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WO2020055129A1 - Composition de traitement ou de prévention du cancer comprenant un dérivé de verbénone - Google Patents

Composition de traitement ou de prévention du cancer comprenant un dérivé de verbénone Download PDF

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Publication number
WO2020055129A1
WO2020055129A1 PCT/KR2019/011769 KR2019011769W WO2020055129A1 WO 2020055129 A1 WO2020055129 A1 WO 2020055129A1 KR 2019011769 W KR2019011769 W KR 2019011769W WO 2020055129 A1 WO2020055129 A1 WO 2020055129A1
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group
cancer
dimethylbicyclo
hept
methoxy
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Korean (ko)
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황종익
김원기
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Korea University Research and Business Foundation
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Korea University Research and Business Foundation
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • 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
    • A61K31/12—Ketones
    • 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
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/308—Foods, ingredients or supplements having a functional effect on health having an effect on cancer prevention

Definitions

  • the present invention relates to a composition for preventing, improving or treating cancer, comprising a berbenone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Cancer is one of the greatest diseases that threaten human health, and is a disease caused by immortalization in an unlimited and unregulated manner through a series of mutations in cell lines.
  • causes of cancer include environmental or external factors such as chemicals, viruses, bacteria, and ionizing radiation, and internal factors such as congenital gene mutations (Klaunig & Kamendulis, Annu Rev Pharmacol Toxicol. , 44: 239-267) , 2004).
  • the present inventors have conducted research to invent an anti-cancer composition that effectively acts against cancer, whereby the berbenone derivative not only inhibits the proliferation of cancer cells, but also has the effect of inhibiting invasion and metastasis. It has been discovered, and the composition for preventing, improving or treating cancer using the same has been completed.
  • Patent Document 001 KR 10-1459612 B
  • the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising a berbenone derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a health functional food composition
  • a health functional food composition comprising a verbenone derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a food additive comprising a berbenone derivative represented by Formula 1 below or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom, a halogen atom selected from F, Cl, Br or I, a hydroxy group, a C 1 to C 3 lower alkyl group, a C 1 to C 3 lower alkoxy Group, amino group, C 1 to C 3 lower alkylamine group, C 1 to C 3 lower alkyldiamine group, aromatic ring having 5 to 8 carbon atoms, aliphatic ring having 5 to 8 carbon atoms, heteroaromatic ring having 5 to 8 ring atoms , And At least one substituent selected from the group consisting of,
  • X, Y and Z are each independently carbon atom or one or more heteroatoms selected from the group consisting of N, O or S atoms,
  • composition for preventing, improving or treating cancer comprising the berbenone derivative of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, while selectively exhibiting low cytotoxicity, proliferation, infiltration and migration of cancer cells It has an excellent inhibitory effect and can be useful for the prevention, improvement or treatment of cancer.
  • 1 is a graph showing the growth inhibitory effect of berbenone derivative in MDA-MB231 breast cancer cells.
  • FIG. 2 is a graph showing the growth inhibitory effect of berbenone derivatives in MDA-MB453 breast cancer cells.
  • 3 is a graph showing the growth inhibitory effect of berbenone derivatives in 4T1 breast cancer cells.
  • FIG. 4 is a graph showing the growth inhibitory effect of LMT356 in various breast cancer cells.
  • 5 is a cell growth analysis results showing the growth inhibitory effect of LMT356 on colon, lung and pancreatic cancer cells.
  • FIG. 6 is a cell growth analysis results showing the growth inhibitory effect of LMT356 on liver cancer cells.
  • Figure 7a is a result of analyzing the death of cancer cells by the treatment of LMT356 by LDH analysis
  • Figure 7b shows the effect of LMT356 on the cell cycle distribution of breast cancer cells.
  • Figure 8a is the result of testing the effect of LMT356 on cell migration by performing wound healing analysis on various doses for 18 hours
  • Figure 8b is an analysis result showing the effect of LMT356 on the infiltration of breast cancer cells.
  • FIG. 11A and 11B are results showing that the tumor volume of mice administered LMT356 is significantly lower than the tumor volume of vehicle-administered mice, and FIG. 11C shows that the LMT356-administered group has a lower tumor weight compared to the vehicle-administered group. This is the result.
  • FIG. 12A and 12C are results of histological analysis of lungs isolated from mice grown by injecting breast cancer cells and confirming that the LMT356-administered group had a significantly reduced tumor size compared to the vehicle-administered group, and FIG. 12B shows the number of tumor nodules This is a result showing a decrease in the LMT356 administration group.
  • One aspect of the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer, comprising a berbenone derivative represented by the following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom, a halogen atom selected from F, Cl, Br or I, a hydroxy group, a C 1 to C 3 lower alkyl group, a C 1 to C 3 lower alkoxy Group, amino group, C 1 to C 3 lower alkylamine group, C 1 to C 3 lower alkyldiamine group, aromatic ring having 5 to 8 carbon atoms, aliphatic ring having 5 to 8 carbon atoms, heteroaromatic ring having 5 to 8 ring atoms , And
  • One or more substituents selected from the group consisting of, X, Y and Z are each independently a carbon atom or one or more heteroatoms selected from the group consisting of N, O or S atoms,
  • more preferable compounds include one of the compounds represented below.
  • the compounds represented by Formula 1 of the present invention may contain one or more asymmetric carbons, and thus may exist as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and respective diastereomers have.
  • These isomers for example, compounds represented by Formula 1, can be separated by column chromatography or HPLC.
  • the stereoisomers of each of the compounds represented by Formula 1 may be stereospecifically synthesized using a known array of optically pure starting materials and / or reagents.
  • the pharmaceutically acceptable salt means a salt commonly used in the pharmaceutical industry, for example, inorganic ionic salts made of calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid , Inorganic acid salts made of perchloric acid and sulfuric acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galactu Organic acid salts, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.
  • inorganic ionic salts made of calcium, potassium, sodium and magnesium, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, i
  • the compound represented by Formula 1 may be prepared, for example, by the method disclosed in Korean Patent Publication No. 10-1459612, and may be chemically synthesized by the method shown in the following reaction scheme.
  • the berbenone derivative represented by Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is used to treat cerebrovascular disease, arteriosclerosis or cardiovascular disease in combination with an effect of treating degenerative brain disease or in combination with a reperfusion treatment. Although it is known to be effective, there is no known therapeutic effect on cancer.
  • Verbenone derivative represented by the formula (1) of the present invention exhibits anti-cancer efficacy inducing apoptosis of the cancer cell line, and has an advantage that it can be stably used without seriously stimulating normal cells or causing harmful effects.
  • the berbenone derivative may be usefully used as an active ingredient in a pharmaceutical composition for preventing or treating cancer.
  • Verbenone derivative represented by the formula (1) was confirmed by a specific experimental example that can induce cell cycle arrest and apoptosis in the S phase for cancer cells.
  • the compounds of the present invention can inhibit the metastasis and invasion of cancer cells.
  • cancer is a disease related to cell death regulation, and collectively refers to a disease in which cells become overproliferated when the balance of normal apoptosis is broken. These abnormal hyperproliferative cells may invade surrounding tissues and organs in some cases to form masses, and may cause destruction or deformation of normal structures in the body, which is commonly referred to as cancer.
  • a tumor refers to a mass that is abnormally grown by autonomous overgrowth of body tissue, and the tumor can be divided into a benign tumor and a malignant tumor.
  • Malignant tumors have a very rapid growth rate compared to benign tumors, and metastasis occurs when they infiltrate surrounding tissues and become life-threatening. These malignant tumors are commonly referred to as 'cancer'.
  • the type of cancer is not particularly limited, but non-limiting examples include colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, stomach cancer, prostate cancer, breast cancer, brain tumor, lung cancer, uterine cancer, bladder cancer and blood cancer. Can be mentioned.
  • the pharmaceutical composition of the present invention has the effect of inhibiting growth, invasion, or metastasis of cancer cells.
  • the breast cancer cell lines MDA-MB231, 4T1, DMA-MB453, SKBR3, MCF-7 and HCC1143 inhibit cell proliferation, infiltration ( invasiton) inhibitory effect and metastasis inhibitory effect were specifically confirmed.
  • the cell proliferation inhibitory effect on the colon cancer cell lines LOVO1, DLD1, CACO2, CT26 and HT29 the cell proliferation inhibitory effect on the lung cancer cell lines A549, NCI-H460, H1975 and H1299 (L), the liver cancer cell line Huh-7 , Hepa1-6, Hep3B and SK-Hep1 cell proliferation inhibitory effect, and pancreatic cancer cell lines BxPC3, Panc1 and CFPAC1 cell proliferation inhibitory effect was specifically confirmed.
  • prevention refers to all actions that inhibit or delay the invention of cancer by administering the pharmaceutical composition of the present invention to an individual
  • treatment refers to cancer by administering the pharmaceutical composition of the present invention to an individual. It means all the actions to improve or improve the symptoms.
  • the berbenone derivative represented by Chemical Formula 1 may be preferably contained in the pharmaceutical composition at 0.1 to 500 ⁇ M, more preferably 1 to 100 ⁇ M.
  • the compound When the compound is used in an appropriate amount within the above range, it is advantageous in that it exhibits economical and anti-cancer effects sufficiently and is more suitable for achieving the object of the present invention.
  • the pharmaceutical composition may contain one or more active ingredients exhibiting the same or similar function in addition to the berbenone derivative represented by Chemical Formula 1.
  • composition according to the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent.
  • the term “pharmaceutically acceptable” means that it is commonly used in the pharmaceutical field, without stimulating the organism when administered, without inhibiting the biological activity and properties of the compound to be administered.
  • the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.
  • Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. You can. These may be used alone or in combination of two or more.
  • composition of the present invention may be used by adding other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , It may be used by additionally adding a binder or a lubricant.
  • other pharmaceutically acceptable additives such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , It may be used by additionally adding a binder or a lubricant.
  • composition of the present invention may be formulated and used in various formulations suitable for oral administration or parenteral administration.
  • Non-limiting examples of the formulation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, preparation powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc. Can be mentioned.
  • a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; Excipients such as Dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearte, sodium stearyl fumarate or polyethylene glycol wax can be used, and sweeteners, fragrances, syrups, etc. can also be used.
  • a liquid carrier such as fatty oil, etc. may be additionally used.
  • Non-limiting examples of the parenteral preparations include injection solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for application, oils, creams, and the like.
  • aqueous solutions In order to formulate the pharmaceutical composition of the present invention for parenteral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, external preparations, etc. may be used, and the non-aqueous solvents, suspensions are propylene glycol, polyethylene Vegetable oils such as glycol and olive oil, and injectable esters such as ethyl oleate may be used.
  • the pharmaceutical composition of the present invention when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed with water with a stabilizer or a buffer to prepare a solution or suspension, and it is used for the preparation of ampoules or vials. It can be formulated for unit administration.
  • a propellant or the like when the pharmaceutical composition of the present invention is formulated as an aerosol agent, a propellant or the like may be combined with an additive so that the water-dispersed concentrate or wet powder is dispersed.
  • animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide Etc. can be formulated as a carrier.
  • the pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present invention may be varied by a method of formulating the pharmaceutical composition, a method of administration, an administration time and / or a route of administration, and the like to achieve the reaction to be achieved by administration of the pharmaceutical composition.
  • Type and extent, type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, components of the drug or other composition used simultaneously or simultaneously with the subject It may vary depending on various factors and similar factors well known in the pharmaceutical field, and a person skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.
  • the dosage for the more preferred effect of the pharmaceutical composition of the present invention is preferably 0.01 mg / kg to 10 g / kg per day, more preferably 0.1 mg / kg to 5 g / kg per day, most preferably 1 mg / kg to 1 g / kg.
  • the concentration of the berbenone derivative represented by Chemical Formula 1 in the pharmaceutical composition, its stereoisomer, or a pharmaceutically acceptable salt thereof is preferably 0.1 to 500 ⁇ M, preferably 1 to 100 ⁇ M.
  • Administration of the pharmaceutical composition of the present invention may be administered once a day, it may be divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way.
  • the route of administration and the mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in the manner, and as long as the pharmaceutical composition can reach the targeted site of interest in any route and mode of administration. Can follow.
  • the pharmaceutical composition may be administered by oral administration or parenteral administration.
  • the parenteral administration method may be administered by, for example, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, intracranial injection, or intrathoracic injection,
  • a method of applying the composition to a disease site, spraying, or inhaling may also be used, but is not limited thereto.
  • the pharmaceutical composition of the present invention may preferably be administered orally or injected, and more preferably, administered orally.
  • composition may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy, and biological response modifiers.
  • composition of the present invention can be administered for human or animal use.
  • the present invention provides a pharmaceutical composition for inhibiting metastasis of cancer comprising a berbenone derivative represented by the following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom, a halogen atom selected from F, Cl, Br or I, a hydroxy group, a C 1 to C 3 lower alkyl group, a C 1 to C 3 lower alkoxy Group, amino group, C 1 to C 3 lower alkylamine group, C 1 to C 3 lower alkyldiamine group, aromatic ring having 5 to 8 carbon atoms, aliphatic ring having 5 to 8 carbon atoms, heteroaromatic ring having 5 to 8 ring atoms , And
  • One or more substituents selected from the group consisting of, X, Y and Z are each independently a carbon atom or one or more heteroatoms selected from the group consisting of N, O or S atoms,
  • the pharmaceutical composition for inhibiting cancer metastasis of the present invention is dedicated to the contents of the pharmaceutical composition for treating or preventing cancer.
  • the present invention provides a health functional food composition for preventing or improving cancer, which includes a berbenone derivative having the structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R 1 , R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom, a halogen atom selected from F, Cl, Br or I, a hydroxy group, a C 1 to C 3 lower alkyl group, a C 1 to C 3 lower alkoxy Group, amino group, C 1 to C 3 lower alkylamine group, C 1 to C 3 lower alkyldiamine group, aromatic ring having 5 to 8 carbon atoms, aliphatic ring having 5 to 8 carbon atoms, and heteroaromatic group having 5 to 8 ring atoms ring, And At least one substituent selected from the group consisting of,
  • X, Y and Z are each independently carbon atom or one or more heteroatoms selected from the group consisting of N, O or S atoms,
  • the health functional food composition of the present invention is dedicated to the contents of the pharmaceutical composition for treating or preventing cancer.
  • improvement means any action that at least reduces the severity of parameters associated with the condition being treated, for example symptoms.
  • the health functional food composition of the present invention When used as a food additive, the composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method.
  • the type of the food is not particularly limited, and includes all foods in the ordinary sense.
  • foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other dairy products including noodles, gums, ice cream, various soups, beverages, And tea, drink, alcoholic beverages, or vitamin complexes.
  • the health functional food composition of the present invention is a beverage composition
  • it may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a normal beverage.
  • natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin; And synthetic sweeteners such as saccharin and aspartame.
  • the proportion of the additional component added can be appropriately determined by the choice of those skilled in the art.
  • the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginsam and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, It may contain alcohol, carbonic acid used in carbonated beverages, and the like.
  • the health functional food composition of the present invention in the foil may contain flesh for the preparation of natural fruit juice, fruit drink or vegetable drink. These ingredients may be used independently or in combination of two or more. The proportions of these additives can also be appropriately selected by those skilled in the art.
  • composition of the present invention contains the compound in an amount of 0.01 to 99% by weight based on the total weight of the composition.
  • the composition as described above is not necessarily limited thereto, and may be changed according to the patient's condition and the type and progress of the disease.
  • the present invention provides a food additive comprising a berbenone derivative having the structure of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient having a cancer prevention or improvement effect.
  • the food additive of the present invention is dedicated to the contents of the health functional food composition for treating or improving cancer.
  • Reagent grade (1S)-(-)-verbenone, aldehydes, methylchloromethylether (MOM-Cl), diisopropylethylamine (DIPEA), potassium hydroxide (KOH) ), And sodium methoxide (sodium methoxide; NaOCH 3 ) were purchased commercially and all reagents and solvents were purchased with high purity and used directly without further purification except for dichloromethane distilled with calcium hydroxide.
  • One reaction was carried out in a vacuum-treated dried glassware under a dry nitrogen atmosphere.
  • TLC Thin-layer chromatography
  • Merck silica gel 60 F 254 UV-visualized silica gel
  • column chromatography uses silica gel (E.Merck silica gel, 70-230, 230-400 mesh).
  • the 1 H-NMR and 13 C-NMR spectra were measured at 500 MHz with a Varian, and the chemical shift was shifted from tetramethysilane (TMS) used as an internal standard reagent (CDCl 3 : d7.26ppm) in ppm. Recorded and the coupling constant was reported in hertz.
  • TMS tetramethysilane
  • FBS Fetal bovine serum
  • a yellow solid phase (1S, 5R) -4- (3,4-dihydroxystyryl) -6,6-dimethyl ratio showing the following physical property values using a method similar to that of Example 5 (Method for preparing 3a compound) Cyclo [3.1.1] hept-3-en-2-one (3c) [(1S, 5R) -4- (3,4-dihydroxystyryl) -6,6-dimethylbicyclo [3.1.1] hept-3-en -2-one (3c)] (yield 84%):
  • a yellow solid phase (1R, 5S) -4- (3,4-dihydroxystyryl) -6,6-dimethyl ratio showing the following physical property values using a method similar to that of Example 5 (Method for preparing 3a compound) Cyclo [3.1.1] hept-3-en-2-one (3d) [1R, 5S) -4- (3,4-dihydroxystyryl) -6,6-dimethylbicyclo [3.1.1] hept-3-en- 2-one (3d)].
  • Cell culture medium was obtained from WELGENE Inc. (Daegu, Korea).
  • Human recombinant TNF- ⁇ was purchased from R & D systems (Minneapolis, MN), and protease inhibitor cocktail was purchased from Roche (Mannheim, Germany).
  • Antibodies to actin, ERK, NF- ⁇ B, p65, p-Akt and p-ERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Antibodies to Akt, PARP, caspase-3 and MMP-9 were obtained from Abcam (Cambridge, UK). All primers for gene cloning and materials for constructing expression vectors were obtained from Cosmogenetech (Seoul, Korea), and DNA sequencing was performed by the same company. All reagents were obtained from Sigma-Aldrich unless otherwise specified.
  • Breast cancer cell lines such as MDA-MB231, MDA-MB453, MCF-7, HCC-1143, 4T1, SKBR3, various lung cancer, liver cancer, colon cancer, pancreatic cancer cell lines and HEK293T cells were obtained from the American Type Culture Collection (Manassas, VA). . The obtained cells were maintained in RPMI1640 or DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. The cells were cultured at 37 ° C. in a humidification chamber containing 5% CO 2 .
  • FBS fetal bovine serum
  • MDA-MB231, MDA-MB453, MCF-7, HCC-1143, SKBR3 (4000 cells / well) and 4T1 (3000 cells / well) were seeded in a 96-well plate, and then transferred to LMT356 for a defined time in a complete culture medium. Treatment. Cell growth was measured using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc. (Rockville, MD)) according to the manufacturer's instructions. After incubating the cells for 2 hours with 10 ⁇ l CCK-8 solution, the absorbance of each well was measured at 450 nm using a microplate reader.
  • CCK-8 Cell Counting Kit-8
  • MDA-MB231 cells were cultured in a 6-well plate at a density of 5 x 10 5 cells / well. The next day, cells were treated with 10 ⁇ M of LMT356 every 24 hours for 3 consecutive days. After harvesting both adherent cells and floating cells and washing with cold PBS, cell cycle analysis was performed. The method fixed the cells with 100% ethanol and stored on ice for 1 hour. The fixed cells were centrifuged for 5 minutes at 3,000 rpm and washed with PBS.
  • PI propidium iodide
  • Cytotoxicity was assessed quantitatively by measuring the amount of LDH released from the plasma-membrane-damaged cells into the culture medium. LDH release into the culture was detected using a cytotoxicity detection kit according to the manufacturer's instructions (Takara Bio Company, Shiga, Japan). Briefly, cells were seeded in 96-well plates with RPMI medium containing 10% FBS and grown for 24 hours. Cells were then incubated with 200 ⁇ l of serum-free RPMI medium containing LMT356 for 24 or 48 hours. Cells treated with vehicle (DMSO) were used as a negative control. Part of the vehicle treated cells was lysed with 1% Triton-X-100 buffer and used as a positive control or a high control.
  • DMSO vehicle
  • the microtiter plate was centrifuged at 250 x g for 10 min, then 100 ⁇ l of the supernatant was transferred to another 96 well plate and 100 ⁇ l of the reaction mixture was added. After incubation at room temperature for 30 minutes, absorbance was measured at 490 nm using a microplate reader. The relative active LDH (%) was calculated by ([A] sample-[A] negative control) / ([A] high control-[A] negative control)) x 100%.
  • mice Female NOD / SCID mice of 5 weeks of age were purchased from KOATECH (Pyeongtaek, Korea). Animals were stored under conditions free of specific pathogens in individually ventilated cage systems. MDA-MB231 cells (1 ⁇ 10 6 cells) were injected subcutaneously into the right flank of the mouse. After 1 day, the mice were randomly divided into 2 groups of 6 mice. One group was injected intraperitoneally with LMT356 (10 mg / kg) daily until the end of the experiment, and the other group was treated with a solvent solution (saline solution containing 5% DMSO and 10% cremophor). Tumor diameter was measured from 3 mm or more and tumor volume was calculated as 0.5 ⁇ length ⁇ width 2 . When the tumor volume reached ⁇ 2 cm 3 , the mouse was anesthetized and surgically excised the tumor and weighed.
  • KOATECH Korean University Animal Experiment Management Committee
  • the lung metastasis model was prepared by intravenously injecting MDA-MB231 cells (1 x 10 6 cells) into the NOD / SCID mice. Mice divided into two groups were injected with vehicle or LMT356 (10 mg / kg) daily. On day 45, mice were sacrificed and lungs were fixed with 4% paraformaldehyde solution, followed by histological analysis. Lung sections (5 ⁇ m) were placed on a glass slide and stained with hematoxylin and eosin. Representative pictures were taken for each group and metastatic tumor nodules were counted. Tumor burden was calculated as the percentage of lung areas with tumors relative to the entire lung area.
  • 1 is a graph showing the growth inhibitory effect of berbenone derivatives in MDA-MB231 breast cancer cells.
  • FIG. 2 is a graph showing the growth inhibitory effect of berbenone derivatives in MDA-MB453 breast cancer cells.
  • 3 is a graph showing the growth inhibitory effect of berbenone derivatives in 4T1 breast cancer cells.
  • the verbenone derivative according to the present invention exhibits a growth inhibitory effect on various breast cancer cells.
  • LMT356 among the tested verbenone derivatives was shown to strongly inhibit the proliferation of cancer cells.
  • LMT356 exhibited a universal growth inhibitory effect on various breast cancer cells. After exposure of various breast cancer cells to different concentrations of LMT356 for 24, 48 and 72 hours, respectively, the effect of LMT356 on the cancer cell growth was observed by CCK-8 analysis.
  • FIG. 4 is a graph showing the growth inhibitory effect of LMT356 in various breast cancer cells.
  • LMT356 inhibited the growth of breast cancer cells of MDA-MB231, 4T1, MDA-MB453, SKBR3, MCF-7 and HCC-1143 in a time and concentration dependent manner.
  • FIG. 5 is a cell growth analysis result showing the growth inhibitory effect of LMT356 on colon cancer, lung cancer, and pancreatic cancer cells
  • FIG. 6 is a cell growth analysis result showing the growth inhibition effect of LMT356 on liver cancer cells.
  • LMT356 exhibits a growth inhibitory effect on various cancer cells such as colon cancer, lung cancer, pancreatic cancer, and liver cancer as well as breast cancer cells.
  • FIG. 7A shows the results of analyzing the death of cancer cells by treatment with LMT356 by LDH analysis.
  • MDA-MB231 cells were exposed to different concentrations of LMT356 (5 and 10 ⁇ M) or vehicle DMSO for 48 hours. Then, the cells were fixed, made permeable, stained with propionate iodide (PI), and the proportion of cells at each stage of the cell cycle determined by the DNA contents was measured.
  • LMT356 5 and 10 ⁇ M
  • vehicle DMSO vehicle DMSO
  • 7B is a result of analyzing the death of cancer cells by the treatment of LMT356 by cytometric analysis.
  • TNBCs are very aggressive, accounting for a major proportion of breast cancer deaths due to metastasis.
  • LMT356 The effect of LMT356 on cell migration was tested by performing wound healing analysis at various doses for 18 hours. Specifically, MDA-MB231, 4T1 and MDA-MB453 cells were cultured at a very close density. The culture monolayer was scratched with a yellow tip. Cells were washed 3 times and incubated with the indicated doses of LMT356. Images were captured at 0 and 18 hours.
  • Figure 8a is the result of testing the effect of LMT356 on cell migration by performing wound healing analysis on various doses for 18 hours.
  • the graph on the right side of FIG. 8A quantitatively shows the percentage of wound closure in various treatment groups.
  • the wound gap of the 2.5 ⁇ M and 5 ⁇ M LMT356 treated groups was significantly greater than that of the untreated group of MDA-MB231, 4T1 and MDA-MB453 cells.
  • the inhibitory effect of the drug on cancer cell infiltration was analyzed by the Transwell infiltration assay. After treating MDA-MB231 and 4T1 cells with different concentrations of LMT356, the cells were allowed to infiltrate into a matrigel coated transwell chamber. Infiltrated cells were counted in five high-power microscope fields, and the resulting count was calculated as the average value. The number of cells infiltrated through the transwell membrane was dose dependently reduced by LMT356 (FIG. 8B). These data indicate that LMT356 affects motility of breast cancer cells.
  • LMT356 inhibits breast tumor growth
  • mice MDA-MB231 cells were implanted into the flanks of immunocompromised mice and LMT356 (10 mg / kg) was administered daily to the mice by intraperitoneal injection.
  • the tumor volume of mice administered LMT356 was significantly lower than that of vehicle-administered mice measured on day 42 (FIGS. 11A and 11B).
  • all tumors were removed from the sacrificed mice and weighed. 11C, it was confirmed that the LMT356-administered group had a significantly lower tumor weight compared to the vehicle-administered group, so that LMT356 inhibited tumor growth in an in vivo-xenograft model.
  • LMT356 inhibits metastasis of MDA-MB231 breast cancer cells
  • LMT356 Due to the effect of LMT356, which strongly blocks in vitro invasion of breast cancer cells, the effect of inhibiting metastasis in vivo was investigated. MDA-MB231 cells were injected into the tail vein of NOD / SCID mice, and then LMT356 was administered. After 45 days, histological analysis of the lungs isolated from the sacrificed mice was performed, confirming that the tumor size of the LMT356 administration group was significantly reduced compared to the vehicle administration group (FIGS. 12A and 12C).
  • 12A is an image of a lung section excised from mice injected daily with vehicle or 10 mg / kg LMT356 for 45 days.
  • mice To investigate whether the vehicle or LMT356 itself had a detrimental effect on mice, a reagent was applied to mice that had had cancer cells removed for the same time. Blood tests and thorough anatomical analysis showed no apparent abnormalities, indicating that this reagent is potentially safe.
  • the verbenone derivative according to the present invention comprising LMT356 inhibits metastasis and growth of various cancers, including breast cancer.

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Abstract

La présente invention concerne une composition pharmaceutique servant à la prévention, au soulagement ou au traitement du cancer, contenant un dérivé de verbénone ou un sel pharmaceutiquement acceptable de ce dernier en tant que principe actif. La composition de la présente invention, servant à la prévention, au soulagement ou au traitement du cancer, comprend un dérivé de verbénone ou un sel pharmaceutiquement acceptable de ce dernier en tant que principe actif, présente une faible cytotoxicité et est néanmoins très efficace dans l'inhibition sélective de la prolifération, de l'infiltration et de la migration de cellules cancéreuses, et peut donc être avantageusement utilisée pour prévenir, soulager ou traiter le cancer.
PCT/KR2019/011769 2018-09-11 2019-09-10 Composition de traitement ou de prévention du cancer comprenant un dérivé de verbénone Ceased WO2020055129A1 (fr)

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