WO2016108489A1 - Nouveau composé de ruthénium et composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif - Google Patents

Nouveau composé de ruthénium et composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif Download PDF

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WO2016108489A1
WO2016108489A1 PCT/KR2015/014074 KR2015014074W WO2016108489A1 WO 2016108489 A1 WO2016108489 A1 WO 2016108489A1 KR 2015014074 W KR2015014074 W KR 2015014074W WO 2016108489 A1 WO2016108489 A1 WO 2016108489A1
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cancer
formula
tumor
compound
ruthenium
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PCT/KR2015/014074
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Korean (ko)
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지기환
조재호
송영호
김동환
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울산대학교 산학협력단
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Priority claimed from KR1020140191962A external-priority patent/KR101683350B1/ko
Priority claimed from KR1020150100362A external-priority patent/KR101744398B1/ko
Priority claimed from KR1020150161880A external-priority patent/KR101776412B1/ko
Application filed by 울산대학교 산학협력단 filed Critical 울산대학교 산학협력단
Publication of WO2016108489A1 publication Critical patent/WO2016108489A1/fr

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    • 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
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C13/00—Cyclic hydrocarbons containing rings other than, or in addition to, six-membered aromatic rings
    • C07C13/28—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof
    • C07C13/32—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings
    • C07C13/54—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings with three condensed rings
    • C07C13/573—Polycyclic hydrocarbons or acyclic hydrocarbon derivatives thereof with condensed rings with three condensed rings with three six-membered rings
    • C07C13/60—Completely or partially hydrogenated phenanthrenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table

Definitions

  • the present invention relates to novel quaternary arene-ruthenium compounds, phenanthrene-ruthenium compounds, benzimidazole-ruthenium and pharmaceutical compositions for the prevention or treatment of cancer diseases containing them as active ingredients.
  • anti-autophagy proteins such as B-cell lymphoma 2 (Bcl-2), picasedelta (PKC ⁇ ), and tissue transglutaminase 2 (TG2).
  • Bcl-2 B-cell lymphoma 2
  • PLC ⁇ picasedelta
  • TG2 tissue transglutaminase 2
  • Induction of autophagy by inhibition of (anti-autophagic proteins) may lead to autophagic cell death of some apoptosis-resistant cancers (eg, breast and pancreatic cancer). Proved.
  • some cases of inhibition of autophagy in cancer cells may increase the susceptibility of cancer to a variety of therapies such as DNA damaging agents, antihormone therapies and radiation therapy. Showed therapeutic benefit.
  • the induction or inhibition of autophagy may provide therapeutic benefits for the patient, so the design and synthesis of autophagy modulators can be used as a new therapeutic strategy for cancer. have.
  • the metal pharmaceutical field is rapidly becoming popular through the therapeutic application of metal-based drugs.
  • Platinum complexes, cisplatin, carboplatin and oxoplatin, are known as the most effective chemoherapeutic agents.
  • cisplatin has serious side effects of nephrotoxicity and neurotoxicity.
  • the present invention seeks to provide novel quaternary arene-ruthenium compounds, phenanthrene-ruthenium compounds, benzimidazole-ruthenium compounds and their use for anticancer treatment.
  • the present invention provides an arene-ruthenium compound represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof:
  • each of X is independently trifluoromethylsulfonate (OTf), nitrate (NO 3 ), toluene-4-sulfonate (toulene-4-sulfonate, OTs), Methanesulfonate (OMs), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO; Is a single bond or a double bond,
  • a nitrogen atom in the aromatic ring of Formula 4 or 5 and ruthenium of Formulas 3a to 3c are respectively bonded, and X is separated to form a compound of Formula 1 or 2.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer diseases, characterized in that it comprises an arene-ruthenium compound represented by Formula 1 or 2 or a pharmaceutically acceptable salt thereof.
  • the present invention provides a phenanthrene-ruthenium compound represented by Formula 6 or a pharmaceutically acceptable salt thereof:
  • A is independently OTf (trifluoromethylsulfonate), nitrate (NO 3 ), OTs (toluene-4-sulfonate), OMs (methanesulfonate), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO,
  • A is separated from the compound of Formula 7, Formula 8 or Formula 9 and ruthenium and a nitrogen atom in the aromatic ring of Formula 10 combine to form a compound of Formula 6.
  • the present invention also provides a pharmaceutical composition for preventing or treating cancer diseases containing the phenanthrene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a benzimidazole-ruthenium compound represented by the following formula (11) or a pharmaceutically acceptable salt thereof:
  • X may be halogen
  • the present invention also provides a pharmaceutical composition for treating or preventing cancer diseases containing the benzimidazole-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the quaternary arene-ruthenium compound according to the present invention showed better anticancer activity compared to known anticancer drugs cisplatin and doxorubicin, and particularly exhibited excellent anticancer activity by strongly inducing autophagy at low concentration in colon cancer cells.
  • the phenanthrene-ruthenium compound according to the present invention inhibits Akt-mTOR signaling pathway and increases caspase activity in cancer cells, thereby inhibiting cancer cell proliferation by activating autophagy of cancer cells and inducing apoptosis cell death. It showed excellent anticancer activity, and showed excellent anticancer activity even at a lower concentration than conventional anticancer agents.
  • the novel benzimidazole-ruthenium compound according to the present invention is a macrophage cell that secretes cytokines, a signaling molecule that regulates the physiological activity of cells. Secrete cytokines that form tumors As it has been confirmed that the effect of inhibiting the growth of cancer cells by increasing the secretion of cytokines that inhibit and increase the anticancer effect, the novel ruthenium compound of the present invention can be usefully used for the prevention or treatment of cancer diseases.
  • FIG. 1 is a schematic diagram for preparing Compounds 1 to 6 according to the present invention.
  • FIG. 2 is a result of the nitromethane -D 3 (Nitromethane-D 3, CD 3 NO 2)
  • the 1 H-NMR (nuclear magnetic resonance, NMR) spectra and the results of FIG. 2 is recorded in a compound L1, B in FIG. 2 is the result of compound 1 according to the present invention.
  • ESI-MS electrospray ionization mass spectrometry
  • FIG. 4 shows UV / Vis spectra results
  • a in FIG. 4 is a result of compounds 1 to 3 according to the present invention
  • b in FIG. 4 is a result of compounds 4 to 6 according to the present invention
  • C in Figure 4 is the result of the receptor (A1 to A3) and the donors (L1 and L2).
  • Figure 5 a is an X-ray crystal structure (X-ray crystal structure) of Compound 1 according to the present invention
  • b in Figure 5 is a space-filling model (CPK model) according to the present invention
  • the structure of Compound 1 is shown (green: ruthenium, red: oxygen, blue: nitrogen and grey: carbon).
  • FIG. 6 is a graph showing the anticancer effect of Compound 3 and Compound 6 according to the present invention in colon cancer cells.
  • FIG. 7 is a graph showing the child action according to the concentration change of Compound 3 and Compound 6 according to the present invention in colon cancer cells.
  • ESI-MS electrospray ionization mass spectrometry
  • FIG. 10 shows apoptosis effect by Compound 12 after treatment with 0, 1, 5, 10 and 20 ⁇ M Compound 12 for 24 hours in AGS and COS7 cell lines.
  • FIG. 10A shows the relative autophagy activity following Compound 12 treatment.
  • 10B shows the results of apoptosis apoptosis according to Compound 12 treatment.
  • FIG. 11 is a result of confirming apoptosis control process by Compound 12
  • Figure 11A is a result of confirming the LC3 and p62 protein levels in the autophagy response induced by Compound 12
  • Figure 11B is Akt and COS7 cell lines in Akt and It is the result of confirming the apoptosis and autophagy effect of the compound 12 by confirming the degree of mTOR expression.
  • Figure 13 shows the results confirming the growth inhibitory activity of compound 12 pre-cultured in AGS cell culture medium.
  • FIG. 14 shows the HR-ESI-MS spectra calculated value (blue) and experimental value (red) of lutenacycle [1M-Cl] + .
  • Figure 16 shows the results of screening genes differentially expressed by RT-PCR method using GeneFishing DEG system in AGS cells treated with 20 ⁇ M cisplatin (Cp) and 10 ⁇ M benzimidazole-ruthenium compound 15 for 24 hours.
  • 19 is a result of treating 20 ⁇ M benzimidazole-ruthenium compound 15 in AGS cells for 0, 12, 24 and 48 hours and confirming the loss of growth inhibitory activity of benzimidazole-ruthenium compound 15 over time.
  • the present invention provides an arene-ruthenium compound represented by the following Chemical Formula 1 or 2 or a pharmaceutically acceptable salt thereof.
  • each of X is independently trifluoromethylsulfonate (OTf), nitrate (NO 3 ), toluene-4-sulfonate (toulene-4-sulfonate, OTs), Methanesulfonate (OMs), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO; Is a single bond or a double bond,
  • a nitrogen atom in the aromatic ring of Formula 4 or 5 and ruthenium of Formulas 3a to 3c are respectively bonded, and X is separated to form a compound of Formula 1 or 2.
  • the present invention is characterized in that the nitrogen atom in the aromatic ring of the formula (4) or (5) and ruthenium of the formula (3c) is bonded, and X is separated to form a compound of the formula (1) or (2), or It provides a pharmaceutically acceptable salt thereof.
  • X is each independently trifluoromethylsulfonate (OTf), nitrate (NO 3 ), toluene-4-sulfonate (toulene-4-sulfonate, OTs), methanesulfonate (methanesulfonate, OMs), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO, wherein Is a single bond or a double bond,
  • the arene-ruthenium compound of the present invention represented by Chemical Formula 1 or 2 may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt.
  • the inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid may be used as the organic acid.
  • Acetic acid glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.
  • hydrochloric acid may be used as the inorganic acid
  • methanesulfonic acid may be used as the organic acid.
  • the arene-ruthenium compound of the present invention represented by Formula 1 or 2 of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a compound of Formula 1 or Formula 2 in a water miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and adding an excess of an organic acid. It may be prepared by addition or precipitation of an acidic aqueous solution of an inorganic acid followed by precipitation or crystallization. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.
  • a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile
  • FIG. 1 A schematic diagram of preparation of the arene-ruthenium compounds 1 to 6 according to the present invention is shown in FIG. 1.
  • the quaternary arene-ruthenium compound of Chemical Formula 1 is represented by [Ru 2 ( ⁇ 4 -C 2 O 4 ) ( ⁇ 6 -p- i PrC 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 ( A1 ), [Ru 2 (dobq) ( ⁇ 6 -p -Pr i C 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 ( A2 ) of formula 3b or [Ru of formula 3c 2 (donq) ( ⁇ 6 -p-Pr i C 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 ( A3 ) and 3,6-di (pyridine), the dipyridyl donor of formula (4) Nitrogen solution containing an equivalent mole of 4-yl) -1,2,4,5-tetraazine (3,6-di (pyridin-4-
  • the quaternary arene-ruthenium compound of Chemical Formula 2 may be prepared by [Ru 2 ( ⁇ - ⁇ 4 -C 2 O 4 ) ( ⁇ 6 -p- i PrC 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 of Chemical Formula 3a.
  • the new arene-ruthenium compounds prepared as described above are prepared by infrared spectroscopy, nuclear magnetic resonance spectra, mass spectroscopy, liquid chromatography, X-ray structure determination, photoluminescence, and elemental analysis calculations and actual measurements of representative compounds. The molecular structure can be confirmed by comparison.
  • the four-nuclear arene-ruthenium compound of Formula 1 or 2 according to the present invention induces autophagy in human AGS (gastric cancer) and HCT-15 (colon cancer) cell lines, thereby inducing It shows excellent anticancer activity by inducing cell death and can be used as an active ingredient of anticancer agent.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer diseases, comprising the arene-ruthenium compound represented by the following Chemical Formula 1 or 2 or a pharmaceutically acceptable salt thereof.
  • each of X is independently trifluoromethylsulfonate (OTf), nitrate (NO 3 ), toluene-4-sulfonate (toulene-4-sulfonate, OTs), Methanesulfonate (OMs), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO; Is a single bond or a double bond,
  • a nitrogen atom in the aromatic ring of Formula 4 or 5 and ruthenium of Formulas 3a to 3c are respectively bonded, and X is separated to form a compound of Formula 1 or 2.
  • the arene-ruthenium compound is a nitrogen atom in the aromatic ring of formula (4) or (5) and ruthenium of the formula (3c) is bonded, the following X is separated to form a compound of formula (1) or (2) or Provided is a therapeutic pharmaceutical composition.
  • X is each independently trifluoromethylsulfonate (OTf), nitrate (NO 3 ), toluene-4-sulfonate (toulene-4-sulfonate, OTs), methanesulfonate (methanesulfonate, OMs), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO, wherein Is a single bond or a double bond,
  • the pharmaceutical composition for preventing or treating cancer diseases containing the arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient is based on the arene-ruthenium compound or 100 parts by weight of the pharmaceutical composition.
  • a pharmaceutically acceptable salt may be included in an amount of 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight, or 10 to 90 parts by weight, but is not limited thereto. It may vary depending on the degree.
  • the pharmaceutical composition for preventing or treating cancer diseases containing the arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient includes a carrier, an excipient, a disintegrant, a sweetener, a coating agent, an expanding agent, It may further comprise one or more adjuvants selected from the group consisting of lubricants, glidants, flavors, antioxidants, buffers, bacteriostatics, diluents, dispersants, surfactants, binders and lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules.
  • solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like
  • lubricants such as magnesium styrate and talc may also be used.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
  • the formulation of the pharmaceutical composition for preventing or treating cancer containing the arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient is granules, powders, coated tablets, tablets, pills, It may be selected from the group consisting of capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal Via the route can be administered to the subject in a conventional manner.
  • Preferred dosages of the pharmaceutical composition for preventing or treating cancer containing the arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient include the condition and weight of the patient, the type and extent of the disease, the form of the drug, the route of administration and It may vary depending on the time period and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 1,000 mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the cancer may be a solid cancer, and more specifically, the arene-ruthenium compound of Formula 1 or 2 according to the present invention may be used for gastric cancer, colorectal cancer, or brain tumor.
  • Brain tumor Low-grade astrocytoma, High-grade astrocytoma, Pituitary adenoma, Meningioma, CNS lymphoma, Oligodendroglioma, Two cranial Craniopharyngioma, Ependymoma, Brain stem tumor, Head & Neck tumor, Larygeal cancer, Oropgaryngeal cancer, Nasal cavity / Sinus cancer PNS tumor, Nasopharyngeal tumor, Salivary gland tumor, Hypoharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest tumor, Small cell lung cancer, Small cell lung cancer cell lung cancer), non-small cell Non small cell lung cancer, Thymoma, Mediastinal tumor, Esophageal cancer, Breast cancer, Male
  • the present invention also provides a phenanthrene-ruthenium compound represented by the following formula (6) or a pharmaceutically acceptable salt thereof:
  • A is independently OTf (trifluoromethylsulfonate), nitrate (NO 3 ), OTs (toluene-4-sulfonate), OMs (methanesulfonate), Cl, Br, I, BF 4 , PF 6 , ClO 4 , CH 3 COO or CF 3 COO,
  • A is separated from the compound of Formula 7, Formula 8 or Formula 9 and ruthenium and a nitrogen atom in the aromatic ring of Formula 10 combine to form a compound of Formula 6.
  • the phenanthrene-ruthenium compound represented by the formula (6) is phenanthrene-ruthenium compound or a pharmaceutical thereof, wherein A is separated from the compound of the formula (8) and the nitrogen atom in the aromatic ring of the formula (10) is bonded It is possible to provide an acceptable salt.
  • the phenanthrene-ruthenium compound of the present invention may be used in the form of a pharmaceutically acceptable salt, and acid salts formed by the pharmaceutically acceptable free acid are useful as salts.
  • the inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid may be used as the organic acid.
  • Acetic acid glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.
  • hydrochloric acid may be used as the inorganic acid
  • methanesulfonic acid may be used as the organic acid.
  • the phenanthrene-ruthenium compound of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of Formula 6 in a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and adding an excess of an organic acid or an inorganic acid. It can be prepared by adding an acidic aqueous solution of and then precipitating or crystallizing. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.
  • a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile
  • phenanthrene-ruthenium compounds such as compounds 11 to 13 of the present invention are HCT-15 (rectal cancer cell line), SK-hep-1 (liver cancer cell line) and AGC (gastric cancer cell line) cells It inhibited Akt-mTOR signaling pathway and increased caspase activity, and showed excellent anticancer activity that inhibited cancer cell proliferation by inducing autophagy and apoptotic cell death of cancer cells, especially cisplatin in AGS cells.
  • the present invention can provide a pharmaceutical composition for preventing or treating cancer diseases, which contains the phenanthrene-ruthenium compound of Formula 6 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the phenanthrene-ruthenium compound, or a pharmaceutically acceptable salt thereof, may exhibit anticancer activity by inducing autophagy and apoptosis of cancer cells.
  • the pharmaceutical composition is 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight or 10 to 90 parts by weight of the phenanthrene-ruthenium compound of Formula 6 based on 100 parts by weight of the pharmaceutical composition It may be included as part, but is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the degree of progression.
  • the pharmaceutical composition is a carrier, excipient, disintegrant, sweetener, coating agent, expanding agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent, dispersant, surfactant, binder and It may further comprise one or more adjuvants selected from the group consisting of lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules.
  • solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like
  • lubricants such as magnesium styrate and talc may also be used.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
  • the formulation of the pharmaceutical composition is a granule, powder, blood
  • It may be selected from the group consisting of tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route.
  • the preferred dosage of the pharmaceutical composition may vary depending on the condition and weight of the patient, the type and extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 1,000 mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the cancer disease may be a solid cancer
  • the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroma, intracranial tumor, epithelial cell tumor, brain stem tumor, Head and neck tumors, laryngeal cancer, oropharyngeal cancer, nasal / sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, breast cancer, Abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penis cancer, prostate cancer, female gen
  • the present invention also provides a benzimidazole-ruthenium compound represented by the following formula (11) or a pharmaceutically acceptable salt thereof:
  • X may be halogen
  • the benzimidazole-ruthenium compound may be a benzimidazole-ruthenium compound represented by Formula 12 or a pharmaceutically acceptable salt thereof:
  • the benzimidazole-ruthenium compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and acid salts formed by the pharmaceutically acceptable free acid are useful as salts.
  • the inorganic acid and organic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, and methanesulfonic acid may be used as the organic acid.
  • Acetic acid glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.
  • hydrochloric acid may be used as the inorganic acid
  • methanesulfonic acid may be used as the organic acid.
  • benzimidazole-ruthenium compounds of the present invention include not only pharmaceutically acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods.
  • the addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of formula 11 in a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and adding an excess of an organic acid or an inorganic acid. It can be prepared by adding an acidic aqueous solution of and then precipitating or crystallizing. The solvent or excess acid may then be evaporated and dried in this mixture to obtain an addition salt or the precipitated salt may be prepared by suction filtration.
  • a water miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile
  • the benzimidazole-ruthenium compound according to the present invention is made by reacting the same amount of a biphasic N, C-donor ligand having dichloro (p-cymene) ruthenium (II) dimer (RuPD) and phenyl-benzimidazole as shown in Scheme 1 below.
  • a biphasic N, C-donor ligand having dichloro (p-cymene) ruthenium (II) dimer (RuPD) and phenyl-benzimidazole as shown in Scheme 1 below.
  • it can be prepared by refluxing for 8 to 20 hours at 50 to 80 °C in the presence of methanol and NaOAc.
  • the benzimidazole-ruthenium compound according to the present invention may be prepared by infrared spectroscopy, nuclear magnetic resonance spectra, mass spectroscopy, liquid chromatography, X-ray structure determination, photoluminescence measurement, and elemental analysis calculations and actual measurements of representative compounds. The molecular structure can be confirmed by comparison.
  • the benzimidazole-ruthenium compound according to the present invention according to the present invention is AGS (gastric cancer cell line), HCT-15 (rectal cancer cell line) and SK-hep-1 (hepatic cancer cell line) It inhibits the proliferation of cells at low micromolar concentrations, and in particular affects macrophages, inhibits the secretion of cytokines that form tumors such as IFN ⁇ and exhibits anticancer effects such as RANTES and IGF-1. It can be used as an active ingredient of an anticancer agent because it shows the effect of inhibiting the growth of cancer cells by increasing the secretion of caine.
  • the present invention provides a pharmaceutical composition for treating or preventing cancer diseases containing the benzimidazole-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a medical use of the benzimidazole-ruthenium compound for the manufacture of an anticancer agent.
  • the present invention also provides a method for treating cancer disease, comprising administering the benzimidazole-ruthenium compound to a subject.
  • the pharmaceutical composition is 0.01 to 90 parts by weight, 0.1 to 90 parts by weight, 1 to 90 parts by weight, or 10 to 10 parts by weight of the benzimidazole-ruthenium compound of Formula 11 based on 100 parts by weight of the pharmaceutical composition It may include 90 parts by weight, but is not limited thereto, and may vary depending on the type and progress of the patient's condition and disease.
  • the pharmaceutical composition is a carrier, excipient, disintegrant, sweetener, coating agent, expanding agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent, dispersant, surfactant, binder and It may further comprise one or more adjuvants selected from the group consisting of lubricants.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, capsules.
  • solid preparations may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the composition.
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin and the like
  • lubricants such as magnesium styrate and talc may also be used.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
  • Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.
  • the formulation of the pharmaceutical composition in the group consisting of granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops or solutions Can be selected.
  • the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhaled, topical, rectal, oral, intraocular or It can be administered to a subject in a conventional manner via the intradermal route.
  • the preferred dosage of the pharmaceutical composition may vary depending on the condition and weight of the patient, the type and extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, but not limited thereto, the daily dosage may be 0.01 to 1,000 mg / kg, specifically 0.1 to 1,000 mg / kg, more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several times, thereby not limiting the scope of the invention.
  • the 'subject' may be a mammal including a human, but is not limited thereto.
  • the cancer disease may be a solid cancer, more specifically the benzimidazole-ruthenium compound of formula 11 according to the present invention is a brain tumor (Brain tumor), low-grade astrocytoma, High-grade astrocytoma, Pituitary adenoma, Meningioma, CNS lymphoma, Oligodendroglioma, Craniopharyngioma, Ependymoma, Brain stem tumor Brain stem tumor, Head & Neck tumor, Larygeal cancer, Oropgaryngeal cancer, Nasal cavity / PNS tumor, Nasopharyngeal tumor, Salivary cancer gland tumor, Hypopharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest Tumor, Small cell lung cancer, Non small cell lung cancer ), Thymic cancer (Thymoma) Mediastinal tumor, Esophageal cancer, Breast cancer, Breast cancer, Male breast cancer, Abdomen-pelvis tumor, Stomach cancer, Hepatom
  • Receptor clips [Ru 2 ( ⁇ - ⁇ 4 -C 2 O 4 ) ( ⁇ 6 -p- i PrC 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 (A1), [Ru 2 (dobq) ( ⁇ 6 -p -Pr i C 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 (A2) and [Ru 2 (donq) ( ⁇ 6 -p-Pr i C 6 H 4 Me) 2 ] [O 3 SCF 3 ] 2 (A3) was synthesized under a dry nitrogen atmosphere according to the standard Schlenk technique.
  • the data source was processed and converted using the HKL200 program.
  • the structures were analyzed using direct methods and purified using full-matrix least-squares of F2 with appropriate software from the SHELXTL program package. UV-Vis spectra were recorded at Cary 100 Conc.
  • Electrospray ionization mass spectrometry (ESI-MS) was performed for accurate analysis of the composition of the compounds according to the present invention.
  • the high energy band observed in the compounds 1 to 6 according to the present invention was also observed in the free L1 and L2, which is an extended aromatic system of the dipyridyl ligand conserved during self-assembly It suggests that transitions occur for the aromatic system.
  • Dinuclear arene-Ru acceptors also have slightly higher energy absorption bands at 250-300 nm, and donor spacers of A3 also generate broadly low energy absorption bands from ⁇ 400 to 550 nm. Confirmed.
  • a suitable single crystal of Compound 1 for X-ray analysis using synchrotron radiation is a slow vaporization of diethyl ether in a methanol solution of Compound 1 Obtained by vapor diffusion.
  • Compound 1 has a tetranuclear rectangular architecture in the result of single crystal X-ray diffaction analysis, and lies in a crystallographic inversion center. It was confirmed.
  • Human gastric cancer cell line AGS and human colon cancer cell line HCT15 were obtained from the American Type Culture Collection (Manassas, VA). All cells were cultured in a medium containing 5% fetal bovine serum (FBS) (RPMI 1640 medium) in the presence of 5% CO 2 at 37 °C.
  • FBS fetal bovine serum
  • IC 50 values are shown in Table 2 below compared to known anti-tumor drugs cisplatin and doxorubicin.
  • HCT-15 cells were cultured in 75-cm 2 culture flasks at 37 ° C., 95% humidity, 5% CO 2 .
  • Polyvinylidene fluoride (HFs) with a 1-mm internal diameter and a molecular weight cutoff point of 500 kDa (Spectrum Laboratories, Houston, TX, USA) were used. Sterilized HFs were washed with normal growth media before filling HCT-15 cells at a density of 5 ⁇ 10 5 cells / mL.
  • Each fiber was heat sealed at a spacing of 1.5 cm with a hot smooth-jawed needle holder and cut into 2-mm tailed segments for ease of handling.
  • Each HFs segment contained about 10 5 cells. The prepared HFs segments were maintained for 24 hours in vitro under normal growth conditions prior to injection.
  • mice All animals were fed with water and food (Purina 5001 Rodent Chow; Purina, St. Louis, MO, USA) following 12 h light-dark cycles. Mice were anesthetized using Zoletil and Rompun. HFs were implanted subcutaneously (subcutaneou, s.c.) and peritoneal (i.p.) of anesthetized mice and sutured with skin staples.
  • mice Two days later, male mice were fed using gavage at 100 mg / kg of body weight for 7 days. Mice were administered 100 ⁇ g kg ⁇ 1 bw / day of compound 3 and 6 for 7 days each.
  • HFs were transferred to 0.5 ml of ethylenediaminetetraacetic acid (EDTA), cut in half in the longitudinal direction with a scalpel and washed with EDTA solution for 3 minutes. After washing for 5 minutes with 0.5 ml of trypsin, it was washed for 3 minutes with medium.
  • EDTA ethylenediaminetetraacetic acid
  • the compound 3 and 6 according to the present invention inhibited the proliferation of tumor cells in the IP region, the growth inhibitory effect by the compound 3 is 14.2%, the growth inhibitory effect by the compound 6 is 21.8% Was observed.
  • the growth inhibitory effect by Compound 3 was 7.9%, and the growth inhibitory effect by Compound 6 was 8.5%, which is lower than that of the IP region.
  • HCT-15 cells were labeled with 0.05 mM MDC (Sigma) in PPM1640 medium for 10 minutes at 37 ° C. After incubation, cells were washed three times with phosphate buffer salt and immediately using a fluorescence microscope (Nikon Eclipse TE 300, Japan) equipped with a filter system (V-2A excitation filter: 380/420 nm, barrier filter: 450 nm). The analysis was carried out. Field of vision and fluorescence images were taken with a digital camera (DP30BW; Olympus).
  • Donor compound 7 was synthesized in the same manner as in Scheme 2 below.
  • 3,6-dibromophenanthrene (89.6 mg, 0.267 mmol), 3-ethynylpyridine (63.3 mg, 0.613 mmol), CuI (2.5 mg, 0.013 mmol), [PdCl 2 (PPh 3 ) 2 ] (11.2 mg, 0.016 mmol), and PPh 3 (4.2 mg, 0.016 mmol) were placed in a round bottom flask and THF (5 mL) and triethylamine (5 mL) were added and bubbled with N 2 for 10 minutes.
  • donor compound 7 and arene-Ru (II) receptors 8, 9 and 10 were respectively added and stirred at room temperature for 6 hours to obtain a clear solution.
  • Diethyl ether was added dropwise to the solution to form a precipitate. The precipitate was washed twice with diethyl ether, centrifuged and dried to crystalline powder to give pure molecular bows 11-13.
  • Phenanthrene donor 7 (Compound 7; 1.14 mg, 0.003 mmol) and Receptor 8 (2.72 mg, 0.003 mmol) were self-assembled in the same manner to obtain Molecular Ball 11 (Compound 11) as a dark red crystalline powder. (Yield 92%, see FIG. 9)
  • Phenanthrene donor 7 (Compound 7; 1.14 mg, 0.003 mmol) and Receptor 9 (2.87 mg, 0.003 mmol) were self-assembled in the same manner to obtain Molecular Ball 12 (Compound 12) as a green crystalline powder. 90% yield)
  • Phenanthrene donor 7 (Compound 7; 1.14 mg, 0.003 mmol) and Receptor 10 (3.17 mg, 0.003 mmol) were self-assembled in the same manner to obtain Molecular Ball 13 (Compound 13), which is a dark green crystalline powder. (Yield 94%)
  • novel compound 11-13 synthesized by the above method was characterized by 1 H and 13 C NMR, electrospray ionization mass spectrometry (ESI-MS) and component analysis, and the solid state structure of complex 5 was determined by single crystal X-ray. Confirmed by analysis.
  • the ⁇ -pyridyl proton of donor 7 was shifted 0.4-0.6 ppm upfield as it complexed with the receptors 8-10, and the 1 H NMR peak associated with the phenanthrene unit was also significantly upfield shifted.
  • electrospray ionization mass spectrometry showed that the isotope distribution peaks of compounds 11, 12, and 13 were found at m / z 709.32, 742.96 and 809.49, respectively, and were consistent with their respective [M-3OTf] 3+ . The results were found to be identical to the theoretical isotope distribution pattern of compounds 11-13.
  • PAL Pohang Accelerator Laboratory
  • the average Ru-N pyridine band distance was 2.11 Hz, which was found to be similar to other tetranuclear structures.
  • Human colon cancer cell HCT-15, human liver cancer cell SK-hep-1, human gastric cancer cell AGS and monkey fibroblast COS-7 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HCT-15, AGS, and COS-7 cells in Dulbecco 'modified Eagle' medium (DMEM) and SK-hep-1 cells in 5% fetal bovine serum and 1% penicillin / streptomycin in RPMI 1640 medium. Incubated at 37 ° C., 5% CO 2 .
  • DMEM Dulbecco 'modified Eagle' medium
  • SK-hep-1 cells in 5% fetal bovine serum and 1% penicillin / streptomycin in RPMI 1640 medium. Incubated at 37 ° C., 5% CO 2 .
  • Each cell was aliquoted into a 96-well plate and incubated overnight at 37 ° C. Thereafter, the cells were treated with each compound for a predetermined time period, and then cultured, and then treated with 10 ⁇ L of a 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) solution 37 Incubated for 4 hours at 5 °C CO 2 .
  • MTT 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide
  • Formazan formed during the incubation period was dissolved by treatment with 100 ⁇ L DMSO and absorbance was measured at 550 nm with a microplate reader.
  • Half-maximal inhibitory concentration (IC 50 ) values for cell growth were determined by fitting a plot of the percentage of viable cells to the log of drug concentration using a linear regression function.
  • Apoptosis analysis was performed using a Tali ® Image-Based Cytometer (Invitrogen).
  • AGS and COS-7 cells were treated with Compound 12 at 0-20 ⁇ M concentration and incubated at 37 ° C., 5% CO 2 for 24 hours. Thereafter, the number of cells was measured using a Countess ® Automated Cell Counter. Cells were harvested with TrypLE TM reagent and stained with Tali ® apoptosis kit.
  • Oh apoptosis was dyed sheath cells to confirm apoptosis in Oh annexin V-Alexa Fluor ® 488 conjugate, was used for propidium iodide (PI) to distinguish dead cells.
  • Percentages of live, apoptotic and dead cells according to the Tali ® analyzer were compared with the data obtained from the flow cytometer, respectively, using the same sample.
  • the cultured cells were washed with cold PBS and lysed with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) to obtain protein extracts. Protein concentration was determined using a protein assay kit (Bio-Rad, Hercules, Calif., USA). Was measured.
  • Bovine serum albumin (BSA) was used as the standard curve, and each protein was digested by the same amount (20 ⁇ g) by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). . Thereafter, it was transferred to an immobilon ® -P polyvinylidene difluoride membrane (Amersham, Arlington Heights, IL, USA) and incubated with primary antibody overnight.
  • AGS cells were dispensed into 96-well plates with a clear bottom.
  • Caspase-3 activity was measured using Caspase-Glo_3 Assay kit (CG3 kit, Promega, Mannheim, Germany) according to the manufacturer's instructions.
  • CG3 analysis is a method of analyzing a luminescence signal generated by cleaving a specific sequence of a light-generating substrate by caspase-3, and fluorescence emission is 485 nm excitation wavelength using Mikrowin 2000 Plate Reader (Tecan, Switzerland). And 527 nm emission wavelength.
  • donor 7 and compound 11-13 showed a strong anticancer effect in all cells as shown in Table 3.
  • the IC 50 value of AGS cells treated with Compound 6 was 3.2 times lower than doxorubicin.
  • Cisplatin (ICsplatin) showed an IC 50 value at a concentration of 100 ⁇ M or more in AGS cells, compared with other cells was found to have the lowest anti-cancer effect in gastric cancer.
  • the AGS cell line which is a human gastric cancer cell, was selected to further analyze the anticancer ability of compounds 11-13.
  • IC 50 concentration of drug required to inhibit 50% of cell viability
  • Green fluorescent protein (GFP) -transduced cells were stained using the Tali ® Viability Kit, and dead cells of AGS gastric cancer cells and COS-7 normal monkey kidney fibroblasts were stained red.
  • the self-extinguishing activity of AGS cells was significantly increased to 27.1, 48.6 and 68.4% in the cell group treated with Compound 12 at 5, 10 and 20 ⁇ M concentrations, respectively, compared to the untreated cell group.
  • COS-7 cells which are normal cells, also increased autodigestion activity by 12.5, 18.9, and 23.3%, respectively, compared to untreated cells.
  • the p62 protein that binds to the LC3 protein is generally a marker used to assess autophagic flux, in which expression is reduced during autophagy and accumulation of p62 protein is induced during autophagy.
  • LC3 which plays an important role in the formation of autodigestion vesicles, is divided into two types, the cytoplasmic LC3-I and the membrane-bound LC3-II. In particular, the conversion of LC3-I to LC-II is increased during the autophagy.
  • Akt-mTOR signaling pathway In human cancers, activation of Akt-mTOR signaling pathway through phosphorylation leads to proliferation and metastasis of cancer cells, whereas inhibition of Akt-mTOR signaling pathway is associated with autophagy inhibition because it activates autophagy.
  • caspase-3 activity was confirmed through substrate cleavage reaction analysis.
  • caspase-3 activity was increased in a concentration-dependent manner in the compound 12 treated cells as shown in FIG.
  • Compound 12 induces cancer cell death by inhibiting the Akt / mTOR signaling pathway and activating autophagy.
  • Compound 12 was treated with DMSO and culture medium (10% fetal bovine serum in DMEM) under the same conditions to confirm the stability of Compound 12.
  • Compound 12 was added to the culture medium and pre-cultured at 37 ° C. for varying times and added to AGS cells for incubation. Compound 12 was preincubated in DMSO under the same conditions as a control.
  • Deuterated NMR solvents were purchased from the Cambridge Isotope Laboratory (Andover, MA, USA) and NMR spectra were obtained using a Bruker 300 MHz spectrometer.
  • Ligand L4 was synthesized in the same manner as in the synthesis of ligand L3 using a mixture of 2-phenylbenzimidazole (294 mg, 1.515 mmol) and KOH (170 mg, 3.031 mmol) in dimethylformamide (DMF).
  • Ligand L5 was synthesized in the same manner as in the synthesis of ligand L3 using a mixture of 2-phenylbenzimidazole (294 mg, 1.515 mmol) and KOH (170 mg, 3.031 mmol) in dimethylformamide (DMF).
  • Ring metal ruthenium compounds 14 to 16 were synthesized by the following Scheme 5.
  • the reaction mixture was stirred at ⁇ 65 ° C. for 12 hours and the reaction process was confirmed using TLC.
  • biphasic phenylbenzimidazole derivative ligand L4 50 mg, 0.081 mmol was dissolved in fresh distilled methanol in a round bottom flask dried under nitrogen atmosphere.
  • reaction mixture was stirred at ⁇ 65 ° C. for 12 hours and the reaction was confirmed using TLC, after complete conversion, the methanol was removed under reduced pressure and dried under vacuum.
  • reaction mixture was stirred at ⁇ 65 ° C. for 12 hours and the reaction was confirmed using TLC, after complete conversion, the methanol was removed under reduced pressure and dried under vacuum.
  • Benzimidazole-ruthenium 16 was identified through single crystal X-ray diffraction (XRD) analysis using a radio accelerator to understand the features, identification and coordination structure around the ruthenium metal.
  • XRD X-ray diffraction
  • a single crystal of suitable red-orange color was obtained in XRD through slow evaporation of the compound 16 methanol solution at room temperature for several days.
  • the ruthenium (II) metal in the ruthenacycle was surrounded by a piano-stool structure containing C, N-chelate and chloride ligands and p-cymene moieties on both sides of the ligand. Interestingly, the two chloride ligands Located in the opposite direction, it was confirmed that each of the trans structure.
  • the lutena cycle is C-H... Cl, C-H... ⁇ and C-H... It was confirmed that stability was increased by several strong and weak non-covalent bonds such as N.
  • FBS heat-inactivated fetal bovine serum
  • DMEM Dulbecco's Modified Eagle Medium
  • Cell suspensions were dispensed in 96-well plates at 1 ⁇ 10 4 cells / well concentration and incubated for 24 hours, then each compound was treated at 0.8, 4, 20 and 100 ⁇ M concentrations for 24, 48 and 72 hours.
  • MTT [3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] solution was dissolved in phosphate buffered saline (PBS, pH 7.2) to 5 mg / mL and filtered with a 0.22 ⁇ m Millipore filter. It was.
  • the percentage of cells that survived was calculated from the percentage of untreated cells.
  • the half maximal inhibitory concentration (IC 50 ) for cell growth inhibition was calculated by approximating logarithmic ratio points of viable cells to logarithm of drug concentration.
  • the IC 50 value was between 6.1-12.5, 7.6-23.7 and 11.1-26.6 ⁇ M for AGS, Sk-hep-1 and HCT-15 cells, respectively.
  • ligand L3-L5 IC 50 values found to be between 9.1-67.2, 39.9-82.5 and 61.9-94.2 ⁇ M for AGS, Sk-hep-1 and HCT-15 cells, respectively. Excellent inhibitory effect was shown.
  • AGS gastric cancer cells
  • Gene expression analysis was performed using ACP-based differential display RT-PCR technique (GeneFishing DEG screening technology) to identify gene expression changes related to the growth inhibition effect of benzimidazole-ruthenium 15 in gastric cancer cells AGS.
  • CDNA was synthesized with reagents of GeneFishing DEG Premix Kit (Seegene, Korea) except reverse transcriptase SuperScript II (Invitrogen, USA).
  • RNA 3 ⁇ g of total RNA was mixed with 2 ⁇ L of 10 ⁇ M cDNA synthesis primer dT-ACP1 (Seegene, Korea) and DEPC treated water to a final volume of 9.5 ⁇ L.
  • the mixture was incubated at 80 ° C. for 3 minutes and immediately placed on ice for 2 minutes and then centrifuged briefly.
  • CDNA synthesis was performed at 42 ° C. for 90 minutes and the reaction was inactivated at 94 ° C. for 2 minutes. After incubation on ice for 2 minutes, the reaction was diluted 5-fold by addition of 80 ⁇ L distilled water.
  • Loosening adjustment primer (Annealing control primer; ACP ) Polymerase chain reaction PCR )
  • DEGs Differentially expressed genes
  • PCR for the second template synthesis was performed in one cycle of incubation at 94 ° C for 5 minutes, 50 ° C for 3 minutes and 72 ° C for 1 minute.
  • amplification was carried out in 40 cycles, such as denaturation at 94 ° C for 40 seconds, annealing at 65 ° C for 40 seconds, and extension at 40 ° C for 40 seconds, followed by 5 minutes at 72 ° C. PCR was performed under the conditions of the last expansion step.
  • the PCR product obtained through the above process was separated using 0.5 ⁇ TAE buffer and 2% agarose gel, and the differently expressed cDNA bands were cut out of the gel and purified.
  • Total RNA was extracted from AGS gastric cancer cells using PureLink TM RNA Mini Kit. Total RNA reverse transcription was performed in 20 ⁇ L volumes, including 1 ⁇ g total RNA, oligo (dT) primers, enzymes and buffers provided in PrimeScript II 1st strand cDNA Synthesis kit (Takara, Japan).
  • Quantitative real-time PCR reaction was performed in MX3005P (Stratagene, USA), using the following primers.
  • RPS21 5'-GCTGCTTCCTTTCTCTCTCTG-3 ', 5'- GCCTGTGACCTTGTCAACCT-3' and ⁇ -actin: 5'-GTCCACCGCAAATGCTTCTA-3 ', 5'-TGCTGTCACCTTCACCGTTC-3'
  • the thermal cycling profile was followed by 40 cycles of 95 ° C. (30 seconds), 53 ° C. (60 seconds) and 72 ° C. (30 seconds) after a 10 minute preincubation step at 95 ° C.
  • Relative quantitative evaluation of RPS21 gene level was performed by comparative CT (cycle threshold) method.
  • H36 mitochondria (KJ994345.1) and ZAM115 mitochondria (KJ185427.1) were found to be genes expressed by ACP11, which also reduced expression in AGS treated with benzimidazole-ruthenium 15 compounds.
  • the downregulated gene RPS21 is known to be a component of eukaryotic lysosomes and is associated with lysosomal protein SA (RPSA) in human cells.
  • RPSA lysosomal protein SA
  • RPSA is known as the non-integrin laminin receptor and is overexpressed on the surface of various cancer cells. Thus, RPSA is considered to play an important role in tumor progression.
  • qRT-PCR was performed using mRNAs of benzimidazole-ruthenium 15 compound treated AGS cells.
  • Cytokines are signaling molecules that play critical roles in many biological processes, such as cell growth, differentiation, gene expression, migration, inflammation, and immunity. During inflammatory reactions, macrophages play a role in activating and collecting cytokines, or directly It kills.
  • arene-Ru derivatives can be used to directly regulate the cancer cell cycle, cytokines produced from these derivatives can indirectly affect cancer cells.
  • 31 cytokines (TNF- ⁇ , IFN ⁇ , G-CSF, GM-CSF, IL-1 ⁇ , IL-8, IP-10, Rantes, VEGF, EGF, IL-6, Resistin, PAI-1, IL-12 , IL-13, Eotaxin-3, PDGF-BB, PIGF-1, ⁇ -NGF, SCF, MCP-1, MIP-1 ⁇ , IL-2, IL-4, IL-10, FGF ⁇ , Leptin, IGF-1
  • benzimidazole-ruthenium 2 (10 ⁇ M) or nothing-treated culture medium was treated with THP-1 human mononuclear leukocytes for 24 hours and Human Cytokine The content of 31 cytokines was determined using an ELISA Plate Array I kit (Signosis, USA).
  • the ELISA analysis was performed according to the manufacturer's instructions, and chemiluminescence detection was performed with a multi-reader.
  • cytokine IFN ⁇ , IL-1 ⁇ , VEGF, EGF, Eotaxin-3, IL-10, TGF- ⁇ and IL-17 ⁇ were increased in AGS cells treated with 10 ⁇ M concentration of Compound 15 as shown in FIG.
  • the secretion of vascular endothelial growth factor (VEGF) was 8.2-fold higher than that of the non-treated compound 15.
  • cytokines known as central mediators of angiogenesis, were secreted more than three times in AGS cells treated with 10 ⁇ M concentration of Compound 15, and this result may be rather helpful for the growth of cancer cells.
  • interleukin- ⁇ IFN ⁇
  • IFN ⁇ interleukin- ⁇
  • benzimidazole-ruthenium 2 was found to reduce the release of rantes and IGF-1 (insulin-like growth factor 1) in macrophages.
  • Lante is highly expressed in various cancers to promote tumor growth and metastasis through cancer cell proliferation and angiogenesis, and IGF-1 has a strong effect at key stages of cancer development such as cancer cell proliferation, apoptosis, angiogenesis and metastasis .
  • benzimidazole-ruthenium 15 reduced the lante and IGF-1 secretion of macrophage, while increasing the secretion of IFN ⁇ , thereby suppressing the growth of cancer cells.
  • the growth inhibitory activity of benzimidazole-ruthenium 15 decreased by 50% after 32 hours of preculture in the cell culture medium.

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Abstract

La présente invention concerne : un nouveau composé arène-ruthénium tétranucléaire et une composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif ; un nouveau composé de phénanthrène-ruthénium et une composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif ; et un nouveau composé de benzimidazole-ruthénium dérivé d'un dérivé benzimidazolyle, et une composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif.
PCT/KR2015/014074 2014-12-29 2015-12-22 Nouveau composé de ruthénium et composition pharmaceutique pour prévenir ou traiter le cancer contenant ledit composé comme principe actif WO2016108489A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
KR1020140191962A KR101683350B1 (ko) 2014-12-29 2014-12-29 신규한 4핵 아렌-루테늄 화합물 및 이를 유효성분으로 함유하는 암 질환 예방 또는 치료용 약학 조성물
KR10-2014-0191962 2014-12-29
KR1020150100362A KR101744398B1 (ko) 2015-07-15 2015-07-15 신규 페난트렌-루테늄 화합물 및 이를 유효성분으로 함유하는 암질환의 예방 또는 치료용 약학조성물
KR10-2015-0100362 2015-07-15
KR1020150161880A KR101776412B1 (ko) 2015-11-18 2015-11-18 신규 벤즈이미다졸-루테늄 화합물 및 이를 유효성분으로 함유하는 암질환의 예방 또는 치료용 약학조성물
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Publication number Priority date Publication date Assignee Title
CN108440602A (zh) * 2018-04-17 2018-08-24 深圳大学 四核铱配合物及其制备方法与应用
CN108689943A (zh) * 2018-06-04 2018-10-23 江苏师范大学 含钌超分子化合物及其制备方法和应用
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CN113387984A (zh) * 2020-03-13 2021-09-14 九江学院 含去质子二甲双胍配体的对称双核钌配合物及其制备方法和应用
CN113387984B (zh) * 2020-03-13 2023-05-23 九江学院 含去质子二甲双胍配体的对称双核钌配合物及其制备方法和应用
CN115925617A (zh) * 2022-08-25 2023-04-07 中山大学·深圳 一种氘代钌配合物及其制备方法和在光催化抗肿瘤中的应用

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