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WO2019031880A1 - Dérivé de kidamycine l1-95-1, et composition pour prévenir ou traiter le cancer le contenant à titre de principe actif - Google Patents

Dérivé de kidamycine l1-95-1, et composition pour prévenir ou traiter le cancer le contenant à titre de principe actif Download PDF

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Publication number
WO2019031880A1
WO2019031880A1 PCT/KR2018/009105 KR2018009105W WO2019031880A1 WO 2019031880 A1 WO2019031880 A1 WO 2019031880A1 KR 2018009105 W KR2018009105 W KR 2018009105W WO 2019031880 A1 WO2019031880 A1 WO 2019031880A1
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cells
breast cancer
cancer
pharmaceutical composition
preventing
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Korean (ko)
Inventor
조용연
홍영수
최경일
장재혁
이철중
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Industry Academic Cooperation Foundation of Catholic University of Korea
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing three or more hetero rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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 kidamycin derivative L1-95-1 and a composition for preventing or treating cancer comprising the same as an active ingredient.
  • Cancer is caused by genetic and environmental factors leading to abnormal cell division because the normal cell division of the cells is not regulated, and it does not die beyond the limit of the number of cell divisions. These transformed cells continue to proliferate into cancerous masses, and the inner core of the cancerous mass is limited in the supply of oxygen and the supply of nutrients for the growth of cancer cells. When oxygen supply is limited, cancer cells express the HIF-1 protein and increase the expression of proteins involved in angiogenesis and proteins involved in energy metabolism. As a result, cancer tissue grows again.
  • breast cancer is one of the most common diseases in women. In the United States, there are about 240,000 breast cancer cases a year and about 40,000 deaths are reported. In Korea, the number of breast cancer patients is rapidly increasing due to westernization of dietary habits and increase of obesity population, and it is estimated that about 18,000 patients are born in a year. Because the breast cancer cell metastasis is not as high as other cancer types, it is a cancer type with a high cure rate of about 93% 5 years survival rate when breast cancer is detected early and appropriate treatment is performed. However, if early detection and appropriate treatment are not done, cancer metastasis will occur through the blood vessels and lymphatic vessels, and the cancer mortality rate will increase. In fact, the survival rate for stage 4 stage breast cancer is 34%, which is much lower than the overall survival rate for breast cancer.
  • breast cancer When breast cancer is treated with an anticancer agent such as taxol or radiation, the growth of the early cancer tissue is stopped and the size of the cancer is reduced. However, after a certain period of time, the cancer may grow again .
  • cancer cells that have regenerated again are treated with the same anticancer drug, the anticancer drug will no longer function, resulting in resistance, and a more malignant trait, such as a higher transfer rate of cancer cells.
  • Triple-negative breast cancer cells are known to be deficient in estrogen and progesterone receptors (ER- / PR-) and not expressed in HER2 (HER2-), suggesting that taxol, tamoxifen, It has the property of being resistant to Her 2 active inhibitor (trastuzumab).
  • triple negative breast cancer patients account for about 12-17% of all breast cancer patients, and about 15.9% of all breast cancer patients in Korea are reported to have triple negative breast cancer.
  • the 5-year survival rate of triple-negative breast cancer patients is reported to be about 77%, which is lower than the 93% seen in other types of breast cancer patients.
  • Triple-negative breast cancer is more aggressive than other types of breast cancer, such as having a histologic grade and invasive ductal carcinoma, compared with other types of breast cancer, and many triple- Like basal-like breast cancer, which has no effect on hormone therapy or trastzumab, has a high degree of chromosomal aberration (Degree of aneuploidy), similar gene-expression profile, cancer subtype).
  • Kida azithromycin (kidamycin) derivative L1-95-1 is Streptomyces sp .
  • its structure has been recently discovered by the present inventors. Therefore, the pharmacokinetics and target proteins for L1-95-1, and the effects on cancer prevention and anti-cancer are not known.
  • the present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a health food composition for preventing or ameliorating cancer, which comprises, as an active ingredient, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a kidamycin derivative L1-95-1 and a composition for preventing or treating cancer comprising the same as an active ingredient.
  • L1-95-1 inhibits cell proliferation and EGF-induced cancer of JB6 Cl41 cells.
  • L1-95-1 selectively induced mitophagy in MDA-MB-231 cells, and the proliferation and proliferation of cancer cells were suppressed by MDA-MB-231 cells. Cancer growth. Therefore, L1-95-1 may be used as an anticancer agent for the treatment of cancer or as a health food for cancer improvement.
  • FIG. 1 shows that L1-95-1 inhibits cell proliferation of JB6Cl41 and transfection by EGF at 500 nM or more.
  • A It is the chemical structure of L1-95-1.
  • B Mouse skin epithelial cells JB6 Cl41 (1 ⁇ 10 3 cells) were inoculated on a 96-well cell culture plate and cell proliferation was measured by MTS assay for 72 hours at 24-hour intervals. Data were expressed as means and standard deviations for 4 measurements and Student's t- test was used for statistical significance (* P ⁇ 0.05).
  • C The epithelial cell line JB6 Cl41 (8 ⁇ 10 3 cells) of the mouse was mixed with BME culture medium containing 0.3% agar and 10% FBS to prepare a suspension.
  • EGF (10 ng / ml)
  • DMSO 0.05, 0.25, 0.5, and 1.25 ⁇ M of L1-95-1
  • Cells were cultured in a 5% CO 2 incubator at 37 ° C for 20 days.
  • Transfection cells were counted using an ECLIPSE Ti inverted microscope using the NIS-Elements AR (V. 4.0) computer software program. Data values were expressed as means and standard deviations for three measurements and Student's t- test was used for statistical significance (* P ⁇ 0.05).
  • FIG. 2 shows that L1-95-1 specifically inhibits cell proliferation and affects the cell cycle of MDA-MB-231, a human triphasic breast cancer cell.
  • A Human breast cancer cells MCF-7 (2 ⁇ 10 3 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 3 cells) And the cell proliferation was measured by MTS assay for 96 hours at intervals of 24 hours. Data were expressed as means and standard deviations for 4 measurements and Student's t- test was used for statistical significance (* P ⁇ 0.05).
  • B Human breast cancer cells MCF-7 (5 ⁇ 10 5 cells) and human triple-negative breast cancer cells MDA-MB-231 (5 ⁇ 10 5 cells) were inoculated into a 60-mm dish and cultured overnight.
  • Each of the cells is treated with 500 nM of L1-95-1 for 48 hours.
  • the cells are harvested and fixed in 70% ethanol / 1 x PBS at -20 ° C for 2 hours. Fixed cells were washed with PBS and treated with RNase A (200 ⁇ g / ml) and propidium iodide (20 ⁇ g / ml) for 15 minutes. The cell cycle was measured by flow cytometry. Data values were expressed as means and standard deviations for three measurements and Student's t- test was used for statistical significance (* P ⁇ 0.05).
  • FIG. 3 shows that L1-95-1 induces autophagy in MDA-MB-231 cells.
  • Human breast cancer cells MCF-7 (3 ⁇ 10 6 cells) and human triple-negative breast cancer cells MDA-MB-231 (3 ⁇ 10 6 cells) were inoculated into a 100-mm dish and cultured overnight. Each of the cells was treated with DMSO and 500 nM of L1-95-1, and morphological changes of the cells were confirmed by optical microscope for 48 hours at 24 hour intervals.
  • (B) Human breast cancer cells MCF-7 (3 ⁇ 10 6 ) and human triple-negative breast cancer cells MDA-MB-231 (3 ⁇ 10 6 ) were inoculated into 100-mm dishes and cultured overnight.
  • Each of the chambers was treated with DMSO and L1-95-1 and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. Lysotracker was treated for 1 hour and Hoechst was treated for 30 minutes. Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program.
  • (D) Human breast cancer cells MCF-7 (2 ⁇ 10 4 ) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 ) were inoculated into 8-chamber slides and incubated at 37 ° C in a 5% CO 2 incubator And cultured overnight.
  • MCherry plasmids were introduced into each cell and cultured in a 5% CO 2 incubator at 37 ° C for 30 hours.
  • the cells were treated with DMSO and L1-95-1 500 nM for 24 h at 37 ° C in a 5% CO 2 incubator.
  • FIG. 4 shows that L1-95-1 selectively induces mitophagy in MDA-MB-231 cells.
  • Human breast cancer cells MCF-7 (3 ⁇ 10 6 cells) and human triple-negative breast cancer cells MDA-MB-231 (3 ⁇ 10 6 cells) were inoculated into 100- CO 2 incubator overnight. Cells were treated with DMSO, 100, 250, 500 nM of L1-95-1 and cultured in a 5% CO 2 incubator at 37 ° C for 3 hours. Cells were collected, proteins were extracted, and Western blotting was performed using the antibodies indicated in the results. ⁇ -actin was used to confirm the amount of protein in the experimental group.
  • Figure 5 shows that L1-95-1 increases ROS development in the mitochondria of MDA-MB-231.
  • Human breast cancer cells MCF-7 (2 ⁇ 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 cells) were inoculated into confocal dishes and incubated in a 5% CO 2 , 37 ° C incubator Lt; / RTI > Each cell was treated with DMSO and L1-95-1 at 500 nM or 1 mM and cultured in a 5% CO 2 , 37 ° C incubator for 24 hours.
  • Mitosox TM Red was treated for 10 minutes at a concentration of 5 ⁇ M, and 10 minutes later, fluorescence was observed using LSM 710 laser scanning confocal microscope.
  • Human breast cancer cells MCF-7 (2 ⁇ 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 cells) were inoculated into confocal dishes and incubated in a 5% CO 2 , 37 ° C incubator Lt; / RTI > Each cell was treated with 500 nM, 1 mM DMSO and L1-95-1 and cultured in a 5% CO 2 , 37 ° C incubator for 24 hours.
  • the present invention provides a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the pharmaceutical composition can inhibit the transformation into cancer cells induced by epidermal growth factor (EGF), reduce the G1 cell cycle and increase the S cell cycle in cancer cells .
  • EGF epidermal growth factor
  • the pharmaceutical composition may induce autophagy in cancer cells, and in particular may induce mitophagy.
  • the pharmaceutical composition can induce ROS (Reactive oxygen species) generation in the mitochondria of cancer cells.
  • ROS reactive oxygen species
  • L1-95-1 referred to in the present invention is a derivative of kidamycin, which is a compound represented by the formula (1).
  • the cancer may be selected from the group consisting of lung cancer, colon cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, Cancer of the head, neck cancer, malignant melanoma, lymphoma, and blood cancer, and may be preferably breast cancer as described in one embodiment of the present invention, and most preferably triple negative breast cancer.
  • triple-negative breast cancer is known to be deficient in the estrogen and progesterone receptors (ER- / PR-) and not in the expression of the HER2 gene, and taxol, tamoxifen ) And Her 2 activity inhibitor (trastuzumab).
  • ER- / PR- estrogen and progesterone receptors
  • HER2 progesterone receptors
  • taxol, tamoxifen HER2 gene, and taxol, tamoxifen
  • Her 2 activity inhibitor trastuzumab
  • triple negative breast cancer patients account for about 12-17% of all breast cancer patients, and about 15.9% of all breast cancer patients in Korea are reported to have triple negative breast cancer.
  • the 5-year survival rate of triple-negative breast cancer patients is reported to be about 77%, which is lower than the 93% seen in other types of breast cancer patients.
  • apoptosis refers to a process of apoptosis programmed into a cell as a mechanism of maintaining homeostasis by regulating the number of cells in development, aging, and tissues.
  • the cells when the cells are damaged by an immune reaction, disease or harmful substance, they also develop before the defense of the cells. Ionizing is also induced by x-ray or UV irradiation, drug treatment using an anticancer drug that damages the gene, and apoptotic cells cause protein cleavage, protein cross- linking, DNA breakdown, and phagocytic recognition.
  • autophagy is an important process that removes damaged proteins or intracellular organelles in a cell to maintain a healthy state and maintain the homeostasis. It always occurs in cells at a certain rate. However, when the cells are insufficiently fed with nutrients, proteins in the cells are damaged to form aggregates, or the like, the cells are required to be energized rapidly, or the cells are adapted to a changing external environment When the environment is changed, it suddenly increases.
  • mitophagy is one of the autophagy, which is an autophagy for specifically removing mitochondria, and by removing old or damaged mitochondria To maintain the integrity of the mitochondria. Mitophagy not only rotates abnormal mitochondria into normal mitochondria but also plays a role in regulating total mitochondrial levels in hypoxia and starvation stress environments, thereby reducing the incidence of ROS, Nutrients, and so on. This function enables the cells to survive even under energy-related stress conditions.
  • composition of the present invention is a pharmaceutical composition
  • suitable carriers excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, lubricants, flavors, antioxidants, buffers, , A diluent, a dispersant, a surfactant, a binder, and a lubricant.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
  • the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.
  • As the suppository base witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.
  • the pharmaceutical composition may be formulated into granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, Can be used.
  • the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, Can be administered to a subject in a conventional manner via the intradermal route.
  • the preferred dosage of the compound may vary depending on the condition and body weight of the subject, the type and degree of disease, the drug form, the administration route and the period, and may be appropriately selected by those skilled in the art.
  • the daily dose may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg, though it is not limited thereto.
  • the administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.
  • the pharmaceutically acceptable salt is not particularly limited, and the compound of the present invention can be formed using organic and inorganic acids.
  • the pharmaceutically acceptable salt include hydrochloric acid, hydrobromic acid, methanesulfonic acid, hydroxyethanesulfonic acid, sulfuric acid, acetic acid, Nitrates, phosphates, borates, tartrates, citrates, succinates, benzoates, ascorbates or salicylates formed with hydrochloric acid, hydrochloric acid, hydrochloric acid, hydrobromic acid,
  • the present invention provides a health food composition for preventing or ameliorating cancer, which comprises, as an active ingredient, a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
  • the health food composition may be provided in the form of powder, granules, tablets, capsules, syrups or beverages.
  • the health food composition may be used in combination with food or food additives other than the compound according to the present invention, And the like.
  • the amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.
  • the effective dose of the compound contained in the health food composition may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes , It is clear that the active ingredient can be used in an amount exceeding the above range since there is no problem in terms of safety.
  • the type of the health food includes meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, alcoholic beverages and vitamin complexes.
  • P-Akt T308
  • p-Akt S473
  • p-P70S6K T389
  • p-mTOR S2448
  • S-6 S235 / 236
  • p-MEK S217 / 221
  • p-ERK T202 / Y204
  • p- RSK T359 / S363
  • total-RSK and anti-rabbit and anti-rabbit anti-rabbit antibodies were purchased from Cell Signaling Technologies (Rockville, Md., USA) mouse secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
  • MDA-MB-231 a human triple-negative breast cancer cell line, and MCF-7, a human breast cancer cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). JB6 Cl41 was cultured in minimal essential medium (MEM) (Corning; Manassas, Va., USA) supplemented with 5% fetal bovine serum (FBS) and antibiotics (100 units / ml penicillin, 100 ⁇ g / ml streptomycin) Lt; / RTI > (100 units / ml penicillin, 100 ⁇ g / ml streptomycin) and insulin (10 ⁇ g / ml streptomycin) in a minimum essential medium (MEM) (Corning; Manassas, VA, USA) MDA-MB-231 was cultured in medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 Units / ml) in Dulbecco
  • the xMark TM Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories , Hercules, Calif., USA) was used to measure the absorbance at 492 nm.
  • the inhibitory effect of L1-95-1 on cell proliferation was measured for 5 days at 24-hour intervals, with 0 hours at 2 hours after inoculation, and L1-95-1 at the indicated concentration every 24 hours And the culture medium was replaced with the culture medium.
  • DMSO untreated group
  • L1-95-1 treated group 100 nM, 250 nM, 500 nM
  • Protein suspension was dissolved in cell lysis buffer [50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40] and the final concentration of 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerophosphate, mM sodium vanadate and 1 mM phenylmethylsulfonyl fluoride (PMSF)].
  • 100 ⁇ l of lysis buffer was added to the prepared MCF-7 and MDA-MB-231 cells, and the cells were disrupted by freezing and thawing, followed by centrifugation at 12,000 rpm for 5 minutes at 4 ° C.
  • Protein concentration was measured according to the protocol of the DC protein assay kit (Bio-Rad) and absorbance was measured at 750 nm using a xMark TM Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories, Hercules, Calif., USA) SDS-PAGE was carried out using polyvinylidene fluoride (SDS-PAGE) and the protein was eluted with a transfer buffer (20 mM Tris-HCl, pH 8.0, containing 150 mM glycine and 20% ; PVDF) membrane.
  • a transfer buffer (20 mM Tris-HCl, pH 8.0, containing 150 mM glycine and 20% ; PVDF
  • T248, p-Akt (T308), and p-Akt (S473) were blocked by blocking with 5% nonfat dry milk in blocking buffer (1X TBS containing 0.05% Tween 20 ), p-p70S6K (T389), p-S6 (S235 / 236), p-MEK (S217 / 221), p-ERK (T202 / Y204)
  • Primary antibodies such as -Akt, total-P70S6K, total-S6, total-MEK, total ERK, total-RSK and ⁇ -actin were hybridized overnight at 4 ° C.
  • Membranes were washed three times with 1X TBS-T buffer and hybridized with a suitable HRP-conjugated secondary antibody.
  • Target proteins were detected using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using a Chemidoc XRS imager system (Bio-Rad Laboratories, Hercules, CA, USA).
  • the cover slides were then attached to the 8-chamber slides using CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA). Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
  • Human breast cancer cells MCF-7 (2 ⁇ 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, L1-95-1 was diluted with each cell culture at a concentration of 500 nM and cultured for 24 hours. After washing three times with PBS, Mitotracker ® Green FM (Invitrogen, OR, USA) was diluted in each cell culture medium at a concentration of 20 nM and treated for 45 minutes.
  • Human breast cancer cells MCF-7 and human triple-negative breast cancer cells MDA-MB-231 were inoculated at 2 ⁇ 10 4 cells in each chamber of an 8-chamber slide and cultured in a 5% CO 2, 37 ° C incubator. Twenty-four hours later, the cells were transfected with 20 ⁇ l of the gelpEI ® DNA Transfection Reagent (Polypuls transfection, NY, USA) and mCherry vector in 150 mM NaCl and cultured for 30 hours. After 30 hours, L1-95-1 was diluted to a concentration of 500 nM in each cell culture medium. After 24 hours, CLEAR-MOUNT with TRIS Buffer (Electron Microscopy Sciences, Hatfield, PA, USA) And a cover slide was attached. Fluorescence was observed using an ECLIPSE Ti inverted microscope and the NIS-Elements AR (V.4.0) computer software program (NIKON Instruments Korea, Gangnam, Seoul, Korea).
  • Human breast cancer cells MCF-7 (2 ⁇ 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, L1-95-1 cells were diluted in cell culture medium (DMSO), 100 nM, 250 nM and 500 nM, respectively, and cultured for 24 hours.
  • DMSO cell culture medium
  • Cells treated with DMSO were then treated with CCCP (carbonyl cyanide 3-chlorophenylhydrazone) at a concentration of 50 ⁇ M in a cell culture medium for the positive control, and treated with MitoProbe TM JC-1 (Life technologies TM , OR, USA) were diluted in each cell culture medium at a concentration of 2 ⁇ M and treated for 15 minutes. After 15 minutes, the culture medium was replaced with calcium-PBS, and fluorescence was observed with a LSM 710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
  • CCCP carbonyl cyanide 3-chlorophenylhydrazone
  • MitoProbe TM JC-1 Life technologies TM , OR, USA
  • Human breast cancer cells MCF-7 (2 ⁇ 10 4 cells) and human triple-negative breast cancer cells MDA-MB-231 (2 ⁇ 10 4 cells) were inoculated into a confocal dish (SPL Life Sciences, Pocheon, South Korea) 2 , and 37 ° C in a constant temperature incubator. After 24 hours, DMSO and L1-95-1 were diluted in the cell culture medium at a concentration of 500 nM, and cultured for 24 hours.
  • Human breast cancer cell MCF-7 (5 ⁇ 10 5 cells) and human triple-negative breast cancer cell line MDA-MB-231 (5 ⁇ 10 5 cells) were inoculated into a 60-mm dish and incubated at 37 ° C in a 5% CO 2 incubator Time. After 24 hours, L1-95-1 was diluted in each cell culture medium (DMSO) and treated group (100 nM, 250 nM, 500 nM) for 24 hours. After washing with PBS, 0.1% Trypsin Cells were recovered using EDTA and washed with 1 x PBS. The cells were fixed with ice-cold 70% ethanol at -20 ° C for 2 hours and recovered by centrifugation at ⁇ 1000 g.
  • DMSO cell culture medium
  • treated group 100 nM, 250 nM, 500 nM
  • Cells were suspended in 250 ⁇ l of 0.6% Triton-X100-1X PBS and RNase A (200 ⁇ g / ml) and propidium iodide (20 ⁇ g / ml) were added and left at 4 ° C for 15 minutes. The cell cycle was measured by flow cytometry (BD FACS Calibur TM flow cytometry, Franklin Lakes, NJ, USA).
  • a medium containing a nutrient source normally used by microorganisms was prepared.
  • YEME medium yeast extract-malt extract medium
  • the primary culture was inoculated in a 1 liter flask with 300 ml of the same medium as above to a final concentration of 5%, and then cultured at 28 ° C for 7 days at 150 rpm. Thirty flasks were cultured at the same time to obtain a culture solution of about 10 L of the strain.
  • the preparation of the compound of formula (I) (L1-95-1) according to the present invention was carried out as follows. 10 L of the culture prepared above was extracted twice with EtOAc of the same amount and the extract was filtered to remove insolubles, and the filtrate was concentrated, and then fractionated with EtOAc and water to obtain 7 g of an organic phase extract. This concentrate was adsorbed onto silica (Cosmosil 75C18-PREP) and subjected to medium pressure liquid chromatography through a reversed phase column (RediSep® Rf Reversed Phase C18 Column) using methanol / To elute the methanol concentration while increasing the ratio. At this time, the fractions containing the kidamycin compound eluted from 80% methanol.
  • This fraction was concentrated under reduced pressure, and acetonitrile and water were then eluted with a gradient of 30:70 to 80:20 by using high performance liquid chromatography (column: Cosmosil C18, length 250 mm, diameter 10 mm) as a solvent at a flow rate of 3 ml / min And the primary eluate was re-eluted with a gradient of 40:60 50:50 concentration concentration to separate the compound L1-95-1 showing a UV absorption peak at 274 nm.
  • the compounds of the examples isolated from the culture medium of W2061 were measured for molecular weight and molecular formula using an ESIMS mass spectrometer (Electrospray Ionization mass spectrometer). Further, 1 H NMR, 13 C NMR, COZY (Correlation Spectroscopy), Heteronuclear Multiple-Bond Coherence (HMBC), and the like were performed through nuclear magnetic resonance (NMR) analysis (Bruker Biospin Advance II 900 NMR spectrometer and Bruker AVANCE HD 800 NMR spectrometer) Distortionless Enhancement by Polarization (DEPT) and Nuclear Overhauser effect spectroscopy (NOESY) spectra were obtained and the molecular structure of the compounds was determined.
  • NMR nuclear magnetic resonance
  • DEPT Distortionless Enhancement by Polarization
  • NOESY Nuclear Overhauser effect spectroscopy
  • Streptomyces sp The substance isolated from the culture broth of the W2061 strain was identified as a new L1-95-1 compound with chidamycin having the above formula, and the L1-95-1 compound was identified by the previously reported chimeric compound (Furukawa, M .; Hayakawa, Iitaka, Y. Tetrahedron 1975, 31, 2989, Furukawa, M .; Iitaka, Y. Tetrahedron Lett 1974, 15, 3287) Structural differences of double bond structure in angolosamine sugar structure Respectively.
  • Kidamycin a L1-95-1 b Position ⁇ c ⁇ H (J in Hz) ⁇ c ⁇ H (J in Hz) 1a 155.7 155.96 2 163.7 163.18 3 108.7 6.38 (s) 108.45 6.43 (s) 4 179.2 180.05 4a 125.8 127.21 5 149.6 149.61 6 125.4 7.96 (d, J 0.6 Hz) 124.74 7.83 (s) 6a 137 136.85 7 183 181.15 7a 125.8 129.6 8 140 127.41 9 133 8.32 (s) 134.42 7.64 (s) 10 138.4 137.57 11 159.7 160.59 11a 116 117.67 12 188.1 188.14 12a 118.9 119.34 13 24 3.01 (s) 23.48 2.90 (s) 14 127.2 127.2 15 12.1 2.00 (s) 11.85 1.97 (s) 16 134.2 7.49 (q, J
  • L1-95-1 was not newly studied as a novel substance newly discovered by the present inventors (FIG. 1A).
  • the effect of L1-95-1 on cell proliferation and carcinogenesis was examined using JB61 Cl41 cells as mouse skin epithelial cells (Fig. 1B).
  • Fig. 1B mouse skin epithelial cells
  • L1-95-1 inhibited the proliferation of JB6 Cl41 cells in a concentration-dependent manner (Fig. 1B).
  • L1-95-1 inhibited the proliferation of JB6 Cl41 cells by about 50% at a concentration of 1 ⁇ M (FIG. 1B).
  • L1-95-1 was used to verify the ability of the cells to inhibit epidermal growth factor (EGF) -induced cell transformation under anchorage-independent conditions.
  • EGF epidermal growth factor
  • JB6 Cl41 cells were colonized by anchorage-independent conditions by EGF stimulation and were transformed into cancer cells (Fig. 1C). However, when L1-95-1 was co-treated with EGF, it was confirmed that the transformation of JB6 Cl41 cells induced by EGF was reduced in a concentration-dependent manner (Fig. 1C). Thus, L1-95-1 inhibits cell proliferation and has the effect of inhibiting EGF-induced cell transformation under anchorage-independent conditions.
  • MCF-7 breast cancer cells were treated with 1 ⁇ M of L1-95-1 to inhibit proliferation of MCF-7 and MDA-MB-231. Inhibiting cell proliferation (Fig. 2A, Left graph).
  • L1-95-1 was treated with MDA-MB-231 for triple-negative breast cancer cells
  • the cell proliferation was inhibited by about 60% at a concentration of 500 nM.
  • L1-95-1 at a concentration of 1 ⁇ M was treated, (Fig. 2A, Right graph). Therefore, it was confirmed that L1-95-1 has specificity for triple negative breast cancer cells in breast cancer cells and effectively inhibits the proliferation of cancer cells 2A).
  • L1-95-1 inhibited cell proliferation, carcinogenesis, and cancer cell proliferation, and accumulation of S-cell cycle was observed but no apoptosis was observed. Therefore, morphological changes were observed in L1-95-1-treated breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231 cells. Cell morphology was not observed in MCF-7 breast cancer cells by treatment with L1-95-1 (Fig. 3A). In contrast, MDA-MB-231 cells induced a change in the shape of cells elongated by treatment with 500 nM of L1-95-1, and they were found in the vacuole ) (Fig. 3A).
  • L1-95-1 induces lysosomal activation and aggregation of LC3-II in triple-negative breast cancer cells, leading to autophagy.
  • mCherry was introduced into cells and analyzed by fluorescence microscopy. L1-95-1 detected both GFP and RFP in breast cancer cell MCF-7.
  • the autophagy-induced signal transduction pathway which is selectively induced by L1-95-1 in triple-negative breast cancer cells MDA-MB-231, was compared with that of MCF-7 in breast cancer cells.
  • L-95-1 was treated with MDA-MB-231 in triplicate negative breast cancer cell MCF-7 and the PI3K-AKT-mTOR signal transduction system associated with autophagy induction and MAPK And analyzed by Western blotting around a signal transduction system.
  • L1-95-1 inhibited phosphorylation of Ser473 of Akt phosphorylated by mTORC2 activated in MDA-MB-231 cells in a concentration-dependent manner (Fig. 4A, fourth panel from top).
  • Thr172 amino acid phosphorylation of AMPKa was increased by L1-95-1 treatment in MDA-MB-231 cells (Fig. 4C).
  • mitochondria an organ that maintains the balance of energy within the cell, has developed an abnormality.
  • 500 nM of L1-95-1 was treated with breast cancer cell MCF-7 and triple negative breast cancer cell MDA-MB-231, respectively, and stained with lysotracker and mitotracker to determine whether activated lysosomes and mitochondria were stained together And analyzed using a confocal microscope.
  • lysotracker staining was not observed by treatment with 500 nM of L1-95-1 in breast cancer cell MCF-7, and it was observed that mitotracker and nucleus were separated and located in different layers (Fig. 4D, Bottom left panel).
  • lysotracker-induced staining was clearly induced by treatment with 500 nM of L1-95-1 in triple negative breast cancer cells MDA-MB-231, and was stained simultaneously with lysotracker and mitotracker, An orange-colored functa was observed (Fig. 4D, bottom right panel).
  • mitosoxin staining was observed with 500 nM of L1-95-1 in triple negative breast cancer cell MDA-MB-231, and mitosoxin staining was stained together with the mitotracker (Fig. 5A, right panel).
  • MDA-MB-231 cells induces mitochondrial damage and affects the potential difference of the mitochondrial inner membrane.
  • JC-1 / red staining was weakly stained in MCF-7 breast cancer cell
  • MDA-MD-231 cells MDA-MD-231 cells
  • JC-1 / green staining was stained in both cells (FIG. 5B, left panel) .

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Abstract

La présente invention concerne un dérivé de kidamycine L1-95-1, et une composition pour prévenir ou traiter le cancer le contenant à titre de principe actif. L1-95-1 a inhibé la prolifération cellulaire et la malignisation induite par l'EGF de cellules JB6 cl41, et on a découvert, par expérimentation à l'aide de cellules du cancer du sein MCf-7 et de cellules du cancer du sein triple négatif MDA-MB-231, que L1-95-1 induit sélectivement la mitophagie dans les cellules MDA-MB-231, de sorte que la prolifération des cellules cancéreuses et la croissance du cancer sont inhibées. Par conséquent, L1-95-1 devrait avoir une applicabilité élevée à titre de médicament anticancéreux pour le traitement du cancer ou à titre d'aliment de santé pour soulager le cancer.
PCT/KR2018/009105 2017-08-10 2018-08-09 Dérivé de kidamycine l1-95-1, et composition pour prévenir ou traiter le cancer le contenant à titre de principe actif Ceased WO2019031880A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0200818A1 (fr) * 1984-12-06 1986-11-12 Microbial Chemistry Research Foundation Dérivés appartenant aux pluramycines à activité antitumorale, procédé pour leur préparation et leur utilisation comme médicament
US4847387A (en) * 1986-07-21 1989-07-11 Kyowa Hakko Kogyo Co., Ltd. Antibiotic compounds DC-92B and DC-92D
JPH01193265A (ja) * 1988-01-28 1989-08-03 Meiji Seika Kaisha Ltd 新規抗腫瘍性抗生物質ｓｆ２５８７物質及びその製造法
EP0452683A2 (fr) * 1990-03-16 1991-10-23 Sapporo Breweries, Ltd. Substance antitumorale de streptomyces et son procédé de préparation

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0200818A1 (fr) * 1984-12-06 1986-11-12 Microbial Chemistry Research Foundation Dérivés appartenant aux pluramycines à activité antitumorale, procédé pour leur préparation et leur utilisation comme médicament
US4847387A (en) * 1986-07-21 1989-07-11 Kyowa Hakko Kogyo Co., Ltd. Antibiotic compounds DC-92B and DC-92D
JPH01193265A (ja) * 1988-01-28 1989-08-03 Meiji Seika Kaisha Ltd 新規抗腫瘍性抗生物質ｓｆ２５８７物質及びその製造法
EP0452683A2 (fr) * 1990-03-16 1991-10-23 Sapporo Breweries, Ltd. Substance antitumorale de streptomyces et son procédé de préparation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KONDO, S. ET AL.: "STRUCTURE OF PLURAMYCIN A AND NEOPLURAMYCIN", THE JOURNAL. OF ANTIBIOTICS, vol. 30, no. 12, 1977, pages 1143 - 1145, XP055577552 *

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