EP4236963A1 - 5alpha-hydroxy-6beta-[2-(1-h-imidazol-4-yl)-ethylamino cholestan-3beta-ol analoge und diese enthaltende pharmazeutische zusammensetzungen zur behandlung von krebs - Google Patents

5alpha-hydroxy-6beta-[2-(1-h-imidazol-4-yl)-ethylamino cholestan-3beta-ol analoge und diese enthaltende pharmazeutische zusammensetzungen zur behandlung von krebs

Info

Publication number
EP4236963A1
EP4236963A1 EP21801547.7A EP21801547A EP4236963A1 EP 4236963 A1 EP4236963 A1 EP 4236963A1 EP 21801547 A EP21801547 A EP 21801547A EP 4236963 A1 EP4236963 A1 EP 4236963A1
Authority
EP
European Patent Office
Prior art keywords
compound
cholestan
imidazol
hydroxy
ethylamino
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21801547.7A
Other languages
English (en)
French (fr)
Inventor
Stéphane SILVENTE
Quentin MARLIER
Arnaud RIVES
Nicolas Caron
Dario MOSCA
Hélène MICHAUX
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dendrogenix
Original Assignee
Dendrogenix
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from BE20205764A external-priority patent/BE1028754B1/fr
Priority claimed from BE20205878A external-priority patent/BE1028852B1/fr
Application filed by Dendrogenix filed Critical Dendrogenix
Publication of EP4236963A1 publication Critical patent/EP4236963A1/de
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the invention relates to the field of sterol compounds and more particularly to analogues of the compound 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol and pharmaceutical compositions comprising them for their use in the treatment of cancer.
  • cancer or "cancerous tumor” encompasses a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. If the cancer cells are not eliminated, the evolution of the disease will more or less quickly lead to the death of the affected person.
  • Cancer management involves surgery, radiotherapy and chemotherapy, which can be used alone or in combination, simultaneously or sequentially.
  • Chemotherapy uses antineoplastic agents which are drugs that prevent or inhibit the maturation and proliferation of neoplasms.
  • Antineoplastic agents work by effectively targeting rapidly dividing cells. Because antineoplastic agents affect cell division, tumors with a high growth rate (such as acute myeloid leukemia and aggressive lymphomas, including Hodgkin's disease) are more susceptible to chemotherapy because a greater proportion of cells targets undergo cell division at any time. Malignant tumors with slower growth rates, such as indolent lymphomas, tend to respond much more modestly to chemotherapy. However, the development of drug resistance is a persistent problem during chemotherapy treatment.
  • AML acute myeloid leukemia
  • anthracycline such as daunorubicin
  • the overall 5-year survival rate is 40% in young adults and about 10% in elderly patients. Response rates vary considerably with aging, from 40% to 55% in patients over 60 and from 24% to 33% in patients over 70. This is even worse for the elderly with unfavorable cytogenetic profiles and death within 30 days of treatment ranges from 10% to 50% with age and worsening.
  • the restriction of the use of these molecules is also due to effects secondary, and in particular the emergence of chronic cardiac toxicity (linked to anthracyclines).
  • the toxic mortality rate linked to intensive chemotherapy is 10 to 20% in patients over 60 years of age.
  • Dendrogenin A is able to restore the sensitivity of chemoresistant tumors to an antineoplastic agent or to increase the effects of antineoplastic agents on tumors, which in turn makes it possible to reduce the effective cytotoxic dose of antineoplastic agents against chemosensitive tumors.
  • the document by 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 for which the alcohol in the 3p position is optionally functionalized with a methanoate or propanoate radical, for the treatment of cancer.
  • the object of the present invention is to provide new compounds and analogues of the compound Dendrogenin A, which are useful for treating cancerous tumours, in particular chemosensitive and/or chemoresistant tumours.
  • the first subject of the invention is a compound of formula (I); or a pharmaceutically acceptable salt of such a compound, in which:
  • Ri is chosen from F, N 3 , OC n H 2n+ i, NR2R3, SR2, SO2R2 , with n ⁇ 8,
  • R2 and R3 are independently chosen from: H, a C1 to C8 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl, arene and heterocyclic groups, for its use as a medicament, and more particularly as a medicament for regressing a mammalian cancerous tumor.
  • a second subject of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one compound of formula (I) for its use in regressing a cancerous tumor of a mammal.
  • solvate is used herein to describe a molecular complex comprising a compound of the invention and containing stoichiometric or sub- stoichiometric values of one or more pharmaceutically acceptable solvent molecules such as ethanol.
  • solvent molecules such as ethanol.
  • hydrate refers when said solvent is water.
  • human refers to a subject of either sex and any stage of development (i.e. newborn, infant, juvenile, adolescent, adult).
  • patient refers to a warm-blooded animal, more preferably a human, who is awaiting reception or receiving medical treatment and/or who will be the subject of a medical procedure.
  • pharmaceutical vehicle means an inert carrier or medium used as a solvent or diluent in which the pharmaceutically active agent is formulated and/or administered.
  • pharmaceutical carriers include creams, gels, lotions, solutions and liposomes.
  • administration means to deliver, the active agent or active ingredient (e.g. the compound of formula (I), in a pharmaceutically acceptable composition, to the patient in which a condition, symptom and/or a disease must be treated.
  • the active agent or active ingredient e.g. the compound of formula (I)
  • treat and “treatment” as used herein include alleviating, alleviating, stopping, curing a condition, symptom and/or disease.
  • analog as used herein means a compound having a similar chemical structure to another reference compound, but differing from it in some component. It may differ by one or more atoms, functional groups, substructures, which are replaced by other atoms, functional groups or substructures. Analogs may have different physical, chemical, biochemical or pharmacological properties. In the present invention, the analogous compounds refer to the compound Dendrogenin A. These analogs have identical or similar pharmacological properties with respect to the reference compound.
  • chemoresistant cancer we mean a cancer in a patient where the proliferation of cancerous cells cannot be prevented or inhibited by means of an antineoplastic agent or a combination of antineoplastic agents usually used to treat this cancer , at a dose acceptable to the patient.
  • Tumors can be intrinsically resistant prior to chemotherapy, or resistance may be acquired during treatment by tumors initially sensitive to chemotherapy.
  • chemosensitive cancer is meant a cancer in a patient which responds to the effects of an antineoplastic agent, i.e. the proliferation of cancer cells can be prevented by means of said antineoplastic agent at a dose acceptable to the patient.
  • the compound of formula (I) belongs to the group of steroids.
  • the numbering of the carbon atoms of the compound of formula (I) therefore follows the nomenclature defined by the IUPAC in Pure & AppL Chem., Vol.61, No.10, pp.1783-1822,1989.
  • the numbering of the carbon atoms of a compound belonging to the group of steroids according to IUPAC is shown below:
  • AML acute myeloid leukemia
  • Dendrogenin A 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol;
  • MCF-7 Michigan Cancer Foundation-7
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • Ch EH Cholesterol Epoxide Hydrolase
  • Neuro2a murine neuroblastoma
  • PBS phosphate buffered saline
  • DMSO dimethyl sulfoxide
  • OD optical density or absorbance
  • CT cholestane-3p,5a,6p-triol
  • 5,6a-EC 5,6a-epoxycholesterol
  • Tam Tamoxifen
  • the invention relates firstly to a compound of formula (I); or a pharmaceutically acceptable salt of such a compound, in which:
  • Ri is chosen from F, N 3 , OC n H 2n+ i, NR2R3, SR2, SO2R2, with n ⁇ 8,
  • R2 and R3 are independently chosen from: H, a C1 to C8 alkyl group, saturated or unsaturated, optionally containing one or more substituents selected from allyl, carbonyl, arene and heterocyclic groups, for its use as a medicament.
  • the invention relates to a compound of formula (I); or a pharmaceutically acceptable salt of such a compound, in which:
  • Ri is chosen from F, N 3 , OC n H 2n+ i, NR2R3, SR2, SO 2 R 2 , with n ⁇ 8,
  • R2 and R3 are independently chosen from: H, a C1 to C8 alkyl group, saturated or unsaturated, optionally containing one or more substituents chosen from allyl, carbonyl, arene and heterocyclic groups, for its use as a medicament for regressing a mammalian cancerous tumor.
  • arene refers to all monocyclic and polycyclic aromatic hydrocarbons
  • heterocyclic refers to monocyclic and polycyclic aromatic compounds comprising as cyclic elements one or more heteroatoms from among O, S and/or N.
  • the radical of carbon 3 can be in position a or p.
  • the p position being a preferred embodiment.
  • the compound of formula (I) is more particularly 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-3p-acetamide (named DX127) .
  • the compound of formula (I) is more particularly 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-3p-amino (named DX125) .
  • the compound of formula (I) is more particularly 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-3p-azide (named DX123) .
  • the compound of formula (I) is 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluoro (named DX111 ).
  • the compound of formula (I) is an O-alkyl analogue such as 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p - methoxyl (DX103) and 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ethoxyl (DX105).
  • the compound of formula (I) is preferably 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methylsulfonyl (named DX129 ).
  • the compound of formula (I) is intended for use in the treatment of cancer of the breast, prostate, colorectal, lung, bladder, skin, uterus , cervix, mouth, brain, stomach, liver, throat, larynx, esophagus, bone, ovary, pancreas, kidney , retina, sinus, nasal cavities, testis, thyroid, vulva, treatment of lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, acute myeloid leukemia or acute lymphocytic leukemia, the multiple myeloma, Merkel cell carcinoma or mesothelioma.
  • the cancer is acinar adenocarcinoma, acinar carcinoma, acrolentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenosquamous carcinoma, adnexal carcinoma, rest tumor adrenal, adrenocortical carcinoma, aldosterone-secreting carcinoma, alveolar soft tissue sarcoma, ameloblastic thyroid carcinoma, angiosarcoma, apocrine carcinoma, Askin tumor, astrocytoma, basal cell carcinoma, basaloid carcinoma, basosquamous carcinoma, bile duct cancer , bone marrow cancer, botryoid sarcoma, bronchioalveolar carcinoma, bronchogenic adenocarcinoma, bronchogenic carcinoma, carcinoma ex pleomorphic adenoma, chloroma, cholangiocellular carcinoma, chondrosarcom
  • the compound is for use in the treatment of a chemosensitive cancer.
  • the compound of formula (I) is intended for use in the treatment of a chemoresistant cancer.
  • the chemoresistant cancer is a hematological or blood cancer, such as leukemia, in particular acute myeloid leukemia or acute lymphocytic leukemia, lymphoma, in particular non-Hodgkin's lymphoma and myeloma multiple.
  • leukemia in particular acute myeloid leukemia or acute lymphocytic leukemia
  • lymphoma in particular non-Hodgkin's lymphoma and myeloma multiple.
  • the cancer is chemoresistant to daunorubicin, cytarabine, fluorouracil, cisplatin, all-trans-retinoic acid, arsenic trioxide, bortezomib, or one of of their combinations.
  • All references to compounds of formula (I) include references to salts, multi-component complexes and their liquid crystals. All references to compounds of formula (I) also include references to polymorphs and their usual crystals.
  • the compound according to the invention may be in the form of pharmaceutically acceptable salts.
  • a pharmaceutically acceptable salt of the compound of formula (I) comprises the acid addition thereof.
  • Suitable acid salts are formed from acids that form non-toxic salts. For example chosen from: acetate, adipate, benzoate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, furamate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, chloride hydrochloride, hydrobromide, bromide, hydroiodide, iodide, isethionate, lactate, malate, maleate, malonate mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccarate, sterate, succinate,
  • compositions of formula (I) can be prepared by one or more of the following three methods:
  • the salt obtained can precipitate and be collected by filtration or can be recovered by evaporation of the solvent.
  • the degree of ionization of the salt obtained can vary from completely ionized to almost non-ionized.
  • the second subject of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one compound according to the invention, as described above for its use in causing a cancerous mammalian tumor to regress.
  • the pharmaceutical composition further comprises at least one other therapeutic agent.
  • this other therapeutic agent is an antineoplastic agent.
  • the antineoplastic agent is a DNA damaging agent such as camptothecin, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, cisplatin, carboplatin, oxaliplatin, cyclophosphamide, chlorambucil, chlormethine, busulfan, treosulfan or thiotepa, an antitumor antibiotic such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, actinomycin D, mitomycin, bleomycin or plicamycin, an antimetabolite such as 5-fluorouracil, cytarabine, fludarabine or methotrexate, an antimitotic such as paclitaxel, docetaxel, vinblastine, vincristine, vindesine or vinore
  • the pharmaceutical composition is used in the treatment of cancer in a patient suffering from a tumor which is chemoresistant to said antineoplastic agent when it is not administered in combination with a compound according to the invention .
  • the pharmaceutical composition is used in the treatment of cancer in a patient suffering from a tumor which is chemosensitive to said antineoplastic agent, and the dose of the antineoplastic agent administered to said patient in combination with a compound according to the invention or one of its pharmaceutically acceptable salts is lower than the dose of the antineoplastic agent when it is not administered in combination with a compound according to the invention.
  • the dose of the antineoplastic agent administered to said patient in combination with a compound according to the invention or one of its pharmaceutically acceptable salts is lower than the dose of the antineoplastic agent administered alone, without any other active ingredient.
  • composition according to the invention may also also comprise other active therapeutic compounds commonly used in the treatment of the pathology set out above.
  • the pharmaceutical composition of the invention can be administered by any route, in particular by route: intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal ), nasal spray inhalation, using tablet, capsule, solution, powder, gel, particle formulation; and contained in a syringe, an implanted device, an osmotic pump, a cartridge, a micropump; or any other means appreciated by the skilled artisan well known in the art.
  • route in particular by route: intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal ), nasal spray inhalation, using tablet, capsule, solution, powder, gel, particle formulation; and contained in a syringe, an implanted device, an osmotic pump, a cartridge, a micropump; or any other means appreciated by the skilled
  • Site-specific administration can be performed, for example, intratumoral, intra-articular, intrabronchial, intra-abdominal, intracapsular, intra-cartilaginous, intracavitary, intracerebellar, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac , intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal or transdermal in a suitable dosage comprising the usual non-toxic and pharmaceutically acceptable vehicles.
  • the pharmaceutical composition is in a form suitable for being administered intravenously, subcutaneously, intraperitoneally or orally.
  • the oral route is particularly preferred.
  • the compound of the invention is also effective on humans.
  • compositions for the administration of the compounds of this invention may be presented in unit dose form and may be prepared by any of the methods well known in the state of the art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients.
  • pharmaceutical compositions are prepared by bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the active object compound is included in an amount sufficient to produce the desired effect on the disease process or state.
  • the pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, capsules, syrups, elixirs, solutions, mouth patches, oral gel, chewing gum, chewable tablets, effervescent powder and effervescent tablets.
  • the pharmaceutical compositions containing the active principle can be in the form of an aqueous or oily suspension.
  • the aqueous suspensions contain the active materials mixed with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;
  • the dispersing or wetting agents may be a natural phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of alkylene oxide ethylene with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol, such as polyoxyethylene monooleate sorbitol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.
  • the oily suspensions can be formulated by suspending the active principle in a vegetable oil, for example peanut, olive, sesame or coconut oil, or in a mineral such as liquid paraffin.
  • Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those mentioned above and flavoring agents can be added to obtain a palatable oral preparation. These compositions can be preserved by adding an antioxidant such as ascorbic acid.
  • Dispersible powders and granules which are suitable for the preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. These formulations may also contain emollient, preservative, flavoring and coloring agents.
  • the pharmaceutical compositions may be in the form of an aqueous or oleaginous suspension which can be injected in a sterile manner.
  • This suspension can be formulated according to the known art using the appropriate dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic diluent or solvent acceptable parenterally, for example a solution in 1,3-butane diol. Acceptable vehicles and solvents that can be used include; water, Ringer's fluid and isotonic sodium chloride solution.
  • sterile fixed oils are traditionally used as a solvent or suspending medium. For this purpose, any fixed oil can be used, including synthetic mono- or diglycerides. Also, fatty acids such as oleic acid are used in the preparation of injectables.
  • compositions of the present invention can also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and which will therefore melt in the rectum to release the drug.
  • a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and which will therefore melt in the rectum to release the drug.
  • materials include cocoa butter and polyethylene glycols.
  • compositions can be administered ocularly by means of solutions or ointments.
  • transdermal delivery of the compounds in question can be achieved by means of iontophoretic and other patches.
  • creams, ointments, jellies, solutions or suspensions are used for topical use.
  • a suitable dosage of the pharmaceutical composition of this invention may generally be from about 0.1 to 50,000 micrograms ( ⁇ g) per kg of body weight. bodily of patient per day, which can be administered in single or multiple doses.
  • the dosage level will preferably be from about 1000 to about 40,000 pg/kg per day, depending on many factors such as the severity of the cancer to be treated, the age and relative health of the subject, the route and the form of administration.
  • this composition can be supplied in the form of tablets containing 1000 to 100000 micrograms of each of the active ingredients, in particular 1000, 5000, 10000, 15000, 20000, 25000, 50000, 75000, 10,0000 micrograms of each active ingredient.
  • This composition can be administered on a 1 to 4 times daily schedule, for example once or twice daily.
  • the dosage regimen may be adjusted to provide an optimal therapeutic response.
  • the invention also discloses a process for the manufacture of the compound of formula (I).
  • the C3 fluorination process includes a Dendrogenin A fluorination step, carried out using a fluorination reagent, for example Diethylaminosulfur trifluoride (DAST) or tetrafluoroborate.
  • DAST Diethylaminosulfur trifluoride
  • tetrafluoroborate A new method for fluorination of sterols.
  • the fluorination reaction with tetrafluoroborate is described in the literature “Org. Let., Vol. 11, No. 21, 2009, 5050-5053, Aminodifluorosulfinium Tetrafluoroborate Salts as Stable and Crystalline Deoxofluorinating Reagents”.
  • the process for the synthesis of 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluorodilactate comprises the following steps:
  • the white powder obtained is the compound 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluorodilactate.
  • the ambient temperature is between 15 and 40°C, for example 25 or 37, preferably 20°C.
  • Figure 2 illustrates the results of an MTT cell viability test performed on MCF-7 breast tumor cells in the presence of the compound 5a-hydroxy-6[3-[2-(1H- imidazol-4-yl)-ethylamino]-cholestan-3p-fluoro.
  • FIG. 3 Figure 3 illustrates the results of Cholesterol Epoxide Hydrolase (ChEH) activity in MCF-7 cells in the presence of the compound 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl )-ethylamino]-cholestan-3p-fluoro.
  • Figure 4 illustrates the pharmacokinetic profile of the compound 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-methoxyl (DX103) in comparison with the compound Dendrogenin A ( DX101).
  • Figure 5 illustrates the pharmacokinetic profile of the compound 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ethoxyl (DX105) in comparison with the compound Dendrogenin A ( DX101).
  • FIG. 6 Figure 6 illustrates the pharmacokinetic profile of the compound 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluoro (DX111) in comparison with the compound Dendrogenin A ( DX101).
  • FIG. 7A illustrates the course of tumor growth and survival rate in mice comparing treatment with DX111 and DX101.
  • FIG. 8 Figure 8 illustrates the pharmacokinetic profile of the compound 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-azide (DX123) in comparison with the compound Dendrogenin A ( DX101).
  • Example 1 Synthesis of the analogous compound 5a-hydroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-fluoro (named DX111)
  • the first step is a synthesis of the compound 3p-fluorocholestane comprising the following steps:
  • the mixture was transferred to a separatory funnel and the organic phase was washed twice with NaHCO 3 sat, 2 other times with a saturated NaCl solution and once with water.
  • the organic phase was dried over MgSO 4 , filtered and then evaporated, allowing a white powder to be obtained. 6.61 g corresponding to 3p-fluorocholestane was obtained. The final yield of the reaction is 99%.
  • the second step consists in synthesizing from 3p-fluoro-cholestane the compound 3p-fluoro-5,6a-Epoxy-cholestane as follows:
  • Vacuum evaporation of the organic solvent was carried out and made it possible to obtain 6.90 g of a white powder comprising: 3p-fluoro-5,6a-Epoxy-cholestane (85% of the white powder) and 3p-fluoro-5 ,6p-Epoxy-cholestane (15% white powder). 3p-Fluoro-5,6a-epoxycholestane was used without further purification.
  • the third step is to synthesize 5a-hydroxy-6-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluoro (DX1 11 in basic form) as follows: [88] 0.80 g of histamine in its basic form (7.2 mmol) was added to a 10 ml butanol solution comprising 1.45 g of the compound 3p-fluoro-5,6a-epoxycholestane (3.6 mmol) with stirring at 130 °C. The mixture was kept under stirring under reflux heating at a temperature of 130°C for 48 hours.
  • TLC thin layer chromatography
  • Example 2 Preparation of a dilactate salt of the compound 5a-hydroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3P-fluoro (DX11 1 in dilactate form):
  • the reaction is stopped by adding water and the products were extracted with an AcOEt/H 2 O system.
  • the organic phase was dried over MgSC and the organic solvents evaporated under vacuum.
  • the cholestan-3-sulphide and cholestan-3-amino derivative products are purified either by column chromatography or by recrystallization.
  • the reaction path to get Dendrogenin A analogs are the same steps developed for the synthesis of Dendrogenin A.
  • R2O2S will be obtained by oxidation of R2S with oxidizing agents such as m-CPBA or H2O2.
  • Dissolve cholesterol add NEt 3 in DCM and add mesyl chloride (MsCI) dropwise in DCM solution at room temperature for 1 h. Stir the reaction for 12 h, then evaporate the organic solvent and crystallize the product with MeOH. The product obtained is a white solid. The product obtained is used to obtain the 3p-sulfide and 3[3-azide derivatives. The product obtained is dissolved in DCM then TMS-SR2 for the 3p-sulfide derivative or TMS-N3 for the 3[3-azide derivative is added to the solution. An addition of BF 3 *Et2O is carried out at ambient temperature. Then, it is stirred for 3 h.
  • MsCI mesyl chloride
  • the culture medium consists of Dulbecco's Modified Eagle Medium (DMEM, marketed by Westburg under the reference LO BE12-604F), comprising 4.5 g/L glucose with L-Glutamine, to which 10% serum is added. of fetal calf (SVF). Neuro2a cells (murine neuroblastoma) are introduced into this culture medium.
  • DMEM Dulbecco's Modified Eagle Medium
  • LO BE12-604F fetal calf
  • a control is also carried out using the protocol described previously without the treatment with 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a-fluoro and 5a -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol.
  • Cell survival is quantified by a trypan blue test with automatic counting by the Biorad TC20 device (TC20TM Automated Cell Counter). The trypan blue test is based on the integrity of cell membranes, which is broken in dead cells. Trypan blue stains dead cells blue. The Biorad TC20 cell counting device counts the proportion of blue and non-blue cells, and reports the percentage of cells. The results are shown in Figure 1.
  • Figure 1 illustrates the ordinate of the percentage of cell survival compared to the control group.
  • Example 6 Effect of 5a-hvdroxy-6B-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-fluoro on the viability of MCF-7 cells [107] A cell viability test was performed on MCF-7 breast tumor cells (Michigan Cancer Foundation-7) overexpressing HER2 (ER(+) cells).
  • the MCF-7 cells are in a cell culture medium identical to Example 5 and are seeded in 12-well plates at 50,000 cells per well. 24 hours after seeding, the cells are treated with the solvate vehicle comprising water and ethanol with a 1% ratio of ethanol, 5a-hydroxy- 6p- [2-(1 H-imidazol-4 -yl)-ethylamino]-cholestan-3p-fluoro and 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol at 1, 2.5 or 5 ⁇ M. The cells are observed under an inverted microscope and photographed via the microscope camera at 24 h and 48 h.
  • the morphological changes of the cells at 1 ⁇ M are very slight. Only a few white vesicles are observed, reflecting the beginning of the autophagy phenomenon, causing cell death after 24 hours of treatment with 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]- cholestan-3-fluoro and 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol. The effects are more marked at 2.5 pM and 5 pM with an increase in the number of dead cells.
  • treatment with 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol shows greater or equal effect after 24 hours observation and similar after 48 hours observation.
  • the medium is aspirated, the cells washed with phosphate buffered saline (PBS) then incubated with MTT (0.5 mg/ml in PBS) for approximately 2 hours.
  • the MTT solution is aspirated and then the purple crystals are dissolved in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • Figure 2 illustrates the ordinate of the percentage of cell viability compared to the control group.
  • the control group is made in a similar way to the groups studied without the addition of the molecules studied in this text.
  • a dose-dependent decrease in cell viability in MTT is measured for 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-fluoro and 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol.
  • the viability is close to 0%. This reflects a capacity for destruction of mammary tumor cells by the compound of formula (I).
  • Example 7 Effect of 5 ⁇ -hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3B-fluoro on the activity of Cholesterol Epoxide Hydrolase (ChEH) in cells MCF-7
  • the compounds 5,6a-epoxycholesterol (5,6a-EC) and 5,6p-epoxycholesterol (5,6p-EC) are oxysterols implicated in the anticancer pharmacology of tamoxifen, a widely used antitumor drug. They are both metabolized to cholestane-3p,5a,6p-triol (CT) by the enzyme cholesterol-5,6-epoxide hydrolase (ChEH), and CT is metabolized by the enzyme HSD11 B2 (1 ip-Hydroxysteroid dehydrogenase 2) to 6-oxo-cholestan-3p,5a-diol (OCDO), a tumor-promoting oncosterone.
  • CT cholesterol-5,6-epoxide hydrolase
  • MCF-7 cells are in a cell culture medium identical to Example 5 and are seeded in 6-well plates at 150,000 cells per well with 3 wells per treatment condition. 24 h after seeding, the MCF-7 cells are treated with [ 14 C]5,6a-EC (stock solution 1000X: 0.6 mM; 20 pCi/pmol; final concentration 0.6 pM) alone or in combination with Tamoxifen (tam ).
  • Tamoxifen is used as a positive control for 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3a- fluoro and 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ol (1 ⁇ M for all molecules).
  • lipid extracts are prepared from the cell pellets by extraction with 100 pL of chloroform, 400 pL of methanol and 300 pL of water. Lipid extracts are analyzed by thin layer chromatography (TLC) using ethyl acetate (EtOAc) as eluent. The analysis is carried out using a plate reader and then by autoradiography. The results are presented in figure 3.
  • Example 8 Synthesis of the compound of formula (I) 5a-hvdroxy-66-[2-(1H-imidazol-4-vD-ethylaminol-cholestan-36-methoxyl (named DX103)
  • a first step consists in dissolving 4.0 grams (g) of cholesterol (10.3 mmol) in 20 milliliters (ml) of tetrahydrofuran (THF). 0.80 g of NaH (60% in oil, 20.0 mmol) was added and left to react for 30 minutes at 60° C., then 1.8 ml of iodomethane (28.9 mmol) were added. The mixture thus obtained was left at 60° C. overnight, namely about 10 h. After cooling the solution, the reaction was quenched by adding 20 ml of water. The mixture was filtered and the THF evaporated under vacuum. The mixture was transferred to a separating funnel and the aqueous phase was extracted three times with ethyl acetate.
  • the second step consists in synthesizing from cholestan-3p-methoxyl the compound 5,6a-epoxy-cholestan-3p-methoxyl as follows:
  • the third step is to synthesize 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-methoxyl (DX103 base form) as follows: 0.81 g of histamine (7.30 mmol) in its basic form was added to a 10 ml butanol solution comprising 1.50 g of the 5,6a-epoxy-cholestan-3p-methoxy compound (3.62 mmol) with stirring. The mixture was kept under stirring under reflux heating at a temperature of 130°C for 48 hours.
  • the progress of the reaction can be monitored by thin layer chromatography (TLC) to follow the conversion of 5,6a-epoxy-cholestan-3p-methoxy.
  • TLC thin layer chromatography
  • the organic phase was dried over anhydrous MgSC.
  • the mixture was purified by column chromatography on a purification automaton.
  • the eluent used is an ethyl acetate-methanol mixture in a ratio of 90-10%.
  • a white powder of 1.32 g of 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-methoxy was obtained.
  • the final yield of the reaction is 69% with a purity greater than 95% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.
  • Example 9 Preparation of a dilactate salt of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-methoxyl (DX103 dilactate form)
  • a dilactate salt of the compound 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-methoxy was prepared as follows:
  • the first step is a synthesis of the compound cholestan-3p-ethoxy comprising the following steps:
  • the third step is to synthesize 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ethoxy (DX105 in basic form) as follows:
  • the organic phase was dried over anhydrous MgSC.
  • the mixture was purified by column chromatography on a purification automaton.
  • the eluent used is an ethyl acetate-methanol mixture in a 90-10 ratio.
  • a white powder of 0.28 g of 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ethoxy was obtained.
  • the final yield of the reaction is 44% with a purity greater than 97% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.
  • Example 11 Preparation of a dilactate salt of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3P-ethoxyl (DX105 in dilactate form)
  • a dilactate salt of the compound 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-ethoxy was prepared as follows:
  • Example 12 Synthesis of the compound of formula (I) 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-octanoxyl (named DX115)
  • the first step is a synthesis of the cholestan-3p-octanoxyl compound comprising the following steps:
  • the second step consists in synthesizing from cholestan-3[3-octanoxyl the compound 5,6a-epoxycholestan-3p-octanoxyl as follows:
  • the third step is to synthesize 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-octanoxyl (DX1 15 in basic form) as follows:
  • the progress of the reaction can be monitored by thin layer chromatography (TLC) to follow the conversion of 5,6a-epoxy-cholestan-3p-octanoxyl.
  • TLC thin layer chromatography
  • the organic phase was dried over anhydrous MgSC.
  • the mixture was purified by column chromatography on a purification automaton.
  • the eluent used is a Ethyl acetate/Methanol mixture in a 95/5 ratio.
  • a white powder of 0.74 g of 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-octanoxyl was obtained.
  • the final yield of the reaction is 55% with a purity greater than 95% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.
  • Example 13 Preparation of a dilactate salt of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-octanoxyl (DX1 15 in dilactate form)
  • a salt dilactate of the compound 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-octanoxyl was prepared as follows:
  • Example 14 Synthesis of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-38-azide (named DX123)
  • the first step is the synthesis of the compound 3-mesylcholestane comprising the following steps:
  • the second step consists in synthesizing from 3[3-mesyl cholesterol the compound 3p-azide-cholestane as follows:
  • reaction was neutralized by adding 100 ml of a 2M NaOH solution.
  • the organics were extracted twice with dichloromethane.
  • the organic phases were combined and rinsed twice with saturated NaCl solution.
  • the organic phase was dried over MgSC, filtered and then evaporated to obtain a solid.
  • the reaction crude was purified by column chromatography eluent 100% hexane. A white-yellowish powder of 13.33 g corresponding to 3p-azide-cholestane was thus obtained.
  • the final yield of the reaction is 65%.
  • the third synthetic step consists in synthesizing from 3p-azoturecholestane the compound 3p-azide-5,6a-epoxycholestane as follows:
  • Vacuum evaporation of the organic solvent was carried out and made it possible to obtain 1.35 g of a white powder corresponding to the mixture of: 3-azide-5,6a-epoxycholestane (83% of the total) and 3p-azide- 5,6 -epoxycholestane (17% white powder). The final product was used without further purification.
  • the fourth step is the synthesis of 5a-hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-azide (DX123 in neutral form) as follows : [162] 864 mg of histamine in its basic form (7.77 mmol) was added to a 20 ml butanol solution comprising 2.02 g of the compound 3p-azide-5,6a-epoxycholestane at 83% (3.9 mmol) under stirring at 130°C. The mixture was kept under stirring under reflux heating at a temperature of 130°C for 48 hours.
  • the progress of the reaction can be monitored by thin layer chromatography (TLC) to follow the conversion of 3p-azide-5,6a-epoxycholestane.
  • TLC thin layer chromatography
  • the organic phase was dried over anhydrous MgSC, filtered and then evaporated to obtain a brown oil.
  • the mixture was purified by column chromatography on silica gel on a purification automaton comprising a prepacked column of 40 g eluting dichloromethane-ethyl acetate 75-25% up to 0-100%.
  • a white powder of 890 mg of 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-azide was obtained.
  • the final reaction yield is 42% with a purity greater than 97% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.
  • Example 15 Preparation of a dilactate salt of the compound 5a-hydroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-azide (DX123 dilactate form)
  • Example 16 Synthesis of a trichloride salt of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3P-amino (named DX125, in trichloride form)
  • the white powder obtained was dissolved with 20 ml of dichloromethane and transferred to a separatory funnel containing 20 ml of an aqueous solution of HCl (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 allowing a white powder to be obtained. The powder was taken up with dichloromethane and filtered one last time to remove the last traces of triphenylphosphine.
  • Example 17 Synthesis of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3B-acetamide (named DX127)
  • the first synthetic step is the reduction of the azide group to an amine in position 3 of the cholestan-3p-azide derivative.
  • the second step consists in synthesizing from cholestan-3-amine the compound cholestan-3-acetamide as follows:
  • the third step is to synthesize 5,6-epoxy-cholestan-3[3-acetamide as follows:
  • Vacuum evaporation of the organic solvent was carried out and made it possible to obtain 1.65 g of a white powder comprising: 5.6a-Epoxy-cholest-3p-acetamide (60% of the white powder) and 5.6 -Epoxy-cholest-3p-acetamide (40% white powder). 5,6a-Epoxy-cholest-3p-acetamide was used without further purification.
  • the fourth step is to synthesize 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-acetamide (DX127 in neutral form) as follows:
  • the organic phase was washed twice with 20 ml of water and three times with 20 ml of a saturated NaCl solution.
  • the organic phase was dried over anhydrous MgSO 4 .
  • the mixture was purified by column chromatography on a purification automaton.
  • the eluent used is a dichloromethane-methanol-ammonia mixture in a ratio of 75-20-5%.
  • a white powder of 0.37 g of 5 ⁇ -hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-acetamide was obtained.
  • the final reaction yield is 30% with a purity greater than 97% measured by NMR (nuclear magnetic resonance) and TLC (thin layer chromatography) analysis.
  • Example 18 Preparation of a dilactate salt of the compound 5a-hvdroxy-6P-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3P-acetamide (DX127 in dilactate form)
  • a dilactate salt of the compound 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-acetamide was prepared as follows:
  • the first step consists in synthesizing from 3p-mesylcholesterol the compound 3p-methylthio-cholestane as follows: ,
  • reaction was neutralized by adding 100 ml of a 2M 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 MgSC, filtered and then evaporated allowing obtaining a solid.
  • the reaction crude was purified by column chromatography on silica gel eluting with 100% hexane. A white powder of 6.04 g corresponding to 3p-methylthio-cholestane was thus obtained.
  • the final yield of the reaction is 63%.
  • the second synthetic step consists in synthesizing from 3
  • Vacuum evaporation of the organic solvent was carried out and made it possible to obtain 2.10 g of a white powder.
  • the reaction crude was purified by column chromatography, eluent initial 100% hexane and then mixtures of hexane and AcOEt.
  • the desired product was purified by column chromatography on silica gel with the eluent hexanes-AcOEt 55-45% v .
  • a white powder of 380 mg corresponding to 3-methylthio-5,6epoxy-cholestane was thus obtained.
  • the final yield of the reaction is 12%.
  • the third step is the synthesis of 5a-hydroxy-6p-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3p-methylsulfonyl (DX129 in basic form) as next :
  • the organic phase was dried over anhydrous MgSC, filtered and then evaporated to obtain a brown oil.
  • the reaction crude was purified by chromatographic column eluent initial AcOEt 100%, then AcOEt-MeOH mixtures.
  • the desired product was purified with AcOEt-MeOH 75-25%.
  • a yellow powder of 190 mg corresponding to 5 ⁇ -hydroxy-6[3-[2-(1H-imidazol-4-yl)-ethylamino]-cholestan-3-methylsulfonyl is obtained.
  • the product is purified a second time by chromatographic column to have a purity greater than 97
  • Example 20 Preparation of a dilactate salt of the compound 5a-hydroxy-6(3-[2-(1H-imidazol-4-yl)-ethylamino1-cholestan-3(3-methylsulfonyl) (DX129 in dilactate form )
  • DX105 shows equivalent bioavailability (even slightly higher) than DX101. On the other hand, it presents a much faster absorption and a much higher maximum concentration, which makes it possible to claim a good prospect in vivo. [21 1]
  • Example 23 Pharmacokinetic study of DX1 11
  • Example 24 Pharmacokinetic study of DX123 [215] The following study is an LC/MS assay in plasma of the various molecules over 3 days (11 measurement points in the end). The graphs are always presented in comparison with the DX101 which is the reference.
  • Example 25 Study of the cytotoxicity of the DX101 analogues according to the invention on 4T 1 cells [219] Cell viability assays were performed on 4T1 murine mammary tumor cells characterized as triple negative (HER2-, ER-, PR-).
  • the culture medium consists of Dulbecco's Modified Eagle Medium (DMEM, marketed by Westburg under the reference LO BE12-604F), comprising 4.5 g/L glucose with L-Glutamine, to which 10% serum is added. of fetal calf (FCS) and 50 U/ml of penicillin/streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf
  • 96-well dishes were seeded with 2000 4T 1 cells per well. After 72 hours (h) of culture under normal conditions, i.e. in an incubator at a temperature of 37°C at 5% bone, we treat the 4T1 cells for 48 hours with DX101, DX103, DX11 1, DX123, DX125, DX127 and DX129 at 100 nM, 1 pM, 2.5 pM and 10 pM.
  • a control condition (CTL) is also carried out in parallel using the protocol described previously without treatment with the molecules DX101, DX103, DX11 1 , DX123, DX125, DX127 or DX129.
  • MTT labeling is carried out at 48 hours. This test is based on the use of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). Tetrazolium is reduced by active living cell mitochondrial succinate dehydrogenase to formazan, a violet-colored precipitate. The quantity of precipitate formed is proportional to the quantity of living cells but also to the metabolic activity of each cell. Thus, a simple assay of the optical density at 550 nm by spectroscopy makes it possible to know the relative quantity of living and metabolically active cells.
  • the medium is aspirated, and the cells are incubated with MTT (0.5 mg/ml in culture medium) for about 3 hours.
  • MTT 0.5 mg/ml in culture medium
  • the MTT solution is aspirated and the purple crystals are dissolved in dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the OD optical density
  • the percentage of viability is then determined in each well compared to the CTL and the IC 5o (concentration for which 50% of living cells remain) is determined for each molecule with the Prism software using a line of nonlinear regression ( og(inhibitor) vs. Response).
  • the percentage of viability is determined using the assay of the activity of the enzyme LDH (lactate dehydrogenase) in the cell supernatants using the non-radioactive cytotoxicity assay kit k/t (Promega).
  • LDH lactate dehydrogenase
  • LDH is an enzyme released in the supernatant of dead cells. The higher the LDH activity in the supernatant, the greater the cell death.
  • the released LDH converts a purple tetrazolium salt to red-colored formazan, absorbing at 490 nm. The intensity of the red color is proportional to the number of dead cells.
  • the supernatants are transferred to a new 96-well plate and are incubated for 30 minutes in the presence of the substrate mix at room temperature.
  • the reaction is stopped using the stop stolution reagent and the absorbance is determined at 490 nM.
  • the percentage of cell death is determined here using a 100% maximum LDH activity control (made from untreated cells incubated in the presence of the lysis solution for 45 minutes at 37°C just before adding of the substrate mix), and the cell viability in each well is then deduced from this percentage.
  • the IC 5 o is then determined as explained in the previous paragraph.
  • the percentage of viability is determined using the CelITox Green Cytotoxicity Assay kit (Promega). This test measures cell death via a change in membrane integrity. This test uses a cyanine-type probe which does not penetrate cells when they are alive, but which binds to the DNA of dead cells which are permeable to the probe, causing it to fluoresce. Accordingly, the higher the fluorescence in the wells, the greater the cell death. After 48 hours of treatment, the cells are incubated for a minimum of 15 minutes in the presence of Celltox green reagent at room temperature and the fluorescence is read at ⁇ emission 485 nm/ ⁇ eX citation 590 nm.
  • the percentage of cell death is determined using the 100% cell death control (made from untreated cells incubated in the presence of the lysis solution for 30 minutes at 37°C before adding the Celltox green reagent), and the cell viability in each well is then deducted from this percentage.
  • the IC 5 O is then determined as explained above.
  • n D represents the number of independent tests with 4 to 10 replicates for each condition.
  • Example 26 Study of the cytotoxicity of DX101 analogues according to the invention on BT-474 cells
  • BT-474 human mammary tumor cells characterized as being triple positive HER2+, ER+, PR+.
  • the BT-474 cells are in a cell culture medium identical to the previous example and are seeded in 24-well plates at 70,000 cells per well, for the determination of cell viability using Trypan blue, or in 96-well plates at 13,000 cells per well for determining cell viability using the MTT or LDH assay.
  • the trypan blue test is based on the integrity of cell membranes, which is broken in dead cells. Trypan blue stains dead cells blue.
  • the Biorad TC20 cell counting device counts the proportion of blue and non-blue cells, and reports the percentage of cells. The percentage of viability is then determined in each well with respect to the untreated cells and the IC 50 is determined as explained in the preceding example. The results are shown in Table 2. Also, the percentage viability of BT-474 cells was determined using the MTT and LDH assay, performed as described in the previous example.
  • n D represents the number of independent tests with 3 to 10 replicates for each condition.
  • Example 27 Effect of the analog compound DX111 on tumor growth in vivo
  • All animal procedures were conducted according to the guidelines of our institution after being approved by the ethics committee.
  • the 4T1 cells were cultured as before, dissociated in trypsin, washed twice in cold PBS and resuspended in 1.5 million/ml PBS.
  • 4T1 tumors were obtained by subcutaneous transplantation of 0.150 million cells in 100 ⁇ L into the flank of female Balb/c mice (9 weeks, January). When the tumors reached a volume of 50-100 mm 3 , the mice were force-fed with 40 mg/kg of DX101 or 40 mg/kg of DX11 1 or of the control vehicle (water).
  • the treatment was carried out every day until the end of the experiment (tumor volume > 1000 mm 3 ).
  • the tumor volume was determined daily using a caliper and calculated using the formula: 1 /z X (Length * Width 2 ).
  • the percentage of tumor growth inhibition was determined according to the following formula: 100 X (1 - (Tumor volume, day 7/ tumor volume day 0) D xm) / (1 - (tumor volume, day 7/ tumor volume day 0) vehicle).
  • Example 28 Study to determine the pharmacokinetics and bioavailability of DX11 1 orally in rats.
  • the results show that the analog compound DX111 has a bioavailability 3 times greater than the reference compound DX101 by reducing the elimination half-life time.
  • the maximum plasma concentration obtained with DX111 is 4 times higher than that of DX101 and clearance is halved.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Steroid Compounds (AREA)
EP21801547.7A 2020-10-29 2021-10-28 5alpha-hydroxy-6beta-[2-(1-h-imidazol-4-yl)-ethylamino cholestan-3beta-ol analoge und diese enthaltende pharmazeutische zusammensetzungen zur behandlung von krebs Pending EP4236963A1 (de)

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BE20205764A BE1028754B1 (fr) 2020-10-29 2020-10-29 ANALOGUES DU 5α-hydroxy-6β-[2-(1H-imidazol-4-yl)éthylamino]cholestan-3β-ol ET COMPOSITIONS PHARMACEUTIQUES LE COMPRENANT POUR UTILISATION DANS LE TRAITEMENT DU CANCER
BE20205878A BE1028852B1 (fr) 2020-12-03 2020-12-03 PRODROGUE DU 5α-hydroxy-6β-[2-(1H-imidazol-4-yl) éthylamino]cholestan-3β-ol ET COMPOSITIONS PHARMACEUTIQUES LE COMPRENANT POUR UTILISATION DANS LE TRAITEMENT DU CANCER
PCT/EP2021/080054 WO2022090427A1 (fr) 2020-10-29 2021-10-28 Analogues du 5alpha-hydroxy-6 beta-[2-(1-h-imidazol-4-yl)ethylamino]-cholestan-3 beta-ol et compositions pharmaceutiques le comprenant pour utilisation dans le traitement du cancer

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EP4236963A1 true EP4236963A1 (de) 2023-09-06

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EP (1) EP4236963A1 (de)
JP (1) JP2023551363A (de)
KR (1) KR20230097077A (de)
AU (1) AU2021372797A1 (de)
CA (1) CA3196518A1 (de)
WO (1) WO2022090427A1 (de)

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CN105611934A (zh) 2013-09-04 2016-05-25 艾菲彻姆 用于治疗化学敏感性或化学耐药性肿瘤的Dendrogenin A和抗肿瘤剂

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US20230391817A1 (en) 2023-12-07
WO2022090427A1 (fr) 2022-05-05
AU2021372797A1 (en) 2023-06-15
CA3196518A1 (fr) 2022-05-05
KR20230097077A (ko) 2023-06-30
JP2023551363A (ja) 2023-12-08
AU2021372797A2 (en) 2023-06-29

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