KR20160011727A - New tetracationic hetero-bimetallacycles compounds and parmaceutical composition for treating or preventing cancer containing the same - Google Patents

Abstract

The present invention relates to a novel cyclic compound containing two metals and a pharmaceutical composition for the prevention or treatment of cancer comprising the same as an active ingredient, wherein the hetero-bimetallic metallacycles (HBMCs) according to the present invention are T98G (Cancer of the head and neck), KB (head and neck cancer), and SNU-80 (thyroid cancer), and inhibits the activity of cancer cells and exhibits excellent anticancer activity.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel cyclic compound containing two metals and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient,

The present invention relates to a novel cyclic compound containing two metals and a pharmaceutical composition for preventing or treating cancer containing the same as an effective ingredient.

Coordination-driven self-assembly, a driving force of coordination, has proven to be an effective approach to the synthesis of large supramolecular metallacyclic structures with well-known shapes and sizes. A directional bonding study is the driving force of a combination of a rigid, ligand-capped transition metal acceptor and a mono- or multidentate donor derived from O- and N-coordination sites. Numerous molecular designs have been reported that use, aesthetically favorable, various shapes, sizes, and symmetries. The square plane Pd (II) and Pt (II) organometallic acceptors with pyridyl or nitrogen donor linkers have been most widely used in the design of metal ring structures due to the tight rectangular planar coordination environment. Organic amides derived from nitrogen donor symmetric polypyridyl ligands have been widely used in self-assembly where coordination bonds are the driving force. Such self-assembled supramolecular designs are being extended to a variety of applications, including host-guest reactions, catalytic reactions, and sensing. In addition, recently, biological properties of such a metal ring structure are beginning to appear. Metal ring compounds and helices have been shown to interact with DNA, while other design structures have shown cytotoxicity to cancer cells and bacteria.

On the other hand, due to the therapeutic application of metal-based drugs, metallopharmaceuticals have emerged as an important area in pharmaceutical chemistry. Organic ligand arrangements in two- or three-dimensional spaces, depending on the accessible redox state of a broad range of coordination numbers and variable metal centers, provide a broad reactive spectrum that can be used for medical purposes. Since the discovery of cisplatin, there has been considerable interest in the use of inorganic compounds in tumor therapy. Despite its successful application as a therapeutic agent for a wide range of cancers, the use of cisplatin has several adverse side effects such as high toxicity and unwanted nephrotoxicity (kidney damage), neurotoxicity (nervous system damage) and bone marrow toxicity (bone marrow suppression). In addition, the efficiency of cisplatin may be lowered by intracellular degradation and tolerance. These disadvantages have led to the development of other platinum drugs such as carboplatin, oxaliplatin and satraplatin, which have low toxicity and are now being used in a variety of cancer treatments. While these derivatives exhibit improved therapeutic efficacy compared to cisplatin, there are also problems associated with the platinum drugs. Thus, considerable efforts are being made to develop drug delivery vectors that can mitigate the problems associated with toxicity, degradation, and resistance. Therrien et al. _{[M (acac) 2] (} M = Pt 2 +, Pd 2 +) are encapsulated in the composite, the anti-tumor activity is increased enhanced anti-reported based coordination bond cage-sandwich (half-sandwich) ruthenium.

Although many Pd and Pt-based metal ring compounds are reported to exhibit host-guest chemistry or other beneficial properties, few compounds exhibit antineoplastic properties. Recently, the present inventors have reported various molecular metal ring compounds constituted by self-assembly studies in which coordination bonds are a driving force, and in vitro anticancer activity was measured thereon. As a result, larger metal ring compounds showed higher activity levels than smaller metal ring compounds, which supports the assumption that large macromolecules are more easily retained within cancer cells.

Korean Patent No. 10-0724586 (registered on May 28, 2007)

The present invention is to provide a novel hetero-bimetallic metallacycles (HBMCs) or a pharmaceutically acceptable salt thereof, a process for preparing the same, and a pharmaceutical composition for treating or preventing cancer diseases containing the same as an active ingredient.

In order to solve the above-mentioned object, the present invention provides a hetero-bimetallic metallacycles (HBMCs) represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate.

The present invention also provides a process for producing the hetero-bimetallic metallacycles (HBMCs) comprising the step of mixing a ligand compound represented by the following formula 2 and a hetero metal compound represented by the formula 3 .

(2)

(3)

In Formula 3, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate.

In addition, the present invention provides a pharmaceutical composition for treating or preventing a cancerous disease containing the above-mentioned hetero-bimetallic metallacycles (HBMCs) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a novel cyclic compound containing two metals and a pharmaceutical composition for the prevention or treatment of cancer comprising the same as an active ingredient, wherein the hetero-bimetallic metallacycles (HBMCs) according to the present invention are T98G (Cancer of the head and neck), KB (head and neck cancer), and SNU-80 (thyroid cancer), and inhibits the activity of cancer cells and exhibits excellent anticancer activity.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the synthesis of Hetero-Bimetallic Metallacycles (HBMCs).
Figure 2 is a comparison ³¹ P NMR spectrum of the complex in nitromethane HBMC _{-d 3 (nitromethane-d 3)} 4 , and Pd 2 receptor compounds.
Figure 3 is a comparison ³¹ P NMR spectrum of nitromethane _{-d 3 (nitromethane-d 3)} HBMC and Pd complex 5 receptors Compound 3 in.
Figure 4 is the calculation (top) and experimental (bottom) ESI-MS spectra of HBMC complex 4 (a) and complex 5 (b).
Figure 5 is a comparative UV-Vis spectrum of ligand 1, Pd receptor compound 2 and HBMC complex 4.
Figure 6 is a comparative UV-Vis spectrum of ligand 1, Pd receptor compound 3 and HBMC conjugate 5.
Figure 7 shows the geometry-optimized chemical structure obtained through the calculation of the molecular mechanics force field of complex 4.
Figure 8 shows the inhibitory effect of complexes 4 and 5 on T98G solid tumors. Percentage of surviving T98G cells was calculated after 24, 48 and 72 h treatment of complex 4 (A) and complex 5 (B). The growth kinetic analyzes (A and B) showed that complexes 4 and 5 had an inhibitory effect on T98G cells in a culture time-dependent manner. The cultured cells were treated with 0.5 to 16 uM concentrations of the complexes. (C) The ability of T98G cells treated with complex 4 and complex 5 to form colonies. (D) decreased growth-inhibiting activity of the pre-reacted complexes in the culture medium. Prior to addition of cultures of T98G cells, complexes 4 and 5 at 6 uM concentration were pre-reacted at 37 ° C. for the indicated times in cell culture medium (DMEM) supplemented with DMSO and 10% FBS.
Figure 9 shows the detection results of intracellular ATP and reactive oxygen species (ROS). (A) Relative intracellular ATP levels of T98G cells in DMEM medium. T98G cells exposed to complexes 4 and 5 showed a further decrease in ATP levels and metabolic activity after 24 hours of treatment. (B) ROS production in T98G cells treated with complexes 4 and 5 is shown. Data were expressed as mean fluorescence intensity of DCF using H2DCFDA probe.
Fig. 10 shows the results of detection of apoptosis in human T98G brain tumor cells. Cells treated with various concentrations (0.05-2 uM) of complexes 4 and 5 were suspended in binding buffer. Annexin V-FITC and propidium iodide solutions were added and the cell solution was incubated for 15 minutes at room temperature and in the dark. Flow cytometry was performed within one hour. In each quadrant, each number represents the percentage of cells.
Figure 11 shows the effect of complexes 4 and 5 on mitochondrial membrane potential in T98G brain tumor cells. The graph shows the red fluorescence intensity curve of the JC-1 reagent on the cells after complex treatment. The abscissa indicates the red fluorescence intensity (PE-A), and the ordinate indicates the number of cells. The indigo blue curve represents the untreated negative control group and the calibration curve represents the positive control group treated with the proton ionophore carbonyl cyanide 3-chlorophenylhydrazone (cccp). Curve migration from the negative control curve to the cccp treated curve indicates a decrease in mitochondrial membrane potential.
Figure 12 shows the effect of complexes 4 and 5 on mitochondrial membrane potential (MMP) and caspase activity after 24 hours of treatment in the T98G brain tumor cell line. (A) To detect MMP changes in T98G cells, the relative values of JC-1 red fluorescence relative to negative control were quantified by flow cytometry with JC-1 reagent. The induction of apoptotic cell death can be confirmed by measuring caspase-3/7 activity using the Promega Caspase-3 / 7Glo assay kit, and the detection of luminescence is directly proportional to the level of caspase inside the cell.
Figure 13 shows the fluorescence of complexes 4 and 5 detected in T98G brain tumor cells after 24 hours of treatment. Fluorescence microscopy (40x magnification) analysis of T98G cells was performed using 2 uM of complexes 4 and 5: (A) bright field image of complex 4, (B) fluorescence image of complex 4, (C ) ≪ / RTI > Complex 5, and (D) Fluorescence image of Complex 5. (E) Fluorescence intensity of complexes 4 and 5 detected at various concentrations in T98G cells.

In order to build up the expertise of the metallosupramolecular structure we have found that by means of an amide-based dipyridyl ligand with two different metal (Pd and Pt) receptors, two new hetero-bimetallic rings The compounds (hetero-bimetallacycles; HBMCs) were designed and synthesized. To confirm the potential biological effects of this system of interaction with biomolecules, we measured the anti-cancer effect of HBMCs against various human cancer cell lines with initiator receptors and donor ligands. Accordingly, the inventors of the present invention confirmed the first cytotoxic activity against amide-based dipyridyl ligands, metal ring systems made from Pd and Pt receptors, and their various cancer cell lines, and completed the present invention.

The present invention provides hetero-bimetallic metallacycles (HBMCs) represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate.

The Hetero-Bimetallic Metal Cycles (HBMCs) of the present invention can be used in the form of pharmaceutically acceptable salts, wherein the acid addition salt formed by a pharmaceutically acceptable free acid useful. 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.

In addition, the hetero-bimetallic metallacycles (HBMCs) of the present invention include 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 in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile, etc., And then precipitating or crystallizing the acid solution. Subsequently, in this mixture, a solvent or an excess acid is evaporated and dried to obtain an additional salt, or the precipitated salt can be produced by suction filtration.

The present invention also provides a process for preparing the hetero-bimetallic metallacycles (HBMCs) comprising the step of mixing a ligand compound represented by the following formula 2 and a hetero metal compound represented by the following formula 3 do.

(2)

(3)

In Formula 3, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate.

The hetero-bimetallic metallacycles (HBMCs) according to the present invention can be easily prepared by reacting the ligand compound represented by the formula (2) and the hetero metal compound represented by the formula (3) in a molar ratio of 1: 1, The reaction may be performed in CH ₂ Cl ₂ / CH ₃ NO ₂ , but is not limited thereto.

As described above, the hetero-bimetallic metallacycles (HBMCs) of the formula (1) prepared according to the present invention can be prepared by a method such as infrared spectroscopy, nuclear magnetic resonance spectrum, mass spectrometry, liquid chromatography, The molecular structure can be confirmed by comparison of determination method, optical rotation measurement method and elemental analysis of representative compounds and measured values.

In addition, the present invention provides a pharmaceutical composition for treating or preventing a cancerous disease containing the above-mentioned hetero-bimetallic metallacycles (HBMCs) or a pharmaceutically acceptable salt thereof as an active ingredient.

Specifically, the Hetero-Bimetallic Metallacycles (HBMCs) or pharmaceutically acceptable salts thereof may exhibit anticancer activity through apoptosis of cancer cells.

Preferably, the cancer disease can be, but is not limited to, brain tumor, head and neck cancer or thyroid cancer.

In one embodiment of the present invention, the pharmaceutical composition comprises 0.01 to 90 parts by weight, 0.1 to 90 parts by weight of hetero-bimetallic metallacycles (HBMCs) of formula (1), 0.1 to 90 parts by weight, To 90 parts by weight, or from 10 to 90 parts by weight, but the present invention is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

In another embodiment of the present invention, the pharmaceutical composition comprises a carrier, an excipient, a disintegrant, a sweetener, a coating agent, a swelling agent, a lubricant, a lubricant, a flavoring agent, an antioxidant, a buffer, a bacteriostatic agent, a diluent, a dispersant, A lubricant, and a lubricant.

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 is in the form of granules, powders, coated tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, Can be selected.

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, 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 body weight of the patient, the type and degree of disease, the type of 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, 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.

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.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

All compounds used in the present invention were purchased from commercial vendors and used without further purification. cis - [(dppf) M (OTf) ₂ ] (M = Pd, Pt) initiators and ligands have previously been reported (Dalton Trans, 2014, 43, 6032). ¹ H, ¹³ C, and ³¹ P {H} NMR spectra were recorded on a Bruker 300 MHz NMR spectrometer. The chemical shift of ³¹ P {H} was reported compared to the open sample H ₃ PO ₄ (δ = 0.0 ppm). Mass spectra were recorded on a Micromass Quattro II triple-quadrupole mass spectrometer using electrospray ionization (ESI) with MassLynx operating system. Elemental analysis was performed using Elemental GmbH Vario EL-3 analyzer. Absorption spectra were recorded using a Cary 100 Conc UV-Visible spectrophotometer.

1. Reagents

Trypan blue, crystal violet, MTT and Hoechst-33342 were purchased from Sigma Chemical Co., Korea. Phosphate buffered saline (PBS), Dulbecco's modified eagle's medium (DMEM), fetal bovine serum (FBS) and trypsin-EDTA were purchased from Hyclone, Korea. H ₂ DCFDA was purchased from Invitrogen, Molecular Probes, Korea. All other compounds used in the present invention were analytical grade and were purchased from Sigma-Aldrich, Korea.

2. Human cell culture

The T98G, KB, SNU-80 and HEK-293 cells used in the present invention were purchased from Korean Cell Line Bank (KCLB), Seoul. We used Dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% non essential amino acid, 1% glutamine, penicillin (100 IU / mL) and streptomycin ) Were cultured in a 75-cm ² culture flask (SPL, Korea) according to the manufacturer's instructions. All cultures were maintained at 37 ° C, 95% relative humidity and 5% CO ₂ .

3. In vitro cell growth inhibition assay (MTT assay)

Cells were inoculated into 96-well plates at a concentration of 1 × 10 ⁵ cells / well in 100 μL of complete medium, and cultured in a 37 ° C., 5% CO ₂ atmosphere for 24 hours for cell attachment. The stock solution (2M) of the complex was made up of a solution of DMSO: water (1: 3) and then diluted to 0.125 uM in incomplete medium for cell line treatment. The calculated concentrations of each complex were added to 100 uL of fresh media solution and dispensed into wells to a final concentration of 300-0.06 uM. All analyzes were repeated with three independent sets. A negative control without drug and a positive control with cisplatin were included in each analysis. After 24 hours of cell exposure to the drug, each well was carefully rinsed with 200 uL PBS buffer. 20 uL MTT solution (5 mg / ml) with 100 uL of fresh complete medium was added to each well to determine cytotoxicity and the plate was allowed to react for 3 hours. After the reaction, the medium was removed and the purple formazan precipitate of each well was disinfected with 100 uL DMSO. Absorbance was measured at 540 nm using a Biotek Synergy HT microplate reader and the survival percentage (%) was calculated, which is directly proportional to the number of metabolically activated cells.

% Survival = OD in sample well / OD in control well &lt; RTI ID = 0.0 &gt;

The results, expressed as inhibitory concentration 50 (IC ₅₀ ), were obtained by applying the values according to the dose response curve trend using the formula provided in Microsoft Excel.

4. Growth kinetics

T98G cells were inoculated on a cell culture plate at 10000 cells / cm ² , and their proliferative kinetics were investigated (24, 48 and 72 h) after varying incubation times after the synthesized complex treatment. Cells were trypsinized and whole cells per plate counted microscopically by trypan blue exclusion method.

5. Clonogenic assay

Colony formation assays basically tested all cells in populations capable of "unlimited" cleavage. 150-400 cells (depending on treatment) were cultured for 10-12 hours before being treated with 37 째 C in DMEM. The cultured cells were also treated according to the synthesized complex dose of 1 to 16 uM. After treatment, the cells were cultured for 8-10 days at 37 &lt; 0 &gt; C in 5% CO ₂ atmospheres in a humid condition in the cow for colony formation. Colonies were fixed with methanol and stained with 1% crystal violet. Colonies over 50 cells were counted and the surviving fraction (SF) of each colony was calculated. The colony formation survival curves were generated by three independent experiments using an east-squares regression with mean survival level.

6. Evaluation of stability of complex

In order to evaluate the stability of the synthesized complex of complex 4 and complex 5, stability was confirmed in both solutions from 0 h to 50 h. To test the stability of the complex, an aqueous solution of either DMSO (6 uM) or DMEM medium (6 uM) was used. In order to confirm inhibition of metabolic growth in T98G tumor cells, the stability of the complex was evaluated by MTT assay.

7. ATP measurement

All multicellular microorganisms use ATP as a means of storing metabolic energy. Thus, ATP quantification is a characteristic signal for detecting metabolically activated cells. For this reason, we have identified intracellular ATP levels in cells treated with complex 4 and complex 5. Various concentrations of complexes were used to carry out the experiments. ATP was measured using a commercial kit (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Korea) and all detection steps were performed according to the manufacturer's protocol.

8. Intracellular ROS detection

2 ' -dichlorofluorescein diacetate (H ₂ DCFDA; Molecular Probes, USA) fluorescent dye probe was used to identify intracellular ROS induced by complex 4 and complex 5 in T98G cells. Upon oxidation, the non-fluorescent compound DCFH-DA was converted to the fluorescent compound dichlorofluorescein (DCF). Thus, the DCF fluorescence was expressed as the ratio and the amount of produced ROS. Briefly, after treatment with the complexes, T98G cells were cultured in a fresh medium containing 10 uM H ₂ DCFDA for 30 min at 37 ° C, washed twice with PBS, excited at 485 nm, The fluorescence was measured using a plate reader (Biotek, Techan, USA) at an emission wavelength of.

9. FACS analysis of annexin V and PI staining

To determine the level of apoptosis in T98G cells treated with complex 4 and complex 5, a flow cytometer was used. To analyze treatment-induced apoptosis, Annexin V-FITC / PI staining was performed followed by FACS analysis. Cells were inoculated and treated with 0.05, 1 and 2 uM concentrations of the complex. Untreated cells and cells treated with cisplatin (which triggered apoptosis) were used as negative control and positive control, respectively. Briefly, after 24 hours of treatment, cells were harvested and applied to Annexin V-FITC / PI staining using the EzWay Annexin V-FITC apoptosis detection kit, all steps following the protocol provided by the manufacturer.

10. Mitochondrial membrane potential detection

Mitochondria play a crucial role in apoptosis induced by many factors, and the decrease in mitochondrial membrane potential (Δψm) is a feature / event of early apoptosis. JC-1 is a selectable marker for mitochondrial membrane potential detection in apoptotic cells. JC-1 is a lipophilic cationic dye. It is present as a monomer in the cell and exhibits green fluorescence at low concentrations (depolarizing the mitochondrial membrane). However, at high concentrations, the dye forms a J-aggregate, exhibits a broad excitation spectrum, exhibits a maximum emission at ~ 590 nm, and the cells exhibit red fluorescence (normal polarized mitochondria). JC-1 staining was performed on T98G cells treated with the MitoProbe JC-1 Assay Kit (Invitrogen, molecular probes) according to the manufacturer's instructions in order to observe the effects of the complex 4 and the complex 5 on the mitochondrial membrane. For positive MMP depolarization, we used a proton ionophore, carbonyl cyanide 3-chlorophenylhydrazone (cccp), which rapidly degrades the membrane potential.

11. Caspase 3/7 activity measurement

Caspases, a family of cysteine proteases, are central modulators of apoptosis. One of the best studied of the cysteine protease family is the acting caspase-3 and caspase-7, which plays an important role in apoptosis and differentiation. To measure apoptosis, Caspase-Glo® 3/7 Assay Kit (Promega, Korea) was used. 100 uL of Caspase-Glo® 3/7 reagent was added to each of 100 uL complex treated cells and control cells and reacted at room temperature. The luminescence of each sample in the plate was recorded with a luminometer (Biotek, Techan, USA).

12. Microscopic analysis of cell uptake and complexes

Cells were inoculated into 96-well plates (SPL, Korea) and cultured for 12 hours for cell attachment. The cells were then exposed to complex 4 and complex 5 at 1-4 μM concentrations in complete culture medium for 48 hours at 37 ° C. After washing with PBS, the supernatants were replaced with fresh media and the complexed cell populations were measured with a fluorescence microplate reader (Biotek, Techan, USA) with excitation and emission filters of 330 and 485 nm, respectively. For microscopic analysis, cells were cultured on glass slides in complete culture medium and exposed to Complex 4 and Complex 5 at 2 uM concentration overnight. The slides were rinsed with PBS and observed under a fluorescence microscope (Ti-U, Nikon) equipped with MBE41300 C-FL filter consisting of excitation Ex340-380 and blocking filter BA435-485.

13. Synthesis of hetero-bimetallic cycle 4

Receptor 1 (0.010 g, 0.020 mmol) and Pd (dppt) ₂ (OTf) ₂ (0.020 g, 0.020 mmol) Receptor 2 was CH ₂ Cl ₂ / CH ₃ NO ₂ (1: 1, 2.0 mL) and the final mixture was stirred at room temperature for 5-6 hours. The reaction mixture was filtered, the solvent was removed under reduced pressure, and the product was washed with diethyl ether. The resulting crude product was redissolved in nitromethane and vapor-dispersed in diethyl ether. Reddish crystalline products were obtained within one day (93%, 0.028 mg). Elemental analysis of C ₁₃₈ H ₉₇ F ₁₂ Fe ₂ N ₁₀ O ₁₆ P ₄ Pd ₂ S ₄ was performed: C, 56.07, H, 3.31, N, 4.74; found: C, 56.88, H, 3.42, N, 4.60; MS (ESI) calculated for [M-3OTf] ³⁺ m / z 836.5, found 836.4; calculated for [M-4OTf] ⁴⁺ m / z 590.1, found 590.1; ^{1 H NMR (300 MHz, nitromethane} -d 3, d, ppm): 10.17 (s, 4H, CONH), 8.46 (d, 8H, H a), 8.43-8.24 (m, 10H, H e / f), 8H, H _d ), 7.20 (d, 8H, H _d ), 8.03-7.94 (m, 32H, Ph), 7.78 (m, 8H, H _b ), 7.71-7.67 , 4.83 (m, 8H, cp), 4.76 (m, 8H, cp); ^{13 C NMR (300 MHz, nitromethane} -d 3, d, ppm): 164.13 (C = O), 151.01, 149.36, 141.39, 135.50, 134.60, 131.26, 129.14, 128.38, 127.66, 124.29, 120.05, 78.40, 77.42, 70.30, 69.40, 30.78.

14. Synthesis of hetero-bimetallic cycle 5

Synthesis was performed in a similar manner to HBMC 4 except that Pd (dppt) ₂ (OTf) ₂ Receptor 3 was used, and the product was isolated as yellow solid crystals in 91% yield. An elemental analysis of C ₁₃₈ H ₉₇ F ₁₂ Fe ₂ N ₁₀ O ₁₆ P ₄ Pt ₂ S ₄ was performed: C, 56.07, H, 3.31, N, 4.74; Found: C, 57.01; H, 3.39; N, 4.81; MS (ESI) calculated for [M-3OTf] ³⁺ m / z 895.5, found 895.4; calculated for [M-4OTf] ⁴⁺ m / z 634.4, found 634.4; _{^{1 H NMR (nitromethane-d 3}} , 300 MHz, d, ppm): 10.48 (s, 4H, CONH), 8.29 (m, 8H, H a), 7.67 (m, 8H, H b), 7.77 (m, 8H, H _c ), 8.15 (m, 2H, H _d ), 8.03-7.98 (m, 40H, Ph), 5.81 (m, 8H, cp), 5.79 (m, 8H, cp); _{^{13 C NMR (nitromethane-d 3}} , 300 MHz, d, ppm): 165.11 (C = O), 152.02, 148.36, 141.38, 135.57, 133.78, 130.92, 129.14, 128.48, 127.67, 124.54, 120.11, 77.41, 78.49, 70.34, 69.77, 31.18.

< Example  1> Hetero-bimetallic Metal ring  compound( Hetero - Bimetallic  Metallacycles; HBMCs ) And Characterization

As shown in FIG. 1, bis (4- (pyridin-4-ylethynyl) phenyl) pyridine-2,6-dicarbazide) pyridine-2,6-dicarboxamide)) donor ligand 1] and a heterometallic cis- M [(dppf) (OTf) ₂ ] [M = Pd ( 2 ), Pt ( 3 ); dppf = 1,1'-bis (diphenylphosphino) ferrocene; HBc complex 4 and complex 5 were prepared by the reaction between [OTF = trifluoromethanesulfonate] receptors by the [2 + 2] method.

In order to confirm the properties of the metal ring compound, NMR spectroscopy was first used. The ¹ H NMR spectra of complex 4 and complex 5 clearly showed that symmetrical species were formed by typical resonance shifts due to metal complexation. By comparing the proton signals at donor ligand 1, we have found that all signals in metal ring complexes 4 and 5 move significantly with coordination bonds. The ³¹ P NMR spectrum of complex 4 exhibited one sharp peak at 34.74 ppm which shifted to ~ 16.5 ppm upfield due to metal coordination with pyridines as compared to the signal of the initiator receptor (Fig. 2). The appearance of a single ³¹ P signal indicates that a symmetrical phosphorus nuclei is present. However, the ³¹ P NMR spectrum of complex 5 showed an abrupt singlet at 12.80 ppm with ¹⁹⁵ Pt satellites, indicating the formation of a single product (FIG. 3).

As a result of electrospray ionization mass spectrometry (ESI-MS), there is further provided evidence that metal ring compounds are formed. m / z = 836.4 ([M -3OTf] 3+) and ^{590.1 ([M-4OTf] 4+} ) and the composite assembly 4 (Fig. 4a) in, m / z = 895.4 ([ M-3OTf] 3+) and The charge state of the assembled complex 5 (Figure 4b) at 634.4 ([M-4OTf] ⁴⁺ ) was clearly observed and analyzed via isotopes. The peaks fit well with the theoretical distribution.

The electronic absorption spectra of the metal ring compounds 4 and 5 as well as their receptors were obtained in a methanol solution at 1 × 10 ^-5 M (FIGS. 5 and 6). For metal ring compound 4, a strong absorption wavelength at 320 nm was observed (Figure 5), with significant blue shifts in the dd band as compared to the initial Pd-receptor. The difference in the lambda _max position for the metal ring compound 4 and Pd-receptor 2 was apparent due to the strong-field coordination of the pyridine ring-lined nitrogen electron pair at the metal center. Similarly, in Metal Ring Compound 5, a strong absorption wavelength at 325 nm was observed (Fig. 6), with significant blue-shift compared to the initiation Pt receptor.

Finally, the structure of the [2 + 2] self assembled HBMC complex 4 was determined using molecular mechanics force field calculations (FIG. 7). UFF force field was used to perform geometry optimization. The Gaussian 09 computational software suite was used. Initially, individual fragments were optimized with chlorine compounds, completing the coordination sphere at the Ru center. The fragments were then combined into full rhomboids and optimized while completing with the structure shown in FIG. The hydrogen atoms of the cyclopentadienyl ring were added to their calculated optimal positions. The ruthenium centers were distributed at 23.8 Å, and the pyridyl-nitrogen atoms of the bridging ligands center ring were distributed at 19.5 Å. Although the structure is very planar, the central pyridyl rings are tilted at twist angles of 24.2 ° and 10.2 ° from the plane bounded by the center of the Pd center, which is a shallow ball-like characteristic of rhomboids .

< Example  2> in cancer cells and normal cells Ligand  And HBMCs The inhibitory effect of

Metal based drugs are widely used in the clinical field. In order to study the biological potential effects of such a system on the interaction between biomolecules, the present inventors have studied ligand compounds 1 (T98G), KB (head and neck cancer), SNU-80 (thyroid cancer) and HEK- , Receptor compounds 2 and 3, and novel HBMCs 4 and 5 were measured. 2- (4-sulfophenyl) -2H-tetrazolium [3- (4,5-dimethylthiazol-2-yl) 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium; MTT) is reduced in living cells, which are converted to colored formazan products through metabolic dehydrogenase enzyme activity. In metabolic activities such as apoptosis or necrosis, MTT cell proliferation assay is widely used as a reliable method for measuring cell proliferation rate. For this reason, MTT assays were used for anticancer sensitivity and radiation sensitivity studies. The data obtained by MTT analysis showed that all complexes had a dose-dependent inhibitory effect on the growth of cancer cells. Complexes 4 and 5 the growth of T98G, KB and SNU-80 4.5 - it was effectively inhibited in a 36.2 uM IC ₅₀ value range (Table 1). The cancer-treating drug, cisplatin, was used as a control. Both complexes 4 and 5 were found to inhibit T98G brain cancer cell growth by 50% in the 4.5 to 5.2 uM range after 24 hours of treatment. Experimental results show that the complexes have lower cytotoxicity at intermediate concentrations (0.32-20.82 uM) for non-malignant HEK-293 cells (Table 1). The IC ₅₀ values of the complexes for HEK-293 cells ranged from 20.6 to 36.2 μM. The results show that complexes 4 and 5 inhibit T98G brain tumor cell growth, which may be due to either growth arrest or apoptosis. Taken together, the results clearly show that complexes 4 and 5 are effectively sensitive to T98G cells in a dose-dependent manner, which may be due to an increase in mitosis (apoptosis) death (associated with cytogenetic damage) . In addition, the inventors of the present invention have conducted extensive studies in order to confirm the effects of the present invention on growth kinetics, survival analysis, reactive oxygen species (ROS) generation assay, mitochondrial membrane potential (MMP) detection, total intracellular ATP detection, Caspase 3/7 activity and annexin V / PI staining assay, clonogenic inhibition, oxidative stress and apoptosis induced by newly synthesized complexes for the detection of apoptosis in T98G cancer cells and apoptosis analysis were performed, respectively.

IC ₅₀ values (uM) of the synthesized metal complexes Complex T98G KB SNU80 HEK293 One 144.0 (+/- 17.5) 124.7 (+ - 9.6) 107.3 (+ - 5.1) 156.6 (+ - 9.9) 2 143.7 (+ - 13.4) 86.6 (+ - 6.9) 80.1 (+ - 7.4) 90.0 (+/- 8.2) 3 32.5 (+/- 5.2) 76.6 (+/- 6.3) 70.1 (+ - 5.4) 65.3 (+/- 5.3) 4 5.2 (+/- 2.0) 15.5 (+/- 2.3) 18.3 (+/- 3.0) 36.2 (+/- 5.1) 5 4.5 (± 2.1) 13.0 (+ - 1.2) 12.0 (+/- 2.8) 35.0 (+ - 3.8) Cisplatin 69.6 (+ - 7.9) 72.6 (+/- 6.2) 49.3 (+ - 5.4) 17.3 (+ - 3.6)

< Example  3> Growth kinetics ( Growth Kinetics )

8 (A) and 8 (B) show growth kinetics patterns of T98G cells. Growth kinetics analysis showed that complexes 4 and 5 had a significant inhibitory effect on T98G cells in a time-dependent manner. The cell suspension was made in a 4 mm medium layer on a 6-well plate and the cultured pions were treated with two complexes at a concentration of 0.5 to 16 uM. It is important to note that cells exposed to complex 4 exhibited a slightly weaker growth inhibitory effect than cells exposed to complex 5. After 24 hours of treatment with complex 5, cell growth inhibition was 32.4% -92.4% (survival rate 7.6% -77.6%), with maximum effect after treatment with complex 5. For T98G cells exposed to complex 4 for 24 hours at various concentrations, 2% -81.8% cells were killed and viability was recorded at 18.2% -98.0%. Taken together, complex 4 and complex 5 inhibited the growth of T98G cells in a dose and culture time-dependent manner. These experiments indicate that both complexes inhibit T98G cancer cell growth and inhibition may be due to growth arrest or apoptosis.

< Example  4> cell Colony  Inhibit formation ability

Figure 8 (C) shows the effect of complexes on T98G cell colony forming ability. Some of the inoculated cells retained the ability to produce colonies after cytotoxic drug treatment. In vitro colony forming methods for measuring clonogenicity have been recognized as valid alternative assays for intracellular tumor growth. When the concentration of the complex was increased, the cell death of the T98G cell group was enhanced, and the ability to form colonies was inhibited. Colony formation assay confirmed the effect of complexes 4 and 5 on colony-forming ability and survival of exponentially grown T98G cells. After treatment of the synthesized complex, the survival fraction of T98G cells was significantly reduced. It is important to note that the treatment of complexes 4 and 5 improves the cell death of the T98G cell line in a concentration-dependent manner and also inhibits the ability to form colonies.

< Example  6> HMBCs Stability of

To confirm the stability of complexes 4 and 5, complexes 4 and 5 of 6 uM solution were pre-reacted at 37 ° C for 0, 10, 25 and 50 h in cell culture medium (DMEM) and DMSO. Cell growth was measured by MTT cell viability assay on T98G tumor cells. As shown in Fig. 8 (D), it was observed that the growth-inhibitory activity of the complexes 4 and 5 was reduced by 50% after 50 hours of pre-reaction in the cell culture medium. In contrast, the growth inhibitory activity was stable up to 25 h after the pre-reaction in the presence of DMSO. The decrease in growth inhibitory activity is expected to be similar to that of the complex from the medium, indicating that complexes 4 and 5 may be stable for <25 h after pre-reaction in cell culture medium. The results indicate that complexes 4 and 5 are stable in DMSO and cell culture medium, at least during periods when the complex has a potent effect on cell viability.

< Example  7> Intracellular ATP  Decrease in level

To confirm the effect of complexes 4 and 5 on the mitochondrial metabolic status, we measured intracellular ATP production, which plays an important role in apoptosis in T98G brain tumor cells. Intracellular ATP was quantitated by Promega Ultra-Glo ™ recombinant luciferase in serum containing cell culture medium. Figure 9 (A) shows that complexes 4 and 5 induced significant changes in ATP production with a similar tendency. After 24 hours of treatment, relative ATP levels decreased further as the concentration of the complex increased. The effect of the 0.05 uM concentration of the complex was negligible, but higher concentrations (2, 4 and 8 uM) significantly reduced mitochondrial ATP production in T98G tumor cells.

< Example  8> intracellular ROS  Improve the level HMBSs  process

The results of intracellular ROS measurement in T98G cells after treatment with the synthesized complex are shown in Fig. 9 (B). Reduced MTT formazan due to inhibition of growth and inhibition of colony formation may increase oxidative stress and inhibit the survival of T98G cells. To confirm that treatment of the synthetic complex improves the steady state of oxidative stress in malignant tumor cells, the cells were labeled with an oxidation-sensitive probe (H2DCFDA), which, according to oxidation by ROS, (DCF) which can be measured at a wavelength of 485 nm and an absorption wavelength of 528 nm. After 24 hours of treatment, complexes 4 and 5 were found to enhance ROS production in T98G cancer cells at 0.5-2 uM concentrations. (MFI-213), 70.5% (MFI-261) and 98.6% (MFI-153) in the concentration of 0.5, 1 and 2 uM, respectively, compared with the untreated control group MFI-304). In addition, when treated with complex 5, the DCF fluorescence in T98G cells was 68.8% (MFI-260), 90.2% (MFI-293) and 120.1% (MFI-339) Respectively. However, cells treated with 0.1 uM did not show a significant change in ROS production. Interestingly, liquid holding conditions had no effect on oxidative stress in the studied cell lines, DCF fluorescence of untreated cells was similar to that of PBS and growth medium (MFI in PBS, DCF = 144; MFI in growth media, DCF = 153).

< Example  9> Intracellular Apoptosis  Inducing HMBCs

Apoptosis best describes programmed cell death and is characterized by apparent morphological characteristics such as cell membrane blobbing, reduced cell volume, activation of caspases, chromatin condensation, and nuclear fragmentation Loses. Since the cell survival after treatment was drastically reduced, the present inventors confirmed through flow cytometry (BD FACS Verse) whether the complexes induce apoptosis. The cell population was measured by fluorescein isothiocyanate (FITC) -labeled annexin V (green fluorescence), and the non-biological dye, propidium iodide (PI) Respectively. In this method, we were able to distinguish between impaired, early apoptotic, late apoptotic and necrotic cells. In Fig. 10, the lower left quadrant (Q1-LL) of the cytograms, which are PI exclusion and FITC-annexin V binding negative (FITC- / PI-), represent viable cells. The right quadrant (Q1-UR) of FITC-annexin V binding and PI uptake (FITC + / PI +) represents late apoptosis, non-vital, necrotic cells. The lower right quadrant (Q1-LR) represents FITC + / PI- apoptotic cells that bind to FITC-annexin V and are intact. All treatment significantly induced intracellular apoptosis in a dose-dependent manner. As shown in Figure 10, treatment of complexes 4 and 5 increased annexin V-FITC binding 2-4 fold. The negative control showed 5% annexin V-FITC binding whereas the 2 uM complexes 4 and 5 showed 20.88% and 21.01%, respectively.

< Example  10> Apoptosis  Inducing HMBCs Mitochondrial membrane potentials associated with &lt; RTI ID = 0.0 & Caspase  Reduced activity

Therapeutics with improved ROS levels often induce changes in mitochondrial membrane permeability in the early stages of apoptosis. Treatment of complexes 4 and 5 generates active species that destroy membrane polarization. Under similar treatment conditions, 24 hours after the reaction, T98G cells were reacted with JC-1 dye for 30 minutes, and the cells were analyzed by FACS analysis. 11 and 12A, mitochondrial membrane potential (MMP) changes in cancer cells can be confirmed. The membrane potentials of complexed 4 and 5 treated T98G cells showed significant changes compared to the untreated control. 1 and 2 uM concentrations of complex 4 induced MMP changes of 11.08% and 34.75% in the cells, respectively. The maximum effect was shown by treatment with complex 5, and 22.60% and 45.23% of MMPs were changed when treated with complex 5 at concentrations of 1 and 2 uM, respectively. Figure 11 shows the observed intracellular band transfer phenomenon after 24 hours of reaction. Treatment with complexes 4 and 5 resulted in mitochondrial membrane depolarization, a sign of apoptosis in a dose-dependent manner in the cells. Caspases 3 and 7 play an important role in cleaving cellular components during apoptosis. Figure 12 (B) shows the activity of caspases in treated cells treated with the control and after 24 hours. In untreated cells, the level of caspase activity detected is related to some apoptotic cells present in the growth population due to natural aging. In treated cells, caspase 3/7 activity increased above basal levels. Conjugates 4 and 5 at 2 uM concentration in T98G cells significantly increased caspase activity by 2.5 and 4 fold, respectively. On the other hand, complexes 4 and 5 significantly increased caspase activity by 6.3 and 9-fold, respectively, even at high concentrations (8 uM).

< Example  11> Fluorescence Detection and Microscopy Analysis

Intracellular exposure of the complex to 1-4 uM concentrations of ethynyl pyridine amide ligand) was also quantitated, and the absorption of complexes 4 and 5 was also studied. The results are shown in FIG. 13, where T98G cells exhibited excellent cell uptake profiles. These results were confirmed by fluorescence microscopy (Fig. 13). As a result, ethynyl pyridine amide ligand), but not in the untreated control group. Cells exposed to complex 4 showed slightly weaker fluorescence than cells exposed to complex 5. Taken together, we confirmed that complexes 4 and 5 accumulate at 2 and 4 μM concentrations in the cytoplasm of T98G cells.

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 (6)

Hetero-Bimetallic Metallacycles (HBMCs) represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In Formula 1, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate. A method for producing hetero-bimetallic metallacycles (HBMCs) according to claim 1, comprising the step of mixing a ligand compound represented by the following formula (2) and a hetero metal compound represented by the following formula (3).
(2)

(3)

In Formula 3, M is independently Pd or Pt, and OTf is trifluoromethanesulfonate. [2] The method of claim 2, wherein the ligand compound represented by Formula 2 and the hetero metal compound represented by Formula 3 are dissolved in CH ₂ Cl ₂ / CH ₃ NO ₂ in a molar ratio of 1: 1 (Hetero-Bimetallic Metal Cycles) (HBMCs). A pharmaceutical composition for treating or preventing a cancerous disease, which comprises the hetero-bimetallic metallacycles (HBMCs) of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. 5. The method according to claim 4, wherein the hetero-bimetallic metallacycles (HBMCs) or pharmaceutically acceptable salts thereof exhibit anticancer activity through apoptosis of cancer cells. A pharmaceutical composition. The pharmaceutical composition according to claim 4 or 5, wherein the cancer disease is brain tumor, head and neck cancer or thyroid cancer.

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