KR20160082765A - Novel Tetranuclear Arene-Ruthenium Compound and Pharmaceutical Composition for Preventing or Treating Cancer Containing the same - Google Patents

Abstract

The present invention relates to a novel tetranuclear arene ruthenium compound, and to a pharmaceutical composition for preventing or treating cancer, comprising the same as an active ingredient. The tetranuclear arene ruthenium compound exhibits anticancer activities in comparison with doxorubicin and cisplatin, publicly known anticancer drugs, and particularly exhibits excellent anticancer activities by strongly inducing autophagy at low concentrations in colon cancer cells, thereby being efficiently used for preventing or treating cancer.

Description

TECHNICAL FIELD The present invention relates to a novel 4-nuclear arene-ruthenium compound and a pharmaceutical composition for preventing or treating cancer diseases containing the same as an effective ingredient.

The present invention relates to a novel 4-nuclear arene-ruthenium compound and a pharmaceutical composition for preventing or treating cancer diseases containing the same as an effective ingredient.

Autophagy plays an important role in regulating cellular 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 is known to be a cell death mechanism in various types of cancer cells that respond to cancer therapy (see Non-Patent Documents 1 and 2).

Many studies have shown that anti-autophagic proteins such as B-cell lymphoma 2, Bcl-2, 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 (e.g., breast and pancreatic cancer) Proved.

In addition, some cases of inhibition of autophagy in cancer cells may increase cancer susceptibility to a variety of therapies such as DNA damaging agents, antihormone therapies, and radiation therapy Showed a therapeutic benefit.

Thus, depending on the cellular feature, the induction or inhibition of autophagy may provide a therapeutic benefit to the patient, so that the design and synthesis of autophagic modulators can be used as a new therapeutic strategy for cancer have.

The field of metal pharmacy is rapidly gaining popularity through the therapeutic application of metal-based drugs. Platinum complexes such as cisplatin, carboplatin and oxoplatin are known to be 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 exhibit low toxicity and high activity in tumors resistant to platinum drugs in recent years, are attracting attention as new metal-based drugs .

However, many studies have been conducted on these ruthenium-based complexes, but research on self-predation has not been reported yet.

1. Cancer Res. 2006, 66, 9349-9351. 2. Autophagy 2010, 6, 322-329.

Accordingly, the present invention provides novel 4-nuclear arene-ruthenium compounds and their use for chemotherapeutic treatment.

In order to accomplish 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:

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3]

(3b)

또는

or

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이고,

ego,

The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2).

The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises an arene-ruthenium compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3]

(3b)

또는

or

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이고,

ego,

The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2).

The quaternary arene-ruthenium compound according to the present invention exhibits better anticancer activity compared to known anticancer drugs cisplatin and doxorubicin, and exhibits excellent anticancer activity by strongly inducing autopoiesis at low concentrations in colon cancer cells, Can be usefully used for preventing or treating cancer.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram showing the preparation of the compounds 1 to 6 according to the present invention. Fig.
2 is a result of the nitromethane _{-D 3 (Nitromethane-D 3,} CD 3 NO 2) The ¹ H-NMR (nuclear magnetic resonance, NMR) spectra and the results of FIG. 2 is recorded in a compound L1, Figure 2b shows the results of Compound 1 according to the present invention.
FIG. 3 shows the results of Electrospray ionization mass spectrometry (ESI-MS) of Compounds 4 to 6 according to the present invention.
Figure 4 shows UV / Vis spectra results, Figure 4 shows the results of Compounds 1 to 3 according to the invention, Figure 4b shows the results of Compounds 4 to 6 according to the invention , And c in Fig. 4 is the result of receptors (A1 to A3) and donors (L1 and L2).
FIG. 5A is an X-ray crystal structure of Compound 1 according to the present invention, and FIG. 5B is a graph showing the X-ray crystal structure according to a space-filling model (CPK model) (Green: ruthenium, red: oxygen, blue: nitrogen and gray: carbon).
6 is a graph showing the anticancer effect of the compound 3 and the compound 6 according to the present invention in colon cancer cells.
FIG. 7 is a graph showing the child behavior of colon cancer cells according to the variation of concentrations of Decompounds 3 and 6 according to the present invention. FIG.

The present invention provides an arene-ruthenium compound represented by the following general formula (1) or (2): or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3]

(3b)

또는

or

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이고,

ego,

The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2).

The present invention also relates to a process for the preparation of an arene-ruthenium compound or a mixture thereof, characterized in that the nitrogen atom in the aromatic ring of formula (4) or (5) is bonded to ruthenium of formula (3c) To provide a pharmaceutically acceptable salt.

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In Formula 3c, each X is independently trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene-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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이다.

to be.

The arene-ruthenium compound of the present invention represented by the general formula (1) or (2) can be used in the form of a pharmaceutically acceptable salt, and the acid addition salt formed by a pharmaceutically acceptable free acid is useful as a salt . As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid and arpartic acid. Preferably, hydrochloric acid is used as the inorganic acid, and methanesulfonic acid is used as the organic acid.

The arene-ruthenium compound of the present invention represented by the above formula (1) or (2) of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which 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 Chemical Formula 1 or Chemical Formula 2 in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, Or by adding an aqueous acid solution of an inorganic acid, followed by precipitation or crystallization. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt may be produced by suction filtration.

A schematic diagram of the preparation of arene-ruthenium compounds 1 to 6 according to the present invention is shown in Fig. Referring to FIG. 1, the 4-nuclear arene-ruthenium compound of Formula 1 is represented by the formula [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)} , of formula 3b _{[Ru 2 (dobq) - [} Ru of _{^{(η 6 p -Pr i C 6}} H 4 Me) 2] [O 3 SCF 3] 2 (A2) ) or (3c ₂ (donq) of 3,6-di (pyridine ^{(η 6 -p-Pr i C} 6 H 4 Me) 2] [O 3 SCF 3] 2 (A3) and dipyridyl donor (donor dipyridyl) of the formula (4) -4-yl) -1,2,4,5-tetrazine (3,6-di (pyridin-4-yl) -1,2,4,5-tetrazine, L1 ) (CH ₃ NO ₂ / CH ₃ OH) and stirred to obtain self-assemblies 1-3 of metal-containing rings.

The 4-nuclear arene-ruthenium compound of formula (2) can also be prepared by reacting [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), of formula 3b _{[Ru 2 (dobq) (η} 6 - p -Pr i C 6 H 4 Me) 2] [O 3 SCF 3] 2 (A2) or [Ru ₂ (donq) of formula 3c ( _{^{η 6 -p-Pr i C 6}} H 4 Me) 2] [O 3 SCF 3] 2 (A3) and 2,5-bis (ethynyl-pyridin-4-yl) furan of formula (5) (2,5-bis (pyridin-4-ylethynyl) furan , L2) is a solution containing equal moles of nitromethane / dimethyl ether _{(CH 3 NO 2 / CH 3} OH) , a self-put and stirred ring (metallacycles containing metal) in the assembly (self- assembly 4 to 6. < / RTI >

The novel arene-ruthenium compounds prepared as described above can be obtained by a method such as infrared spectroscopy, nuclear magnetic resonance spectroscopy, mass spectrometry, liquid chromatography, X-ray crystallography, To confirm the molecular structure.

As can be seen from the following Examples, the 4-nuclear arene-ruthenium compound of Formula 1 or 2 according to the present invention induces autophagic action in human AGS (stomach cancer) and HCT-15 (colon cancer) cell lines, Exhibit excellent anticancer activity by inducing apoptosis, and can be used as an active ingredient of an anticancer agent.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating cancer diseases, which comprises an arene-ruthenium compound represented by the following general formula (1) or (2) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3]

(3b)

또는

or

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이고,

ego,

The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2).

The arene-ruthenium compound is characterized in that the nitrogen atom in the aromatic ring of the formula (4) or (5) is disubstituted with the ruthenium of the formula (3c) to form a compound of the formula (1) or A pharmaceutical composition for therapeutic use is provided.

[Chemical Formula 1]

(2)

이고,

ego,

는,

Quot;

[Chemical Formula 3c]

이고,

ego,

는 단일결합 또는 이중결합이며,In Formula 3c, each X is independently trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene-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,

는,In the formula 1

Quot;

[Chemical Formula 4]

이며,

Lt;

은,(2)

silver,

[Chemical Formula 5]

이다.

to be.

In one embodiment of the present invention, the pharmaceutical composition for prevention or treatment of cancer diseases containing the above-mentioned arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient is characterized in that the aforementioned arene-ruthenium compound or its The pharmaceutically acceptable salt may be contained 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 the present invention is not limited thereto. It depends on the degree.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer diseases containing the above-mentioned arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient may be a carrier, an excipient, a disintegrant, a sweetener, And one or more auxiliaries selected from the group consisting of lubricants, lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders and lubricants.

Specific examples of carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules These solid preparations can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In another embodiment of the present invention, the pharmaceutical composition for preventing or treating cancer comprising the above-mentioned Arene-ruthenium compound or a pharmaceutically acceptable salt thereof as an active ingredient is a granule, a powder, a coating, a tablet, Capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops, or liquids.

According to one embodiment of the present invention, the pharmaceutical composition may be administered orally, intraarterally, intraperitoneally, intramuscularly, intraarterally, intraperitoneally, intrasternally, transdermally, nasally, inhaled, topically, rectally, ≪ / RTI > can be administered to the subject in a conventional manner.

A preferable dosage of the pharmaceutical composition for prevention or treatment of cancer containing the above arene-ruthenium compound or its pharmaceutically acceptable salt as an active ingredient depends on the condition and body weight of the patient, the kind and degree of the disease, the drug form, And may be suitably selected by those skilled in the art. According to one embodiment of the present invention, the daily dose 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, though it is not limited thereto. The administration may be performed once a day or divided into several times, and thus the scope of the present invention is not limited thereto.

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 particularly, the arene-ruthenium compound of formula (I) or (II) according to the present invention may be used for the treatment of gastric cancer, colorectal cancer, (CNS), Brain tumor, Low-grade astrocytoma, High-grade astrocytoma, Pituitary adenoma, Meningioma, CNS lymphoma, Oligodendroglioma, Neuromuscular blockers have been shown to be useful in the treatment of craniopharyngiomas, ependymomas, brain stem tumors, head & neck tumors, larygal cancers, oopgaryngeal cancer, PNS tumor, Nasopharyngeal tumor, Salivary gland tumor, Hypopharyngeal cancer, Thyroid cancer, Oral cavity tumor, Chest Tumor, Small cell lung cancer (Small cell lung cancer), non-small cell In the case of non-small cell lung cancer, thymoma, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, Abdomen-pelvis tumor, Liver cancer, gall bladder cancer, billiary tract tumor, pancreatic cancer, small intestinal tumor, anal cancer, bladder cancer, kidney cancer Renal cell carcinoma, male genital cancer, penile cancer, prostate cancer, female genital cancer, cervix cancer, endometrial cancer, For the treatment of endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female vaginal cancer, female urethral cancer or skin cancer. It is preferable to use it. And more preferably for the treatment of gastric or colon (large bowel) cancers.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

1. General

Receptor clip of ^{_{[Ru 2 (μ-η 4}} -C 2 O 4) (η 6 -p- i PrC 6 H 4 Me) 2] (acceptor clip) [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 ( L1 ) (2,5-bis (pyridin-4-ylethynyl) furan) ( L2 ) was prepared by a known method (Chem. Soc. 2010, 132, 950- 952 and Chem. 2000, 39, 2547-2557) and dried according to a previously known method (Pergamon Press, Oxford, UK, 1988) and then distilled.

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

The chemical shift (?) In the ¹ H NMR spectrum was recorded as relative ppm values with tetramethylsilane (Me ₄ Si) as the international standard (0.0 ppm). The mass spectra were recorded on a Micromass Quattro II triplequadrupole mass spectrometer using electrospray ionization with a MassLynx operating system. Elemental analyzes were performed using an Elemental GmbH Vario EL-3 instrument.

Diffraction data for a single crystal of Compound 1 according to the present invention was obtained from the ADSC Quantum 210 CCD diffractometer at 100 K and Supramolecular Crystallography Beamline 2D at l = 0.80000 A Were collected from synchrotron radiation (Pohang Accelerator Laboratory (PAL), Pohang, Korea).

Data sources were processed and transformed 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. The UV-Vis spectrum was recorded on the Cary 100 Conc.

< Example  1> Compound Synthesis

1. Synthesis of Compound 1

A mixture of the donor L1 (2.36 mg, 0.01 mmol) and the ruthenium acceptor Al (8.56 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethyl ether (CH ₃ NO ₂ / CH ₃ OH, 1: Lt; / RTI &gt; for 24 hours. 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 with a yield of 92% as a brown powder.

^{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 3C NMR (75 MHz, [D ₃ ] Nitromethane):? = 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 -3 OTf] ³⁺ and 397.42 [ 1- ⁴ OTf] 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. Compound 2 Synthesis

A mixture of the donor L1 (2.36 mg, 0.01 mmol) and the ruthenium acceptor A2 (9.06 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethyl ether (CH ₃ NO ₂ / CH ₃ OH, 1: 1) Lt; / RTI &gt; for 24 hours. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain a solid. The resulting solid was washed with diethyl ether and dried to obtain Compound 2 with a powder of Wine-red color with a yield of 90%.

^{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):? = 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 -4 OTf] 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.

3. Synthesis of Compound 3

A mixture of the donor L1 (2.36 mg, 0.01 mmol) and the ruthenium acceptor A3 (9.57 mg, 0.01 mmol) was added to 2 mL of nitromethane / dimethyl ether (CH ₃ NO ₂ / CH ₃ OH, 1: 1) Lt; / RTI &gt; for 24 hours. 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 with a yield of 91%.

^{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):? = 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.

4. Compound 4 Synthesis

A solution of the donor L2 (2.70 mg, 0.01 mmol) in CH ₃ NO ₂ (1 mL) was added dropwise to a solution of ruthenium A1 (8.56 mg, 0.01 mmol) in CH ₃ OH (1 ml). After the dropwise addition, the mixture was stirred at 40 DEG C for 36 hours. 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 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):? = 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 -3 OTf] 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.

5. Compound 5 Synthesis

The CH ₃ NO ₂ (1 mL) solution of the donor L2 (2.70 mg, 0.01 mmol) was added dropwise to CH ₃ OH solution (1 ml) of a ruthenium receptor A2 (9.06 mg, 0.01 mmol) . After the dropwise addition, the mixture was stirred at 40 DEG C for 36 hours. 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 5 as a wine-red solid in 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 -3 OTf] ³⁺ , 439.55 [ 5 -4 OTf] 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.

6. Synthesis of Compound 6

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 ruthenium acceptor A3 (9.57 mg, 0.01 mmol). After the dropwise addition, the mixture was stirred at 40 DEG C for 36 hours. 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 6 as a sea-green solid with a yield of 90%.

^{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):? = 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 -3 OTf] 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.

< Example  2> Compound analysis

One. NMR  analysis

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

The chemical shift (?) In the ¹ H NMR spectrum was recorded as relative ppm values with tetramethylsilane (Me ₄ Si) as the international standard (0.0 ppm).

Referring to FIG. 2, the Hα nuclei of pyridine rings relative to L1 showed upfield shifts (Δδ = 0.17-0.51 ppm), and most spectra showed ruthenium (II) center (Pyridine-N) coordination to the 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 present invention, The signal for the proton at δ = 5.84 ppm, the signal for the naphthoquinone proton of compound 3 according to the invention was observed as sharp singlets at δ = 7.28 ppm.

2. Mass spectrum 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 of the [4 + 2] fragmented ions.

Multiple charge fragmentation ions for the compound 1 according to the invention was observed in ^{4+ m / z 579.57 [1 -3TfO} ] 3+ and m / z = 397.42 [1 -4TfO ], multi for compound 2 according to the invention charge ion fragmentation was observed in ^{4+ m / z 422.44 [2 -4TfO} ], this peak were analyzed isotopically.

The appearance of the expected peaks as well as the isotopic patterns clearly confirmed the formation of [2 + 2] subassemblies of compounds 1 and 2 according to the present invention.

ES1 from the results of mass spectrum of compound 4 to 6 according to the invention 4 m / z 602.38 [4 -3OTf] 3+, 5 are 636.06 [5 -3OTf] ³⁺ and 439.55 [5 -4OTf] ^4+, 6 Showed a peak at 669.05 [ 6 -3 OTf-] 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 a methanol solution.

As a result, as shown in Fig. 4, the high energy bands observed in the compounds 1 to 6 according to the present invention were also observed in free Ll and L2, indicating that the extended aromatic system of the dipyridyl ligand suggesting that transitions may occur for aromatic systems.

The dinuclear arene-Ru acceptors also have slightly higher energy absorption bands at 250-300 nm and the A3 donq spacers also exhibit broadly low energy absorption bands from ~ 400-550 nm Respectively.

4. Molecular structure of Compound 1

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

Suitable single crystals of Compound 1 for X-ray analysis using synchrotron radiation can be prepared by slowly adding diethyl ether to a methanol solution of Compound 1 , And vapor diffusion.

As shown in FIG. 5, in the result of a single crystal X-ray diffraction analysis, Compound 1 has a tetranuclear rectangular architecture and lies in a crystallographic inversion center Respectively.

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

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

< Example  3> Extracellular antitumor activity analysis

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 RPMI 1640 medium containing 5% fetal bovine serum (FBS) in the presence of 5% CO ₂ at 37 ° C.

2. 3- (4,5- Dimethyl thiazole Yl) -2,5- Diphenyltetrazolium  Bromimide (colorimetric 3- (4,5- dimethylthiazole -2- yl ) -2,5- 두henyltetrazolium bromide , MTT) analysis

Two cells prepared in Example 1 of Example 3 were treated with Compounds 1 to 6 of the present invention, and cell viability was evaluated by MTT assay. Cells were inoculated into 96-well plates, incubated at 37 ° C, and stimulated with each compound for 24 hours. Cells of each well were incubated with 10 μL of MTT solution for 4 hours at 37 ° C in the presence of 5% CO ₂ , and then 100 μL of dimethyl sulfoxide (DMSO) was added. Then, absorbance concentration values were measured at a wavelength of 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 inhibitory concentration (IC ₅₀ ) value for cell growth was plotted as a plot of the alveolar fraction of cell viability versus the logarithm of the drug concentration using a linear regression function. Was obtained by fitting.

The IC ₅₀ values are listed in Table 2 below, in comparison with cisplatin, doxorubicin, which is a known antineoplastic drug.

Sample
IC ₅₀ [μM] ^[a] AGS HCT -15 One > 200 > 200 2 180.4 ± 11.64 > 200 3 29.6 ± 0.58 29.1 ± 0.64 4 > 200 > 200 5 105.0 ± 10.16 > 200 6 22.8 ± 1.00 26.8 ± 2.50 L1 > 200 > 200 L2 152.2 ± 5.13 > 200 A3 125.2 ± 7.64 > 200 Cisplatin 107.4 ± 1.87 12.4 ± 1.85 Doxorubicin 3.0 ± 0.09 15.2 + 1.43

As a result, Compounds 3 and 6 according to the present invention showed considerable efficacy. The efficacy of Compounds 3 and 6 (IC ₅₀ = 29.6 μM, 22.8 μM) was more potent for AGS cancer cells than cisplatin (IC ₅₀ = 107.4 μM), and compounds 3 and 6 (IC ₅₀ = 29.1 μM, 26.8 μM) was similar to the efficacy of cisplatin (IC ₅₀ = 12.4 μM) and doxorubicin (IC ₅₀ = 15.2 μM).

< Example  4> Analysis of Anticancer Activity in Animal Models

1. Cells and Hollow  Fiber ( Hollow fiber , HFs ) Ready

HCT-15 cells were cultured in a 75-cm ² culture flask at 37 &lt; 0 &gt; C, 95% humidity, 5% CO ₂ . Polyvinylidene fluoride (HFs), 1-mm internal diameter and a molecular weight cutoff point of 500 kDa (Spectrum Laboratories, Houston, TX, USA) were used. Sterile HFs were washed with normal growth media (normal growth media), before filling the HCT-15 cells at a density of ⁵ × 10 5 cells / mL.

Each fiber was heat sealed at 1.5 cm intervals with a hot smooth-jawed needle holder and cut into segments with a 2-mm tail for ease of handling. HFs were each segment comprises from about 10 ⁵ cells. Prepared HFs segments were maintained in vitro for 24 hours in normal growth conditions prior to injection.

2. In vivo transplantation

For in vivo transplantation of the 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) according to 12 h light-dark cycles. Mice were anesthetized with Zoletil and Rompun. HFs were implanted subcutaneously, s.c. and peritoneal (ip) in anesthetized mice and sutured using a skin staple.

Two days later, male mice were fed with 100 mg / kg gavage for body weight for 7 days. And mice were given 100 μg kg ^-1 bw / day of Compounds 3 and 6 for 7 days, respectively.

To analyze the viable cell count in HFs, HFs were transferred to 0.5 ml of ethylenediaminetetraacetic acid (EDTA), cut in half lengthwise using a scalpel and washed with EDTA solution for 3 minutes. Then, the cells were washed with 0.5 ml of trypsin for 5 minutes, followed by washing with medium for 3 minutes.

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

3. Hollow  Fiber analysis ( hollow fiber assay )

As a result, as shown in FIG. 6, the proliferation of tumor cells at the IP site was inhibited by the compounds 3 and 6 according to the present invention, the growth inhibitory effect by Compound 3 was 14.2%, the growth inhibitory effect by Compound 6 was 21.8% Respectively. 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 was lower than that in IP region, which is considered to be due to the lack of the developed blood vessels.

< Example  5> Self-predation  Confirming the effect of active action

After the antitumor activity of Compound 3 and Compound 6 was confirmed through in vitro and in vitro experiments, the activity of Compound 3 and Compound 6 in HCT-15 cells And to evaluate the induction of self-predation. Monodansylcadaverine (MDC) staining was used to detect autophagic vacuoles accumulating during autophagic aging.

HCT-15 cells were labeled with 0.05 mM MDC (Sigma) in PPM1640 medium for 10 min at 37 &lt; 0 &gt; C. After incubation, the cells were washed three times with phosphate buffered saline and immediately subjected to fluorescence microscopy (Nikon Eclipse TE 300, Japan) equipped with a filter system (V-2A excitation filter: 380/420 nm, Respectively. Field and fluorescence images were taken with a digital camera (DP30BW; Olympus).

As a result, as shown in Fig. 7, by the treatment of Compound 3 and Compound 6 , the number of MDCs that self-trapped liposomes markedly increased significantly.

Both Compound 3 and Compound 6 strongly induced autophagic activity at low concentrations (0 to 5 μM), and at higher concentrations, autophoretic activity was reduced. These results were very similar to those of increasing cell death and decreasing cell proliferation.

Meanwhile, Compound 3 or 6 according to the present invention can be formulated into various forms according to the purpose. The following examples illustrate some formulations containing the compound according to the present invention as an active ingredient, but the present invention is not limited thereto.

< Formulation example  1> Purification (direct pressurization)

After 5.0 mg of Compound 3 or 6 was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesium stearate were mixed and pressed to prepare tablets.

 < Formulation example  2> Purification (wet assembly)

5.0 mg of Compound 3 or 6 was sieved, and 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water, and an appropriate amount of this solution was added, followed by atomization. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The fine particles were pressed to prepare tablets.

< Formulation example  3> Powder and Capsule

5.0 mg of Compound 3 or 6 was sieved and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was extruded through a hard No. 5 &lt; / RTI &gt; gelatin capsules.

&Lt; Formulation Example 4 >

100 mg of Compound 3 or 6 , 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ .12H ₂ O and 2974 mg of distilled water were mixed. The mixed solution was filled in an ampoule made of transparent glass, sealed in an upper lattice by dissolving the glass, sterilized by autoclaving at 120 DEG C for 15 minutes or longer, and injected.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

An arene-ruthenium compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(2)

ego,

Quot;
[Chemical Formula 3]

(3b)

or
[Chemical Formula 3c]

ego,
In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,
In the formula 1

Quot;
[Chemical Formula 4]

Lt;
(2)

silver,
[Chemical Formula 5]

ego,
The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2). The method according to claim 1,
The arene-ruthenium compound is a compound wherein the nitrogen atom in the aromatic ring of formula (4) or (5) is bonded to the ruthenium of formula (3c) to form a compound of formula (1) or Or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(2)

ego,

Quot;
[Chemical Formula 3c]

ego,
In Formula 3c, each X is independently trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene-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,
In the formula 1

Quot;
[Chemical Formula 4]

Lt;
(2)

silver,
[Chemical Formula 5]

being. A pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises an arene-ruthenium compound represented by the following formula (1) or (2) or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

(2)

ego,

Quot;
[Chemical Formula 3]

(3b)

or
[Chemical Formula 3c]

ego,
In the above formulas (3a), (3b) and (3c), each X is independently selected from the group consisting of trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene- 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,
In the formula 1

Quot;
[Chemical Formula 4]

Lt;
(2)

silver,
[Chemical Formula 5]

ego,
The nitrogen atom in the aromatic ring of formula (4) or (5) and the ruthenium of formula (3a) to (3c) are bonded to each other to form a compound of formula (1) or (2). The method of claim 3,
The arene-ruthenium compound is characterized in that the nitrogen atom in the aromatic ring of formula (4) or (5) is bonded to ruthenium of formula (3c) and the following X is removed to form a compound of formula (1) or A pharmaceutical composition for therapeutic use.
[Chemical Formula 1]

(2)

ego,

Quot;
[Chemical Formula 3c]

ego,
In Formula 3c, each X is independently trifluoromethylsulfonate (OTf), nitrate (NO ₃ ), toluene-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,
In the formula 1

Quot;
[Chemical Formula 4]

Lt;
(2)

silver,
[Chemical Formula 5]

being. The method according to claim 3 or 4,
The pharmaceutical composition for the prevention or treatment of cancer according to any one of claims 1 to 3, wherein the allen-ruthenium compound or a pharmaceutically acceptable salt thereof exhibits anticancer activity by activating autophagy and increasing apoptosis. The method according to claim 3 or 4,
Wherein said cancer is a solid cancer. The method according to claim 6,
The solid tumors may be colon cancer, stomach cancer, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial, cytoplasm, brain stem tumor, head and neck tumor, laryngeal cancer, Cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, cancer of the gallbladder, gall bladder cancer, Cancer of the female genital organs, cancer of the female genital organs, cancer of the female reproductive system, cancer of the uterine cervix, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female gynecological cancer, female gynecologic cancer, Or skin cancer. &Lt; / RTI &gt;

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