (I) also include references to its polymorphs and their ordinary crystals.

[0055] The compounds according to the present invention may be in the form of pharmaceutically acceptable salts. A pharmaceutically acceptable salt of the compound of formula (I) includes its acid addition.

[0056] Suitable salts are formed from acids that form non-toxic salts, such as acetate, adipine, benzoate, bicarbonate, carbonate, bisulfate, sulfate, borate, cansylate, citrate, cyclamate, edisylate, esylate, formate, furamate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, chloride hydrochloride, hydrobromide, bromide, and hydroiodide. Selected from iodide, isethionate, lactate, maleate, malonate mesylate, methyl sulfate, naphthylate, 2-naphthylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, dihydrogen phosphate, pyroglutamate, sugarate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, trifluoroacetate, and xinafoate. Preferably, pharmaceutically acceptable salts of the compound of formula (I) are formed from lactase.

[0057] A pharmaceutically acceptable salt of the compound of formula (I) can be prepared by one or more of the following three methods: (i) Reacting the compound of formula (I) with a desired acid; (ii) Using a desired acid or base to remove an acid-instability or base-instability protecting group from a suitable precursor of the compound of formula (I), or to open the ring of a suitable cyclic precursor, such as a lactone or lactam; or (iii) Converting one salt of the compound of formula (I) to another salt by reaction with a suitable acid or base, or by using a suitable ion exchange column.

[0058] These three reactions typically occur in solution. The resulting salt can be precipitated and recovered by filtration or by evaporation of the solvent. The degree of ionization of the resulting salt can vary from fully ionized to nearly non-ionized.

[0059] A second object of the present invention is a pharmaceutical composition comprising at least one of the compounds described above according to the present invention in a pharmaceutically acceptable vehicle for use in reducing cancerous tumors in mammals.

[0060] According to one embodiment, the pharmaceutical composition also includes at least one other therapeutic agent.

[0061] According to a preferred embodiment, the other therapeutic agent is an antineoplastic agent.

[0062] According to one embodiment, the antineoplastic agent is a DNA damaging agent, such as camptothecin, irinotecan, topotecan, amsacrin, etoposide, etoposide phosphate, teniposide, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, chlorambucil, chlormethine, busulfan, treosulfan or thiotepa, or an antitumor antibiotic, such as daunorubicin, doxorubicin, epirubicin, idarubicin mitoxantrone, or val This includes rubicin, actinomycin D, mitomycin, bleomycin or plicamycin, antimetabolites such as 5-fluorouracil, cytarabine, fludarabine or methotrexate, antimitotics such as paclitaxel, docetaxel, vinblastine, vincristine, vindesine or vinorelbine, or various antineoplastic agents such as bortezomib, all-trans retinoic acid, arsenic trioxide, or combinations thereof.

[0063] According to one embodiment, the pharmaceutical composition is used to treat cancer in patients with tumors that are resistant to chemotherapy unless administered in combination with the compound according to the present invention.

[0064] According to one embodiment, the pharmaceutical composition is used to treat cancer in a patient suffering from a tumor that is chemically sensitive to the antineoplastic agent, and the dose of the antineoplastic agent administered to the patient in combination with the compound according to the present invention or a pharmaceutically acceptable salt thereof is less than the dose of the antineoplastic agent administered without combination with the compound according to the present invention. In particular, the dose of the antineoplastic agent administered to the patient in combination with the compound according to the present invention or a pharmaceutically acceptable salt thereof is lower than the dose of the antineoplastic agent administered alone without other active ingredients.

[0065] The pharmaceutical composition according to the present invention may further contain other therapeutic compounds commonly used for the treatment of the above-mentioned pathological conditions.

[0066] According to one embodiment, the pharmaceutical composition of the present invention may be administered using tablets, capsules, solutions, powders, gels or particle formulations, particularly via any route including intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, pulmonary, transmucosal (oral, intranasal, vaginal, rectal), or nasal spray inhalation; it may be contained in syringes, implantable devices, osmotic pumps, cartridges or micropumps; or it may be administered by any other means well known in the art and understood by those skilled in the art. Site-specific administration can be performed, for example, in an appropriate dose including a normal, non-toxic, and pharmaceutically acceptable vehicle, within tumors, joints, bronchi, abdominal cavities, capsules, cartilage, cavities, cerebellum, ventricles, colons, cervix, stomachs, livers, hearts, bones, pelvis, pericardium, abdominal cavities, pleura, prostates, lungs, rectums, kidneys, retinas, tendon synovial sheaths, thoracic cavities, uteruses, blood vessels, bladders, lesions, vaginas, rectums, buccal, sublingual, nasal cavities, or percutaneously. Preferably, the pharmaceutical composition is in a form suitable for intravenous, subcutaneous, intraperitoneal, or oral administration, with oral administration being particularly preferred.

[0067] In addition to warm-blooded animals such as mice, rats, dogs, cats, sheep, horses, cattle, and monkeys, the compounds of the present invention are also effective in humans.

[0068] According to one embodiment, a pharmaceutical composition for administering the compound of the present invention may be provided in unit dose form and may be prepared by any of the methods well known in the prior art. All methods include the step of arranging the active ingredient in combination with a support constituting one or more auxiliary components. Generally, the pharmaceutical composition is prepared by arranging the active ingredient in combination with a liquid support or a finely divided solid support or both, and then, if necessary, shaping the product into a desired formulation. In the pharmaceutical composition, the active target compound is included in an amount sufficient to produce a desired effect on a disease process or condition. The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, such as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, capsules, syrups, elixirs, solutions, oral patches, oral gels, chewing gums, chewable tablets, effervescent powders and effervescent tablets. The pharmaceutical composition containing the active ingredient may be in the form of an aqueous or oily suspension.

[0069] According to one embodiment, the aqueous suspension contains the active material in mixture with excipients suitable for the production of the aqueous suspension. These excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum. Dispersants or wetting agents may be natural phosphatides, such as lecithin, or condensation products of alkylene oxides and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxides and long-chain aliphatic alcohols, such as heptadecaethyleneoxyketanol, or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol anhydride, such as polyethylene sorbitol monooleate. The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavorings, and one or more sweeteners, such as sucrose or saccharin.

[0070] According to one embodiment, an oily suspension can be formulated by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in a mineral oil, such as liquid paraffin. The oily suspension may contain a thickener, such as beeswax, hard paraffin, or cetyl alcohol. By adding sweeteners and flavorings as described above, an oral formulation with a pleasant taste can be obtained. These compositions can be preserved by adding antioxidants such as ascorbic acid. Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide an active ingredient mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives.

[0071] Syrups and elixirs can be formulated with sweeteners, such as glycerol, propylene glycol, sorbitol, or sucrose. These formulations may also contain softeners, preservatives, flavorings, and colorings.

[0072] Pharmaceutical compositions may be in the form of aqueous or oily suspensions that can be injected in a sterile manner. These suspensions can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents as described above. Sterile preparations for injection may also be sterile solutions or suspensions for injection in non-toxic diluents or solvents that are parenterally acceptable, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents include water, Ringer's solution, and isotonic sodium chloride solutions. Furthermore, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any non-volatile oil, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are used in the preparation of injectable products.

[0073] The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration of the pharmaceutical. These compositions can be prepared by mixing the pharmaceutical with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the pharmaceutical. Examples of such materials include cocoa butter and polyethylene glycol.

[0074] Furthermore, the pharmaceutical composition can be administered to the eye by solution or ointment. Transdermal administration of the compound under consideration can also be achieved by iontophoresis patches, etc. For topical use, creams, ointments, jellies, solutions, or suspensions are used.

[0075] In the treatment of mammals or patients with cancer or at risk of developing cancer, an appropriate dose of the pharmaceutical composition of the present invention may generally be about 0.1 to 50,000 micrograms (μg) per kg of patient body weight per day, which may be administered in single or multiple doses. The dose level is preferably about 1,000 to about 40,000 μg / kg / day, depending on many factors, such as the severity of the cancer being treated, the age and relative health status of the subject, the route of administration, and the form of administration. For oral administration, the composition may be provided in the form of tablets containing 1,000 to 100,000 micrograms of each active ingredient, particularly 1,000, 5,000, 10,000, 15,000, 20,000, 25,000, 50,000, 75,000, or 100,000 micrograms of each active ingredient. The composition may be administered 1 to 4 times a day, for example, on a schedule of once or twice a day. The dosage regimen can be adjusted to provide the optimal therapeutic response. The present invention also discloses a method for producing a compound of formula (I).

[0076] According to one embodiment, the C3 fluorination process includes a step of fluorinating dendrogenin A using a fluorinating reagent, such as diethylaminosulfur trifluoride (DAST) or tetrafluoroborate. The fluorination reaction using DAST is described in 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 using tetrafluoroborate is described in Org. Lett., Vol. 11, No. 21, 2009, 5050-5053, "Aminodifluorosulfinium Tetrafluoroborate Salts as Stable and Crystalline Deoxofluorinating Reagents".

[0077] According to one embodiment, the synthesis process of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate includes the following: - A step of dissolving the compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-cholestane in anhydrous ethanol and adding lactic acid thereto; - The mixture is stirred at room temperature for 3 hours; - A process of evaporating organic solvents. The resulting white powder is compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate.

[0078] According to one embodiment of the process, the ambient temperature is 15-40°C, for example, 25 or 37°C, preferably 20°C.

[0079] The present invention will be better understood, and other purposes, details, features, and advantages will become clearer, from the following description of some specific embodiments of the invention, which are given merely to illustrate without limitation, with reference to the accompanying drawings. [Examples]

[0080] Various experiments were conducted to evaluate the properties of the compound of formula (I).

[0081] Preferred compounds according to the present invention corresponding to general formula I, their synthesis and activity are described below:

[0082] [ka]

[0083] Other compounds not listed within the general formula range form an essential part of the compound according to the present invention.

[0084] [Example 1]: Synthesis of the analog compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX111) The first step is the synthesis of compound 3β-fluorocholestane, which includes the following steps:

[0085] [ka]

[0086] 5.00 g of diethylaminosulfur trifluoride (d=1.22 g / ml, 31.0 mmol) was dissolved in 200 ml of anhydrous DCM. 6.66 g of cholesterol (17.2 mmol) was dissolved in 100 ml of anhydrous dichloromethane and added dropwise to the fluoro reagent at 0°C. The resulting mixture was left to stand for 5 hours with magnetic stirring, during which time it was warmed to room temperature. After this period, the reactants were neutralized by adding 100 ml of saturated NaHCO3 solution. The mixture was transferred to a separatory funnel, and the organic phase was washed twice with saturated NaHCO3, twice with saturated NaCl solution, and once with water. The organic phase was dried over MgSO4, filtered, and then evaporated to obtain a white powder. 6.61 g equivalent to 3β-fluorocholestane was obtained. The final reaction yield was 99%.

[0087] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.40-5.39(d,1H),4.47-4.30(m,1H),2.45-2.42(t,2H),2.03-1 .95(m,3H),1.90-0.95(m,26H),0.92-0.91(d,3H),0.87-0.85(dd,6H),0.68(s,3H).

[0088] The second step involves synthesizing the compound 3β-fluoro-5,6α-epoxycholestane, starting from 3β-fluorocholestane, as follows.

[0089] [ka]

[0090] 4.96 g (22.1 mmol) of 77% pure meta-chloroperbenzoic acid was dissolved in 100 ml of dichloromethane and added dropwise to a mixture of 6.61 g (17.0 mmol) of 3β-fluorocholestane dissolved in 50 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed twice with an aqueous solution containing 10% by weight of Na2S2O3, twice with a saturated NaHCO3 solution, and with a saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. Vacuum evaporation of the organic solvent was performed to obtain 6.90 g of white powder containing 3β-fluoro-5,6α-epoxycholestane (85% of the white powder) and 3β-fluoro-5,6β-epoxycholestane (15% of the white powder). The 3β-fluoro-5,6α-epoxycholestane was used without further purification.

[0091] 1 H-NMR(500 MHz, CDCl3): δ(ppm)4.82-4.64(m,1H),2.91-2.90(d,1H),2.28-2.21(m,1H),2.10-2.06(m,1H ),1.97-1.70(m,6H),1.59-0.92(m,23H),0.89-0.88(d,3H),0.87-0.85(dd,6H),0.61(s,3H).

[0092] The third method involves synthesizing 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (the base form DX111) as follows.

[0093] [ka]

[0094] 0.80 g of histamine in its base form (7.2 mmol) was added to 10 ml of a butanol solution containing 1.45 g of compound 3β-fluoro-5,6α-epoxycholestane (3.6 mmol) while stirring at 130°C. The mixture was refluxed with stirring and heated at 130°C for 48 hours.

[0095] The conversion of 3β-fluoro-5,6α-epoxycholestane can be tracked by monitoring the progress of the reaction using thin-layer chromatography (TLC).

[0096] After cooling, the mixture was diluted with 15 ml of methyl tert-butyl ether. The organic phase was washed three times with 15 ml of water.

[0097] The organic phase was dried over anhydrous MgSO4, filtered, and then evaporated to obtain a brown oil. The mixture was purified by silica gel column chromatography in a purifier containing a 20 g packed column eluted with 100% ethyl acetate. 0.86 g of a white powder of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The final reaction yield, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 41%, and the purity was over 97%.

[0098] 1 H-NMR(500 MHz, CDCl3): δ(ppm)7.54(s,1H),6.80(s,1H),5.05-4.88(m,1H),3.03-2.96(m,1H),2.77-2.73(m,3H),2.46(s,1 H),2.27-2.20(q,1H),2.00-1.98(d,2H),1.86-0.94(m,31H),0.91-0.89(d,3H),0.87-0.85(d,6H),0.67(s,3H).

[0099] [Example 2]: Preparation of the dilactate salt (DX111 in dilactate form) of compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane:

[0100] The dilactate salt of compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was prepared by the following method.

[0101] [ka]

[0102] 267.2 mg of lactic acid (2.97 mmol) was added with stirring to a 15 ml solution of 0.76 g of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 1.03 g of a white powder of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0103] 1H-NMR(500 MHz,MeOD-4d):δ(ppm)7.58(s,1H),6.79(s,1H),4.73-4.57(m,1H),3.86-3.82(dd ,2H),3.35-3.31(dd,2H),3.18-3.13(m,1H),3.03-2.98(m,1H),2.77-2.75(t,2H) ,2.70(s,1H),2.12-2.05(dd,1H),1.78-1.76(d,1H),1.70-1.68(d,1H),1.63-0.8 5(m,30H),0.78-0.73(d,2H),0.68-0.66(d,3H),0.61-0.60(dd,6H),0.49(s,3H).

[0104] [Example 3]: Preparation of 3α-amino and 3α-sulfide derivatives or analogs of formula (I):

[0105] The process is as follows:

[0106] [ka]

[0107] Cholesterol is stirred in tetrahydrofuran (THF) in the presence of NaH for 5 minutes at 70°C, and p-toluenesulfonyl chloride (p-TsCl) is added. The mixture is stirred at 70°C for 4 hours. Water is added, the reaction mixture is filtered, and the organic solvent is evaporated. The reaction product is extracted with dichloromethane / water (DCM / H2O) and dried over MgSO4. The organic solvent is removed by vacuum evaporation. The obtained product is used directly in the next step. The obtained product is dissolved in THF while stirring with 1.1 equivalents of NuH (nucleophile - hydrogen) for 12 hours. NuH corresponds to R2SH or NHR2R3 at 70°C. The reactants are quenched by the addition of water, and the product is extracted using an siRNA / H2O system. The organic phase is dried over MgSO4, and the organic solvent is evaporated under vacuum. Cholestane 3-sulfide and cholestane 3-amino derivatives are purified by either column chromatography or recrystallization. The reaction pathway for obtaining the dendrogenin A analog is the same as the process developed for the synthesis of dendrogenin A.

[0108] The product R2O2S is obtained by oxidation of R2S with an oxidizing agent such as m-CPBA or H2O2.

[0109] Example 4: Preparation of 3β-amino and 3β-sulfide derivatives or analogs of formula (I): The process is as follows:

[0110] [ka]

[0111] Cholesterol is dissolved, Et3N in DCM is added, and mesyl chloride (MsCl) in the DCM solution is added dropwise at room temperature over 1 hour. The reaction mixture is stirred for 12 hours, then the organic solvent is evaporated and the product is crystallized from MeOH. The obtained product is a white solid. The obtained product is used to obtain 3β-sulfide and 3β-azide derivatives. The obtained product is dissolved in DCM, then TMS-SR2 for the 3β-sulfide derivative or TMS-N3 for the 3β-azide derivative is added to the solution. The addition of BF3*Et2O is carried out at room temperature. The mixture is then stirred for 3 hours.

[0112] The 3β-azide is reduced to 3β-amino by the action of LiAlH4 in Et2O, and then converted to the product of formula (I) by the reaction of R2X (X=Br, Cl, or I) in Et2O (or pyridine) as a solvent. The reaction pathway for obtaining the dendrogenin A analog is the same as the process developed for the synthesis of dendrogenin A. The sulfonyl derivative R2O2S is obtained by oxidizing R2S with a common oxidizing agent. This method is described in detail in the reference: Organic Letters, 2009, 11, 3, 567-570, "Practical Synthesis of 3β-Amino-5-cholestene and Related 3β-Halides Involving i-Steroid and Retro-i-Steroid Rearrangements" (https: / / doi.org / 10.1021 / ol802343z).

[0113] [Example 5]: Cytotoxicity study of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX111)

[0114] For this experiment, a cell culture medium was prepared. The culture medium consisted of Dulbecco's modified Eagle medium (DMEM, reference number LO BE12-604F, sold by Westburg) containing 4.5 g / L of glucose including L-glutamine, to which 10% fetal bovine serum (FCS) was added. Neuro2a (mouse neuroblastoma) cells were introduced into this culture medium.

[0115] Neuro2a cells were seeded at a rate of 10,000 cells per well in a 24-well dish. After 72 hours (h) of incubation under normal conditions, i.e., in a 37°C incubator with 5% CO2, the Neuro2a cells were treated with 100 nM, 1 μM, and 10 μM of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol for 48 hours. Controls (CTLs) were also carried out using the same protocol without treatment with 3α-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol. Cell viability was quantified by an automated trypan blue test using a Biorad TC20 machine (TC20® automated cell counter). The trypan blue test is based on the integrity of the cell membrane, which is destroyed in dead cells. Trypan blue stains dead cells blue. The Biorad TC20 cell counter counts the proportion of blue and non-blue cells and reports the cell proportions. The results are shown in Figure 1. Figure 1 shows the percentage of cell viability relative to the control group on the y-axis.

[0116] Figure 1 shows that, in response to treatment with 100 nM 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane, the percentage of viable cells remained unchanged compared to the control group (CTLs). Furthermore, at concentrations of 1 μM and 10 μM, cell viability was 75% and 30%, respectively. Similar activity was observed between the two test compounds. In conclusion, the cytotoxic activity of the compound 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane of formula (I) is observed against Neuro2a tumor cells at concentrations of 1 μM and 10 μM.

[0117] [Example 6]: Effect of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane on the viability of MCF-7 cells

[0118] A cell viability test was performed on MCF-7 (Michigan Cancer Foundation-7) mammary tumor cells (ER(+) cells) that overexpress HER2. MCF-7 cells were placed in the same cell culture medium as in Example 5 and seeded at 50,000 cells / well in 12-well plates. 24 hours after seeding, cells were treated with 1, 2.5, or 5 μM of vehicle solvates containing ethanol-water and ethanol (ethanol ratio 1‰), 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane, and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol. Cells were observed under an inverted microscope and photographed with a microscope camera at 24 and 48 hours. Morphological changes in cells at 1 μM were very small. Only a small number of white vesicles were observed, reflecting the initiation of autophagy, and cell death was induced after 24 hours of treatment with 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol. At 2.5 μM and 5 μM concentrations, this effect was more pronounced with increasing numbers of dead cells. Indeed, after 24 hours of treatment with 2.5 μM of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane, numerous white vesicles and detached cells were observed. After treatment with 5 μM 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane for 24 hours, 99% of the observed cells were supernatant, reflecting cell death, while 1% of the cells were adhesive and showed white vesicles. After treatment with 2.5 μM 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane for 48 hours, a stronger inhibitory effect on cell proliferation was observed than after 24 hours, with more cells becoming rounded, reflecting cell death. The inhibitory effect on cell proliferation is indicated by inhibition of cell proliferation. After treatment with 5 μM 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane for 48 hours, all cells were supernatant.Treatment with 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane showed a greater or equal effect to treatment with 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol after 24 hours of observation, and was comparable after 48 hours of observation.

[0119] Cell viability is measured by labeling with MTT at 48 hours. This test is based on the use of the tetrazolium salt MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide). Tetrazolium is reduced to formazan, a purple precipitate, by mitochondrial succinate dehydrogenase in active living cells. The amount of precipitate formed is proportional to the amount of living cells, but also to the metabolic activity of each cell. Therefore, a simple measurement of optical density at 540 nm by spectroscopy makes it possible to determine the relative amount of living cells to metabolically active cells. After 48 hours, the medium is aspirated, the cells are washed with phosphate-buffered saline (PBS), and then incubated with MTT (0.5 mg / ml in PBS) for approximately 2 hours. The MTT solution is aspirated, and the purple crystals are dissolved in dimethyl sulfoxide (DMSO). Optical density (OD) is measured at 540 nm.

[0120] The results of this test are shown in Figure 2. Figure 2 shows the percentage of cell viability relative to the control group on the y-axis. The control group was prepared in the same manner as the tested group, without the addition of the molecules tested in this text. Compared to the control, the dose-dependent decrease in cell viability in MTT was measured for 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol. At a concentration of 5 μM, viability was close to 0%. This reflects the ability of the compound of formula (I) to kill mammary tumor cells. These results are consistent with the observations made above at 24 and 48 hours.

[0121] [Example 7]: Effect of 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane on cholesterol epoxide hydrolase (ChEH) activity in MCF-7 cells

[0122] The compounds 5,6α-epoxycholesterol (5,6α-EC) and 5,6β-epoxycholesterol (5,6β-EC) are oxysterols involved in the anticancer pharmacology of tamoxifen, a widely used antitumor drug. Both are metabolized by the enzyme cholesterol-5,6-epoxydohydrolase (ChEH) to cholestane-3β,5α,6β-triol (CT), which is then metabolized by the enzyme HSD11B2 (11β-hydroxysteroid dehydrogenase 2) to the protumorogenic oncosterone 6-oxocholestane-3β,5α-diol (OCDO).

[0123] The objective of the following experiment is to demonstrate the ability to block ChEH of 3α-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and thus limit the metabolism of oncosterone, a tumor-promoting metabolite.

[0124] MCF-7 cells were placed in the same cell culture medium as in Example 5, seeded at 150,000 cells / well in a 6-well plate, with 3 wells per treatment condition. 24 hours after seeding, the MCF-7 cells were [ 14 Treat with C]5,6α-EC (1000 × stock solution: 0.6 mM; 20 μCi / μmol; final concentration 0.6 μM) alone or in combination with tamoxifen (tam). Tamoxifen is used as a positive control for 3α-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane and 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol (1 μM for all molecules).

[0125] After 24 hours of treatment, the culture medium was collected, and lipid extracts were prepared from the cell pellet by extraction with 100 μL of chloroform, 400 μL of methanol, and 300 μL of water. The lipid extracts were analyzed by thin-layer chromatography (TLC) using ethyl acetate (siRNA) as the eluent. The analysis was performed by plate reader and then by autoradiography. The results are shown in Figure 3. Nearly complete metabolism from epoxide to CT and OCDO was observed (wells 2 and 4), complete inhibition of ChEH activity by tamoxifen and near-complete inhibition by 3α-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (trace of CT) was observed. Similar results were observed using 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol.

[0126] In conclusion, 3α-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane has similar ChEH inhibitory activity to 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol.

[0127] [Example 8]: Synthesis of compound 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX103) of formula (I)

[0128] [ka]

[0129] The first step involved dissolving 4.0 grams (g) of cholesterol (10.3 mmol) in 20 milliliters (ml) of tetrahydrofuran (THF). 0.80 g of NaH (60% in the oil, 20.0 mmol) was added and the mixture was reacted at 60°C for 30 minutes, followed by the addition of 1.8 ml (28.9 mmol) of iodomethane. The resulting mixture was left standing at 60°C overnight, i.e., for about 10 hours. After cooling the solution, the reaction was neutralized by adding 20 ml of water. The mixture was filtered, and the THF was evaporated under vacuum. The mixture was transferred to a separatory funnel, and the aqueous phase was extracted three times with ethyl acetate. The resulting organic phases were combined, dried over MgSO4, and then evaporated to obtain oil. The obtained oil was dissolved in 2 ml of Et2O, and MeOH was added until a white precipitate formed. The powder was filtered off, washed with cold MeOH, and dried. This yielded 3.40 g of 3β-methoxycholestane (corresponding to a yield of 82%) as a white powder.

[0130] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.36(s,1H),3.35(s,3H),3.09-3.02(q,1H),2.40-2.36(d,1H),2.18-2.13(t ,1H),2.03-1.81(m,5H),1.60-1.00(m,24H),0.92-0.91(d,3H),0.87-0.85(dd,6H),0.68(s,3H).

[0131] The second step involves synthesizing the compound 3β-methoxy-5,6α-epoxycholestane, starting from 3β-methoxycholestane, as follows.

[0132] [ka]

[0133] 1.80 g (8.90 mmol) of meta-chloroperbenzoic acid was dissolved in 70 ml of dichloromethane and added dropwise to a mixture of 2.50 g (6.24 mmol) of 3β-methoxycholestane dissolved in 20 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed with an aqueous solution containing 10 wt% Na2S2O3, a saturated NaHCO3 solution, and a saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The organic solvent was evaporated under vacuum to obtain a clear, viscous oil. 5 ml of Et2O was added to dissolve the oil, then 25 ml of EtOH was added, and the mixture was heated three times to its boiling point, and finally kept at 0°C overnight to promote precipitation. The white powder was filtered off, washed with cold MeOH, and dried. In this way, 1.73 g of 3β-methoxy-5,6α-epoxycholestane was obtained, corresponding to a yield of 67% (enantiomer excess ≥90%).

[0134] 1 H-NMR(500 MHz, CDCl3): δ(ppm)3.45-3.39(m,1H),3.33(s,3H),2.90-2.89(d,1H),2.00-0.94(m,31H),0.89-0.88(d,3H),0.86-0.85(dd,6H),0.60(s,3H).

[0135] The third step involves synthesizing 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (DX103 base form) as follows.

[0136] [ka]

[0137] 0.81 g of histamine (7.30 mmol) in its base form was added with stirring to 10 ml of a butanol solution containing 1.50 g of compound 3β-methoxy-5,6α-epoxycholestane (3.62 mmol). The mixture was refluxed with stirring and heated at 130°C for 48 hours. The conversion of 3β-methoxy-5,6α-epoxycholestane can be tracked by monitoring the progress of the reaction using thin-layer chromatography (TLC). After cooling, the mixture was diluted with 10 ml of methyl tert-butyl ether. The organic phase was washed twice with 10 ml of water, and then once with 10 ml of saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The mixture was purified by column chromatography using a purifier. The eluent used was a 90% / 10% mixture of ethyl acetate and methanol. 1.32 g of a white powder of 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The final reaction yield, measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 69%, and the purity was over 95%.

[0138] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.62(s,1H),6.88(s,1H),3.71-3.65(m,1H),3.34(s,3H),2.98-2.97(d,1H),2.78-2.77(m,3H),2 .45(s,1H),2.03-2.00(m,1H),1.94-1.83(m,3H),1.65-1.01(m,27H),0.95-0.94(d,3H),0.91-0.89(d,6H),0.71(s,3H).

[0139] [Example 9]: Preparation of the dilactate salt (DX103 dilactate form) of compound 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0140] The dilactate salt of compound 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was prepared as follows.

[0141] [ka]

[0142] 21.0 mg of lactic acid (1.89 mmol) was added with stirring to a 15 ml solution of 0.50 g (0.95 mmol) of 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 0.52 g of a white powder of 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0143] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.61(s,1H),6.84(s,1H),3.93-3.89(q,2H),3.62-3.57(m,1H),3.39-3.09(m,8H),3.21-3.16 (m,1H),2.84-2.74(m,3H),1.91-1.81(m,2H),1.70-0.79(m,31H),0.73-0.72(d,3H),0.68-0.66(d,6H),0.56(s,3H).

[0144] [Example 10]: Synthesis of compound 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX105) of formula (I) The first step is the synthesis of compound 3β-ethoxycholestane, which includes the following steps:

[0145] [ka]

[0146] 4.00 g of cholesterol (10.3 mmol) was dissolved in 20 ml of THF. 0.82 g of NaH (60% of the oil, 20.0 mmol) was added and the mixture was reacted at 60°C for 30 minutes, then 1.9 ml of iodoethane (28.9 mmol) was added. The resulting mixture was left overnight at 60°C. After cooling the solution, the reaction was neutralized by adding 20 ml of water. The mixture was filtered, and the THF was evaporated under vacuum. The mixture was transferred to a separatory funnel, and the aqueous phase was extracted three times with ethyl acetate. The resulting organic phases were combined, dried over MgSO4, and then evaporated to obtain oil. The resulting oil was dissolved in 2 ml of Et2O, and MeOH was added until a white precipitate formed. The powder was filtered off, washed with cold MeOH, and dried. This yielded 2.12 g of 3β-ethoxycholestane (corresponding to a yield of 49%) in a white powder.

[0147] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.35(s,1H),3.53-3.51(q,2H),3.17-3.14(m,1H),2.38-2.35(d,1H),2.22-2.1 7(t,3H),2.02-1.79(m,5H),1.60-0.94(m,27H),0.92-0.91(d,3H),0.87-0.85(dd,6H),0.67(s,3H).

[0148] The second step involves synthesizing the compound 3β-ethoxy-5,6α-epoxycholestane, starting from 3β-ethoxycholestane, as follows.

[0149] [ka]

[0150] 1.44 g (equivalent to 6.43 mmol) of meta-chloroperbenzoic acid was dissolved in 50 ml of dichloromethane and added dropwise to a mixture of 2.0 g (4.82 mmol) of 3β-ethoxycholestane dissolved in 10 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed with an aqueous solution containing 10 wt% Na2S2O3, a saturated NaHCO3 solution, and a saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The organic solvent was evaporated under vacuum to obtain a clear, viscous oil. 5 ml of Et2O was added to dissolve the oil, then 25 ml of EtOH was added, and the mixture was heated three times to its boiling point, then kept at 0°C overnight to promote precipitation. The white powder was filtered off, washed with cold MeOH, and dried. In this way, 0.72 g of 3β-ethoxy-5,6α-epoxycholestane was obtained, corresponding to a 35% yield (enantiomer excess ≥90%).

[0151] 1 H-NMR (500 MHz, CDCl3): δ(ppm)3.55-3.46(m,3H),2.89-2.88(d,1H),2.04-0.93(m,34H),0.89-0.88(d,3H),0.86-0.85(dd,6H),0.60(s,3H).

[0152] The third step involves synthesizing 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (base form DX105) as follows.

[0153] [ka]

[0154] 0.31 g of histamine in its base form (equivalent to 2.74 mmol) was added with stirring to 5 ml of a butanol solution containing 0.51 g of compound 3β-ethoxy-5,6α-epoxycholestane (1.18 mmol). The mixture was refluxed with stirring and heated at 130°C for 48 hours. The conversion of 3β-ethoxy-5,6α-epoxycholestane can be tracked by monitoring the progress of the reaction using thin-layer chromatography (TLC). After cooling, the mixture was diluted with 5 ml of methyl tert-butyl ether. The organic phase was washed twice with 5 ml of water, and then once with 5 ml of saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The mixture was purified by column chromatography using a purifier. The eluent used was a 90 / 10 ethyl acetate / methanol mixture. 0.28 g of a white powder of 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The final reaction yield, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 44%, and the purity was over 97%.

[0155] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.62(s,1H),6.89(s,1H),3.82-3.76(m,1H),3.57-3.52(q,2H),3.05-3.00(m,1H),2.85-2.80(m,3H) ,2.50(s,1H),2.03-1.83(m,5H),1.65-1.51(m,7H),1.42-1.01(m,22H),0.96-0.94(d,3H),0.91-0.89(d,6H),0.72(s,3H).

[0156] [Example 11]: Preparation of the dilactate salt (DX105 in dilactate form) of compound 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0157] The dilactate salt of compound 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was prepared by the following method.

[0158] [ka]

[0159] 166.2 mg of lactic acid (1.85 mmol) was added with stirring to a 5 ml solution of 0.50 g (0.92 mmol) of 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 0.20 g of a white powder of 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0160] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.61(s,1H),6.84(s,1H),3.92-3.89(q,2H),3.60-3.57(m,1H),3.39-3.09(m,7H),2 .84-2.74(m,3H),1.91-1.81(m,2H),1.70-0.79(m,34H),0.73-0.72(d,3H),0.67-0.65(d,6H),0.54(s,3H).

[0161] [Example 12]: Synthesis of compound 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX115) of formula (I) The first step is the synthesis of compound 3β-octanoxycholestane, which includes the following steps.

[0162] [ka]

[0163] 4.00 g of cholesterol was dissolved in 20 ml of tetrahydrofuran. 0.84 g of NaH was added and the mixture was reacted at 60°C for 30 minutes, followed by the addition of 3.0 g of isooctane. The resulting mixture was left overnight at 60°C. After cooling the solution, the reaction was neutralized by adding 20 ml of water. The mixture was filtered, and the THF was evaporated under vacuum. The mixture was transferred to a separatory funnel, and the aqueous phase was extracted three times with ethyl acetate. The resulting organic phases were combined, dried over MgSO4, and then evaporated to obtain oil. The obtained oil was dissolved in 2 ml of Et2O, and MeOH was added until a white precipitate formed. The powder was filtered off, washed with cold MeOH, and dried. This yielded a white powder containing 2.5 g (equivalent to 48%) of 3β-octanoxycholestane.

[0164] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.35(s,1H),3.45-3.43(q,2H),3.15-3.10(q,1H),2.37-2.35(d,1H),2.21-2.16(t,1H), 2.02-1.95(m,2H),1.90-1.84(m,3H),1.58-0.97(m,39H),0.92-0.91(d,3H),0.87-0.86(dd,6H),0.67(s,3H).

[0165] The second step involves synthesizing the compound 3β-octanoxy-5,6a-epoxycholestane, starting from 3β-octanoxycholestane, as follows.

[0166] [ka]

[0167] 0.90 g (equivalent to 4.0 mmol) of meta-chloroperbenzoic acid was dissolved in 40 ml of dichloromethane and added dropwise to a mixture of 1.50 g (3.0 mmol) of 3β-octanoxycholestane dissolved in 10 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed with an aqueous solution containing 10 wt% Na2S2O3, a saturated NaHCO3 solution, and a saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The organic solvent was evaporated under vacuum to obtain a clear, viscous oil. 5 ml of Et2O was added to dissolve the oil, then 25 ml of MeOH was added, and the mixture was heated three times to its boiling point, and finally kept at 0°C overnight to promote precipitation. The white powder was filtered off, washed with cold MeOH, and dried. In this way, 1.19 g of 3β-octanoxy-5,6a-epoxycholestane was obtained, corresponding to a yield of 77% (enantiomer excess ≥90%).

[0168] 1 H-NMR(500 MHz, CDCl3):δ(ppm)3.51-3.37(m,3H),2.88-2.87(d,1H),2.02-1.87(m,4H),1.84- 1.76(m,1H),1.69-1.67(m,1H),1.58-1.45(m,7H),0.89-0.88(m,42H),0.60(s,3H).

[0169] The third step involves synthesizing 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (the base form DX115) as follows.

[0170] [ka]

[0171] 0.48 g of histamine in its base form (equivalent to 4.31 mmol) was added with stirring to 10 ml of a butanol solution containing 1.1 g of compound 3β-octanoxy-5,6α-epoxycholestane (2.14 mmol). The mixture was refluxed with stirring and heated at 130°C for 48 hours. The conversion of 3β-octanoxy-5,6α-epoxycholestane can be tracked by monitoring the progress of the reaction using thin-layer chromatography (TLC). After cooling, the mixture was diluted with 10 ml of methyl tert-butyl ether. The organic phase was washed twice with 10 ml of water, and then once with 10 ml of saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The mixture was purified by column chromatography using a purifier. The eluent used was a 95 / 5 ethyl acetate / methanol mixture. 0.74 g of a white powder of 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The final reaction yield, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 55%, and the purity was over 95%.

[0172] 1 H-NMR(500 MHz, MeOD-4d): δ(ppm)7.59(s,1H),6.86(s,1H),3.79-3.74(q,1H),3.49-3.47(q,2H),2.95-2.90(m,1H),2.78-2. 70(m,3H),2.40(s,1H),2.01-1.84(m,5H),1.62-1.54(m,9H),1.39-1.02(m,30H),0.95-0.89(d,12H),0.70(s,3H).

[0173] [Example 13]: Preparation of the dilactate salt (DX115 in dilactate form) of compound 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0174] The dilactate salt of compound 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was prepared by the following method.

[0175] [ka]

[0176] 166.2 mg of lactic acid (1.85 mmol) was added with stirring to a 5 ml solution of 0.57 g (0.92 mmol) of 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 0.59 g of a white powder of 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0177] 1 H-NMR (500 MHz,MeOD-4d):δ(ppm)7.71(s,1H),6.94(s,1H),4.02-3.98(q,2H),3.72-3.6 5(q,1H),3.41-3.31(m,3H),3.21-3.16(m,1H),2.95-2.92(t,2H),2.86-2.85 (d,1H),2.04-1.99(t,1H),1.96-1.93(d,1H),1.83-1.59(m,7H),1.49-1.04( m,38H),0.98-0.89(m,2H),0.85-0.84(d,3H),0.81-0.77(m,9H),0.66(s,3H).

[0178] [Example 14]: Synthesis of compound 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (named DX123)

[0179] The first step is the synthesis of compound 3-mesylcholestane, which includes the following steps:

[0180] [ka]

[0181] 40 g of cholesterol (0.1 mol) and 22 ml of Et3N (d=0.88 g / ml, 0.19 mol) were dissolved in 340 ml of anhydrous dichloromethane in a 1 L flask at 0°C. 10 ml of methanesulfonyl chloride (1.48 g / ml, 0.13 mol) was dissolved in 40 ml of anhydrous dichloromethane and added dropwise to the cholesterol solution. The resulting mixture was left overnight under magnetic stirring and then warmed to room temperature. After this time, the reaction was monitored by TLC and concentrated under vacuum to 2 / 3 of the initial volume. The addition of 500 ml of MeOH produced 46.4 g of a white precipitate corresponding to the desired product (97% yield).

[0182] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.42-5.41(d,1H),4.55-4.49(q,1H),3.00(s,3H),2.56-2.45(m,2H),2.05-1.9 6(m,3H),1.92-1.75(m,3H),1.60-0.93(m,23H),0.92-0.90(d,3H),0.87-0.85(dd,6H),0.67(s,3H).

[0183] The second step involves synthesizing the compound 3β-azidocholestane, starting from 3β-mesylcholesterol, as follows.

[0184] [ka]

[0185] The following were added sequentially to a 500 ml flask at room temperature: 23.27 g of 3β-mesylcholesterol (50.1 mmol), 100 ml of anhydrous dichloromethane, 7.5 ml of trimethylsilyl azide (d=0.868 g / ml, 56.5 mmol), and finally 12.5 ml of boron trifluoride diethyl etherate (d=1.15 g / ml, 101.3 mmol). The resulting mixture was then magnetically stirred for 3 hours. After this period, the reaction mixture was neutralized by adding 100 ml of 2 M NaOH solution. The organic product was extracted twice with dichloromethane. The organic phases were combined and rinsed twice with saturated NaCl solution. The organic phases were dried over MgSO4, filtered, and then evaporated to obtain a solid. The crude reaction product was purified by column chromatography eluting with 100% hexane. This yielded 13.33 g of a yellowish-white powder corresponding to 3β-azidocholestane. The final reaction yield was 65%.

[0186] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.39-5.38(d,1H),3.23-3.17(q,1H),2.30-2.28(d,2H),2.03-1.97(m,2H ),1.91-1.81(m,3H),1.60-0.94(m,24H),0.92-0.91(d,3H),0.87-0.86(dd,6H),0.68(s,3H).

[0187] The third synthesis step involves starting with 3β-azidocholestane and synthesizing the compound 3β-azido-5,6α-epoxycholestane as follows.

[0188] [ka]

[0189] 950 mg (4.24 mmol) of 77% pure meta-chloroperbenzoic acid was dissolved in 15 ml of dichloromethane, and this was added dropwise to a solution of 1.3 g (3.16 mmol) of 3β-azidocholestane dissolved in 15 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed twice with 10 wt% Na2S2O3 aqueous solution, twice with saturated NaHCO3 solution, and once with saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The organic solvent was evaporated under vacuum to obtain 1.35 g of white powder corresponding to a mixture of 3-azido-5,6α-epoxycholestane (83% of the total) and 3β-azido-5,6β-epoxycholestane (17% of the white powder). The final product was used without further purification.

[0190] 1 H-NMR(500 MHz, CDCl3):δ(ppm)3.63-3.56(q,1H),2.94-2.93(d,1H),2.13-2.08(t,1H ),1.97-0.94(m,30H),0.89-0.88(d,3H),0.86-0.85(dd,6H),0.61(s,3H).

[0191] The fourth step is the synthesis of 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (the neutral form of DX123) as follows.

[0192] [ka]

[0193] 864 mg of histamine in its base form (7.77 mmol) was added to 20 ml of a butanol solution containing 2.02 g of 83% compound 3β-azido-5,6α-epoxycholestane (3.9 mmol) while stirring at 130°C. The mixture was refluxed with stirring and heated at 130°C for 48 hours. The conversion of 3β-azide-5,6α-epoxycholestane can be tracked by monitoring the progress of the reaction using thin-layer chromatography (TLC). After cooling, the mixture was diluted with 15 ml of methyl tert-butyl ether. The organic phase was washed three times with 15 ml of water. The organic phase was dried over anhydrous MgSO4, filtered, and then evaporated to obtain a brown oil. The mixture was purified by silica gel column chromatography in a purifier containing a 40 g pre-packed column eluted with 75 / 25% to 0 / 100% dichloromethane / ethyl acetate. 890 mg of a white powder of 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The final reaction yield, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 42%, and the purity was over 97%.

[0194] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.55(s,1H),6.81(s,1H),3.73-3.67(q,1H),2.90-2.85(m,1H),2.72-2.62(m,3H),2.33(s,1H),2.05-2.00(t, 1H),1.96-1.94(m,1H),1.84-1.77(m,1H),1.74-1.72(m,1H),1.62-0.97(m,27H),0.89-0.88(d,3H),0.85-0.84(d,6H),0.64(s,3H).

[0195] [Example 15]: Preparation of the dilactate salt (DX123 dilactate form) of compound 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0196] [ka]

[0197] 63.5 mg of lactic acid (0.77 mmol) was added with stirring to a 4 ml solution of 210 mg of 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 263.5 mg of a white powder of 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0198] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.67(s,1H),6.92(s,1H),4.01-3.97(m,2H),3.73-3.67(q ,1H),3.34-3.29(m,1H),3.19-3.13(m,1H),2.91-2.88(t,2H),2.81(s,1H),2.20 -2.15(t,1H),1.94-1.92(d,1H),1.77-1.75(m,3H),1.66-1.58(m,4H),1.47-0.9 8(m,26H),0.95-0.87(m,2H),0.83-0.82(d,3H),0.77-0.76(dd,6H),0.65(s,3H).

[0199] [Example 16]: Synthesis of the trichloride salt (DX125 in trichloride form) of compound 3β-amino-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0200] The reaction for synthesizing the trichloride salt of 3β-amino-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane from 3β-azido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane is as follows:

[0201] [ka]

[0202] A solution of 300 mg (0.56 mmol) of 3β-azido-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane in 8.0 ml of THF was added with 730 mg (2.8 mmol) of triphenylphosphine while stirring at 70 °C. The mixture was refluxed with stirring and heated at 70 °C for 2 hours. Then, 0.5 ml of water (equivalent to 2,78 mmol) was added and stirring was continued at 70 °C for another 2 hours. The progress of the reaction was monitored by thin layer chromatography (TLC), and then the solvent mixture was evaporated. The resulting white powder was dissolved in 20 ml of dichloromethane and transferred to a separating funnel containing 20 ml of aqueous HCl solution (1 ml of 37% HCl in 19 ml of water). The aqueous phase was washed three times with dichloromethane. The aqueous phase was dried under vacuum to obtain a white powder. The powder in dichloromethane was taken up and finally filtered to remove the last trace amount of triphenylphosphine. By this procedure, 350 mg of the trichloride salt of 3β-amino-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane was obtained in quantitative yield and with a purity of over 95%.

[0203] 1 H-NMR (500 MHz, MeOD-4d): δ (ppm) 8.90 (s, 1H), 7.56 (s, 1H), 3.67 - 3.60 (q, 1H), 3.56 - 3.41 (m, 4H), 3.28 - 3.27 (d, 1H), 2.61 - 2.56 (t, 1H), 2.08 - 2.05 (d, 1H), 1.99 - 1.11 (m, 28H), 1.06 - 1.00 (dd, 1H) 0.96 - 0.94 (d, 3H), 0.89 - 0.88 (dd, 6H), 0.78 (s, 3H).

[0204] [Example 17]: Synthesis of the compound 3β-acetamido-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX127)

[0205] The first synthetic step is the reduction of the azide group at the 3-position of the cholestane 3β-azide derivative to an amine.

[0206] [Chemical formula]

[0207] 5.21 g (12.7 mmol) of cholestan-3β-azide was dissolved in 60 ml of tetrahydrofuran (THF), and then a total of 2.32 g (61.1 mmol) of LiAlH4 was added in portions of about 480 mg five times at intervals of 15 minutes. The mixture thus obtained was stirred magnetically for 3 hours. After this period, the reaction was quenched by adding a few drops (added gently) of 5% aqueous Na2CO3 solution. The organic phase was extracted three times with EtOAc, and the organic phases were combined. The combined organic phase was dried over MgSO4, filtered, and then evaporated to give a solid. In this way, 3.78 g of a sufficiently pure white powder corresponding to 3β-aminocholestane was obtained. The final reaction yield was 77%.

[0208] 1 1H-NMR (500 MHz, CDCl3): δ (ppm) 5.32 - 5.31 (d, 1H), 2.63 - 2.57 (q, 1H), 2.17 - 2.13 (m, 1H), 2.08 - 1.93 (m, 4H), 1.85 - 1.81 (m, 2H), 1.72 - 1.68 (m, 1H), 1.43 - 0.84 (m, 33H), 0.68 (s, 3H).

[0209] The second step consists of synthesizing the compound cholestan-3β-acetamide starting from 3-aminocholestane as follows.

[0210]

Chemical formula

[0211] 3.78 g (9.8 mmol) of 3β-aminocholestane was dissolved in 20 ml of anhydrous dichloromethane, and then 16 ml (198 mmol) of anhydrous pyridine and 5.0 g (49.0 mmol) of acetic anhydride were added to the reaction mixture. The resulting mixture was stirred and kept at room temperature overnight. The mixture was washed three times with 0.1 M aqueous HCl solution, the organic phase was dried over anhydrous MgSO4, filtered, and dried under vacuum. The resulting oil was dissolved in 30 ml of chloroform, 90 ml of MeOH was added, and the mixture was heated three times to boiling point until the volume of the solvent decreased by 2 / 3, and finally maintained at 0°C to promote precipitation. A white powder was obtained, which was filtered off, washed with cold MeOH, and dried. In this way, 2.41 g of cholestane 3β-acetamide was obtained, corresponding to a yield of 58%.

[0212] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.36-5.35(d,1H),5.32-5.30(d,1H),3.73-3.65(q,1H),2.32-2.29(d,1H ),2.09-1.79(m,9H),1.60-0.95(m,22H),0.92-0.90(d,3H),0.87-0.85(dd,6H),0.67(s,3H).

[0213] The third step involves synthesizing 5,6-epoxycholestane 3β-acetamide as follows.

[0214] [ka]

[0215] 1.19 g (5.3 mmol) of 77% pure meta-chloroperbenzoic acid was dissolved in 10 ml of dichloromethane and added dropwise to a mixture of 1.61 g (3.8 mmol) of cholestane 3β-acetamide dissolved in 25 ml of dichloromethane. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed twice with an aqueous solution containing 10% by weight of Na2S2O3, and twice with saturated NaHCO3 solution and saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. Vacuum evaporation of the organic solvent was performed to obtain 1.65 g of white powder containing 5,6α-epoxycholestane 3β-acetamide (60% of the white powder) and 5,6β-epoxycholestane 3β-acetamide (40% of the white powder). 5,6α-epoxycholestane 3β-acetamide was used without further purification.

[0216] 1 H-NMR (500 MHz, CDCl3): δ(ppm)5.29-5.28(d,1H),4.05-3.99(q,1H),2.89-2.88(d,1H),2.08-0.84(m,43H),0.60(s,3H).

[0217] The fourth step involves synthesizing 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane 3β-acetamide (the neutral form of DX127) as follows.

[0218] [ka]

[0219] 0.47 g of histamine, in its base form (equivalent to 4.26 mmol), was added with stirring to 20 ml of a butanol solution containing 1.65 g of compound 5,6α-epoxycholestane 3β-acetamide, equivalent to 0.99 mmol. The mixture was refluxed with stirring and heated at 130°C for 48 hours. The reaction could be monitored by thin-layer chromatography (TLC) to track the conversion of 5,6α-epoxycholestane 3β-acetamide. After cooling, the mixture was diluted in 20 ml of methyl tert-butyl ether. The organic phase was washed twice with 20 ml of water, then three times with 20 ml of saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The mixture was purified by column chromatography using a purifier. The eluent used was a 75 / 20 / 5% dichloromethane / methanol / ammonia mixture. 0.37 g of a white powder of 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane 3β-acetamide was obtained. The final reaction yield, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography), was 30%, and the purity was over 97%.

[0220] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.56(s,1H),6.81(s,1H),4.15-4.08(q,1H),2.91-2.88(m,1H),2.74-2.68(m,3H),2.35(s, 1H),1.99-1.94(m,2H),1.87-1.77(m,5H),1.66-0.97(m,28H),0.90-0.88(d,3H),0.85-0.84(d,6H),0.65(s,3H).

[0221] [Example 18]: Preparation of the dilactate salt (DX127 in dilactate form) of compound 3β-acetamido-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0222] The d-lactate salt of the compound 3β-acetamido-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane was prepared by the following method.

[0223] [Chemical formula]

[0224] 120.6 mg of lactic acid (1.34 mmol) was added with stirring to a 5 ml solution of 370 mg of 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane 3β-acetamide in absolute ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated in vacuo, 490 mg of a white powder of 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane 3β-acetamide d-lactate salt was obtained.

[0225] 1 H-NMR (500 MHz, MeOD-4d): δ (ppm) 7.69 (s, 1H), 6.91 (s, 1H), 4.08 - 4.03 (m, 1H), 3.37 - 3.27 (m, 1H), 3.18 - 3.12 (m, 2H), 2.91 - 2.88 (t, 2H), 2.77 (s, 1H), 2.07 - 2.02 (t, 1H), 1.93 - 1.90 (d, 1H), 1.79 (s, 3H), 1.76 - 0.88 (m, 36H), 0.82 - 0.81 (d, 3H), 0.75 - 074 (dd, 6H), 0.63 (s, 3H).

[0226] [Example 19]: Synthesis of the compound 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestane (named DX129)

[0227] The first step consists of synthesizing the compound 3β-methylthiocolestane starting from 3β-mesylcholesterol as follows.

[0228] [Chemical formula]

[0229] In a 500 ml flask, 10.62 g (22.9 mmol) of 3-mesylcholesterol, 50 ml of dichloromethane, 5.0 g (41.6 mmol) of trimethyl(methylthio)silane, and 8.0 ml (d=1.15 g / ml, 64.8 mmol) of boron trifluoride diethyl etherate were added sequentially at room temperature. The resulting mixture was then magnetically stirred for 3 hours. After this period, the reaction mixture was neutralized by adding 100 ml of 2 M NaOH solution. The organic phase was extracted twice with dichloromethane. The organic phases were combined and rinsed twice with saturated NaCl solution. The organic phase was dried over MgSO4, filtered, and then evaporated to obtain a solid. The crude reaction product was purified by silica gel column chromatography, which eluted with 100% hexane. In this way, 6.04 g of a white powder corresponding to 3β-methylthiocholestane was obtained. The final reaction yield was 63%.

[0230] 1 H-NMR(500 MHz, CDCl3):δ(ppm)5.33(s,1H),2.71-2.65(m,1H),2.30-2.26(m,2H),2.11(s ,3H),2.02-0.94(m,29H),0.92-0.91(d,3H),0.87-0.85(dd,6H),0.67(s,3H).

[0231] The second synthesis step involves starting with 3β-methylthiocholestane and synthesizing the compound 3β-methylsulfonyl-5,6-epoxycholestane as follows.

[0232] [ka]

[0233] 6.30 g (28.1 mmol) of 77% pure meta-chloroperbenzoic acid was dissolved in 40 ml of dichloromethane, and a solution of 2.9 g (6.8 mmol) of 3-methylthiocholestane in 20 ml of dichloromethane was added dropwise. The resulting mixture was stirred and kept at room temperature for 3 hours. The resulting mixture was washed twice with 10 wt% aqueous Na2S2O3, three times with saturated NaHCO3 solution, and once with saturated NaCl solution. The organic phase was dried over anhydrous MgSO4. The organic solvent was evaporated under vacuum to obtain 2.10 g of white powder. The crude reaction product was purified by column chromatography, first eluting with 100% hexane, then with a mixture of hexane and siRNA. The desired product was purified by silica gel column chromatography, eluting with 55% / 45% hexane / siRNA. In this way, 380 mg of white powder corresponding to 3-methylthio-5,6-epoxycholestane was obtained. The final reaction yield was 12%.

[0234] 1 H-NMR(500 MHz, CDCl3):δ(ppm)3.25-3.20(m,1H),3.02-3.01(d,1H),2.83(s,3H),2.11-2.09(m,1H),1.98-1.9 3(m,3H),1.87-1.79(m,3H),1.57-0.93(m,24H),0.89-0.88(d,3H),0.86-0.85(dd,6H),0.61(s,3H).

[0235] The third step is the synthesis of 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane (base form DX129) as follows.

[0236] [ka]

[0237] 338 mg of histamine in its base form (3.04 mmol) was added to 5 ml of a butanol solution containing 350 mg of the compound 3-methylsulfonyl-5,6-epoxycholestane (0.75 mmol) while stirring at 130°C. The mixture was refluxed with stirring and heated at 130°C for 48 hours. The progress of the reaction could be monitored by thin-layer chromatography (TLC) to track the conversion of 3-methylsulfonyl-5,6-epoxycholestane. After cooling, the mixture was diluted with 5 ml of methyl tert-butyl ether. The organic phase was washed three times with 15 ml of saturated sodium chloride. The organic phase was dried over anhydrous MgSO4, filtered, and then evaporated to obtain a brown oil. The crude reaction product was purified by column chromatography, first eluting with 100% Â, then with an  / MeOH mixture. The desired product was purified with a 75% / 25%  / MeOH mixture. 190 mg of yellow powder equivalent to 3-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane was obtained. The product was purified a second time by column chromatography to obtain a purity of over 97%, as measured by NMR (nuclear magnetic resonance) and TLC (thin-layer chromatography) analysis.

[0238] 167.4 mg of white powder was obtained. The final reaction yield was 39%.

[0239] 1 H-NMR(500 MHz,MeOD-4d):δ(ppm)7.63(s,1H),6.89(s,1H),3.49-3.40(m,1H),3.04-3.02(m,1H),2.89(s,3H),2.81-2.78(m,3H),2.50(s,1H),2.44-2 .40(t,1H),2.02-2.01(m,1H),1.94-1.92(m,1H),1.88-1.83(m,1H),1 .76-1.00(m,30H),0.90-0.89(d,3H),0.85-0.84(d,6H),0.66(s,3H).

[0240] [Example 20]: Preparation of the dilactate salt (DX129 in dilactate form) of compound 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane

[0241] [ka]

[0242] 51.6 mg of lactic acid (1.34 mmol) was added with stirring to a 5 ml solution of 165.0 mg of 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane in anhydrous ethanol. Stirring was continued at room temperature for 3 hours. When the organic solvent was evaporated under vacuum, 216.6 mg of a white powder of 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane dilactate salt was obtained.

[0243] 1 H-NMR (500 MHz,MeOD-4d):δ(ppm)7.77(s,1H),6.98(s,1H),3.52-3.47(m,1H),3.45-3.4 1(m,1H),3.18-3.14(m,1H),2.96-2.94(t,2H),2.89-2.87(m,4H),2.59-2.55 (t,1H),2.02-2.00(m,1H),1.97-1.95(m,1H),1.86-1.80(m,2H),1.75-1.65( m,5H)1.57-0.95(m,29H),0.90-0.89(d,3H),0.84-0.82(dd,6H),0.72(s,3H).

[0244] [Example 21]: Pharmacokinetic study of DX103

[0245] The following study is an LC / MS assay of various molecules in plasma over three days (ultimately 11 measurement points). Graphs are always shown in comparison to the DX101 reference.

[0246] [Table 1]

[0247] Plasma sampling at 0 (not injected), 5, 10, 15, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours (at point 11).

[0248] Figure 4 shows the pharmacokinetic profiles of DX103 compared to DX101. The results are as follows:

[0249] [Table 2]

[0250] Conclusion: The DX103 profile shows faster absorption into the body and slightly lower bioavailability compared to DX101.

[0251] [Example 22]: Pharmacokinetic study of DX105

[0252] The following study is an LC / MS assay of various molecules in plasma over three days (ultimately 11 measurement points). Graphs are always shown in comparison to the DX101 reference.

[0253] [Table 3]

[0254] Plasma sampling at 0 (not injected), 5, 10, 15, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours (at point 11).

[0255] Figure 5 shows the pharmacokinetic profiles of DX105 compared to DX101. The results are as follows:

[0256] [Table 4]

[0257] DX105 exhibits bioavailability comparable to (or slightly higher than) that of DX101. However, it shows much faster absorption and significantly higher maximum concentrations, suggesting good in vivo potential.

[0258] [Example 23]: Pharmacokinetic study of DX111

[0259] The following study is an LC / MS assay of various molecules in plasma over three days (ultimately 11 measurement points). Graphs are always shown in comparison to the DX101 reference.

[0260] [Table 5]

[0261] Plasma sampling at 0 (not injected), 5, 10, 15, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours (at point 11).

[0262] Figure 6 shows the pharmacokinetic profiles of DX111 compared to DX101. The results are as follows:

[0263] [Table 6]

[0264] This oral pharmacokinetic study shows that DX111 has three times higher absorption than DX101. Furthermore, DX111 also has a higher maximum concentration and faster absorption.

[0265] [Example 24]: Pharmacokinetic study of DX123

[0266] The following study is an LC / MS assay of various molecules in plasma over three days (ultimately 11 measurement points). Graphs are always shown in comparison to the DX101 reference.

[0267] [Table 7]

[0268] Plasma sampling at 0 (not injected), 5, 10, 15, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours (at point 11).

[0269] Figure 8 shows the pharmacokinetic profiles of DX123 compared to DX101. The results are as follows:

[0270] [Table 8]

[0271] This initial oral pharmacokinetic analysis shows that DX123 has twice the bioavailability compared to DX101. These results suggest promising in vivo potential for DX123.

[0272] [Example 25] Cytotoxicity test of DX101 analog according to the present invention on 4T1 cells

[0273] Cell viability tests were performed using mouse 4T1 mammary gland tumor cells characterized as triple-negative (HER2-, ER-, PR-).

[0274] For this experiment, a cell culture medium was prepared. The culture medium consisted of Dulbecco's modified Eagle medium (DMEM, sold by Westburg as LO BE12-604F) containing 4.5 g / L of glucose with L-glutamine, to which 10% fetal bovine serum (FCS) and 50 U / ml of penicillin / streptomycin were added. 4T1 cells were introduced into this medium.

[0275] 4T1 cells were seeded at a rate of 2000 cells per well in a 96-well plate. After culturing under normal conditions, i.e., in an incubator at 37°C and 5% O2 for 72 hours (h), the 4T1 cells were treated with 100 nM, 1 μM, 2.5 μM, and 10 μM concentrations of DX101, DX103, DX111, DX123, DX125, DX127, and DX129 for 48 hours. Control conditions (CTLs) without treatment with molecules DX101, DX103, DX111, DX123, DX125, DX127, or DX129 were also performed in parallel using the protocol described above.

[0276] Cell viability is measured by three different methods. The first method involves MTT labeling at 48 hours. This test is based on the use of tetrazolium salt MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide). Tetrazolium is reduced to formazan, a purple precipitate, by mitochondrial succinate dehydrogenase in active living cells. The amount of precipitate formed is proportional to the amount of living cells, but also to the metabolic activity of each cell. Therefore, a simple assay of optical density at 550 nm by spectroscopy makes it possible to determine the relative amounts of living cells and metabolically active cells. After 48 hours, the medium is aspirated and the cells are incubated with MTT (0.5 mg / ml in the culture medium) for approximately 3 hours. The MTT solution is aspirated and the purple crystals are dissolved in dimethyl sulfoxide (DMSO). Optical density (OD) is measured at 550 nm. The percentage of viability relative to CTLs is then determined for each well, and IC is calculated. 50 The concentration at which 50% of cells survive is determined for each molecule using a nonlinear regression curve (log (inhibitor) vs. response) in Prism software.

[0277] In the second method, the percentage of viability is determined by assaying the activity of the enzyme LDH (lactate dehydrogenase) in the cell supernatant using a non-radioactive cytotoxicity assay kit (Promega). LDH is an enzyme released into the supernatant of dead cells. The higher the LDH activity in the supernatant, the greater the cell death. In this enzyme assay, the released LDH converts the purple tetrazolium salt into red formazan, which is absorbed at 490 nm. The intensity of the red color is proportional to the number of dead cells. After 48 hours of treatment, the supernatant is transferred to a new 96-well plate and incubated at room temperature for 30 minutes in the presence of the substrate mix. The reaction is stopped with a stop solution reagent, and the absorbance is determined at 490 nM. Here, the percentage of cell death is determined using an LDH activity control with up to 100% activity (prepared from untreated cells incubated at 37°C for 45 minutes in the presence of cell lysis solution immediately before adding the substrate mix), and then the cell viability of each well is estimated from this percentage. Next, IC 50 This is decided as explained in the previous paragraph.

[0278] In the third method, viability is determined using the CellTox Green Cytotoxicity Assay Kit (Promega). This assay measures cell death by changes in membrane integrity. This assay uses a cyanine probe that binds to the DNA of dead cells, which is permeable to the probe but does not penetrate living cells, making the DNA fluorescent. As a result, the higher the fluorescence in the well, the greater the cell death. After 48 hours of treatment, the cells are incubated in the presence of Celltox green reagent at room temperature for at least 15 minutes, and λ 発光 485nm / λ 励起 Fluorescence is read at 590 nm. The percentage of cell death is determined using a 100% cell death control (prepared from untreated cells incubated at 37°C for 30 minutes in the presence of cell lysis solution before adding Celltox green reagent), and then the cell viability of each well is estimated from this percentage. Then, IC is performed. 50 We will decide as explained earlier.

[0279] IC of these tests50 The results are shown in Tables 1a, 1b, and 1c. In these tables, - a The significance level is the LogIC of the compound. 50 and DX101's LogIC 50 The results were calculated by comparison with (minimum n=3), followed by a one-way ANOVA test and then Dunn's post-hoc test. -n b This represents the number of independent tests with 4 to 10 repetitions for each condition.

[0280] [Table 9]

[0281] [Table 10]

[0282] [Table 11]

[0283] Regarding DX111, IC 50 Tables 1a, 1b, and 1c show that DX123 exhibits significantly lower activity than DX101 (by a maximum coefficient of 2.5) and higher cytotoxic activity than DX101. In addition, the activity of DX123 tends to be higher than that of DX101, while the activity of DX125, DX127, and DX129 is lower than that of DX101.

[0284] [Example 26]: Cytotoxicity test of DX101 analog according to the present invention on BT-474 cells

[0285] Cell viability tests were also performed on BT-474 human mammary tumor cells (characterized as triple-positive HER2+, ER+, PR+). BT-474 cells were in the same cell culture medium as in the previous examples and seeded at 70,000 cells / well in 24-well plates for cell viability determination using trypan blue, or at 13,000 cells / well in 96-well plates for cell viability determination using MTT or LDH assays. After culturing under normal conditions, i.e., in a 37°C incubator with 5% O2 for 96 hours (h), BT-474 cells were treated with 100 nM, 1 μM, 2.5 μM, and 10 μM of DX101, DX103, DX105, DX111, DX123, and DX127 for 48 hours. Using the aforementioned protocol, a control is also performed that does not involve processing with DX101, DX103, DX105, DX111, DX123, and DX127.

[0286] After 10 minutes of trypsin digestion at 37°C, cell viability was quantified by the trypan blue test, also performed using an automated counting system with a Biorad TC20 machine (TC20® automated cell counter). The trypan blue test is based on the integrity of the cell membrane destroyed by dead cells. Trypan blue stains dead cells blue. The Biorad TC20 cell counter counts the percentage of blue and non-blue cells and reports the cell percentage. The percentage of viability relative to untreated cells is then determined for each well, and IC is calculated. 50 The viability of BT-474 cells was determined as described in the previous example. The results are shown in Table 2. In addition, the viability of BT-474 cells was determined using the MTT and LDH assays performed as described in the previous example.

[0287] The results are shown in Tables 2a, 2b, and 2c. In these tables, - a The significance level is the LogIC of the compound. 50 and DX101's LogIC 50 The p-values ​​were calculated by comparison with (minimum n=3), and by a one-way ANOVA test followed by Dunn's post-hoc test (except for the LDH trial, where the p-value was calculated by a t-test). -nb This represents the number of independent tests with 3 to 10 repetitions for each condition.

[0288] [Table 12]

[0289] [Table 13]

[0290] [Table 14]

[0291] Tables 2a, 2b, and 2c show that the activities of molecules DX103, DX105, DX111, and DX127 are similar to those of DX101, and that in this series, the activity of DX123 tends to be superior to that of DX101. Therefore, since all of these molecules have biological data similar to or better than that of DX101, they are considered good candidates for industrial development.

[0292] [Example 27]: Effect of analog compound DX111 on tumor growth in vivo

[0293] All animal procedures were performed in accordance with the guidelines of the inventors' facility after approval by the ethics committee. 4T1 cells were cultured as before, dissociated with trypsin, washed twice with cold PBS, and resuspended in 1,500,000 / ml PBS. 4T1 tumors were obtained by subcutaneously transplanting 150,000 cells per 100 μL into the flank of female Balb / c mice (9 weeks old, 1 month old). The tumors were 50–100 mm in size. 3 Once the tumor volume reached a certain level, mice were administered 40 mg / kg of DX101 or 40 mg / kg of DX111 or a control vehicle (water) via gastric tube. Treatment was performed daily until the end of the experiment (tumor volume > 1000 mm²). 3 The tumor volume is determined daily using calipers, and the formula is: 1 / 2 X (length * width). 2The calculation was performed using the following formula: 100X(1-(tumor volume on day 7 / tumor volume on day 0)). DX111 ) / (1-(tumor volume on day 7 / tumor volume on day 0) ビヒクル This was determined using ).

[0294] The survival rates of animals were compared using the Kaplan-Meier method.

[0295] Figure 7A shows that DX111 exhibits superior tumor growth inhibition compared to DX101 (**p<0.01, one-way ANOVA and Tukey post-hoc test). Further determination showed that tumor growth inhibition on day 7 was 67% in DX111-treated animals and 48% in DX101-treated animals.

[0296] Furthermore, the analysis of animal survival rates, also shown in Figure 7B, indicates that the median survival time was superior for animals treated with DX111 (Log-rank Mantel-Cox test, *p<0.05 and ns, not significant; Log-rank test for trend, **p<0.01). Additionally, after 15 days of treatment, the survival rate for animals treated with DX111 was 25%, while the survival rate for animals treated with DX101 was 0%. The median survival time after treatment with DX101 (40 mg / kg) was 9 days, compared to 10 days for DX111 (40 mg / kg).

[0297] In conclusion, the effect of DX111 on tumor growth inhibition is far greater in vivo and strongly impacts animal survival rates.

[0298] [Example 28]: A study to determine the pharmacokinetics and bioavailability of DX111 orally in rats.

[0299] [Table 15]

[0300] Plasma sampling at 0 (uninjected), 15, 30 minutes, 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, and 72 hours.

[0301] The results are shown in Table 3.

[0302] [Table 16]

[0303] Surprisingly, the results show that the analog compound DX111 has three times higher bioavailability than the reference compound DX101 by reducing its elimination half-life. The maximum plasma concentration obtained with DX111 is four times that of DX101, and the clearance is halved.

[0304] While the present invention has been described in relation to several specific embodiments, it is very clear that it is by no means limited thereto, and that all technical equivalents and combinations of the described means are also included, where they fall within the context of the present invention. The use of the verbs "contain," "equip," or "include," and their conjugations, does not preclude the existence of elements or processes other than those described in the claims.

Claims

1. A compound of formula (I) for use as a pharmaceutical to reduce cancerous tumors in mammals: 【Chemistry 1】 During the ceremony, R1 is at the β position, R １ but, F, N ３ 、 OC ｎ H ２ｎ＋１ 、 NR ２ R ３ Here, R ２ is H or COC ｎ H ２ｎ＋１ and R ３ = H SO ２ R ２ Here, R ２ is H or C ｎ H ２ｎ＋１ That is, Selected from, n ≤ 8, and the compound of formula (I) is not 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane-3β-ol, When R1 is F, the compound of formula (I) is 3β-fluoro-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane, When R1 is N3, the compound of formula (I) is 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-3β-azide. A compound or a pharmaceutically acceptable salt of such a compound.

2. The compound for use according to claim 1, wherein the compound of formula (I) is 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-3β-acetamide.

3. The compound for use according to claim 1, wherein the compound of formula (I) is 5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]-3β-amine.

4. The compound of formula (I) above, 3β-methoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane 3β-ethoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane A compound for use according to claim 1, selected from 3β-octanoxy-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane.

5. The compound for use according to claim 1, wherein the compound of formula (I) is 3β-methylsulfonyl-5α-hydroxy-6β-[2-(1H-imidazole-4-yl)ethylamino]cholestane.

6. The compound for use according to any one of claims 1 to 5, wherein the cancerous tumor is a chemosensible carcinoma.

7. The compound for use according to any one of claims 1 to 5, wherein the cancerous tumor is a chemotherapy-resistant cancer.

8. The compound for use according to claim 7, wherein the chemotherapy-resistant cancer is hematological or blood cancer.

9. The compound for use according to claim 7 or 8, wherein the cancer is resistant to chemotherapy with daunorubicin, cytarabine, fluorouracil, cisplatin, all-trans retinoic acid, arsenic trioxide, bortezomib, or a combination thereof.

10. A pharmaceutical composition for use in reducing cancerous tumors in mammals, comprising at least one compound according to any one of claims 1 to 5 in a pharmaceutically acceptable vehicle.

11. A pharmaceutical composition for use according to claim 10, comprising at least one other therapeutic agent.

12. The pharmaceutical composition for use according to claim 11, wherein the other therapeutic agent is an antineoplastic agent.

13. A pharmaceutical composition for the treatment of cancer in a patient suffering from a tumor that is chemically sensitive to the antineoplastic agent, wherein the dose of the antineoplastic agent administered to the patient in combination with the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof is less than the dose of the antineoplastic agent administered without combination with the compound according to any one of claims 1 to 5.

14. A pharmaceutical composition according to any one of claims 10 to 13, characterized in that it is in a form suitable for administration via any route.

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