KR20190074542A - Novel ruthenium complex, preparation method thereof and Pharmaceutical composition for use in preventing or treating cancer containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a novel ruthenium complex compound, a method for manufacturing the same, and a pharmaceutical composition for preventing or treating cancer containing the same as an active component. The novel ruthenium complex according to the present invention exhibits an excellent killing effect on cancer cells and can excellently inhibit resistance occurrence mechanism of cancer stem cells, thereby being able to be usefully used as the pharmaceutical composition for preventing or treating cancer or a composition for suppressing resistance to an anticancer agent, and the pharmaceutical composition for preventing or treating cancer in which an existing anticancer agent resistance problem is improved by using with other chemotherapeutic agents.

Description

TECHNICAL FIELD The present invention relates to a novel ruthenium complex, a process for producing the same, and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient.

The present invention relates to a novel ruthenium complex, a process for producing the same, and a pharmaceutical composition for preventing or treating cancer containing the ruthenium complex as an active ingredient.

Platinum-based cisplatin derivatives are currently the most promising chemotherapeutic drugs used to treat cancer. However, prolonged administration of platinum derivatives has been reported to cause not only side effects but also drug resistance and cancer recurrence (Non-Patent Document 1).

Recently, ruthenium-based metal complexes have been proposed as an alternative to platinum derivatives. Recently, in-vitro and in vivo studies of ruthenium metal complexes have shown that ruthenium complexes have strong affinity for serum albumin and transferrin, similar to Fe, Lt; RTI ID = 0.0 > Ru < / RTI >

On the other hand, the rapid division of cells requires a high concentration of iron, and ruthenium complexes with Fe-like properties are highly likely to accumulate in cancer tissues that are faster than normal cells. In the metastatic cancers, Fe transport proteins such as transferrin and ferritin (Non-Patent Document 2).

On the other hand, when the transition metal complex is accumulated in the mitochondria or ER, the active oxygen species (ROS) is effectively produced through the fenton-type oxidation and the reduction reaction. Therefore, the redox circulation is an important characteristic of the transition metal. The metal simultaneously deactivates antioxidant proteins and allows free radicals to be produced in cancer cells under an acidic microenvironment.

Excessive ROS production is one of the characteristics of cancer cells, and increased metabolism of peroxisome and mitochondria is a major cause of ROS production in tumors. Cancer cells effectively use ROS for cell proliferation, angiogenesis and metastasis. Cancer cells can increase the expression of antioxidant proteins, such as glutathione and thioredoxin, to precisely control excessive ROS, It can withstand the limit of toxicity.

In addition, Cancer Stem Cell has been well proven to express high levels of antioxidant enzyme proteins as well as non-enzymatic antioxidant proteins compared to proliferative cancer cells (Non-Patent Document 3). The mechanism of survival of cancer cells described above allows CSC to develop drug resistance to chemotherapeutic drugs.

Therefore, it is expected that therapeutic strategies such as not only suppressing the antioxidant capacity of cancer cells but also simultaneously increasing ROS production may induce irreversible oxidative stress and subsequent cell death.

Previous studies have shown that it is possible to effectively kill cancer cells by inhibiting the expression of specific antioxidant proteins and over the limits of cancer cells through a cell apoptosis mechanism (Non-Patent Document 4) , Or a therapeutic agent capable of effectively inhibiting the development of resistance to cancer and cancer stem cells has not been developed, and continuous research and efforts are required.

Therefore, the inventors of the present invention have been able to solve the problems of resistance to cancer and the occurrence of side effects using platinum-based chemotherapeutic agents, and in particular, they are capable of effectively inhibiting the resistance mechanism of cancer stem cells and further, The inventors of the present invention have found that the novel Ru complex according to the present invention not only remarkably suppresses the resistance mechanism of cancer stem cells but also induces ROS production remarkably and has an excellent cancer cell killing effect Thus, the present invention has been completed.

Shen D-W, Pouliot LM, Hall MD, Gottesman MM. 2012. Pharmacological reviews 64: 706-721. Brookes et al., 2006; Kwok and Richardson 2002 Nagano et al., 2013; Trachootham et al., 2009 Dharmaraja 2017, Sznarkowska et al., 2017

It is an object of the present invention to provide a novel transition metal complex which can be used as an active ingredient of an anticancer agent having a cancer cell killing effect or can be provided as an active ingredient of a resistance generation inhibitor with excellent inhibition of the cancer tolerance development mechanism.

Another object of the present invention is to provide a method for producing the transition metal complex.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer containing the transition metal complex as an active ingredient.

Another object of the present invention is to provide a composition for inhibiting anticancer drug resistance which comprises the transition metal complex as an active ingredient.

It is still another object of the present invention to provide a health functional food for preventing or ameliorating cancer containing the transition metal complex as an active ingredient.

In order to solve the above object,

The present invention provides a complex represented by the following general formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;

N is an integer from 0 to 5;

y is an integer from 1 to 3;

z is 0 to 3;

Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

,

, 및

;

,

,

,

,

, And

;

또는

이고;R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ and R ^3j is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano ,

Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.

The present invention also provides a complex represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

(2)

In Formula 2,

X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;

N is an integer from 0 to 5;

y is an integer from 1 to 2;

z is 0 to 1;

Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

, 및

;

,

,

,

,

,

, And

;

또는

이고;R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ , R ^3j and R ^3k is independently hydrogen, a substituted or unsubstituted C _1-6 branched Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein the substituted alkyl and substituted alkoxy are optionally substituted with one or more groups selected from the group consisting of halogen, oxo, nitro, and cyano ≪ / RTI >

Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.

Further, the present invention relates to a process for preparing a compound represented by the following formula 1,

Preparing a compound represented by the formula (4) from a compound represented by the formula (3);

Preparing a complex represented by the general formula (1) from the compound represented by the general formula (4) prepared in the above step,

[Reaction Scheme 1]

In the above Reaction Scheme 1,

X, n, y, z, a ligand, R ¹ , R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are as defined in Formula 1.

The present invention also relates to a process for producing a compound represented by the formula

Preparing a compound represented by the formula (6) from a compound represented by the formula (5);

Preparing a complex represented by the general formula (2) from the compound represented by the general formula (6) prepared in the above step;

[Reaction Scheme 2]

In the above Reaction Scheme 2,

X, n, y, z, a ligand, R ¹ , R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are as defined in Formula 2.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a complex compound represented by Chemical Formula 1 or Chemical Formula 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

The present invention also provides a composition for inhibiting anticancer drug resistance, which comprises a complex represented by the above formula (1) or (2), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the composition for inhibiting anticancer drug resistance, and a chemotherapeutic agent.

The novel Ru complex according to the present invention not only exhibits an excellent killing effect on cancer cells but also can inhibit the mechanism of resistance of cancer stem cells to excellent effect and is useful as a pharmaceutical composition for preventing or treating cancer , Or a composition for inhibiting anticancer drug resistance, and a pharmaceutical composition for preventing or treating cancer, wherein the existing anticancer drug resistance problem is improved by using it in combination with other chemotherapeutic agents.

The FACS analysis of FIG. 1 is a graph showing absorbance by CD44-positive MCF7 cells at different time points (30, 60 and 90 minutes) after 10 占 본 of Example 1 treatment of the present invention.
Figure 2 shows intracellular localization observed with confocal microscopy after treatment of Example 1 of the invention with Cd133 positive HCT-116 cells: (A) Localization location in mitochondria - (a) nuclear staining b) luminescence of Example 1 (c) emission of mitochondrion red (d) superimposed image; (B) Localization position in ER - (a) Nuclear dyeing (b) Emission of Example 1 (c) Emission of ER-tracker red (d) Superimposed image.
FIG. 3 is a graph showing the degree of intracellular ROS generation in CD44-positive MCF7 and CD133-positive HCT-116 cells treated with different concentrations of 1, 5, and 10 μM of Example 1. FIG. The bar graph shows the average fluorescence intensity of 2 ', 7'-dichlorofluorescein diacetate. The error bars represent the standard deviation of three independent measurements (** P <0.01, *** P <0.001 Vs Control (Control)).
4 is an image and a graph showing the results of high-content analysis to find MPTP, cytoplasmic calcium and caspase 3/7 in treated CD133-positive HCT-116 cells of Example 1 performed using AOTF . Cancer cells were treated with different concentrations of Ru-1 (1, 5 and 10 [mu] M) for 16 hours in a humidified atmosphere of 5% CO2 for 16 hours, fluorescence emission of Calcine AM, calcium orange and magic red caspase 3/7 Were monitored using AOTF at 525 nm, 580 nm and 630 nm, respectively. The green fluorescence image corresponds to the cell image of MPT, the orange fluorescence image represents cytoplasmic calcium, and the red fluorescence image represents caspase 3/7. B. The bar graph shows the mean fluorescence intensity of Calcine AM, Calcium Indicator Orange and Caspase 3/7.
5 is an image and a graph showing the induction of apoptosis and the expression of anti-apoptotic proteins in CD133-positive HCT116 cells treated with Example 1 of the present invention: A. Bax (1 column-DAPI, 2 column-Bax, 3 column - superposition); Expression of B. Bak (1 column-DAPI, 2 columns-Bak, 3 columns-overlap); C. Expression of Bcl-2 (one column -DAPI, two columns-Bcl-2, three columns-overlap); D. Western blot analysis of Bcl-2, Bax and Bak in the control or Example 1 treated cells; And E. graphs are graphs showing the relative expression of Bcl-2, Bax and Bak according to Example 1 treatment versus control. The error bars represent the standard deviation of three independent measurements (* P <0. ** P <0.01, Vs Control).
6 is an image of Embodiment 1 of the present invention docked with GRP 78 obtained from the CDOCKER program of Discovery Studio.
Figure 7A shows the effect of (a) GRP78, ((a)) on HEK293 cells transfected with pTag-YFP-GRP78-C, pDs-CLU- Red2-N1 and pAC- ATR- b) Clusterin, (c) an image showing the expression of ATR; B is a bar graph showing the mean fluorescence intensities of GFP, RFP and GFP in plasmid transfected HEK293 cells at different concentrations of the Invention Example 1; C is a graph showing mRNA expression of GRP78 in CD133-positive HCT116 cells treated with Example 1 of the present invention. Here, mRNA expression was analyzed by RT-PCR; D shows the expression of GRP78 in CD133 positive HCT116 cells treated with the control or Inventive Example 1 (0, 6, and 12 [mu] M). Expression fluorescence of GRP-78 is consistent with the fluorescence of the ER tracker red; E is a bar graph showing the mean fluorescence intensity of GRP-78 in CD133 positive HCT cancer cells treated with different concentrations of Example 1; F is a Western blot showing the expression of GRP78 in the control or CD133-positive HCT116 cells treated with Example 1; And G is a bar graph showing the relative expression of GRP-78 relative to the control. The error bars represent the standard deviation of three independent measurements (** P <0.01, Vs Control).
Figure 8 shows A. Treatment protocol: CD133-positive HCT116 cells were intravenously injected into mice at day 0, and mice treated with Example 1 of the present invention at 14-18 days. The mice were sacrificed on the last day of the experiment; B. Photographs of experimental mice (control, low dose and high dose treatments); C. Photographs of tumors isolated from experimental mice Control, low dose and high dose treatments); D. Tumor volume of experimental mice during treatment; E. Tumor isolated from experimental mice The weight of xenografted tumor; F. An image showing fluorescence emission of the CD133 antibody labeled with Alexaflour 680 in a control mouse or a treated experimental mouse according to the present invention Example 1; And G is a graph showing the fluorescence intensity of the CD133 antibody labeled with Alexaflour680.

Hereinafter, the present invention will be described in detail.

The present invention provides a complex represented by the following general formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;

N is an integer from 0 to 5;

y is an integer from 1 to 3;

z is 0 to 3;

Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

,

, 및

;

,

,

,

,

, And

;

또는

이고;R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ and R ^3j is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano ,

Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.

In one embodiment of the present invention, the complex compound represented by Formula 1 may be a complex compound of Formula 1 '.

[Formula 1 ']

Here, R ¹ , X, and n are the same as defined in Formula 1.

In one embodiment of the present invention, the complex compound represented by the general formula (1)

; 또는

; or

일 수 있다.

Lt; / RTI &gt;

On the other hand, in the ligand of the Ru complex of the present invention, the ligand of the bidentate ligand can be used in the complex represented by the general formula (1).

In one aspect of the invention, the Ru complex is ionized in the weakly acidic state of the cytoplasm, from which Ru (II) complexes accumulate, particularly in the mitochondria or ER of cancer cells. This accumulation continues to lead to the production of ROS (reactive oxygen species). Generally, such ROS production is inhibited or regulated by the cancer cell by regulating antioxidant protein expression. In addition to ROS generation, the Ru complex of the present invention inhibits the expression of the antioxidant protein itself, thereby greatly increasing the oxidative stress applied to the cell, The death of cancer cells Apoptosis is actively induced and eventually kills cancer and cancer stem cells.

Accordingly, the Ru complex of the present invention is useful as an active ingredient of a pharmaceutical composition for the prevention or treatment of cancer, and further, as described in the present invention, can be used as an excellent anticancer agent, .

On the other hand, in one aspect of the present invention, the novel Ru complex of the present invention exhibits an excellent effect of suppressing the mechanism of tolerance to cancer in addition to the killing effect on cancer and cancer stem cells. Specifically, it has been found that tolerance to an anticancer agent is generated by an increase in the expression of GRP-78 by cadmium-selenide (CSC), as a mechanism of development of cancer resistance in the past. Therefore, the inhibitory activity against GRP-78 is an important pathway to inhibit or prevent the development of anticancer drug resistance.

In one embodiment of the present invention, the novel Ru complexes of the present invention were found to significantly inhibit GRP-78 expression and activity. In particular, it has been experimentally confirmed that the Ru complex of the present invention not only inhibits the expression of GRP-78 but also binds to GRP-78 dimer and its activity is inhibited. Were identified and confirmed that the resistance mechanism of the cancer stem cells was markedly suppressed. Therefore, the novel Ru complex of the present invention can be used as a composition for inhibiting anticancer drug resistance, and is particularly useful as an anticancer adjuvant that can be used in combination with conventional chemotherapeutic agents such as platinum-based chemotherapeutic agents Can be usefully used.

The present invention also provides a complex represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

(2)

In Formula 2,

X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;

N is an integer from 0 to 5;

y is an integer from 1 to 2;

z is 0 to 1;

Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

, 및

;

,

,

,

,

,

, And

;

또는

이고;R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ , R ^3j and R ^3k is independently hydrogen, a substituted or unsubstituted C _1-6 branched Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein the substituted alkyl and substituted alkoxy are optionally substituted with one or more groups selected from the group consisting of halogen, oxo, nitro, and cyano &Lt; / RTI &gt;

Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.

In one aspect of the present invention, the complex compound represented by Formula 2 may be a complex compound represented by Formula 2 'below.

[Formula 2 ']

Wherein R ¹ , X and n are the same as defined in the above formula (2).

In one embodiment of the present invention, the complex compound represented by the general formula (2)

Lt; / RTI &gt;

On the other hand, in the complex represented by the general formula (2), lig (ligand) of the Ru complex of the present invention can use a ligand of a tridentate ligand. In particular, in the complex represented by the above formula (2), ligands represented by Lig can be used without limitation as long as they are trivalent ligand ligands, and the present invention includes these.

In one aspect of the invention, the Ru complex is ionized in the weakly acidic state of the cytoplasm, from which Ru (II) complexes accumulate, particularly in the mitochondria or ER of cancer cells. This accumulation continues to lead to the production of ROS (reactive oxygen species). Generally, such ROS production is inhibited or regulated by the cancer cell by regulating antioxidant protein expression. In addition to ROS generation, the Ru complex of the present invention inhibits the expression of the antioxidant protein itself, thereby greatly increasing the oxidative stress applied to the cell, The death of cancer cells Apoptosis is actively induced and eventually kills cancer and cancer stem cells. In particular, it was proved in the present invention that the cancer killing effect of cisplatin, which is a conventional white cancer chemotherapeutic agent (anticancer agent), is better than that of cancer cells (see Experimental Example 1 and Table 2 below).

Accordingly, the Ru complex of the present invention is useful as an active ingredient of a pharmaceutical composition for the prevention or treatment of cancer, and further, as described in the present invention, can be used as an excellent anticancer agent, .

On the other hand, in one aspect of the present invention, the novel Ru complex of the present invention exhibits an excellent effect of suppressing the mechanism of tolerance to cancer in addition to the killing effect on cancer and cancer stem cells. Specifically, it has been found that tolerance to an anticancer agent is generated by an increase in the expression of GRP-78 by cadmium-selenide (CSC), as a mechanism of development of cancer resistance in the past. Therefore, the inhibitory activity against GRP-78 is an important pathway to inhibit or prevent the development of anticancer drug resistance.

In one embodiment of the present invention, the novel Ru complexes of the present invention were found to significantly inhibit GRP-78 expression and activity. In particular, it has been experimentally confirmed that the Ru complex of the present invention not only inhibits the expression of GRP-78 but also binds to GRP-78 dimer and its activity is inhibited. Were identified and confirmed that the resistance mechanism of the cancer stem cells was markedly suppressed. Therefore, the novel Ru complex of the present invention can be used as a composition for inhibiting anticancer drug resistance, and is particularly useful as an anticancer adjuvant that can be used in combination with conventional chemotherapeutic agents such as platinum-based chemotherapeutic agents Can be usefully used.

In one aspect of the present invention, the complex of formula (I) or (II) of the present invention can be used in the form of a pharmaceutically acceptable salt. Examples of the salt include pharmaceutically acceptable acids formed by free acid Additional salts are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Examples of such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chloro Such as benzenesulfonate, benzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1-sulfonate, naphthalene-2-sulfonate, mandelate and the like.

The acid addition salt according to the present invention can be prepared by a conventional method. For example, the compound of formula (1) or (2) is dissolved in an organic solvent such as methanol, ethanol, acetone, dichloromethane, acetonitrile, etc., Filtration and drying of the resulting precipitate, or distillation of the solvent and excess acid under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).

Furthermore, the present invention includes not only the complex compound represented by Chemical Formula 1 or Chemical Formula 2 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers and hydrates thereof which can be prepared therefrom.

Further, the present invention relates to a process for preparing a compound represented by the following formula 1,

Preparing a compound represented by the formula (4) from a compound represented by the formula (3);

Preparing a complex represented by the general formula (1) from the compound represented by the general formula (4) prepared in the above step,

[Reaction Scheme 1]

In the above Reaction Scheme 1,

X, n, y, z, a ligand, R ¹ , R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are as defined in Formula 1.

In one aspect of the method of the present invention for preparing a reaction scheme 1, the above process may be carried out in accordance with the production process represented by the following reaction scheme 1 '.

[Reaction Scheme 1 ']

Hereinafter, the method for producing the complex represented by the formula (1) will be described in detail.

In the above Reaction Scheme 1,

Step (a) includes, but is not limited to, the method in which the target compound (a) is prepared from 1,10-phenansroline-5,6-dione, but in one embodiment, fluorene- The reaction can be carried out by refluxing the aldehyde, vodaldehyde, ammonium acetate, and glacial acetic acid.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, Preferably, the reaction time is 3 hours to 8 hours.

Step (b) can also be performed by reacting the compound prepared in step (a), cis- [Ru (bpy) ₂ Cl ₂ ] .2H ₂ O, in ethylene glycol.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, in one, from 90 to 150 ℃ preferably under N ₂ atmosphere, and the reaction time can be carried out in about 5 hours to about 10 hours.

In the meantime, the step (c) of Scheme 1 'is not limited to the method in which the objective compound of step (c) can be prepared from 1,10-phenansroline-5,6-dione, , 4- (dimethylamino) -1-naphthaldehyde, fluorene-2-carboxaldehyde, ammonia acetate, and glacial acetic acid.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, Preferably, the reaction time is 3 hours to 8 hours.

The present invention also relates to a process for producing a compound represented by the formula

Preparing a compound represented by the formula (6) from a compound represented by the formula (5);

Preparing a complex represented by the general formula (2) from the compound represented by the general formula (6) prepared in the above step;

[Reaction Scheme 2]

In the above Reaction Scheme 2,

X, n, y, z, a ligand, R ¹ , R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are as defined in Formula 2.

In one embodiment of the process for preparing Reaction Scheme 2 of the present invention, the process may be carried out in accordance with the production process represented by Reaction Scheme 2 'below.

[Reaction Scheme 2 ']

Hereinafter, the method for preparing the complex represented by the formula (2) will be described in detail.

In the above Reaction Scheme 2,

Step (a) includes, but is not limited to, the method in which the step (a) target compound can be prepared from 1,2,4,6-trichloro-1,3,5-triazine, but in one embodiment, N, N-diethylaniline.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, Preferably, the reaction temperature may be 50 to 90 ° C, and the reaction time may be 5 to 12 hours.

Further, step (b) can be carried out by refluxing the compound prepared in step (a), 3,5-dimethylpyrazole, K metal in THF.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, , And the reaction time may be from 5 hours to 12 hours.

In the meantime, the step (c) of Scheme 2 'is not limited thereto as long as the target compound can be prepared from 2,2': 6 ', 2''- terpyridine in step (c) in one embodiment, it may be done by reflux for a hotel pyridine, RuCl ₃ .3H ₂ O in ethanol.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, Preferably, the reaction time is 1 hour to 5 hours.

In addition, if step (d) is a method capable of producing the Ru complex of the present invention, which is the final target compound from the compound prepared in step (c) and the compound prepared in step (b) In one embodiment, however, it may be carried out by reacting the compound prepared in step (c) with the compound prepared in step (b) in ethylene glycol.

Here, the reaction conditions, for example, the solvent, the reaction temperature and the reaction time can be adjusted without particular limitation, considering the yield and the degree of side reaction of the target compound produced by the ordinary skilled artisan, One, preferably reflux under an atmosphere of N ₂ , the reaction time can be carried out from 12 hours to 36 hours, or overnight.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising as an active ingredient a complex compound represented by Chemical Formula 1 or Chemical Formula 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

In one aspect of the present invention, the pharmaceutical composition contains the Ru complex of the present invention, that is, the complex compound represented by Chemical Formula 1 or Chemical Formula 2 as an active ingredient, and particularly, as shown in the experimental examples of the present invention, Or cancer stem cell death inducing effect and ROS generation inducing effect), and exhibits an effect of significantly inhibiting the expression of enzymatic and non-enzymatic antioxidant protein, which is a ROS defense mechanism of cancer or cancer stem cells.

Therefore, it has been confirmed that the Ru complex of the present invention is excellent in the cell infiltration into cancerous or cancer stem cells, and in particular, it is ionized in the weakly acidic condition of the cytoplasm, , Or ER, and it was confirmed that ROS production was remarkable. In addition, since the expression of the enzymatic and non-enzymatic antioxidant protein is significantly inhibited and the expression of pro-apotopsis protein is significantly increased, irreversible oxidative stress is induced in cancer or cancer stem cells, Apotopsis effect. &Lt; / RTI &gt; Furthermore, in addition to the in vitro experiments of cancer cells and cancer stem cells (CSC) of colon cancer or breast cancer, it has been proved that it can be used as an excellent anticancer agent in animal model experiments in vivo. As a result, It is understood that the pharmaceutical composition for the prevention or treatment of cancer is useful.

On the other hand, in the above cancer, the cancer is included without limitation as long as the cancer can be treated based on the experimentally proven effects of the present invention as described above, but preferably the cancer is selected from the group consisting of a false myxoma, intrahepatic bile duct cancer, , Cancer of the liver, cancer of the thyroid gland, colon cancer, testicular cancer, myelodysplastic syndrome, glioma, oral cancer, oral cancer, bacterial sarcoma, acute myelogenous leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian carcinoma, , Pituitary adenoma, multiple myeloma, gallbladder cancer, biliary cancer, colon cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, diffuse large B cell lymphoma, bladder enlargement cancer, bladder cancer, peritoneal cancer, pituitary cancer, , Pneumocystis carcinoma, non-small cell lung cancer, non-Hodgkin's lymphoma, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small bowel cancer, meningioma, Malignant lymphoma, malignant mesothelioma, malignant melanoma, ovarian cancer, vulvar cancer, ureter cancer, urethral cancer, primary site unidentified cancer, gastric lymphoma, malignant melanoma, malignant melanoma, Cancer of the uterus, endometrial cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, gastrointestinal cancer, gastric cancer, gastric cancer, gastric carcinoma, gastrointestinal stromal cancer, Wilms's tumor, breast cancer, sarcoma, Cancer of the lung, squamous cell carcinoma of the lung, squamous cell carcinoma of the lung, squamous cell carcinoma of the lung, rhabdomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, squamous cell carcinoma, Laryngeal cancer, pleural cancer, and thymus cancers.

Furthermore, preferably said cancer is acute myelogenous leukemia (AML); Chronic myelogenous leukemia (CML); Acute lymphocytic leukemia (ALL); Chronic lymphocytic leukemia (CLL); Hodgkin's disease (HD); Non-Hodgkin's lymphoma (NHL); B-cell lymphoma; T-cell lymphoma; Multiple myeloma (MM); Amyloidosis; Valgendrogmaglobulinemia; Myelodysplastic syndrome (MDS); Small lymphocytic lymphoma (SLL); Marginal lymphoma; Asymptomatic multiple myeloma; And bone marrow proliferative syndrome.

As used herein, the term "cancer" is used to establish unregulated or aberrantly regulated cell proliferation, reduced cellular differentiation, impaired ability to invade surrounding tissues, and / &Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; The term "cancer" includes, but is not limited to, solid tumors and blood mediated tumors (hematologic malignant tumors). The term "cancer" includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. The term "cancer" further includes primary and metastatic cancers.

The solid tumor may be pancreatic cancer; Invasive bladder cancer containing bladder cancer; Colorectal cancer; Breast cancer including thyroid cancer, stomach cancer, metastatic breast cancer; Prostate cancer, including androgen-dependent and androgen-independent prostate cancer; Kidney cancer, including, for example, metastatic renal cell carcinoma; Liver cancer including hepatocellular carcinoma and intrahepatic bile duct; Lung and bronchial carcinoma, including non-small cell lung cancer (NSCLC), squamous cell lung cancer, bronchoalveolar alveolar carcinoma (BAC), adenocarcinoma of the lung, and small cell lung cancer (SCLC); Ovarian cancer, including, for example, advanced epithelial or primary peritoneal cancer; Cervical cancer; For example, uterine cancer including uterine body and cervix; Endometrial cancer; Gastric cancer; Esophagus cancer; For example, head and neck cancer including squamous cell carcinoma of the head and neck, nasopharyngeal cancer, oral cavity and pharynx; Melanoma; Neuroendocrine carcinoma, including metastatic neuroendocrine tumors; For example, brain tumors including glioma / glioblastomas, inverse dysplastic glomerulonephrosis, adult glioblastoma multiforme, and adult retroformed astrocytoma; Metastatic neuroendocrine tumors; Bone cancer; And neuroendocrine involvement including soft tissue sarcoma.

The hematological malignancies include acute myelogenous leukemia (AML); Chronic myelogenous leukemia (CML), including accelerated CML and CML aspirate (CML-BP); Acute lymphocytic leukemia (ALL); Chronic lymphocytic leukemia (CLL); Hodgkin's disease (HD); Non-Hodgkin's lymphoma (NHL), including follicular lymphoma and mantle cell lymphoma; B-cell lymphoma (including diffuse large B-cell lymphoma (DLBCL)); T-cell lymphoma; Multiple myeloma (MM); Amyloidosis; Valgendrogmaglobulinemia; (Including anemia of intractable anemia (RAE), hyperammonemia (RAEB), and transformation with accompanying RAEB (RAEB-T)) with mastitis formation syndrome (MDS) ; Small lymphocytic lymphoma (SLL); Marginal lymphoma; Asymptomatic multiple myeloma; And myeloproliferative syndrome.

The present invention also provides a composition for inhibiting anticancer drug resistance, which comprises a complex represented by the above formula (1) or (2), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

In one aspect of the present invention, the novel Ru complex of the present invention has an effect as an anticancer agent as described above, and also has an effect of inhibiting the expression of GRP-78 of CSC (cancer stem cell) It is experimentally proved that the generation can be suppressed well. In particular, it has been experimentally proved that not only the proportional inhibitory activity is proved by increasing the concentration but also the expression and activity of GRP-78 are remarkably suppressed even at a very low concentration. As a result, A cancer resistant to an anticancer agent, or a cancer stem cell.

In another aspect of the present invention, as described in the background art, a conventional chemotherapeutic agent (anticancer agent) is a resistance mechanism of CSC (cystic fibrosis), which is resistant to or less effective as a drug treatment And there was also a problem of drug side effects. Among the conventional combination therapies, the problem of tolerance and side effects of transition metal complexes, for example, platinum complexes, has been reported to limit the tolerance of CSCs.

From this point of view, the effect of inhibiting the resistance mechanism of CSC of the novel Ru complexes proved by the present invention is very important. From this, it can be seen that the Ru complex of the present invention can be used in combination with a conventional chemotherapeutic agent, Method will be useful as a therapeutic agent capable of inhibiting cancer or cancer stem cell resistance to an anticancer drug.

Accordingly, the present invention provides a pharmaceutical composition for prevention or treatment of cancer comprising the composition for inhibiting anticancer drug resistance, and a chemotherapeutic agent.

As described above, it has been proved that the present invention inhibits GRP-78 expression and activity in a dose-dependent manner. From this fact, not only has an excellent cell killing effect been confirmed in cancer cells or cancer stem cells, , The novel Ru complex of the present invention can be used as an anticancer agent itself or as a therapeutic agent which can be used in combination with a conventional chemotherapeutic agent (anticancer agent).

Herein, the cancer includes all the carcinomas described herein as cancer, and refers to one or more of them.

In the pharmaceutical composition according to the present invention described above, the complex compound represented by the formula (1) or (2), a stereoisomer thereof or a pharmaceutically acceptable salt thereof may be administered in various forms of oral and parenteral administration In the case of formulation, it may be prepared by using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and troches, , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants (such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine and may optionally contain binders such as starch, agar, alginic acid or sodium salts thereof Release or boiling mixture and / or absorbent, colorant, flavor, and sweetening agent.

The pharmaceutical composition containing the complex compound represented by Formula 1 or Formula 2 as an active ingredient can be administered parenterally, and parenteral administration is performed by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection .

In this case, in order to formulate the composition for parenteral administration, a complex or a pharmaceutically acceptable salt thereof of Formula 1 or Formula 2 is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, which is then mixed with an ampule or vial Unit dosage form. The compositions may contain sterilized and / or preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, Or may be formulated according to the coating method.

It should be understood by those skilled in the art that the formulations described above are only for the ordinary formulation examples, and that the formulations of the present invention are not limited thereto.

Further, the dose of the complex of the present invention represented by the above formula (1) or (2) or its pharmaceutically acceptable salt to the human body may be varied depending on the age, weight, sex, dosage form, , 0.1-1000 mg / day, preferably 1-500 mg / day, on the basis of an adult subject having a body weight of 70 kg, and may be administered once a day at a predetermined time interval according to the judgment of a doctor or pharmacist It may be divided into several doses.

Furthermore, the present invention provides a health functional food composition for preventing or ameliorating cancer, which comprises the complex compound represented by Chemical Formula 1 or Chemical Formula 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

The cancer refers to all the above-mentioned cancer diseases, including, but not limited to, caustic myxoma, intrahepatic bile duct cancer, hepaticoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, , Chronic myelogenous leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, Lymphoma, leukemia, retinoblastoma, choroidal melanoma, diffuse large B cell lymphoma, bladder cancer, bladder cancer, peritoneal cancer, pituitary cancer, adrenal cancer, non-sinus cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, Neuroblastoma, neuroblastoma, renal cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, pancreatic cancer, pancreatic cancer, pancreatic cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small bowel cancer, Cancer of the vulva, cancer of the urethra, cancer of the urethra, cancer of the primary site, cancer of the primary site, gastric lymphoma, gastric cancer, gastric carcinoma, gastrointestinal stromal cancer, Wilms' tumor, breast cancer, sarcoma, The present invention relates to a method for the treatment and prophylaxis of cancer of the uterine cervix, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, , Cancer of the tonsil, squamous cell carcinoma, lung cancer, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, and thymic carcinoma.

Further, the present invention relates to a method for treating cancer, comprising administering to a subject a therapeutically effective amount of a complex of Formula 1 or Formula 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, to provide.

Herein, the therapeutically effective amount refers to an amount that is sufficient to treat, prevent or ameliorate the symptoms or the condition of the subject upon administration into the body, depending on the administration method. In addition, the amount may vary depending on the body weight, age, sex, condition, and family history of the subject to be administered. In the present invention, the treatment method can set different doses depending on different conditions.

The "effective amount" is an amount effective, for example, to treat cancer. In another embodiment, an "effective amount" of a compound is an amount that is capable of causing cancer or cancer stem cell death.

The complexes and compositions according to the methods of the present invention may be administered using any amount and any route of administration effective for treating the disease. The exact amount required will vary from subject to subject depending on the species, age, and general condition of the subject, the severity of the infection, the particular agent, its mode of administration, and the like. The complexes of the present invention are frequently formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" means a physically discrete unit of formulation suitable for the subject to be treated, as used herein. It will also be appreciated that the total daily usage of the complexes and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific effective dosage level for any particular subject or organism will depend on a variety of factors including: the severity of the disease and disorder to be treated; The activity of the specific compound employed; Specific composition; Age, weight, general health, gender and diet of the subject; The time of administration, the route of administration, and the rate of excretion of the particular compound employed; Duration of treatment; The particular compound used alone or coadministered, and other factors well known in the medical arts.

The term "subject ", as used herein, means an animal, such as a mammal, such as a human.

The ruthenium complex of the present invention exhibits anticancer activity and has been confirmed to be a metal complex that targets intracellular organelles for drug resistance protein and anticancer activity, so that multidrug resistance can be overcome in cancer stem cells.

In general, the cancerous tumor microenvironment (pH 6.5-6.8) is more acidic than normal tissue (pH 7.4). At this tumor acidic pH, it is presumed that the physiologic conditions make it easier to protonate the Ru complex of the invention and more easily lead to the internal mitochondrial membrane (IMM) negative potential.

Surprisingly, the inventive Ru complexes coexist in both mitochondria and ER. When the transition metal complexes are accumulated in the mitochondria or ER, it is possible to effectively produce reactive oxygen species (ROS) through the pentotonic oxidation-reduction reaction. This property is considered to be a characteristic feature that the transition metal simultaneously deactivates the antioxidant protein, Cancer cells can withstand the metabolic and pharmacological load to the limit of ROS toxicity. CSC also exhibits high levels of antioxidant enzymes as well as non-enzymatic antioxidant proteins as compared to proliferating cancer cells, enabling CSC to develop drug resistance to chemotherapy drugs from this survival mechanism.

Therefore, therapeutic strategies such as simultaneously suppressing the antioxidant capacity of cancer cells as well as increasing ROS production can induce irreversible oxidative stress and subsequent apoptosis, and the Ru complex according to the present invention can be administered in a dose- -116 and CSC of MCF-7 cells, indicating excellent cytotoxicity.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

Ruthenium trichloride trihydrate and other reagents or chemicals were purchased from Sigma Aldrich, South Korea. Analytical grade solvents were purchased from Alfa Aesar or Sigma Aldrich South Korea and degassed with 100% dry nitrogen for 20 minutes before use in the reaction. Deionized water was used for the preparation of the sodium chlorate solution. Acetonitrile and toluene commercial solvents were used for column purification and purchased from Daejung Chemicals, Korea.

< Example  Synthesis of ruthenium complex-1 (RU-1): [Ru (bdpta) (tpy)] (ClO ₂ ) ₂

A mixture of terpyridine 2 (300 mg, 1.29 mmol) and 0.336 g of RuCl ₃ .3H ₂ O (1.29 mmol) in anhydrous ethanol (80 ml) was refluxed at 80 ° C for 3 hours. Excess ethanol was concentrated on a rotary evaporator to give a red solid which was washed with diethyl ether and hexane. A red solid [Ru (tpy) Cl ₃ ] was obtained, dried and used in the next step of the synthesis without further spectral analysis. 400 mg of [Ru (tpy) Cl ₃ ] material, 25 ml of dry ethylene glycol and 0.9 mmol of bdpta ligand were added and refluxed overnight at 170 ° C under a N ₂ atmosphere. Then, the reaction mixture was cooled to room temperature and added to 1M NaClO _4. The solvent was then evaporated in a rotary evaporator. The crude ruthenium complex was purified twice by column chromatography over neutral alumina (CH ₃ CN / toluene, 40/60, v / v). The color of the dark red-brown band was collected, the addition of a saturated NaClO _4, and then, the solvent was distilled ClO ₄ ^- to give the product as a salt. The solid was then washed with diethyl ether, hexane and dried under vacuum.

Yield: 400 mg, 51.4%. Anal. Calcd for C ₃₈ H ₃₉ N ₁₁ Ru (%): C, 60.78; H, 5.24; N, 20.52. Found: C, 60.82; H, 5.40; N, 20.75. . ^{1 H NMR (400 MHz, Methanol} -d 4) δ 10.38 - 10.31 (m, 1H, tpy), 8.84 (dd, 1H, tpy), 8.69 (dd, J = 8.2, 1.4 Hz, 1H, tpy), 8.43 1H, tpy), 7.89 (ddd, J = 7.1, 5.7, 1.3 Hz), 8.36 (m, 2H, aniline ring Ar proton), 8.35-8.25 1H, tpy), 7.66 (td, J = 7.7,1.8 Hz, 1H, tpy), 7.20-7.10 (m, 2H, tpy), 6.97-6.89 (m, 2H, aniline ring Ar proton), 6.52 , J = 7.7, 1.1 Hz, 1H, tpy), 6.31 (s, 2H, pyrazole CH proton), 3.61 (q, J = 7.1 Hz, 4H, alkyl CH 2), 3.34 (s, 6H, pyrazole ring 2- CH ₃ ), 3.07 (s, 6H, pyrazole ring 2-CH ₃ ), 1.34-1.21 (m, 6H, alkyl CH ₃ ). ^{13 C NMR (600 MHz, DMSO} -d 6): δ 12.68, 14.05, 16.18, 110.81, 111.45, 121.07, 121.24, 123.82, 131.60, 136.91, 137.98, 144.02, 149.23, 151.70, 152.59, 155.45, 156.34, 164.01. MALDI-TOF mass spectra: 752.07 (C ₃₈ H ₃₉ N ₁₁ Ru), UV-Visible spectrum (CH ₃ CN):? Max 380 nm and 515 nm. HRMS (ESI): Calcd for C ₃₈ H ₃₉ N ₁₁ Ru: 750.8770, found: 750.8780.

Example 2 Synthesis of ruthenium complex-2 (RU-2): [Ru (bpy) ₂ (Fip)] (ClO ₂ ) ₂

Imidazo [4,5-f] [1,10] phenanthroline (250 mg, 0.65 mmol) and cis- [Ru (bpy) ₂ Cl ₂ ]. 2H ₂ O (315 mg, 0.65 mmol) were mixed and dissolved in 40 ml of ethylene glycol. Then the reaction mixture at 120 ℃ under N ₂ environment was refluxed for 7 hours. The completion of the reaction was confirmed by TLC. The reaction mixture was then cooled to room temperature and diluted with 100 ml of DI water. Saturated NaClO ₄ solution was added to the mixture, and the mixture was slowly stirred to obtain a dark reddish orange solid. The resulting crude product was filtered and neutral alumina was purified twice by column chromatography using a stationary phase and toluene / acetonitrile (40/60, v / v) as mobile phase solvent. A dark red orange band was collected and the solvent was concentrated under reduced pressure. Then the product was added slowly to a saturated (50%) NaClO ₄ solution ClO ₄ ^- was obtained in the salt.

Yield: 450 mg. [Ru (bpy) ₂ (Fip)] (ClO ₂ ) ₂ in acetone / DMSO mixture was slowly evaporated to determine the X-ray crystal structure. Anal. Calcd for C ₄₆ H ₃₂ N ₈ Ru (%): C, 69.25; H, 4.04; N, 14.04. Found: C, 69.55; H, 4.24; N, 14.10. ^{1 H NMR (400 MHz, DMSO} -d 6) δ 14.36 (s, 1H, NH), 9.13 (d, J = 8.3 Hz, 2H), 8.86 (dd, J = 15.5, 8.2 Hz, 4H), 8.55 ( 2H), 7.72-7.55 (m, 3H), 8.16-7.89 (m, 7H), 7.85 (d, J = 5H), 7.51 - 7.38 (m , 2H), 7.35 (t, J = 6.7 Hz, 2H), 4.15 (s, 2H, CH 2). ^{13 C NMR (600 MHz, DMSO} -d 6): δ 36.94, 121.23, 121.77, 123.73, 124.95, 125.85, 126.13, 126.77, 127.00, 127.59, 128.21, 128.44, 138.41, 140.82, 143.80, 144.32, 144.50, 151.97, 153.62, 157.11. MALDI-TOF mass spectra: 797.10 (C ₄₆ H ₃₂ N ₈ Ru), UV-Visible spectrum (CH ₃ CN): λmax 337 nm and 463 nm. HRMS (ESI) Calcd for C ₄₆ H ₃₂ N ₈ Ru: 797.8880, found: 797.8889.

Example 3 Synthesis of ruthenium complex 3 (RU-3): [Ru (bpy) ₂ (Ipna)] (ClO ₂ ) ₂

(200 mg, 0.51 mmol) and cis- [1, &lt; RTI ID = 0.0 &gt; Ru (bpy) ₂ Cl ₂ ]. 2H ₂ O (250 mg, 0.51 mmol) was mixed and dissolved in 40 ml of ethylene glycol. Then the reaction mixture at 120 ℃ under a N ₂ environment was refluxed for 7 hours. The completion of the reaction was confirmed by TLC. The reaction mixture was then cooled to room temperature and diluted with 100 ml of DI water. Saturated NaClO ₄ solution was added to the mixture, and the mixture was slowly stirred to obtain a dark reddish orange solid. The resulting crude product was filtered, washed with water, and the neutral alumina was fixed and purified twice by column chromatography using toluene / acetonitrile (20/80, v / v) as mobile phase solvent. The dark red band was collected and the solvent was concentrated under reduced pressure. Then the product was added slowly to a saturated (50%) NaClO ₄ solution ClO ₄ ^- was obtained in the salt.

Yield: 350 mg, Anal. Calcd for C ₄₅ H ₃₅ N ₉ Ru (%): C, 67.32; H, 4.39; N, 15.70. Found: C, 67.72; H, 4.45; N, 15.72. ^{1 H NMR (400 MHz, DMSO} -d 6) δ 14.37 (s, 1H), 9.16 (dd, J = 8.2, 1.3 Hz, 1H), 9.09 (dd, J = 8.3, 1.3 Hz, 1H), 9.05 - 8.16 (m, 2H), 8.16-7.99 (m, 5H), 7.98-8.98 (m, 1H), 8.87 (dd, J = 14.6, 8.2 Hz, 4H), 8.35-8.29 7.90 (m, 2H), 7.87 (d, J = 5.6 Hz, 2H), 7.68-7.56 (m, 6H), 7.35 (td, J = 7.9, 3.6 Hz, 3H), 2.96 (s, 6H). ^{13 C NMR (600 MHz, DMSO} -d 6): δ 45.21, 113.42, 121.21, 121.76, 124.93, 126.15, 127.64, 128.30, 129.33, 132.31, 138.48, 150.31, 152.00, 153.38, 157.10, and 157.32. MALDTI-TOF mass spectra: 802.2 ( C 45 H 35 N 9 Ru), UV-Visible spectrum (CH 3 CN): λmax 365nm and 464nm, HRMS (ESI) Calcd for C 45 H 35 N 9 RuClO 4: 903.2059, found : 903.2065.

The chemical structural formulas of the ruthenium complexes of the present invention prepared in Example 1-3 are shown in Table 1 below.

Example Chemical structural formula One

2

3

<Experimental Example 1> Evaluation of toxicity to cancer cells and cancer stem cells

To evaluate the toxicity of the Ru complex of the present invention to cancer cells and cancer stem cells, the following experiment was conducted.

MTT assay evaluated the toxicity of the Example 1-3 Ru complexes to cancer cells and cancer stem cells.

Specifically, the thiourea blue tetrazolium bromide (MTT) dye-based cytotoxicity assay was performed according to the manufacturer's protocol (Vybrant® MTT Cell Proliferation Assay Kit (V-13154), Thermofischer Scientific, USA)

MCF-7 and HCT-116 cells were used as cancer cells and CD44-positive MCF7 cells and CD133-positive HCT116 cells were used as CSCs, respectively.

Herein, the breast cancer cell line MCF-7 and the colon cancer cell line HCT-116 were purchased from Korean Cell Line Bank (KCLB, Seoul)

CDC-positive MCF7 cells and CD133-positive HCT116 cells, which are CSCs thereof, were isolated from MCF-7 and HCT-116 cell lines using CSC (cancer stem cell) markers. Specifically, MCF- (CD44 and CD133 microbead kit (MACS Miltenyi Biotec)) using the CD133 marker in the HCT-116 cell line.

Each cancer cell line or cancer stem cell prepared from the above-mentioned procedure was individually seeded in a 96-well plate and incubated in DMEM medium supplemented with 10% FBS at 37 占 폚 for 24 hours. Cells were then treated with various concentrations (0-100 [mu] M) of Example 1-3 Ru complex and incubated for 24 hours. The DMEM medium was then removed and the cells were incubated with 100 [mu] L of MTT solution for 3 hours at 37 [deg.] C. 75 μL of MTT solution was removed and 50 μL of DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured using a multiplot reader (Molecular Devices, Gemini XS). Cisplatin was used as a positive control, and the IC ₅₀ values of Example 1-3 and cisplatin for the cancer cell lines were calculated, and the results are shown in Table 2 below.

Treating compound IC ₅₀ ([mu] M) MCF-7 CD44 + MCF-7 HCR-116 CD133 + HCR-116 Example 1 8.9 ± 0.72 10.1 ± 1.03 11.9 ± 0.16 15.1 ± 0.13 Example 2 19.3 ± 2.84 27.67 + - 4.12 22.3 ± 1.32 30.6 ± 3.71 Example 3 67 ± 7.67 72.35 + - 9.17 65.6 ± 6.4 77.9 ± 4.26 Positive control group
(Cisplatin) 44.47 + 5.09 62.18 + - 8.48 39.45 + 5.16 59.34 + - 6.52

As can be seen from Table 2, cytotoxicity against cancer cells and cancer stem cells was observed in the order of Example 1> Example 2> cisplatin> Example 3.

Particularly, it was confirmed that the ruthenium metal complex of Example 1 had the highest toxicity to cancer cells and cancer stem cells. In Example 1, MCF-7, CD44 + MCF-7, HCT-116 and CD133 + HCT- The IC ₅₀ values were found to be 8.9 μM, 10.1 μM, 11.9 μM and 15.1 μM, respectively.

Compared with cisplatin, Example 1 exhibited an excellent cancer cytotoxic effect on MCF7 cell line and CD44 positive MCF7 cell with 5 to 6 times, and HCT116 cell line and CD133 positive HCT116 cell showed 3 to 4 times superior cancer stem cell killing effect The results are confirmed.

Therefore, the novel ruthenium complex according to the present invention has excellent cancer cell and cancer stem cell killing effect. As a result, it is possible to provide a pharmaceutical composition for prevention or treatment of cancer containing the active ingredient, And the novel ruthenium complex of the present invention shows an excellent killing effect on the cancer stem cell which is a main cause of drug resistance, and can be also useful as a chemotherapeutic drug used for the treatment or prevention of cancer have.

&Lt; Experimental Example 2 >

Since the efficacy of the successful chemotherapy of the drug depends on the uptake and accumulation in the intracellular organ, the following experiment was conducted in order to evaluate the cellular uptake of the ruthenium complex of the present invention.

<2-1> Evaluation of cell inflow of Example 1

To evaluate the cell uptake level of the Ru complex of the present invention, CD44-positive MCF-7 cells, which are cancer stem cells, were treated with 10 μM of Example 1 and then the intracellular fluorescence intensity of the ruthenium complex at 620 nm was measured using FACS , And a time interval (30, 60, 90 minutes). The results are shown in Fig. 1 (a).

1 (a), it can be seen that the fluorescence intensity of the ruthenium complex of Example 1 of the present invention significantly increased at the time of 30 minutes or more and reached the saturation state at the time point of 60 minutes. In Example 1 of the present invention, It was confirmed that MCF-7 was rapidly absorbed into CSC (cancer stem cells).

&Lt; 2-2 > Evaluation of cell inflow of Example 2 and Example 3

Experiments were carried out under the same conditions as in <2-1> above for CD44-positive MCF-7 cells (CSC), but the second or third embodiment of the present invention was used, and FACS analysis results are shown in FIG. 1 (b) . (Fig. 17).

As can be seen in FIG. 1 (b), the cell infiltration into the cord stem cells was also observed in Example 2 at the elapsed time of 90 minutes. However, compared with Example 2 or Example 3, (Permeability) is confirmed (Example 1> Example 2> Example 3).

Therefore, the Ru complex of the present invention is excellent in the influx of cancer stem cells into the cancer stem cells, and it is possible to induce the killing effect of cancer cells more excellently. As a result, it is possible to provide a pharmaceutical composition for preventing or treating cancer, May be useful as a chemotherapeutic adjuvant for the prevention or treatment of cancer.

<Experimental Example 3> Localization of Drug Subcellular Localization

In order to analyze the mechanism of action of the Ru complex of the present invention, an intracellular localization position analysis was performed, and cell-specific dyes were used in CD44 positive MCF-7 and HCT-116 cells CSC.

<3-1> Localization of intracellular localization of Example 1

CD133-positive HCT-116, and CD44-positive MCF-7 were placed in cover glasses and incubated at 37 ° C for 24 hours. The cells were then treated with 10 [mu] M of Inventive Example 1 ruthenium complex and cultured at 37 [deg.] C for 2 hours. After drug treatment, the cells were washed with 1X PBS and incubated with MitoTracker® Red CMXRos (Thermofischer scientific, USA) or ER-ID® Red (Enzo Life Sciences, Inc., USA) with Hoechst 33258 nuclear dye (Sigma Aldrich) ) For 30 minutes. Finally, the cells were fixed with 4% formaldehyde for 15 minutes at room temperature.

The fluorescence intensity of the tracker dye and the ruthenium complex in the present invention was observed using a confocal microscope (Nikon, Eclipse Ti-U / Yokogawa, CSU-X1) Respectively. (Figure 2A) (Figure 2B) (S20 and S21).

2 (a) and 2 (b), the overlapping confocal fluorescence microscope image clearly shows that the complex of Example 1 is mainly distributed in mitochondria and ER of cytoplasm of CD133-positive HCT-116. Also, it was confirmed that the CD44-positive MCF-7 effectively contained the complex of Example 1 in mitochondria and ER.

&Lt; 3-2 > Analysis of intracellular localization positions of Example 2 and Example 3

Similar to the above <3-1>, intracellular localization analysis was performed on CD44 positive MCF-7 for Example 2 or Example 3, and the results are shown in FIGS. 9 (a) and (b).

9 (a) and (b), Example 2 was found to be distributed relatively densely in ER than in mitochondria, but Example 3 showed uniform distribution in both ER and mitochondria.

From these results, it can be seen that the Ru complex of the present invention acts through apoptosis pathway mediating ER and mitochondria.

EXPERIMENTAL EXAMPLE 4 Evaluation of ROS Generation in Cancer Cells

In order to evaluate ROS production in the amyloid cells of the Ru complex of the present invention, the following experiment was conducted.

&Lt; 4-1 > Evaluation of ROS generation in Example 1

CSC (cancer stem cells) of MCF-7 and HCT-116 cells were seeded in 12-well culture plates and cultured overnight at 37 ° C, 5% CO ₂ . Cells were then treated for 6 hours with or without the Ru complex of the invention (treated with 1 μM, 5 μM, 10 μM of Example 1), incubated at 37 ° C. in a 5% CO ₂ environment Respectively. At the end of the treatment, the cells were fixed with 4% formaldehyde and incubated at room temperature for 10 minutes. After washing with 1 x PBS, the cells were treated with 20 [mu] M 2 ', 7'-dichlorofluorescein acetate (DCFDA) and incubated at room temperature for 15 min. The RO induced 2', 7'- Fluorescence intensity of fluorescein was measured at excitation and emission wavelengths of 495 and 529 nm, and the results are shown in Figures 3 (a) and (b).

3 (a) and 3 (b), it was confirmed that Example 1 of the present invention induces ROS production in concentration ratio (0, 1, 5 and 10 μM) in CSC (cancer stem cells). The oxidation-sensitive DCFH-DA produces green fluorescence when ROS is generated. FIG. 3 (b) is a bar graph showing the average fluorescence intensity of DCFH-DA staining in cells treated with 1 μM, 5 μM, and 10 μM concentration of Example 1, confirming that the fluorescence intensity was increased in proportion to concentration do.

&Lt; 4-2 > Evaluation of ROS generation in Examples 2 and 3

In the same manner as in the above <4-1>, the production of ROS was evaluated using CSC (cancer stem cells) of MCF-7 cells according to concentrations of Example 2 or Example 3. The results are shown in FIG. 10 (a) And (b).

10 (a) and 10 (b), it was confirmed that Example 2 and Example 3 induce ROS generation in a concentration-dependent manner, and it can be confirmed that maximum ROS generation is induced at 12 and 60 μM, respectively.

Therefore, in the cancer cells and the cancer stem cells, the ruthenium complex of the present invention significantly induces the production of ROS from the redox circulation, and from this, the oxidative stress and the apoptosis effect of the cells can be excellently attained. A pharmaceutical composition for preventing or treating cancer, and a chemotherapeutic adjuvant for the prophylactic or therapeutic treatment of cancer having drug resistance.

Experimental Example 5 High-content MPT, cytoplasmic Ca ²⁺  And caspase 3/7 screening assay

High - content analysis was performed to evaluate induction of mitochondrial permeability transition (MPTP), cytoplasmic calcium level and activation of caspase - 3 in the inner mitochondrial membrane, and the mechanism of apoptosis was analyzed based on this.

&Lt; 5-1 > The MPT of Example 1, cytoplasmic Ca ²⁺  And caspase 3/7 screening assay

The CSCs of HCT-116 and MCF-7 were cultured in 12-well culture flasks respectively and maintained in DMEM medium supplemented with 10% FBS for 24 hours at 37 ° C and 5% CO ₂ atmosphere. Next, the cells were maintained for 16 hours without or with the Ru complex of the invention Example 1 (1, 5 and 10 μM concentration) treatment. After washing with 1 x PBS buffer, the cells were incubated with a calcium indicator orange (Invitrogen, CA, USA) in a dark room for 45 minutes at room temperature. Cells were washed with 1X PBS and incubated with calcein-acetoxymethyl ester (Invitrogen, CA, USA) for 30 min at 37 ° C. The caspase-3 substrate was then added and the cells were incubated for 6 min at 37 [deg.] C in the dark. CoCl _{2 was} then added and incubated in a dark room at 37 ° C for 10 minutes. Finally, the cells were washed with 1X PBS and image analysis was performed using an acousto-optic modulation filter (AOTF) equipped with a microscope as previously described (Kim et al). Images were analyzed using commercially available software (MetaMorph, version 7.7, Molecular Devices, CA, USA) and the results are shown in Figures 4 (a) and (b).

Referring to Figures 4 (a) and (b), fluorescence emission of Calcine AM, Calcium Orange and Magic Red Caspase 3/7 was monitored at 525 nm, 580 nm and 630 nm, respectively, using AOTF. The green fluorescence image corresponds to the cell image of MPT, the orange fluorescence image represents cytoplasmic calcium, and the red fluorescence image represents caspase 3/7. The bar graph in (b) shows the mean fluorescence intensity of Calcine AM, Calcium Indicator Orange and Caspase 3/7.

MPTP was assessed by measuring the fluorescence intensity of calcein AM transferred to the mitochondria. The observed results show that the treatment of Example 1 significantly reduced the fluorescence intensity of calcein-AM accumulated in the mitochondria of CSC in a concentration-dependent manner compared to the control (p < 0.01). A sharp decrease in the fluorescence intensity of calcein-AM accumulated in the mitochondria was observed in the treatment of Example 1 at 2 [mu] M and saturate in the treatment of Example 1 at 4 [mu] M.

In addition, increased levels of cytoplasmic calcium and caspase-3 activation were observed in a concentration-dependent manner in response to ruthenium complex treatment.

From the above results, we can see the cell death mechanism of Example 1 in CSC of HCT-116 and MCF-7.

Cellular localization studies clearly showed that Example 1 complexes were mostly distributed in mitochondria and ER. Thus, the ROS induced by Example 1 can mediate calcium release from the ER, which may play a role in the activation of caspase-mediated cell death.

Increased ROS and cytoplasmic calcium are also involved in mitochondrial permeability transition (MPTP) formation. Formation of MPTP in the inner mitochondrial membrane leads to membrane depolarization, matrix swelling and rupture of the outer membrane. It causes total release of Cyt-c and subsequent activation of caspase-3. The high-content analysis performed in this experiment clearly showed that MPCTP formation, cytoplasmic calcium and active caspase 3 were increased in the CSC of HCT-116 and MCF-7 cells in proportion to the treatment of Example 1, .

&Lt; 5-2 > The MPTs of Examples 2 and 3, cytoplasmic Ca ²⁺  And caspase 3/7 screening assay

(CD44 (+) MCF-7) of MCF-7 were performed in the same manner as in <5-1> above and the results were shown in FIGS. 11 (a) and b).

11 (a) and (b), it was found that the cells of Example 2 and Example 3 also showed a cell death mechanism, but Example 1 was found to be most sensitive to the treatment concentration.

<Experimental Example 6> Expression of anti-apoptotic proteins inducing apoptosis

In order to evaluate the cancer cell killing effect upon treatment of the Ru complex of the present invention, the expression of apoptosis inducing or anti-apoptotic protein was analyzed.

Specifically, immunofluorescence analysis for Bcl-2, Bax, Bak and GRP78 was performed and performed as previously described (Ahn et al., 2014). Fluorescence images were obtained using an Olympus FLUOVIEW 500 confocal microscope.

Western blot analysis was also performed as previously described (Parthasarathy et al., 2013).

<6-1> Example 1 Expression Analysis of Apoptosis-Inducing Protein in Treated HCT-116 CSC

FIG. 5 shows the induction of apoptosis and expression of anti-apoptotic proteins in CD133-positive HCT116 cells treated with Example 1. FIG.

Figure 5A shows immunofluorescence analysis of Bcl-2 expression in control and HCT-116 CSC treated with Example 1. The protein level of Bcl-2 was further assessed by Western blotting and shown in Figure 5D.

As a result, it was confirmed that the expression of Bcl-2 was significantly inhibited by the treated CSC of Example 1 as compared with the control.

5B and 5C, on the other hand, show the expression of Bax and Bak in the CSC treated with the control and Example 1, respectively. The oligomer structure of Bax and Bak was observed in the treated cells of Example 1. However, oligomerization of Bax and Bak was not well observed in control cells. The apoptotic proteins Bax and Bak are generally present in the cytoplasm, but during apoptosis they oligomerize the mitochondrial outer membrane and cause MOMP.

That is, oligomerization of Bak / Bax leads to mitochondrial permeability (MOMP) which causes release of cytochrome c (Cyt c).

FIG. 5D shows Western blot analysis of Bak and Bax, showing that Bax and Bak levels were significantly increased in the CSC cells treated with Example 1 (p < 0.01) compared to the control.

Therefore, activation of Bak and Bax inducing apoptosis was observed according to the treatment of Example 1 of the present invention, and the oligomerization of Bak / Bax caused mitochondrial permeability (MOMP), which causes release of cytochrome c (Cyt c) Respectively. In the cytoplasm, Cyt c forms an apoptosome with protease activity factor 1 (Apaf1) and protease caspase-9. This polyphosphatase complex activates caspase-3 and caspase-7.

\ MOMP can not be returned for cell survival, and furthermore, the reduced anti-apoptotic Bcl-2 expression observed in the Example 1 treated cells maintains Cyt-c release from the mitochondria.

In addition, since activated Bax actively regulates calcium release in the ER, the transient overexpression of Bax results in the release of ER Ca ²⁺ with an increase in mitochondrial Ca ²⁺ and cytochrome c release, resulting in Apoptosis is induced, anti-apoptosis is suppressed, and a remarkable killing effect of cancer cells is exhibited.

From this, the ruthenium complex of the present invention induced the formation of MPTP and MOMP in the mitochondria of cancer cells and confirmed the effective intracellular cytotoxic mediated action of cancer cells.

&Lt; Experimental Example 7 >

Molecular modeling studies have been conducted to provide more information on how the Ru complexes of the invention bind to GRP 78.

First, two possibilities were considered for the docking of Ligand Example 1 with GRP 78:

1) Example 1 shows the ATPase domain binding of GRP 78; or

2) Binding to a dimer interface using amino acid residues of both monomer units A and B

Could not be accommodated in the ATP domain of the first scenario and successfully modeled with the dimer interface of GRP 78 in the second scenario as large as the size of the complex of Example 1.

The corresponding docking results are shown in FIG. 6 in a 3D view of the interaction of Example 1 with GRP 78.

The result of this computer simulation docking was confirmed to be the biological activity of the Ru complex of the present invention, as a GRP 78 inhibitor with potent anticancer activity.

ROS-induced oxidative processes induce structural changes in proteins and are therefore likely to be ubiquitinated. To confirm this, we used the Accelrys Discovery Studio (v 4.0) as the X-ray crystal structure of GRP 78 (PDB: As a result of the docking, Example 1 of the present invention intercalates the dimer structure of GRP-78 by forming strong noncovalent interaction with a specific amino acid residue of GRP-78 at the interface of the dimer structure of the protein.

The observed docking results imply that the ROS generated at the interface of Example 1 and at the GRP-78 interface could modify the structure of GRP-78 and subsequently modify the ubiquitin mediated fusion.

&Lt; Experimental Example 8 >

In order to evaluate the inhibitory effect of the novel Ru complex according to the present invention on the development of cancer resistance against the anticancer agent, the following experiment was conducted.

It has been reported that the resistance of cancer cells to an anticancer drug is induced through a pathway such as an increase in GAP-78 expression and an increase in activity of cancer stem cells (CSC), and thus the effect of the novel Ru complex of the present invention is evaluated .

<8-1> Plasmid preparation and transfection of GRP-78, CLU and ATR

Genomic DNA of control human samples was isolated using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, Calif.) According to the manufacturer's instructions.

GRP-78: forward (50 pmol; 5'-AAAGCTTTTATGAAGCTCTCCCTGGTGG-3 ') and

GRP-78: reverse (50 pmol; 5'-AGGATCCCTACAACTCATCTTTTTCTGCTG-3 ') or

CLU: forward (50 pmol; 5'-AAAGCTTATGCAGGTTTGCAGCCAGC-3 ') and

CLU: reverse direction (50 pmol; 5'-GGATCCTTTTATTTTATTTTCCTCACTCTCCT-3 ')

ATR: forward (50 pmol, 5'-AAGCTTATGGCCACGGCGGAGC-3 ') and

ATR: reverse (50 pmol; 5'-GGATCCTTTTATTTTATTTTCCTCACTCTCCT-3 ')

200 ng of DNA was amplified with a specific primer of &lt; RTI ID = 0.0 &gt;

It has the following temperature profile: 4 minutes at 95 占 폚; 95 DEG C for 30 seconds, 59 seconds for 66 DEG C for 30 seconds, 72 DEG C for 1 minute and 30 cycles; The last extension is at 72 ° C for 5 minutes.

The GRP78, CLU and ATR primers were designed to amplify the GRP78, CLU and ATR transcripts of each gene.

PCR amplification was confirmed by analysis of the 1.9 kb GRP78, 1.5 kb CLU and 850 bp ATR fragments using 1.2% agarose gel with standard molecular weight markers.

The PCR amplified GRP78, CLU and ATR fragments were restricted to HindIII / BamHI enzymes. The restricted GRP78, CLU and ATR fragments were cloned into pTag-YFP-C (4.7 kb; Evrogen, Moscow, Russia), pDs- Red2- N1 (4.7 kb; Clontech, USA) and pAC- USA).

The conjugated fusion gene vectors were individually transformed into E. coli E. coli DH5 [alpha] cells via chemical modification as proposed by the manufacturer (Invitrogen, Carlsbad, Calif.).

Transformants (pTag-YFP-GRP78-C, pDs-CLU-Red2-N1 and pAC-ATR-GFP1-N1) were selected on LB agar plates supplemented with kanamycin (30 ug / mL). The plasmid from the resistant colonies was isolated using a plasmid medium kit (Qiagen). Plasmid cloned with GRP78, CLU and ATR genes were co-transfected into HEK-293 cells.

Specifically, human embryonic kidney cells (HEK-293) were obtained from Korean Cell Line Bank (KCLB1, Seoul, Korea) and cultured in DMEM supplemented with 10% FBS. First, HEK-293 cells (5xlO ⁵ cells / well) were seeded in 24-well culture plates containing antibiotic-free DMEM and FBS for 24 hours. The next day, HEK-293 cells were co-transfected with plasmid DNA (GRP78, CLU and ATR) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Cell culture plates were maintained in a CO ₂ incubator at 37 &lt; 0 &gt; C for 6 hours. The cells were then washed and grown in culture in a CO ₂ incubator at 37 ° C for 48 hours. GRP78, and cultured as in Example 1 of 10 μM for 16 hours and the CLU ATR amplification cells from the 5% CO ₂ incubator 37 ℃. Then, fluorescence microscopic photographs of HEK-293 cells were observed for quantitative analysis. Data collection and quantitative analysis were performed as previously described (Naoghare et al., 2008).

<8-2> Quantitative RT-PCR

HEK293 cells were treated with Example 1 for 16 hours and then maintained in a DMEM medium at 37 ° C in a CO ₂ incubator. Total RNA was then extracted according to the manufacturer's protocol (Dynabeads MRNA Purification Kit, Invitrogen, USA). The integrity of the isolated RNA was assessed qualitatively by the ratio of absorbance at 260/280 nm and then qualitatively assessed by 1% agarose RNA gel electrophoresis.

The concentration of RNA was measured at 260 nm using Nanodrop (Thermo Scientific, U.S.A). Total RNA, cDNA specific for GAPDH and GRP78 were prepared using the QuantiTect Reverse Transcription Kit (Qiagen, USA) according to the manufacturer's protocol. Two picomole primers were used for amplification of the target m-RNA. GAPDH expression was used to determine the amount of the starting template of the sample, followed by quantitative PCR reaction.

The following primers were used: GAPDH sense CATGAGAAGTATGACAACAGCCT, antisense AGTCCTTCCACGATACCAAAGT; GRP78 sense TGCCGCTTTGCAGGTGTATT, antisense CCGATGCTCAGAGCTTTCTCC.

The SYBR green method was used to perform RT-PCR (SYBR green qPCR kit, Thermo Scientific, USA). RT-PCR was performed using an Applied Biosystems Real-Time PCR instrument (7300).

Data were normalized to GAPDH and analyzed by the 2 ^-ΔΔC T method.

<8-3> Quantum dot-antibody conjugation

Antibodies specific for GRP78 were conjugated to Q-dot 705 (Quantum Dot Conjugation Kit; Invitrogen, Carlsbad, Calif., USA). To initiate antibody and quantum dot conjugation, the antibody was first treated with dithiothreitol (DTT) to induce disulfide bond disruption and the antibody was converted to a reduced state. The antibody was then incubated with maleimide-functionalized Quantum dot for 1 hour at room temperature.

The conjugate was then treated with 2-mercaptoethanol to remove the maleimide group. Unconjugated quantum dot was eluted using the column provided in the kit.

The Q-dot antibody conjugate was diluted 1: 200 with 1% BSA for immunofluorescence analysis. The excitation and emission used for fluorescence analysis were 405/705 nmdl, respectively.

As a result, confocal analysis confirmed that the maximum of GRP-78 expression was superimposed within the ER region. In addition, it was confirmed that the expression of GRP78 decreased according to the concentration of Example 1. The lifetime of the ROS is in microseconds, and therefore the lifetime of the ROS is higher in the vicinity of the ruthenium complex of the present invention.

It can be concluded that the binding of GRP-78 with Example 1 can cause a structural change of GRP-78 leading to ubiquitin mediated degradation.

Western blot analysis also confirmed that the dose of Example 1 depended on the protein level of GRP-78 in a dependent manner.

The increase in expression of GRP-78 is found in CD44 + MCF-7 cells, and CD24-CD44 + Grp78 + cells have been reported to show better chemotherapy and tolerance than CD24-CD44 + cells in head and neck cancer cell lines. Likewise, knockdown of GRP78 has been reported to exhibit enhanced sensitivity to cisplatin and radiation therapy.

Thus, it is clear that the inhibitory effect of Example 1 on GRP-78 is a very useful property that reduces the likelihood of tumor recurrence in cancer treatment and overcomes the drug resistance of cancer.

Experimental Example 9 Human colon carcinoma xenograft model

In order to evaluate the cancer cell killing effect of the Ru complex of the present invention, the following experiment was conducted on a human colon carcinoma xenograft model.

This in vivo animal experiment was conducted with the approval of the Institutional Animal Care and Use Committee of Seoul National University and a 7-week-old Swiss female athymic mouse was purchased from Daehan Biolink (Seoul, Korea). All mice were administrated at Seoul National University College of Pharmacy (Seoul, Korea) without specific pathogens and maintained at 12 - 24 hours at 24 - 26 ℃.

CD133-positive HCT-116 cells (3 × 10 ⁶ cells) isolated from HCT-116 cell culture were mixed with 100 μl of LDEV-Free reducing growth factor matrix matrix (Geltrex; Thermofischer Scientific, USA) Lt; / RTI &gt; When the tumors reached a volume of 100 mm ³ , mice were divided into three groups of six mice each.

First, the control group was injected intraperitoneally with 1 x PBS containing 4% DMSO;

The other two groups of mice were intraperitoneally injected with Example 1 (Example 1 dissolved in DMSO and diluted with 1x PBS) at a dose of 0.03 mg / kg / body weight or 0.06 mg / kg / body weight daily.

Treatment was repeated for 2 weeks and the experiment was stopped when the tumor size of the control reached about 1500 mm ³ .

The tumor size and weight were monitored daily during treatment.

Tumor volume (mm ³ ) was calculated as follows:

Volume = (maximum diameter (mm)) (vertical diameter (mm)) ² x 0.5.

<9-1> In vivo anticancer activity of Example 1

As described above, the possibility of in vivo chemotherapy of the Example 1 complex for CD133 + HCT-116 cell xenotransplantation in athymic nude mice was investigated.

Figure 8A describes an experimental protocol for the xenotransplantation model. 0.03 mg or 0.06 mg of the Example 1 Ru complex / kg / body weight was administered daily to the experimental group during the 2-week treatment period, and as a result, the tumor weight and volume of the mouse group treated with Example 1 were compared with the control mice group It is confirmed that it is remarkably reduced (Fig. 8B and C).

Tumor weight was significantly reduced from 55% to 80% compared to the control (Fig. 8D).

In addition, the tumor weights in the control group, the experimental group (low dose) and the experimental group (high dose) were 3.2g, 1.6g and 1.15g, respectively, showing the remarkable anticancer efficacy of Example 1 (Fig. 8E).

Meanwhile, the mice of the experimental group treated in Example 1 maintained normal body weight during the whole treatment process, and no mortality was observed. As a result, it was confirmed that the Ru complex of the present invention was not toxic to normal cells.

In general, the failure of chemotherapy and the recurrence of cancer depend on the number of cancer stem cells (CSC) and its number. In particular, the superior anticancer activity of the inventive Ru complexes described above in respect of CSC is superior to that of new chemotherapeutic agents, &Lt; / RTI &gt;

The experimental mice treated in Example 1 showed a reduction in fluorescence intensity of 2X and 4X, respectively, in the low and high dose treated groups (Fig. 8G) compared to the control group, indicating that the Ru complex of the present invention, CSC &lt; / RTI &gt; and exhibits excellent cytotoxicity.

Therefore, the Ru complex of the present invention can be usefully used as an anticancer agent and an adjuvant (resistance-inhibiting composition) for existing chemotherapy. Furthermore, the Ru complex of the present invention confirmed the excellent in vivo treatment potential for the colon cancer xenograft model. As a result, it was found that the Ru complex of the present invention is excellent as an alternative to the conventional chitosan such as a platinum-based chemotherapeutic agent, It can be seen that

Claims (12)

A complex represented by the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

In Formula 1,
X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;
N is an integer from 0 to 5;
y is an integer from 1 to 3;
z is 0 to 3;
Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

, And

;
R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ and R ^3j is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano ,
Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.
The method according to claim 1,
The complex represented by the general formula (1) is a complex of the following general formula (1 '), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:
[Formula 1 ']

Here, R ¹ , X, and n are the same as defined in Formula 1.
A complex represented by the following formula (2), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
(2)

In Formula 2,
X is at least one member selected from the group consisting of Cl ^- , PF ₆ ^- , Br ^- , BF ₄ ^- , ClO ₄ ^- , CF ₃ SO ₃ ^- and SO ₄ ^-2 ;
N is an integer from 0 to 5;
y is an integer from 1 to 2;
z is 0 to 1;
Each of the ligands is independently a ligand selected from the group consisting of

,

,

,

,

,

, And

;
R ¹ is

or

ego;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are each independently hydrogen, substituted or unsubstituted C _1-6 branched or straight chain alkyl, or substituted or unsubstituted C _1-6 branched or straight- Wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano;

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f , R ^3g , R ^3h , R ³ⁱ , R ^3j and R ^3k is independently hydrogen, a substituted or unsubstituted C _1-6 branched Or substituted or unsubstituted C _1-6 branched or straight chain alkoxy wherein the substituted alkyl and substituted alkoxy are optionally substituted with one or more groups selected from the group consisting of halogen, oxo, nitro, and cyano &Lt; / RTI &gt;
Each of R ^4a , R ^4b , R ^4c , R ^4d , R ^4e , R ^4f , R ^4g and R ^4h is independently hydrogen, a substituted or unsubstituted C _1-6 branched or straight chain alkyl, Substituted C _1-6 branched or straight chain alkoxy wherein said substituted alkyl and substituted alkoxy are substituted with one or more substituents selected from the group consisting of halogen, oxo, nitro, and cyano.
The method of claim 3,
The complex compound represented by the general formula (2) is a complex compound of the following general formula (2 '), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:
[Formula 2 ']

Wherein R ¹ , X and n are the same as defined in the above formula (2).
As shown in Scheme 1 below,
Preparing a compound represented by the formula (4) from a compound represented by the formula (3);
A process for preparing a complex represented by the general formula (1) as set forth in claim 1, which comprises: preparing a complex represented by the general formula (1) from the compound represented by the general formula (4)
[Reaction Scheme 1]

In the above Reaction Scheme 1,
X, n, y, z, a ligand, R ¹ , R ^2a , R ^2b , R ^2c , R ^2d , R ^2e and R ^2f are as defined in formula (1)
As shown in Reaction Scheme 2 below,
Preparing a compound represented by the formula (6) from a compound represented by the formula (5);
A method for preparing a complex represented by the following formula (2), comprising: preparing a complex represented by the formula (2) from a compound represented by the formula (6)
[Reaction Scheme 2]

In the above Reaction Scheme 2,
X, n, y, z, Lig, R 1, R 2a, R 2b, R 2c, R 2d, R 2e and R ^2f are the same as defined in formula (2) the third term.
A pharmaceutical composition for preventing or treating cancer, which comprises the complex of claim 1 or 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
8. The method of claim 7,
The cancer may be selected from the group consisting of a cancer selected from the group consisting of a cancer selected from the group consisting of a caudal myxoma, an intrahepatic bile duct cancer, a hepatoblastoma, a liver cancer, a thyroid cancer, a colon cancer, a testicular cancer, a myelodysplastic syndrome, a glioblastoma, a oral cancer, a larynx cancer, a bacterial sarcoma, an acute lymphocytic leukemia, Cholangiocarcinoma, cholangiocarcinoma, chronic myelogenous leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, diffuse large B cell lymphoma, Barter's bulge, ovarian cancer, ovarian cancer, ovarian cancer cell, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, Cancer, bladder cancer, peritoneal cancer, pituitary cancer, adenocarcinoma, non-sinus cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, childhood leukemia, small bowel cancer, Malignant melanoma, malignant lymphoma, malignant mesothelioma, malignant melanoma, ovarian cancer, vulvar cancer, ureter cancer, urethral cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue tumor, Cancer of the uterus, uterine cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, uterine cancer, breast cancer, sarcoma, penile cancer, Cancer of the lungs, squamous cell carcinoma of the lung, squamous cell carcinoma of the lung, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid, vaginal cancer, spinal cord cancer, neuroendocrine tumor, pancreatic cancer, salivary cancer, Kaposi sarcoma, Paget's disease, Skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, and thymic carcinoma.
A composition for inhibiting anticancer drug resistance, which comprises the complex of claim 1 or 3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
A pharmaceutical composition for prevention or treatment of cancer comprising the composition for inhibiting anticancer drug resistance according to claim 9, and a chemotherapeutic agent.
11. The method of claim 10,
Wherein the chemotherapeutic agent is a platinum-based chemotherapeutic agent.
12. The method of claim 11,
Wherein said platinum-based chemotherapeutic agent is cisplatin, carboplatin, iproplatin, or oxaliplatin or a pharmaceutically acceptable salt thereof.

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