WO2016108489A1

WO2016108489A1 - Novel ruthenium compound, and pharmaceutical composition for preventing or treating cancer, containing same as active ingredient

Info

Publication number: WO2016108489A1
Application number: PCT/KR2015/014074
Authority: WO
Inventors: 지기환; 조재호; 송영호; 김동환
Original assignee: 울산대학교 산학협력단
Priority date: 2014-12-29
Filing date: 2015-12-22
Publication date: 2016-07-07

Abstract

The present invention relates to: a novel tetranuclear arene-ruthenium compound, and a pharmaceutical composition for preventing or treating cancer, containing the same as an active ingredient; a novel phenanthrene-ruthenium compound, and a pharmaceutical composition for preventing or treating cancer, containing the same as an active ingredient; and a novel benzimidazole-ruthenium compound derived from a benzimidazolyl derivative, and a pharmaceutical composition for preventing or treating cancer, containing the same as an active ingredient.

Description

Novel ruthenium compound and pharmaceutical composition for preventing or treating cancer disease containing the same as an active ingredient

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.

Autophagy plays an important role in regulating cell homeostasis and is considered as one of the most important molecular responses that contribute to cell survival, growth, differentiation and host defense responses. Recently, autophagy has been known to be a cell death mechanism that occurs in various types of cancer cells in response to cancer treatment (see Non-Patent Documents 1 and 2).

Many studies have shown anti-autophagy proteins, such as B-cell lymphoma 2 (Bcl-2), picasedelta (PKCδ), and tissue transglutaminase 2 (TG2). 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.

In addition, 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.

Thus, depending on the cellular features, 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.

However, cisplatin has serious side effects of nephrotoxicity and neurotoxicity.

Therefore, the development of metal-based drugs without side effects is required, and ruthenium-based complexes, which show low toxicity and high activity in tumors resistant to platinum drugs, are emerging as new metal-based drugs. I am getting it.

However, although much research has been conducted on these ruthenium-based complexes, studies on autophagy have not been reported.

The present invention seeks to provide novel quaternary arene-ruthenium compounds, phenanthrene-ruthenium compounds, benzimidazole-ruthenium compounds and their use for anticancer treatment.

In order to achieve the above object, the present invention provides an arene-ruthenium compound represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof:

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3a]

[Canvas 3b]

or

[Formula 3c]

ego,

In Formulas 3a, 3b, and 3c, 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,

Of Formula 1

Is,

[Formula 4]

Is,

Of Formula 2

silver,

[Formula 5]

ego,

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.

In addition, 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:

[Formula 6]

In the above formula,

Is

[Formula 7]

,

[Formula 8]

or

[Formula 9]

ego,

In Formula 7, Formula 8 or Formula 9, 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,

Is

[Formula 10]

ego,

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:

[Formula 11]

In Formula 11, 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.

1 is a schematic diagram for preparing Compounds 1 to 6 according to the present invention.

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.

3 is an electrospray ionization mass spectrometry (ESI-MS) of the compounds 4 to 6 according to the present invention.

FIG. 4 shows UV / Vis spectra results, a in FIG. 4 is a result of compounds 1 to 3 according to the present invention, and 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).

In 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).

6 is a graph showing the anticancer effect of Compound 3 and Compound 6 according to the present invention in colon cancer cells.

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.

8 is an electrospray ionization mass spectrometry (ESI-MS) spectrum of Compound 11-13, wherein A is [5-3OTf] ³⁺ , B is [6-3OTf] ³⁺ , and C is [7-3OTf] ³⁺ spectrum The result is.

9 shows the results of confirming the X-ray crystal structure of Compound 11 in two directions.

FIG. 10 shows apoptosis effect by Compound 12 after treatment with 0, 1, 5, 10 and 20 μM 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.

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.

12 shows the results of confirming caspase-3 activity in Compound 12 treated AGS cells.

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] ⁺ .

15 is an X-ray crystal structure of benzimidazole-ruthenium compound 16.

Figure 16 shows the results of screening genes differentially expressed by RT-PCR method using GeneFishing DEG system in AGS cells treated with 20 μM cisplatin (Cp) and 10 μM benzimidazole-ruthenium compound 15 for 24 hours.

FIG. 17 shows the mean ± standard error (n = 3), with 1.175-20 μM benzimidazole-

ruthenium compound

15 and 20 μM cisplatin (Cp) treated with AGS cells for 24 hours and RPS21 expression confirmed by qRT-PCR analysis. ), * P <0.05 and ** p <0.01 shows a significant difference compared to the control.

18 is a result of confirming the cytokine secretion changes of THP-1 human macrophages treated with 10 μM benzimidazole-ruthenium compound 15 for 24 hours, [a] is a value showing the change ratio compared to the control 1.

19 is a result of treating 20 μM 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.

Hereinafter, the present invention will be described in more detail.

The present invention provides an arene-ruthenium compound represented by the following

Chemical Formula

1 or 2 or a pharmaceutically acceptable salt thereof.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3a]

[Canvas 3b]

or

[Formula 3c]

ego,

Is a single bond or a double bond,

Of Formula 1

Is,

[Formula 4]

Is,

Of Formula 2

silver,

[Formula 5]

ego,

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.

In addition, 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.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3c]

ego,

In Formula 3c, 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,

Of Formula 1

Is,

[Formula 4]

Is,

Of Formula 2

silver,

[Formula 5]

ego,

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. Preferably, hydrochloric acid may be used as the inorganic acid, and 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 schematic diagram of preparation of the arene-ruthenium compounds 1 to 6 according to the present invention is shown in FIG. 1. Referring to FIG. 1, the quaternary arene-ruthenium compound of Chemical Formula 1 is represented by [Ru ₂ (μ−η ⁴ -C ₂ O ₄ ) (η ⁶ -p- i PrC ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ ( A1 ), [Ru ₂ (dobq) (η 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) (η 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-yl) -1,2,4,5-tetrazine, L1 ) It was put into methane / dimethyl ether (CH ₃ NO ₂ / CH ₃ OH) and stirred to obtain as self-assembly 1 to 3 metallacycles.

In addition, the quaternary arene-ruthenium compound of Chemical Formula ₂ may be prepared by [Ru ₂ (μ-η ⁴ -C ₂ O ₄ ) (η ⁶ -p- i PrC ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ of Chemical Formula 3a. ( A1 ), [Ru ₂ (dobq) (η ⁶ -p- Pr ⁱ C ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ ( A2 ) of Formula 3b or [Ru ₂ (donq) ( η ⁶ -p-Pr ⁱ C ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ ( A3 ) and 2,5-bis (pyridin-4-ylethynyl) furan of formula 5 (2,5-bis (pyridin-4-ylethynyl) furan ( L2 ) was added to the same molar solution containing nitromethane / dimethyl ether (CH ₃ NO ₂ / CH ₃ OH) and stirred to self-assembly (self-assembly of metallacycles) assembly) to 4-6.

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.

In addition, as can be seen in the following examples, 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.

Accordingly, 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.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3a]

[Canvas 3b]

or

[Formula 3c]

ego,

Is a single bond or a double bond,

Of Formula 1

Is,

[Formula 4]

Is,

Of Formula 2

silver,

[Formula 5]

ego,

A nitrogen atom in the aromatic ring of

Formula

1 or 2.

In addition, 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.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3c]

ego,

Is a single bond or a double bond,

Of Formula 1

Is,

[Formula 4]

Is,

Of Formula 2

silver,

[Formula 5]

to be.

In one embodiment of the present invention, 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.

In another embodiment of the present invention, 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.

Specifically, 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. And the like, and such 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. In addition to simple excipients, 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. As the 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.

In another embodiment of the present invention, 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.

According to one embodiment of the invention 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.

In the present invention, the 'subject' may be a mammal including a human, but is not limited thereto.

In one embodiment of the present invention, 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 breast cancer, Abdomen-pelvis tumor , Liver cancer, gallbladder cancer, biliary tract tumor, pancreatic cancer, small intestinal tumor, rectal cancer, bladder cancer, kidney cancer ( Renal cell carcinoma, Male genital cancer, Penile cancer, Prostatic cancer, Female genital cancer, Cervical cancer, Endometrial cancer (Endometrial cancer), Ovarian cancer, Uterine sarcoma, Vaginal cancer, Vulva cancer, Urethral cancer or Skin cancer It is preferable to use. Even more preferably, it may be used for the treatment of gastric cancer or colon (colon) cancer, but is not limited thereto.

The present invention also provides a phenanthrene-ruthenium compound represented by the following formula (6) or a pharmaceutically acceptable salt thereof:

[Formula 6]

In the above formula,

Is

[Formula 7]

,

[Formula 8]

or

[Formula 9]

ego,

Is

[Formula 10]

ego,

More preferably, 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. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

In addition, 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.

According to one embodiment of the present invention, 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. Showed an IC ₅₀ value at a concentration of 100 μM or more, while the IC ₅₀ value of AGS cells treated with Compound 12 was 3.2 times lower than that of doxorubicin, and thus showed high anticancer activity even at a lower concentration than conventional anticancer drugs. .

Therefore, 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.

In one embodiment of the present invention, 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.

In another embodiment of the invention, 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.

In another embodiment of the invention, 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.

According to one embodiment of the invention, 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.

In one embodiment of the present invention, 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 genital tumor, cervical cancer, endometrial cancer It may be selected from the group consisting of ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer and skin cancer, and more preferably, it may be used for the treatment of rectal cancer, liver cancer or stomach cancer. It is not to be limited to.

[Formula 11]

In Formula 11, X may be halogen.

In particular, the benzimidazole-ruthenium compound may be a benzimidazole-ruthenium compound represented by Formula 12 or a pharmaceutically acceptable salt thereof:

[Formula 12]

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. Preferably, hydrochloric acid may be used as the inorganic acid, and methanesulfonic acid may be used as the organic acid.

In addition, the 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.

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. In particular, it can be prepared by refluxing for 8 to 20 hours at 50 to 80 ℃ in the presence of methanol and NaOAc.

Scheme 1

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.

In addition, as can be seen in the following examples, 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 IFNγ 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.

Accordingly, 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.

In one embodiment of the present invention, 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.

In another embodiment of the invention, 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.

In one embodiment of the present invention, 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, Hepatoma, Gallbladder cancer Gall bladder cancer, biliary tract tumor, pancreatic cancer, small intestinal tumor, large intestinal tumor, anal cancer, bladder cancer , Renal cell carcinoma, male genital cancer, penile cancer, prostatic cancer, female genital cancer, cervix cancer , Endometrial cancer, Ovarian cancer, Uterine sarcoma, Vaginal cancer, Vulva cancer, Urethral cancer or Skin cancer It is preferred to use for the treatment of). Even more preferably, it may be used for the treatment of gastric cancer, liver cancer, lung cancer or rectal cancer, but is not limited thereto.

Hereinafter, examples will be described in detail to help understand the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<< 실시예Example 1> 1> 아렌Aren -루테늄 화합물 제조 및 항암활성 확인-Preparation of ruthenium compound and anticancer activity

1. 출발물질 합성 및 NMR 분석방법1. Starting material synthesis and NMR analysis

Receptor clips [Ru ₂ (μ-η ⁴ -C ₂ O ₄ ) (η ⁶ -p- i PrC ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ (A1), [Ru ₂ (dobq) (η ⁶ -p -Pr ⁱ C ₆ H ₄ Me) ₂ ] [O ₃ SCF ₃ ] ₂ (A2) and [Ru ₂ (donq) (η 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. 3,6-di (pyridin-4-yl) -1,2,4,5-tetrazine (3,6-di (pyridin-4-yl) -1,2,4,5-tetrazine) (L1) And 2,5-bis (pyridin-4-ylethynyl) furan (L2) are known methods (Chem. Soc. 2010, 132, 950-). 952 and Chem. 2000, 39, 2547-2557), dried according to known methods (Pergamon Press, Oxford, UK, 1988) and then distilled.

¹ H and ¹³ C NMR spectra were recorded on a Bruker 300 MHz spectrometer.

Chemical shifts (δ) in the ¹ H NMR spectrum were recorded as relative ppm values with tetramethylsilane (Me ₄ Si) as international standard (0.0 ppm). Mass spectra were recorded on a Micromass Quattro II triplequadrupole mass spectrometer using electron spray ionization with a MassLynx operating system. Elemental analyses were performed using the Elemental GmbH Vario EL-3 instrument.

The diffraction data in a single crystal of Compound 1 according to the present invention is 100 K on the ADSC Quantum 210 CCD diffractometer and l = 0.80000 A in the supramolecular crystallography beamline 2D. Collected in synchrotron radiation (Pohang Accelerator Laboratory (PAL), Pohang, Korea).

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.

2. 화합물 합성2. Compound Synthesis

2-1. Compound 1 Synthesis

A mixture of donor L1 (2.36 mg, 0.01 mmol) and ruthenium acceptor Al (8.56 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethylether (CH ₃ NO ₂ / CH ₃ OH, 1: 1) and room temperature Stirred for 24 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to obtain Compound 1 as a brown powder in 92% yield.

¹ H NMR (300 MHz, [D ₃ ] Nitromethane): δ = 8.47 (d, J = 6.0 Hz, 8H), 8.37 (d, J = 6.0 Hz, 8H), 5.96 (d, J = 6.3 Hz, 8H ), 5.81 (d, J = 6.3 Hz, 8H), 2.85-2.98 (m, 4H), 2.27 (s, 12H), 1.39 ppm (d, J = 6.9 Hz, 24H); ¹ ₃ C NMR (75 MHz, [D ₃ ] Nitromethane): δ = 166.9, 158.0, 150.0, 136.8, 119.7, 99.1, 93.9, 78.1, 77.7, 27.0, 17.0, 12.9; MS (ESI) for 1 (C ₇₂ H ₇₂ F ₁₂ N ₁₂ O ₂₀ Ru ₄ S ₄ ): 597.57 [1-3OTf] ³⁺ and 397.42 [1-4OTf] ⁴⁺ ; C, H, N analysis (%) calcd for C ₇₂ H ₇₂ F ₁₂ N ₁₂ O ₂₀ Ru ₄ S ₄ (2185.93): C 39.56, H 3.32, N 7.69; found: C 39.23, H 3.63, N 7.37.

2-2. Compound 2 Synthesis

A mixture of donor L1 (2.36 mg, 0.01 mmol) and ruthenium acceptor A2 (9.06 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethylether (CH ₃ NO ₂ / CH ₃ OH, 1: 1) and room temperature Stirred for 24 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to obtain Compound 2 as a wine-red powder in 90% yield.

¹ H NMR (300 MHz, [D ₃ ] Nitromethane): δ = 8.66 (d, J = 6.4 Hz, 8H), 8.49 (d, J = 6.4 Hz, 8H), 6.01 (d, J = 6.1 Hz, 8H ), 5.75-5.89 (m, 12H), 2.87-3.03 (m, 4H), 2.25 (s, 12H), 1.40 (d, J = 6.9 Hz, 24H); ¹³ C NMR (75 MHz, [D ₃ ] Nitromethane): δ = 180.5, 150.0, 119.4, 100.1, 97.3, 94.9, 79.3, 78.1, 27.2, 17.0, 12.9; MS (ESI) for 2 (C ₈₀ H ₇₆ F ₁₂ N ₁₂ O ₂₀ Ru ₄ S ₄ ): 422.44 [2-4OTf] ⁴⁺ ; C, H, N analysis (%) calcd for C ₈₀ H ₇₆ F ₁₂ N ₁₂ O ₂₀ Ru ₄ S ₄ (2286.05): C 42.03, H 3.35, N 7.35; found: C 41.83, H 3.27, N 6.90.

2-3. Compound 3 Synthesis

A mixture of donor L1 (2.36 mg, 0.01 mmol) and ruthenium acceptor A3 (9.57 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethylether (CH ₃ NO ₂ / CH ₃ OH, 1: 1) and room temperature Stirred for 24 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to obtain compound 3 as a sea-green powder in 91% yield.

¹ H NMR (300 MHz, [D ₃ ] Nitromethane): δ = 8.81 (d, J = 6.3 Hz, 8H), 8.44 (d, J = 6.3 Hz, 8H), 7.28 (s, 8H), 5.82 (d , J = 6.2 Hz, 8H), 5.63 (d, J = 6.2 Hz, 8H), 2.86-3.01 (m, 4H), 2.20 (s, 12H), 1.38 (d, J = 7.0 Hz, 24H); ¹³ C NMR (75 MHz, [D ₃ ] Nitromethane): δ = 172.5, 163.3, 154.7, 147.3, 132.4, 128.7, 127.9, 103.8, 99.3, 84.0, 83.4, 32.7, 22.8, 18.3; C, H, N analysis (%) calcd for C ₈₈ H ₁₀₄ F ₁₂ N ₁₂ O ₂₀ Ru ₄ S ₄ (2386.17): C 44.30, H 3.38, N 7.04; found: C 43.18, H 3.44, N 6.68.

2-4. Compound 4 Synthesis

A CH ₃ NO ₂ (1 mL) solution of donor L2 (2.70 mg, 0.01 mmol) was added dropwise to a CH ₃ OH solution (1 ml) of the ruthenium receptor A1 (8.56 mg, 0.01 mmol). After dropping the mixture was stirred at 40 ° C. for 36 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to give Compound 4 as a yellow solid in a yield of 88%.

¹ H NMR (300 MHz, [D ₄ ] Methanol): δ = 8.03 (d, J = 6.0 Hz, 8H), 7.53 (d, J = 6.0 Hz, 8H), 6.89 (s, 4H), 5.92 (d , J = 6.1 Hz, 8H), 5.75 (d, J = 6.1 Hz, 8H), 2.74-2.88 (m, 4H), 2.21 (s, 12H), 1.35 (d, J = 6.7 Hz, 24H); ¹³ C NMR (75 MHz, [D ₄ ] Methanol): δ = 172.5, 153.9, 139.1, 134.7, 128.3, 122.3, 103.9, 99.0, 92.7, 89.1, 83.3, 32.6, 22.7, 18.2; MS (ESI) for 4 (C ₈₄ H ₇₆ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ ): 602.38 [4-3OTf] ³⁺ ; C, H, N analysis (%) calcd for C ₈₄ H ₇₆ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ (2254.04): C 44.76, H 3.40, N 2.49; found: C 43.97, H 3.72, N 2.68.

2-5. Compound 5 Synthesis

A solution of CH ₃ NO ₂ (1 mL) of donor L2 (2.70 mg, 0.01 mmol) was added dropwise to a solution of CH ₃ OH (1 ml) of ruthenium receptor A2 (9.06 mg, 0.01 mmol). After dropping the mixture was stirred at 40 ° C. for 36 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to give Compound 5 as a wine-red solid with a yield of 86%.

¹ H NMR (300 MHz, [D _4] Methanol): δ = 8.27 (d, J = 6.3 Hz, 8H), 7.49 (d, J = 6.3 Hz, 8H), 6.93 (s, 4H), 6.03 (d , J = 6.2 Hz, 8H), 5.76-5.84 (m, 12H), 2.80-2.95 (m, 4H), 2.19 (s, 12H), 1.35 (d, J = 7.0 Hz, 24H); ¹³ C NMR (75 MHz, [D ₄ ] Methanol): δ = 185.5, 154.2, 139.0, 134.7, 128.4, 124.2, 121.9, 119.9, 105.5, 103.0, 100.3, 92.3, 88.1, 85.0, 83.5, 32.8, 22.7, 18.3; MS (ESI) for 5 (C ₉₂ H ₈₀ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ ): 635.72 [ 5 -3OTf] ³⁺ , 439.55 [ 5 -4OTf] ⁴⁺ ; C, H, N analysis (%) calcd for C ₉₂ H ₈₀ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ (2354.16): C 46.94, H 3.43, N 2.38; found: C 46.47, H 3.77, N 3.01.

2-6. Compound 6 Synthesis

A solution of CH ₃ NO ₂ (1 mL) of donor L2 (2.70 mg, 0.01 mmol) was added dropwise to a solution of CH ₃ OH (1 ml) of ruthenium receptor A3 (9.57 mg, 0.01 mmol). After dropping the mixture was stirred at 40 ° C. for 36 h. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The obtained solid was washed with diethyl ether and dried to give compound 6 as a sea-green solid in 90% yield.

¹ H NMR (300 MHz, [D ₄ ] Methanol): δ = 8.42 (d, J = 6.6 Hz, 8H), 7.43 (d, J = 6.6 Hz, 8H), 7.24 (s, 8H), 6.92 (s , 4H), 5.84 (d, J = 6.4 Hz, 8H), 5.61 (d, J = 6.4 Hz, 8H), 2.75-2.89 (m, 4H), 2.09 (s, 12H), 1.32 (d, J = 6.8 Hz, 8H); ¹³ C NMR (75 MHz, [D ₄ ] Methanol): δ = 172.5, 153.3, 138.8, 134.5, 128.2, 121.6, 119.9, 112.8, 105.2, 101.3, 92.4, 87.7, 86.0, 84.2, 32.2, 22.7, 17.5; MS (ESI) for 6 (C ₁₀₀ H ₈₄ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ ): 669.07 [ 6 -3OTf] ³⁺ ; C, H, N analysis (%) calcd for C ₁₀₀ H ₈₄ F ₁₂ N ₄ O ₂₂ Ru ₄ S ₄ (2456.04): C 48.94, H 3.45, N 2.28; found: C 48.03, H 3.60, N 2.62.

3. 화합물 분석3. Compound Analysis

3-1. NMR analysis

¹ H and ¹³ C NMR spectra were recorded on a Bruker 300 MHz spectrometer.

Chemical shifts (δ) in the ¹ H NMR spectrum were recorded as relative ppm values with tetramethylsilane (Me ₄ Si) as international standard (0.0 ppm).

Referring to FIG. 2, the Hα nuclei of the pyridine rings relative to L1 showed upfield shifts (Δδ = 0.17-0.51 ppm), and most spectra were ruthenium (II) centers. It was caused by the loss of electron density due to the coordination of pyridine-N to (Ru (II) centers).

The protons corresponding to the capped p-cymene moiety occurred as two doublets at about δ = 5.63-6.01 ppm and the benzoquinone of compound 2 according to the invention The signal for proton was δ = 5.84 ppm and the signal for naphthoquinone proton of Compound 3 according to the invention was observed as sharp singlets at δ = 7.28 ppm.

3-2. Mass spectral analysis

Electrospray ionization mass spectrometry (ESI-MS) was performed for accurate analysis of the composition of the compounds according to the present invention.

In the results of the ESI-MS analysis of FIG. 3, the formation of [2 + 2] self-assembled

tetranuclear macrocycles

1 and 2 resulted in multiply charged ions. fragmented ions).

Multiple charge fragmentation ions for compound 1 according to the invention were observed at m / z 579.57 [ 1 -3TfO] ³⁺ and m / z = 397.42 [ 1 -4TfO] ⁴ ⁺ and multiple for compound 2 according to the invention Charge fragmentation ions were observed at m / z 422.44 [ 2 -4TfO] ⁴⁺ and these peaks were analyzed isotopically.

As with isotopic patterns, the appearance of expected peaks clearly confirmed the formation of the [2 + 2] self-assembly of

compounds

1 and 2 according to the present invention.

In the results of the ES1 mass spectra of the compounds 4 to 6 according to the invention, 4 is m / z 602.38 [ 4 -3OTf] ³⁺ , 5 is 636.06 [ 5 -3OTf] ³⁺ and 439.55 [ 5 -4OTf] ⁴⁺ , 6 Peaked at 669.05 [ 6 -3OTf-] ³⁺ .

3-3. UV- vis analysis

The results of the UV-vis absorption spectra for compounds 1 to 6 and their corresponding metal acceptors (A1 to A3) and donors (L1 and L2) according to the invention were recorded in methanol solution.

As a result, as shown in Figure 4, 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.

3-4. Molecular Structure of Compound 1

The molecular structure of Compound 1 according to the present invention was confirmed by X-ray crystallography.

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.

As shown in FIG. 5, 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.

Perspective drawings of Compound 1 were also determined and the lengths and angles of the bonds selected in Table 1 below.

Ru(1)-N(1)와 Ru(1)#-N(1)#Ru (1) -N (1) and Ru (1) #-N (1) #	2.110(10)2.110 (10)	Ru(1)-O(1)Ru (1) -O (1)	2.102(8)2.102 (8)
Ru(1)-O(2)Ru (1) -O (2)	2.112(9)2.112 (9)	Ru(2)-N(2)와 Ru(2)#-N(2)#Ru (2) -N (2) and Ru (2) #-N (2) #	2.098(9)2.098 (9)
Ru(2)-O(3)Ru (2) -O (3)	2.092(9)2.092 (9)	Ru(2)-O(4)Ru (2) -O (4)	2.118(8)2.118 (8)
O(1)-Ru(1)-O(2)O (1) -Ru (1) -O (2)	78.7(3)78.7 (3)	O(1)-Ru(1)-N(1)O (1) -Ru (1) -N (1)	82.2(4)82.2 (4)
O(2)-Ru(1)-N(1)O (2) -Ru (1) -N (1)	81.3(3)81.3 (3)	O(3)-Ru(2)-O(4)O (3) -Ru (2) -O (4)	78.8(3)78.8 (3)
O(3)-Ru(2)-N(2)O (3) -Ru (2) -N (2)	82.8(4)82.8 (4)	O(4)-Ru(2)-N(2)O (4) -Ru (2) -N (2)	84.6(3)84.6 (3)

4. 세포 외 항암 활성 분석4. Extracellular anticancer activity assay

4-1. Cell preparation

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 ℃.

4-2. 3- (4,5- Dimethylthiazole -2-yl) -2,5- Diphenyltetrazolium Bromide (colorimetric 3- (4,5- dimethylthiazol -2- yl ) -2,5- diphenyltetrazolium bromide, MTT) analysis

After treatment with the compounds 1 to 6 of the present invention to the two cells prepared in 1 of Example 3 was evaluated for cell viability by MTT assay. Cells were seeded in 96-well plates, incubated at 37 ° C. and then stimulated with each compound for 24 hours. Cells of each well were incubated with 10 μL of MTT solution for 4 hours in the presence of 37 ° C., 5% CO ₂ , followed by addition of 100 μL of dimethyl sulfoxide (DMSO). Then, absorbance concentration values were measured at 550 nm using an Enzyme-Linked Immunosorbent Assay (ELISA) reader. Absorbance density values were calculated from the absorbance ratios of untreated and treated cells. The maximum-half inhibitory concentration (IC ₅₀ ) value for cell growth is plotted as the logarithm of the percentage of cell survival versus logarithm of drug concentration using a linear regression function. Was obtained by fitting.

The IC ₅₀ values are shown in Table 2 below compared to known anti-tumor drugs cisplatin and doxorubicin.

SampleSample	IC₅₀[μM]^[a] IC ₅₀ [μM] ^[a]
SampleSample	AGSAGS	HCT-15HCT-15
1One	>200> 200	>200> 200
22	180.4±11.64180.4 ± 11.64	>200> 200
33	29.6±0.5829.6 ± 0.58	29.1±0.6429.1 ± 0.64
44	>200> 200	>200> 200
55	105.0±10.16105.0 ± 10.16	>200> 200
66	22.8±1.0022.8 ± 1.00	26.8±2.5026.8 ± 2.50
L1L1	>200> 200	>200> 200
L2L2	152.2±5.13152.2 ± 5.13	>200> 200
A3A3	125.2±7.64125.2 ± 7.64	>200> 200
시스플라틴Cisplatin	107.4±1.87107.4 ± 1.87	12.4±1.8512.4 ± 1.85
독소루비신Doxorubicin	3.0±0.093.0 ± 0.09	15.2±1.4315.2 ± 1.43

As a result, compounds 3 and 6 according to the present invention showed considerable efficacy. Compounds 3 and _{6 (IC 50 = 29.6 μM,} 22.8 μM) of efficacy were more potent better than cisplatin (IC ₅₀ = 107.4 μM) against AGS cancer cells, HCT-15

cancer compounds

3 and 6 in the cells (IC ₅₀ = 29.1 μM, 26.8 μM) were similar to those of cisplatin (IC ₅₀ = 12.4 μM) and doxorubicin (IC ₅₀ = 15.2 μM).

5. 동물 모델 내 항암 활성 분석5. Anticancer Activity Analysis in Animal Models

5-1. Cells and Hollow Hollow fiber, HFs ) Preparations

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.

5-2. In vivo transplantation

For in vivo transplantation of HFs prepared in Example 1, 6 week old nude mice were used.

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.

Two days later, male mice were fed using gavage at 100 mg / kg of body weight for 7 days. Mice were administered 100 μg kg ⁻¹ bw / day of

compound

3 and 6 for 7 days each.

To analyze the number of viable cells in HFs, 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.

All washes were pooled and centrifuged at 500 g for 5 minutes to collect cells. The number of viable cells was confirmed using trypan blue exclusion assay.

5-3. Hollow Fiber analysis

As a result, as shown in Figure 6, 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. In the SC region, 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.

6. 화합물의 6. Compound 자가포식Self-feeding 작용 활성 효과 확인 Action Active Effect Confirmation

Scrapes the inside, after confirming the in vitro anti-tumor activity of Compound 3 and Compound 6 by experiments, the compound 3 in the HCT-15 cells with Compound 6 The induction of autophagy was evaluated. Monodansylcadaverine (MDC) staining was used to detect autophagic vacuoles that accumulate during autophagy ripening.

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).

As a result, as shown in FIG. 7, the number of MDCs labeled with autophagy vacuoles was significantly increased by the treatment of

compounds

3 and 6 .

Both

compounds

3 and 6 strongly induced autophagy activity at low concentrations (0-5 μM), and at higher concentrations autophagy activity decreased. These results were very similar to increasing cell death and decreasing cell proliferation.

<< 실시예Example 2> 페난트렌-루테늄 화합물 제조 및 항암활성 확인 2> Preparation of phenanthrene-ruthenium compound and anticancer activity

All chemicals were purchased and used without further purification, and all solutions were diluted and used according to standard methods.

The areren-ruthenium acceptor clips used in this experiment are described in conventional literature (Therrien B, Suss-Fink G, Govindaswamy P, Renfrew AK, Dyson PJ. 2008, 47, 3773-3776 .; Barry NPE, Furrer J, Therrien B. 2010, 93, 1313-1328), 3,6-dibromophenanthrene is known from the prior art (Nakamura Y, Tsuihiji T, Mita T, Minowa T, Tobita S, Shizuka H, Nishimura J. 1996, 118, 1006-1012 ).

¹ H and ¹³ C NMR spectra were recorded on a Bruker 300 MHz spectrometer. Mass spectra for self-assembly were recorded on a Micromass Quattro II triple-quadrupole mass spectrometer using electron spray ionization with the MassLynx operating system.

2. 2. 디피리딜Dipyridyl 페난트렌 공여체(화합물 7) 합성 Synthesis of Phenanthrene Donor (Compound 7)

Donor compound 7 was synthesized in the same manner as in Scheme 2 below.

Scheme 2

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.

The reaction mixture was refluxed under nitrogen for 2 days and cooled, then the solvent was removed under reduced pressure. The remaining residue was purified by flash column chromatography to give compound 7 as a white solid (yield 48%, see FIG. 8).

Mp: 221-223 ° C. Anal. calcd. for C ₂₈ H ₁₆ N ₂ : C, 88.40; H, 4. 24; N, 7.36. Found: C, 88.12; H, 4. 22; N, 7.40. ¹ H NMR (300 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 9.03 (d, J = 1.5 Hz, 1H), 8.82 (dd, J = 1.8, 0.9 Hz, 1H), 8.56 (dd, J = 4.8 , 1.8 Hz, 1H), 8.07 (dd, J = 7.9, 1.8 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.82 (dd, J = 8.3, 1.5 Hz , 1H), 7.51 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H). ¹³ C NMR (75 MHz, CDCl ₃ ) δ 152.17, 148.53, 138.99, 132.33, 129.82, 129.69, 129.04, 127.83, 126.74, 123.45, 121.04, 120.75, 93.57, 86.91. HRMS for C ₂₈ H ₁₆ N ₂ : calcd., 380.1313; observed, 380.1314.

<< 실시예Example 7> 화합물 11 내지 13 합성 7> Synthesis of Compounds 11-13

Compounds 11 to 13 were synthesized in the same manner as in Scheme 3 below.

Scheme 3

To 1.5 mL of nitromethane / methanol (1: 1 volume ratio), 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.

3-1. Compound 11 Synthesis

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)

Mp: 233-235 ° C. (dec.). Anal. calcd. for C ₁₁₂ H ₉₂ F ₁₂ N ₄ O ₂₀ Ru ₄ S ₄ : C, 52.25; H, 3. 60; N, 2.18. Found: C, 52.39; H, 3.59; N, 2.17. ¹ H NMR (300 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 8.41 (s, 2H), 8.37 (s, 2H), 8.24 (d, J = 7.1 Hz, 2H), 8.03 (d, J = 5.7 Hz, 2H), 7.53-7.36 (m, 8H), 6.07 (d, J = 6.3 Hz, 4H), 5.84 (d, J = 6.3 Hz, 6H), 2.91 (d, J = 6.9 Hz, 2H), 2.22 (s, 6 H), 1.35 (d, J = 6.9 Hz, 12 H). ¹³ C NMR (75 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 184.77, 155.68, 152.58, 142.90, 133.28, 130.20, 129.95, 129.68, 128.69, 127.05, 124.31, 124.03, 120.62, 119.80, 105.49, 102.82, 100.41, , 97.07, 84.69, 84.65, 82.76, 32.54, 22.46, 18.29. ESI-MS for C ₁₀₉ H ₉₂ F ₃ N ₄ O ₁₁ Ru ₄ S: calcd., 709.42 [11-3 OTf] ⁺³ ; observed, 709.32.

3-2. Compound 12 Synthesis

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)

Mp: 238-239 ° C. (dec.). Anal. calcd. for C ₁₂₀ H ₉₆ F ₁₂ N ₄ O ₂₀ Ru ₄ S ₄ : C, 53.89; H, 3.62; N, 2.09. Found: C, 53.81; H, 3.63; N, 2.10. ¹ H NMR (300 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 8.50 (d, J = 1.7 Hz, 2H), 8.38 (s, 2H), 8.20 (d, J = 4.7 Hz, 2H), 8.16 ( d, J = 7.9 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.44-7.32 (m, 6H), 7.26 (s, 4H), 5.84 (d, J = 6.3 Hz, 4H), 5.59 (d, J = 6.3 Hz, 4H), 2.89 (d, J = 6.9 Hz, 2H), 2.14 (s, 6H), 1.34 (d, J = 6.9 Hz, 12H). ¹³ C NMR (75 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 171.91, 154.93, 151.80, 142.60, 138.66, 133.19, 130.32, 129.86, 129.72, 128.60, 127.03, 126.44, 123.83, 120.72, 119.81, 112.59, 104.59 , 101.26, 96.42, 85.65, 85.00, 83.73, 31.94, 22.40, 17.43. ESI-MS for C ₁₁₇ H ₉₆ F ₃ N ₄ O ₁₁ Ru ₄ S: calcd., 742.77 [12-3OTf] ⁺³ ; observed, 742.95.

3-3. Compound 13 Synthesis

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%)

Mp: 240-241 ° C. (dec.). Anal. calcd. for C ₁₃₆ H ₁₀₄ F ₁₂ N ₄ O ₂₀ Ru ₄ S ₄ : C, 56.82; H, 3.65; N, 1.95. Found: C, 56.89; H, 3. 64; N, 1.96. ¹ H NMR (300 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 8.74 (dd, J = 5.8, 3.3 Hz, 4H), 8.60 (s, 2H), 8.21 (d, J = 5.6 Hz, 2H), 8.16 (s, 2H), 8.06 (dd, J = 5.8, 3.3 Hz, 4H), 7.98 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.34 (s, 2H ), 7.19 (dd, J = 8.1, 5.7 Hz, 2H), 7.11 (d, J = 8.2 Hz, 2H), 6.04 (d, J = 6.3 Hz, 4H), 5.75 (d, J = 6.3 Hz, 4H ), 3.05 (dt, J = 13.9, 6.9 Hz, 2H), 2.26 (s, 6H), 1.39 (d, J = 6.9 Hz, 12H). ¹³ C NMR (75 MHz, CD ₃ NO ₂ / CD ₃ OD) δ 170.33, 155.58, 151.69, 141.96, 134.87, 134.26, 133.06, 130.03, 129.82, 129.43, 128.54, 128.32, 126.83, 126.35, 123.52, 120.53, 119.80 , 108.27, 104.42, 101.34, 96.15, 85.75, 84.80, 83.11, 32.07, 22.56, 17.92. ESI-MS for C ₁₃₃ H ₁₀₄ F ₃ N ₄ O ₁₁ Ru ₄ S: calcd., 809.46 [13-3 OTf] ⁺³ ; observed, 809.49.

3-4. Characterization of Compounds 11-13

The 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 typical resonance shifts in proportion to the metal ligand binding of the free donor and acceptor in compound 11-13 molecules synthesized by this procedure via ¹ H NMR spectra in CD ₃ OD / CD ₃ NO ₂ (1/1) Confirmed.

Referring to FIG. 8, the α-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.

In addition, electrospray ionization mass spectrometry (ESI-MS) 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.

X-ray structure analysis

The molecular structure of Compound 11 was clearly confirmed by single crystal X-ray analysis.

Diffraction data from loops attached to single crystals were collected on an ADSC quantum 210 CCD diffractometer using synchrotron radiation (λ = 0.80000 Hz) at 100 K of Macromolecular Crystallography Beamline 2D at Pohang Accelerator Laboratory (PAL).

Initial data was processed and reduced using the HKL2000 program. The structure was identified by direct method and refined by fullmatrix least-squares refinement on F ² using appropriate software in the SHELXTL program package.

X-ray data of Compound 11: C ₁₁₃ H ₉₆ F ₁₂ N ₄ O ₂₁ Ru ₄ S ₄ , M = 2606.46, triclinic, P , α = 16.457 (3) ', β = 18.067 (4)', c = 22.099 (4) Å, α = 68.36 (3) °, β = 81.25 (3) °, γ = 64.86 (3) °, V = 5527.2 (19) Å ³ , Z = 2, T = 100 K, μ (synchrotron ) = 0.504 mm-1, ρcalcd = 1.566 g cm- ³ , 26273 reflections measured, R1 = 0.0670 and wR2 = 0.2255 for 26273 reflections (I> 2σ (I)), R1 = 0.1163 and wR2 = 0.2834 (all data), GoF = 1.233, 1498 parameters and 195 restraints, CCDC: 1062887.

All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were placed at ideal positions in geometry.

As a result, it was confirmed that two phenanthrene units are stacked by strong pi-pi interactions at a distance of approximately 3.5 Å, as shown in FIG. To form molecules of the same shape.

The average Ru-N pyridine band distance was 2.11 Hz, which was found to be similar to other tetranuclear structures.

4. 암세포에 대한 세포독성 확인4. Confirmation of cytotoxicity against cancer cells

4-1. Cell Culture and Reagents

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 .

4-2. Cell viability assay

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 μL of a 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) solution 37 Incubated for 4 hours at 5 ℃ CO ₂ .

Formazan formed during the incubation period was dissolved by treatment with 100 μL 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.

4-3. Self-extinguishing vacuole Labeling

The autodigestive activity of compounds 11 to 13 was confirmed by fluorescent MDC (monodansylcadaverine) staining.

Cells were stained with 0.05 mM MDC (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes at 37 ° C. in RPMI 1640 medium, washed three times with PBS, and immediately filtered with V-2A excitation filter. , 380/420 nm; barrier filter, 450 nm) with a fluorescence microscope (Nμikon Eclipse TE 300, Tokyo, Japan).

4-4. Cell image analysis

Apoptosis analysis was performed using a Tali ^® Image-Based Cytometer (Invitrogen). AGS and COS-7 cells were treated with Compound 12 at 0-20 μM 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 ^™ 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.

4-5. Weston Blot

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 μg) 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.

Thereafter, the secondary antibody bound to Horadish peroxidase was bound to the membrane and visualized using a chemiluminescence (ECL) kit (Amersham).

4-6. Caspase -3 activity analysis

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.

4-7. Confirmation of cytotoxicity against cancer cells

In order to confirm the cytotoxicity of each organometallic multinuclear allen-ruthenium (II) compound synthesized in the previous process against cancer cells, compounds 7 which are donors to HCT-15, SK-hep-1 and AGS cells, which are human cancer cells, Compounds 11-13 were treated and IC ₅₀ values of each cell were measured.

As a result, donor 7 and compound 11-13 showed a strong anticancer effect in all cells as shown in Table 3. In particular, 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 μM or more in AGS cells, compared with other cells was found to have the lowest anti-cancer effect in gastric cancer.

From the above results, the AGS cell line, which is a human gastric cancer cell, was selected to further analyze the anticancer ability of compounds 11-13.

화합물compound	IC₅₀ [μM]^[a] IC ₅₀ [μM] ^[a]
화합물compound	HCT-15HCT-15	SK-hep-1SK-hep-1	AGS AGS
77	>100> 100	>100> 100	>100> 100
1111	>100> 100	>100> 100	>100> 100
1212	3.8±0.153.8 ± 0.15	9.0±0.189.0 ± 0.18	2.5±0.342.5 ± 0.34
1313	70.1±1.6770.1 ± 1.67	62.9±2.0762.9 ± 2.07	68.6±1.3368.6 ± 1.33
CisplatinCisplatin	72.4±9.4272.4 ± 9.42	41.1±5.9241.1 ± 5.92	>100> 100
DoxorubicinDoxorubicin	22.4±4.8822.4 ± 4.88	13.6±3.0213.6 ± 3.02	7.96±1.407.96 ± 1.40

^[a] IC ₅₀ : concentration of drug required to inhibit 50% of cell viability

5. 화합물 12의 세포독성 확인5. Confirmation of Cytotoxicity of Compound 12

In order to confirm whether the cytotoxic effect of Compound 12 was caused by autophagy or apoptotic cell death in the AGS cells, the previous autosomal activity was confirmed.

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.

As a result, referring to FIG. 10A, 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 μM 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.

In particular, it was confirmed that the autodigestion activity of AGS cells treated with Compound 12 at a concentration of 20 μM was 1.4-fold higher than that of normal cells.

10B, apoptosis cell death was induced in a concentration-dependent manner of Compound 12 in AGS cells, whereas COS-7 normal cells treated with the same amount of Compound 12 had a greater percentage of cells surviving than AGS cells. As a result, it was confirmed that the sensitivity of Compound 12 was low in normal cells.

From the above results, it was confirmed that Compound 6 selectively showed cytotoxicity of cancer cells through induction of autophagy activity and apoptotic cell death without altering autophagy or apoptosis of normal fibroblasts.

6. 화합물 12의 6. Of Compound 12 자가포식Self-feeding 및 세포사멸 유도 확인 And induction of apoptosis

Western blot analysis was performed on AGS cells activated with autophagy with compound 12 to analyze cellular mechanisms by confirming the expression of p62, a selective target of autophagy, and the intracellular distribution of LC3 protein, a reliable autodigestion marker.

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.

Referring to FIG. 11A, it was confirmed that the expression of p62 protein and the conversion of LC3-I to LC3-II were significantly increased in AGS cells treated with Compound 12 than untreated AGS cells.

In addition, to confirm apoptosis induced by Compound 12 in AGS cells, phosphorylation of Akt and mTOR proteins was confirmed by Western blot.

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.

Accordingly, the effect of compound 12 on the Akt-mTOR signal pathway was confirmed.

As a result, the expression of p-Akt was significantly reduced in AGS cells treated with Compound 12 as compared to AGS cells not treated with Compound 12 as shown in FIG. 11B, and the expression of mTOR was also decreased.

On the other hand, in order to confirm whether compound 12 induces apoptosis by caspase, caspase-3 activity was confirmed through substrate cleavage reaction analysis.

As a result, caspase-3 activity was increased in a concentration-dependent manner in the compound 12 treated cells as shown in FIG.

From the results, it was confirmed that Compound 12 induces cancer cell death by inhibiting the Akt / mTOR signaling pathway and activating autophagy.

7. 화합물 12의7. Of Compound 12 안정성 stability 확인 Confirm

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.

10 μM 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.

As a result, as shown in Figure 13 10 μM Compound 12 showed stability up to 50 hours in DMSO, it was confirmed that the inhibitory activity is reduced by 50% when Compound 12 is pre-cultured for 24 hours in the culture medium.

<< 실시예Example 3> 3> 벤즈이미다졸Benzimidazole -루테늄 화합물 제조 및 항암활성 확인-Preparation of ruthenium compound and anticancer activity

1. 화학물질 및 화합물 분석1. Chemical and Compound Analysis

Chemicals 2-phenylbenzimidazole [2-phenylbenzimidazole], 1,2-di (bromomethyl) benzene [2-di (bromomethyl) benzene], 1,3-di (bromomethyl) benzene [1,3 -di (bromomethyl) benzene], 1,4-di (bromomethyl) benzene [1,4-di (bromomethyl) benzene], sodium acetate anhydrous, ruthenium metal salt [(η6-cymene ) RuCl ₂ ] ₂ was purchased from Sigma-Aldrich and used without further purification.

Deuterated NMR solvents were purchased from the Cambridge Isotope Laboratory (Andover, MA, USA) and NMR spectra were obtained using a Bruker 300 MHz spectrometer.

¹ H NMR chemical shifts have been reported to be related to the residual protons of deuterated CDCl ₃ (7.26 ppm) and deuterated DMSO-d6 (2.50 ppm).

Triple Quandrupole LC-Mass spectrometry (Finnigan TSQ Quantum Ultra EMR) using electrospray ionization was used to obtain ESI MS data of ligand and lutenacycle, and analyzed by MassLynx software suite system of Korea Basic Science Institute (Seoul). It was.

2. 2. 페닐벤즈이미다졸Phenylbenzimidazole 양팔형 리간드(L3 내지 L5) 합성 Bilateral Ligand (L3 to L5) Synthesis

Compounds L3 to L5 were synthesized by the following schemes.

Scheme 4

2-1. Ligand L3 Synthesis

A mixture of 2-phenylbenzimidazole (294 mg, 1.515 mmol) and KOH (170 mg, 3.031 mmol) in 7 mL of dimethylformamide (DMF) was added to a round bottom flask and stirred at 40 ° C. for 1 hour. Thereafter, 1,4-di (bromomethyl) benzene (200 mg, 0.757 mmol) was added to the reaction mixture, which was stirred for 24 hours at 40 ° C. Then, 100 mL of distilled water was poured into the residue. The milky precipitate was filtered off and washed with excess distilled water.

After a few days, colorless crystalline material was obtained from ethanol at room temperature in 81% (299 mg) yield.

¹ H NMR (300 MHz, CDCl ₃ ): 5.34 (s, 4H, -CH _2- ), 6.91-6.95 (m, 2H, ArH), 7.07 (d, 2H, J = 9.0 Hz, ArH), 7.22- 7.25 (m, 1H, ArH), 7.27-7.30 (m, 2H, ArH), 7.33-7.36 (m, 2H, ArH), 7.38-7.44 (m, 4H, ArH), 7.46-7.51 (m, 2H, ArH), 7.58-7.61 (m, 4H, ArH), 7.89 (d, 2H, J = 6.0 Hz, ArH) and 8.08-8.11 (m, 1H, ArH). Anal. Calcd for C ₃₄ H ₂₆ N ₄ (Mwt. 490.59): C, 83.24; H, 5. 34; N, 11.42. Found: C, 83.22; H, 5. 34; N, 11.29. ESI-MS for L3: m / z = 491.10 [M] ⁺ .

2-2. Ligand L4 Synthesis

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).

After a few days, a white solid material was obtained from methanol in 80% (312 mg) yield.

¹ H NMR (300 MHz, DMSO-d ₆ ): 5.43 (s, 4H, -CH _2- ), 6.52 (s, 1H, ArH), 6.93 (d, 2H, J = 6.0 Hz, ArH), 7.17- 7.39 (m, 11H, ArH), 7.44-7.47 (m, 6H, ArH) and 7.72 (d, 2H, J = 6.0 Hz, ArH). Anal. Calcd for C ₃₄ H ₂₆ N ₄ (Mwt. 490.59): C, 83.24; H, 5. 34; N, 11.42. Found: C, 83.34; H, 5. 36; N, 11.32. ESI-MS for L4: m / z = 491.19 [M] ⁺ .

2-3. Ligand L5 Synthesis

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).

After a few days, a white solid material was obtained from methanol in 87% (324 mg) yield.

¹ H NMR (300 MHz, DMSO-d ₆ ): 5.51 (s, 4H, -CH _2- ), 6.91 (s, 4H, ArH), 7.18-7.26 (m, 4H, ArH), 7.39-7.26 (m , 4H, ArH), 7.48-7.51 (m, 6H, ArH) and 7.65-7.71 (m, 6H, ArH). Anal. Calcd for C ₃₄ H ₂₆ N ₄ (Mwt. 490.59): C, 83.24; H, 5. 34; N, 11.42. Found: C, 83.12; H, 5.41; N, 11.14. ESI-MS for L5: m / z = 491.21 [M] ⁺ .

3. 고리금속 3. Ring metal 루테니움Ruthenium 화합물 합성 Compound synthesis

Ring metal ruthenium compounds 14 to 16 were synthesized by the following Scheme 5.

Scheme 5

3-1. Ring metal

Ruthenium

Compound 14 Synthesis

One equivalent of the biphasic phenylbenzimidazole derivative ligand L3 (50 mg, 0.081 mmol) was dissolved in fresh distilled methanol in a round bottom flask dried under nitrogen atmosphere.

Anhydrous sodium acetate (1.2 mmol) 2.3 equiv. At room temperature was added to the mixture and stirred, followed by addition of 1 molar concentration of [(η ⁶ -cymene) RuCl ₂ ] ₂ (0.5 mmol).

The reaction mixture was stirred at ˜65 ° C. for 12 hours and the reaction process was confirmed using TLC.

After complete conversion, the methanol was removed under reduced pressure and dried under vacuum.

The dried solid was dissolved in dichloromethane (10 mL), the orange red solution was filtered, concentrated to 2 mL and crystallized by addition of methanol.

After a few days, the crystalline product was obtained in 91.9% (77.32 mg) yield.

Anal. _{_{_{Calcd for C 54 H 52 Cl 2}}} N 4 Ru 2 · CH 3 OH (Mwt. 1062.11): C, 62.20; H, 5.31; N, 5.28. Found: C, 62.12; H, 5. 25; N, 5.53. ¹ H NMR (300 MHz, DMSO-d6) δ 0.76 (d, 6H, J = 6.0 Hz), 0.82 (d, 6H, J = 6.0 Hz), 2.06 (d, 6H, J = 6.0 Hz), 2.11- 2.19 (m, 2H, -CH-), 5.70 (d, 2H, J = 6.0 Hz, ArCH), 5.74-5.80 (m, 4H, ArCH), 6.06.6.09 (m, 2H), 6.15-6.17 (m , 4H, ArCH / CH2), 6.87-6.95 (m, 4H), 7.02-7.10 (m, 3H), 7.42-7.45 (m, 3H), 7.49-7.53 (m, 4H), 7.77 (d, 2H, J = 6.0 Hz), 8.13 (d, 2H, j = 6 Hz) and 8.29-8.34 (m, 2H); ESI-MS for ruthenacycle 14: m / z = 995.28 [1M-Cl] ⁺ .

3-2. Ring metal

Ruthenium

Compound 15 Synthesis

One equivalent of the 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.

Anhydrous sodium acetate (1.2 mmol) 2.3 equiv. At room temperature was added to the mixture and stirred, followed by addition of 1 molar concentration of [(η ⁶ -cymene) RuCl ₂ ] ₂ (0.5 mmol) at room temperature.

The 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.

A yellowish brown benzimidazole-ruthenium (Compound 15) was obtained in 89.9% (75.41 mg) yield.

Anal. _{_{_{Calcd for C 54 H 52 Cl 2}}} N 4 Ru 2 · CH 3 OH + H 2 O (Mwt. 1080.20): C, 61.16; H, 5.41; N, 5.19. Found: C, 60.86; H, 5.11; N, 5.18. ¹ H NMR (300 MHz, DMSO-d6) δ 0.66 (d, 6H, J = 6.0 Hz), 0.76 (d, 6H, J = 6.0 Hz), 1.98 (s, 6H, CH ₃ ), 2.06-2.15 ( m, 2H, -CH-), 5.33 (d, 2H, J = 6.0 Hz, ArCH), 5.74 (d, 2H, ArCH), 5.66 (d, 2H, J = 6.0 Hz), 5.86 (s, 4H, -CH2-), 6.07 (d, 2H, J = 6.0 Hz, ArCH), 6.69-6.75 (m, 4H, ArCH), 6.97 (br, 1H), 7.04-7.09 (m, 3H, ArCH), 7.34 ( t, 2H, J = 3 Hz, ArCH), 7.77 (t, 2H, J = 6.0 Hz), 7.62 (d, 2H, J = 9 Hz), 8.05 (d, 2H, J = 9.0 Hz) and 8.30 ( d, 2H, J = 9.0 Hz, ArCH); ESI-MS for ruthenacycle 15: m / z = 995.13 [2M-Cl] ⁺ .

2-3. Ring metal

Ruthenium

Compound 15 Synthesis

One equivalent of the biphasic phenylbenzimidazole derivative ligand L5 (50 mg, 0.081 mmol) was dissolved in fresh distilled methanol in a round bottom flask under nitrogen atmosphere.

After a few days, the crystalline product was obtained in 93.5% (81.32 mg) yield.

Anal. _{_{_{Calcd for C 54 H 52 Cl 2}}} N 4 Ru 2 · 2CH 3 OH (Mwt. 1094.15): C, 61.47; H, 5.53; N, 5.12. Found: C, 61.75; H, 5. 26; N, 5.51. ¹ H NMR (300 MHz, DMSO-d6) δ 0.62 (d, 6H, J = 6.0 Hz), 0.75 (d, 6H, J = 6.0 Hz), 1.99 (s, 6H, C H ₃ ), 2.05-2.11 (m, 2H, -CH-), 5.23 (d, 2H, J = 9.0 Hz, ArCH), 5.57 (d, 2H, J = 9.0 Hz, ArCH), 5.66 (t, 2H, J = 6.0 Hz), 5.66 (d, 2H, J = 6 Hz), 5.87 (s, 4H, -CH2-), 6.03 (d, 2H, J = 6.0 Hz, ArCH), 6.86 (t, 2H, J = 9 Hz, ArCH) , 6.93 (br, 2H, ArCH), 7.04 (t, 2H, J = 9.0 Hz, ArCH), 7.34 (t, 2H, J = 6 Hz, ArCH), 7.43 (t, 2H, J = 6.0 Hz), 7.57 (d, 2H, J = 9 Hz), 7.65 (d, 2H, J = 9.0 Hz), 7.85 (d, 2H, J = 9 Hz, ArCH) and 8.26 (d, 2H, J = 9.0 Hz, ArCH ); ESI-MS for ruthenacycle 15: m / z = 995.15 [3M-Cl] ⁺ .

2-4. Compound analysis

The structure of benzimidazole-ruthenium synthesized by the above procedure was confirmed by various analytical methods. Comparing all lutenacycle downfield shifts with the corresponding ligands L3 to L5, ¹ H NMR chemical shifts of compound 14-16 were identified, with new double peaks in the range δ 0.50-0.80 ppm, δ 2.40-2.60 ppm As one singlet and one multiplet in the range δ 2.00-2.10 ppm were identified, it was confirmed that ruthenium p-cymene moieties exist in the alkyl moiety for each compound.

In addition, eight proton resonances with ligand proton resonances in the aryl moiety were identified, due to the presence of two p-cymene moieties.

HR-ESI-MS data confirmed the additional structure of benzimidazole-ruthenium 14-16. Mass spectrum of benzimidazole-ruthenium m / z One chloride ligand loss signal in m / z 995.28 [2M-Cl] ⁺ and [3M-Cl] ⁺ moieties and m / z 480.17 [2M-2Cl] ²⁺ and [3M-2Cl In the ²⁺ moiety, two chloride ligand loss signals confirmed the double ring metal composition, and the observed experimental spectra patterns and theoretically calculated isotope distributions coincide with each other as shown in FIG. 14.

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.

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.

Through structural purification, the benzimidazole-ruthenium structure as shown in FIG. 15 could be confirmed.

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.

In addition, the lutena cycle is C-H... Cl, C-H... π and C-H... It was confirmed that stability was increased by several strong and weak non-covalent bonds such as N.

4. 종양세포 성장억제 효과 확인4. Confirmation of tumor cell growth inhibition effect

The cancer cell toxicity of the ruthenium-based compounds RuPD, ligands L3 to L5 and benzimidazole-ruthenium 14 to 16 synthesized in the preparation was confirmed.

Human gastric carcinoma (AGS), SK-hep-1 (human hepatocellular carcinoma) and HCT-15 (human colorectal carcinoma) cell lines containing 10% heat-inactivated fetal bovine serum (FBS) and 1% phenylsilin / streptomycin Growth was performed at 37 ° C., 5% CO ₂ conditions using RPMI 1640 and Dulbecco's Modified Eagle Medium (DMEM) 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 μM concentrations for 24, 48 and 72 hours.

Stock solutions of 2 mg / mL compound, oxaliplatin and doxorubicin were prepared with dimethylsulfoxide (DMSO) and stored at -20 ° C, and cisplatin was dissolved in 0.9% NaCl (5 mM).

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 μm Millipore filter. It was.

After 10 μL of MTT solution was added to each well and incubated for 3 hours at 37 ° C., 5% CO ₂ conditions, MTT solution was removed and DMSO 100 μL was added to each well to lyse cells and multi-reader (Tecan, Switzerland) The cell viability was confirmed by obtaining an absorbance concentration value at 550 nm.

The percentage of cells that survived was calculated from the percentage of untreated cells.

Using a linear regression function, 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.

As a result, as shown in Table 1, after 72 hours of exposure, RuPD and ligands L3 to L5 inhibited the growth of cancer cells similarly or decreased to 48 hours of exposure, while those of benzimidazole-ruthenium 14 to 16 and cisplatin were almost similar. Or increased in proportion to the exposure time.

In addition, when benzimidazole-ruthenium 14-16 was treated for 72 hours, the IC ₅₀ value was between 6.1-12.5, 7.6-23.7 and 11.1-26.6 μM for AGS, Sk-hep-1 and HCT-15 cells, respectively. As determined to be much higher than ligand L3-L5 (IC ₅₀ values found to be between 9.1-67.2, 39.9-82.5 and 61.9-94.2 μM for AGS, Sk-hep-1 and HCT-15 cells, respectively) Excellent inhibitory effect was shown.

In particular, most of the samples showed a specific effect on gastric cancer cells (AGS), the inhibitory effect of AGS cells increased with the exposure time. However, ligands L4 and L5 increased the drug dose for significant inhibitory effects at 72 hours (IC ₅₀ = 67.2 and 28.6 μM) than at 48 hours of exposure (IC ₅₀ = 16.6 and 7.6 μM).

From these results it can be suggested that L4 and L5 decrease in stability after 48 hours.

In addition, benzimidazole-ruthenium 14 to 16 showed a very high inhibitory effect in AGS gastric cancer cells, especially 15 compounds showed a very good anti-cancer effect (IC ₅₀ = 6.1 μM, 72 h exposure) in proportion to the exposure time. .

Although cisplatin (IC ₅₀ = 2.6 μM) showed a higher anticancer effect than benzimidazole-ruthenium 15, it showed a much better anticancer effect than oxaliplatin (IC ₅₀ = 25.9 μM).

5. 활성 유전자 확인5. Identify active genes

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.

5-1. RNA extraction and template cDNA synthesis

Total RNA was extracted from tumor cells treated with or without the benzimidazole-ruthenium 15 compound using PureLink ^™ RNA Mini Kit (Ambion, USA).

CDNA was synthesized with reagents of GeneFishing DEG Premix Kit (Seegene, Korea) except reverse transcriptase SuperScript II (Invitrogen, USA).

3 μg of total RNA was mixed with 2 μL of 10 μM cDNA synthesis primer dT-ACP1 (Seegene, Korea) and DEPC treated water to a final volume of 9.5 μL.

The mixture was incubated at 80 ° C. for 3 minutes and immediately placed on ice for 2 minutes and then centrifuged briefly.

4 μL of 5 × RT buffer (Noble Bio, Korea), 5 μL of 2 mM dNTP (Takara, Japan), 0.5 μL of 40 U / μL RNase inhibitor and 200 U / μL M-MLV reverse transcriptase (Noble Bio, Korea) 1 μL was added to the final 20 μL volume.

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 μL distilled water.

5-2. Loosening adjustment primer (Annealing control primer; ACP ) Polymerase chain reaction PCR )

Differentially expressed genes (DEGs) were screened according to the manufacturer's instructions using the GeneFishing ^™ DEG kit (Seegene, Korea).

Two 20 μL of the final reactant containing 3 μL of the first diluted template cDNA, 1 μL of 10 μM dT-ACP2, 10 μL of SeeAmp ^™ ACP ^™ master mix and 2 μL of 5 μM arbitrary ACP in one cycle of one-step PCR at 50 ° C The first template cDNA was synthesized.

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.

After synthesis of the second template cDNA, 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.

SolGent Co. DNA sequencing was performed in Daejeon, Korea, and the sequences were analyzed using the BLASTX search program of the National Center for Biotechnology Information (NCBI) GenBank.

5-3. Quantitative RT- PCR analysis

Total RNA was extracted from AGS gastric cancer cells using PureLink ^™ RNA Mini Kit. Total RNA reverse transcription was performed in 20 μL volumes, including 1 μg 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 β-actin: 5'-GTCCACCGCAAATGCTTCTA-3 ', 5'-TGCTGTCACCTTCACCGTTC-3'

Real-time PCR using SYBR Premix Ex Taq II (Takara, Japan), 25 μL final volume with 2 μL cDNA template, 12.5 μL Master Mix, 1 μL each primer (10 μM stock solution), 8.5 μL sterile distilled water PCR reaction was performed.

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.

5-4. result

As a result of gene expression analysis as described above, as shown in Figure 16, the differential expression of the mRNA fragments identified on the agarose gel is three down-regulated in AGS cells treated or not treated with benzimidazole-ruthenium 15 compound The gene was identified.

Analysis using BLAST (NCBI GenBank) confirmed that the gene expressed by ACP1 was lysosomal protein S21 (RPS21, CR542132.1), and the S21 protein was added to Compound 15 in AGS cells treated with benzimidazole-ruthenium 2 compound. It was confirmed that it is reduced by.

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.

In addition, the downregulated gene RPS21 is known to be a component of eukaryotic lysosomes and is associated with lysosomal protein SA (RPSA) in human cells.

The 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.

To confirm the expression level of the RPS21 gene, qRT-PCR was performed using mRNAs of benzimidazole-ruthenium 15 compound treated AGS cells.

As a result, as shown in FIG. 17, the dose-dependent expression of RPS21 mRNA was reduced, and in the cells treated with Compound 15 at a concentration of 2.5 μM, the expression of RPS21 mRNA was reduced by 50% compared with the control group. When treated at 20 μM concentration, 88% expression was shown to be inhibited compared to the control (p <0.01).

On the other hand, cisplatin-treated experimental group showed increased expression of RPS21, similar to genefishing results.

6. 분비된 사이토카인 분석6. Secreted Cytokine Analysis

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.

Since arene-Ru derivatives can be used to directly regulate the cancer cell cycle, cytokines produced from these derivatives can indirectly affect cancer cells.

Therefore, the effect of benzimidazole-ruthenium 15 compound on cytokine secretion of macrophages was confirmed.

31 cytokines (TNF-α, IFNγ, G-CSF, GM-CSF, IL-1α, IL-8, IP-10, Rantes, VEGF, EGF, IL-6, Resistin, PAI-1, IL-12 , IL-13, Eotaxin-3, PDGF-BB, PIGF-1, β-NGF, SCF, MCP-1, MIP-1α, IL-2, IL-4, IL-10, FGFβ, Leptin, IGF-1 In order to confirm the secretion of TGF-β, Adipo and IL-17α), benzimidazole-ruthenium 2 (10 μM) 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.

As a result, cytokine IFNγ, IL-1α, VEGF, EGF, Eotaxin-3, IL-10, TGF-β and IL-17α were increased in AGS cells treated with 10 μM 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.

As described above, cytokines, known as central mediators of angiogenesis, were secreted more than three times in AGS cells treated with 10 μM concentration of Compound 15, and this result may be rather helpful for the growth of cancer cells.

However, among the cytokines secreted from the macrophages by compound 15, interleukin-γ (IFNγ) was a cytokine exhibiting anticancer effect and increased by 4.5 times compared with the control group. IFNγ regulates the proliferation and differentiation of cancer cells or modulates an immunomodulatory response to directly exhibit anti-tumorigenic effects.

In addition, 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 .

From the above results, it was confirmed that benzimidazole-ruthenium 15 reduced the lante and IGF-1 secretion of macrophage, while increasing the secretion of IFNγ, thereby suppressing the growth of cancer cells.

7. 화합물 안정성 확인7. Check compound stability

To confirm the stability of benzimidazole-ruthenium 15 compound, 20 μM concentration of compound 15 was incubated in cell culture medium and DMSO for 0, 12, 24 and 48 hours at 37 ° C. AGS cancer cell growth was confirmed by the process.

As a result, as shown in FIG. 19, the growth inhibitory activity of benzimidazole-ruthenium 15 decreased by 50% after 32 hours of preculture in the cell culture medium.

In contrast, growth inhibition activity in DMSO remained stable up to 24 hours.

The results showed that benzimidazole-ruthenium 15 was stable up to 24 hours in cell culture medium and DMSO.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

An arene-ruthenium compound represented by the following Chemical Formula 1 or 2 or a pharmaceutically acceptable salt thereof:

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3a]

[Formula 3b]

or

[Formula 3c]

ego,

In Formulas 3a, 3b, and 3c, 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,

Of Formula 1
Is,

[Formula 4]

Is,

Of Formula 2
silver,

[Formula 5]

ego,

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 method of claim 1,

The arene-ruthenium compound is characterized in that the nitrogen atom in the aromatic ring of formula (4) or (5) is combined with ruthenium of formula (3c), and the following X is separated to form a compound of formula (1) or (2). Or pharmaceutically acceptable salts thereof:

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3c]

ego,

In Formulas 3a, 3b, and 3c, 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,

Of Formula 1
Is,

[Formula 4]

Is,

Of Formula 2
silver,

[Formula 5]

being.
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.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3a]

[Formula 3b]

or

[Formula 3c]

ego,

In Formulas 3a, 3b, and 3c, 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,

Of Formula 1
Is,

[Formula 4]

Is,

Of Formula 2
silver,

[Formula 5]

ego,

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 method of claim 3, wherein

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, cancer prevention or Therapeutic pharmaceutical composition.

[Formula 1]

[Formula 2]

ego,

Is,

[Formula 3c]

ego,

In Formulas 3a, 3b, and 3c, 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,

Of Formula 1
Is,

[Formula 4]

Is,

Of Formula 2
silver,

[Formula 5]

being.
The method according to claim 3 or 4,

The arene-ruthenium compound or a pharmaceutically acceptable salt thereof is a pharmaceutical composition for preventing or treating cancer disease, characterized in that to activate the autophagy action to increase apoptosis to show anticancer activity.
The method according to claim 3 or 4,

The cancer is a solid composition for the prevention or treatment of cancer cancer.
The method of claim 6,

The solid cancer is colon cancer, gastric cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendrocyte, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal / sinus cancer , Nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, liver cancer, gallbladder cancer, biliary cancer, pancreatic cancer, Small bowel cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer Or skin cancer is a pharmaceutical composition for the prevention or treatment of cancer.
A phenanthrene-ruthenium compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 6]

In the above formula,

Is

[Formula 7]

,

[Formula 8]

or

[Formula 9]

ego,

In Formula 7, Formula 8 or Formula 9, 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,

Is

[Formula 10]

ego,

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 of claim 8, wherein the phenanthrene-ruthenium compound represented by Formula 6 is characterized in that A is separated from the compound of Formula 8 and that a nitrogen atom in the aromatic ring of Formula 10 is bonded. Pharmaceutically acceptable salts.
A pharmaceutical composition for preventing or treating cancer diseases, comprising the phenanthrene-ruthenium compound of claim 8 or a pharmaceutically acceptable salt thereof as an active ingredient.
The pharmaceutical composition for preventing or treating cancer diseases according to claim 10, wherein the phenanthrene-ruthenium compound or a pharmaceutically acceptable salt thereof exhibits anticancer activity by inducing autophagy and apoptosis of cancer cells. .
The pharmaceutical composition for preventing or treating cancer diseases according to claim 11, wherein the cancer disease is solid cancer.
13. The method of claim 12, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendrocyte, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal / sinus sinus Cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric 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, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, external woman A pharmaceutical composition for treating or preventing cancer diseases, characterized in that selected from the group consisting of genital cancer, female urethral cancer and skin cancer.
Benzimidazole-ruthenium compound represented by Formula 11 or a pharmaceutically acceptable salt thereof:

[Formula 1]

In Formula 11, X is halogen.
A pharmaceutical composition for treating or preventing cancer diseases, comprising the benzimidazole-ruthenium compound of claim 14 or a pharmaceutically acceptable salt thereof as an active ingredient.
The method according to claim 15, wherein the benzimidazole-ruthenium compound or a pharmaceutically acceptable salt thereof affects macrophages to inhibit the secretion of cytokines that form tumors, and increases the secretion of cytokines that exhibit anticancer effects. A pharmaceutical composition for treating or preventing cancer diseases by inhibiting growth of cancer cells.
The pharmaceutical composition for treating or preventing cancer diseases according to claim 16, wherein the cancer disease is solid cancer.
18. The method of claim 17, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendrocyte, intracranial carcinoma, epithelial cell tumor, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal / sinus sinus Cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, gastric 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, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, outside of woman A pharmaceutical composition for treating or preventing cancer diseases, characterized in that selected from the group consisting of genital cancer, female urethral cancer and skin cancer.