Tetranuclear ligands, nucleic acid cleavage agents, anticancer agents, or tetranuclear metal complexes

Abstract

To provide a tetranuclear metal complex that can be easily synthesized and has effective anticancer action.SOLUTION: The present invention provides a tetranuclear metal complex represented by chemical formula (V).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a tetranuclear ligand or a tetranuclear metal complex having the tetranuclear ligand, etc.

Background Art

[0002] There is cisplatin among the metal complexes clinically used as chemotherapeutic agents for cancer. Cisplatin shows an anticancer effect by directly binding to the DNA of cancer cells and distorting the three-dimensional structure of DNA. However, cisplatin may show side effects such as vomiting and nephrotoxicity, and in recent years, cisplatin-resistant cancer has also been reported (Non-Patent Document 1). Therefore, a chemotherapeutic agent to replace cisplatin is required.

[0003] For example, bleomycin binds to iron in cancer cells to activate oxygen, thereby cleaving DNA strands and suppressing the growth of cancer cells (Non-Patent Document 2). Bleomycin is widely used in clinical medicine as an excellent chemotherapeutic agent for squamous cell carcinoma such as human skin, head and neck, and cervical cancer, and malignant lymphoma. However, bleomycin is a water-soluble glycopeptide antibiotic obtained from the actinomycete Streptomyces verticillus, and a chemotherapeutic agent obtained by a simple synthesis method that does not depend on microorganisms is required.

[0004] Also, for example, an iron complex of an N4Py ligand that mimics the active center of bleomycin has been reported to react with hydrogen peroxide (H2O2) to form an active species, which oxidatively cleaves DNA and shows anticancer activity (Non-Patent Document 3). However, since this iron complex has high cleavage activity even under conditions where H2O2 is not present, it does not have selectivity between normal cells and cancer cells. Therefore, the development of a metal complex having selectivity between normal cells and cancer cells is required.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] Cancer cells have higher concentrations of H2O2 and reducing substances than normal cells. Therefore, if we can synthesize metal complexes that exhibit cancer cytotoxicity, we can use them to develop anticancer drugs with fewer side effects that selectively kill cancer cells. We found that a dinuclear copper complex with a dinuclear ligand, in which dpa is introduced at the 2,6 positions of p-cresol via amide groups, significantly accelerates DNA oxidative cleavage upon activation by hydrogen peroxide or oxygen molecule activation using sodium ascorbate (AscNa) as a reducing agent. However, this complex had low cytotoxicity and needed improvement.

[0007] The present invention aims to provide a tetranucleated ligand or tetranuclear metal complex that has little effect on normal cells and effectively cleaves the DNA of cancer cells. [Means for solving the problem]

[0008] In order to improve the cytotoxicity against cancer cells, (1) DNA oxidative cleavage activity, (2) improvement of cell introduction of the complex, etc. are required. In the present invention, a tetranuclear copper complex of a ligand dimerized using various linkers and having a substituent introduced into the pyridyl group was developed to solve the problems. The tetranuclear ligand according to the present invention is represented by the following chemical formula (I).

[0009] [Chemical formula]

[0010] Here, (i) X is H, OMe, Cl, Br, I, Me, NY2, CO2Y or COYY', and (ii) Y and Y' are H or alkyl.

[0011] Further, the tetranuclear metal complex according to the present invention is represented by the following chemical formula (IV).

[0012] [Chemical formula]

[0013] Here, M is Cu, Fe, Zn, Co, Mn, Re, Ru, Rh, Pd, Pt or Ce. [Advantages of the Invention]

[0014] According to the present invention, a tetranuclear ligand or a tetranuclear metal complex that has little effect on normal cells and has an accurate DNA cleavage action on cancer cells can be easily obtained. [Brief Description of the Drawings]

[0015] [Figure 1] It is a diagram showing the 1H NMR spectrum of the tetranuclear ligand according to the present invention. [Figure 2] It is a diagram showing the ESI-MS spectrum of the tetranuclear ligand according to the present invention. [Figure 3] It is a diagram showing the 1H NMR spectrum of the tetranuclear ligand according to the present invention. [Figure 4]It is a diagram showing the ESI-MS spectrum of the tetranuclear ligand according to the present invention. [Figure 5] It is a diagram showing the 1H NMR spectrum of the tetranuclear ligand according to the present invention. [Figure 6] It is a diagram showing the ESI-MS spectrum of the tetranuclear ligand according to the present invention. [Figure 7] It is a diagram showing the ESI-MS spectrum of the tetranuclear complex according to the present invention. [Figure 8] It is a diagram showing the ESI-MS spectrum of the tetranuclear complex according to the present invention. [Figure 9] It is a diagram showing the ESI-MS spectrum of the tetranuclear complex according to the present invention. [Figure 10] It is a diagram showing the complex concentration dependence of the oxidative cleavage reaction of the tetranuclear metal complex according to the present invention. [Figure 11] It is a diagram showing the complex concentration dependence of the oxidative cleavage reaction of the tetranuclear metal complex according to the present invention. [Figure 12] It is a diagram showing the complex concentration dependence of the oxidative cleavage reaction of the tetranuclear metal complex according to the present invention. [Figure 13] It is a diagram showing the complex concentration dependence of the oxidative cleavage reaction of the tetranuclear metal complex according to the present invention. [Figure 14] It is a diagram showing the complex concentration dependence of the oxidative cleavage reaction of the tetranuclear metal complex according to the present invention. [Figure 15] It is a diagram showing the cytotoxicity of the tetranuclear metal complex according to the present invention.

Mode for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating the understanding of the principle of the present invention, and the scope of the present invention is not limited to the following embodiments. Other embodiments in which those skilled in the art appropriately substitute the configurations of the following embodiments are also included in the scope of the present invention.

[0017] As a result of diligent research, the inventors have discovered, as a new finding, that the tetranuclear metal complex of the tetranuclear ligand shown in the following formula has high nucleic acid cleavage activity, and have completed the present invention based on this fact.

[0018] [ka]

[0019] Here, (i) X is H, OMe, Cl, Br, I, Me, NY2, CO2Y, or COYY', and (ii) Y, Y' is H or alkyl. Also, n is between 1 and 8.

[0020] Furthermore, the tetranucleating ligand shown in the following formula also possesses high nucleic acid cleavage activity. n is between 1 and 8.

[0021] [ka]

[0022] Here, (i) X is H, OMe, Cl, Br, I, Me, NY2, CO2Y, or COYY', and (ii) Y, Y' is H or alkyl. Also, n is between 1 and 8.

[0023] In the present invention, a tetranuclear ligand represented by the following chemical formula (II) or (III) is preferred.

[0024] [ka]

[0025] [ka]

[0026] Furthermore, the inventors have discovered, as a new finding, that the tetranuclear metal complex shown in the following formula has high nucleic acid cleavage activity. Here, M is Cu, Fe, Zn, Co, Mn, Re, Ru, Rh, Pd, Pt, or Ce, and n is 1 to 8.

[0027] [ka]

[0028] Furthermore, the tetranuclear metal complex shown in the following formula also exhibits high nucleic acid cleavage activity. n is between 1 and 8.

[0029] [ka]

[0030] In the present invention, tetranuclear metal complexes represented by the following chemical formula (V) or (VI) are preferred.

[0031] [ka]

[0032] [ka]

[0033] In the above, the nucleic acid to be cleaved is either DNA or RNA.

[0034] Furthermore, the tetranuclear ligand and tetranuclear metal complex according to the present invention have high nucleic acid cleavage activity and can therefore be used, for example, as a tool for analyzing gene structure. In addition, the tetranuclear ligand and tetranuclear metal complex according to the present invention can cleave the nucleic acids of cancer cells and can therefore be used as anticancer agents. Normal cells have scavenging enzymes such as catalase, which can decompose hydrogen peroxide into water and oxygen, but cancer cells have almost no enzymes such as catalase and cannot decompose H2O2 like normal cells, resulting in a higher H2O2 concentration in cancer cells compared to normal cells. Furthermore, due to homeostasis, cancer cells also have a high concentration of reducing substances. The tetranuclear metal complex of the tetranuclear ligand according to the present invention can cleave nucleic acids simply by the reaction of the compound with H2O2 or reducing substances, and can specifically cleave the nucleic acids of cancer cells. Therefore, according to the present invention, there is little effect on normal cells. In addition, the tetranuclear metal complex according to the present invention binds H2O2 at two locations, two of the four metal ions (for example, four copper ions), so it has a high H2O2 activation ability. Therefore, even when used in vivo, it reacts with trace amounts of hydrogen peroxide to exhibit high nucleic acid cleavage activity. Furthermore, it reacts with the tetranuclear metal complex reducing substance according to the present invention to reduce four metal ions (for example, four copper ions), and then activates oxygen molecules to generate reactive oxygen species. Because it has four metal ions, it can easily provide the three electrons necessary for the activation of oxygen molecules even within cells, easily generating hydroxyl radicals HO·, which exhibit high nucleic acid cleavage activity.

[0035] The tetranucleated ligands and tetranuclear metal complexes according to the present invention can be used for various cancers, and are not particularly limited, but can be used for the treatment of colorectal cancer, gastric cancer, esophageal cancer, colon cancer, liver cancer, pancreatic cancer, breast cancer, lung cancer, gallbladder cancer, bile duct cancer, biliary tract cancer, rectal cancer, ovarian cancer, uterine cancer, kidney cancer, bladder cancer, prostate cancer, osteosarcoma, brain tumor, leukemia, myasthenia, skin cancer, malignant melanoma, malignant lymphoma, tongue cancer, myeloma, thyroid cancer, metastatic skin cancer, cutaneous melanoma, and the like.

[0036] The administration method of the anticancer agent having a tetranuclear ligand and a tetranuclear metal complex according to the present invention is not particularly limited and may be administered orally or parenterally. Furthermore, it can be prepared in an appropriate dosage form depending on the administration method, and can be prepared in various formulations such as injections, capsules, tablets, granules, powders, pills, fine granules, rectal administration preparations, oily suppositories, aqueous suppositories, etc.

[0037] Various formulations can be prepared by adding pharmacologically acceptable additives, such as excipients, binders, lubricants, disintegrants, surfactants, and flow enhancers, as appropriate. Excipients include lactose, fructose, glucose, corn starch, and sorbitol; binders include methylcellulose, ethylcellulose, acacia gum, gelatin, hydroxypropylcellulose, and polyvinylpyrrolidone; lubricants include talc, magnesium stearate, and polyethylene glycol; disintegrants include starch, sodium alginate, gelatin, calcium carbonate, calcium citrate, dextrin, magnesium carbonate, and synthetic magnesium silicate; surfactants include sodium lauryl sulfate, soy lecithin, sucrose fatty acid ester, and polysorbate 80; and flow enhancers include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

[0038] The dosage of the anticancer agent having a tetranuclear ligand and a tetranuclear metal complex according to the present invention is appropriately determined considering the method of use, the patient's age, sex, the severity of symptoms, etc., but for example, for adults, it is 10 to 800 mg per day, preferably 100 to 200 mg, which can be administered once a day or in several divided doses. [Examples]

[0039] (1) Synthesis of 1,3-bis(N-(N-(8-(2,6-bis(N,N-bis(2-pyridylmethyl)carbamoyl)-1-hydroxy)-4-benzamide)-3,6-dioxaoctyl))benzamide (H2L1) Isophthalic acid (10.9 mg, 65.6 μmol) was dissolved in DMSO (1 mL) and placed in a 25 mL round-bottom flask with a stirrer. HATU (54.3 mg, 142 μmol) and DIPEA (42 μL) were added while stirring at 60°C. N-(8-amino-3,6-dioxaoctyl)-2,6-bis(N,N-bis(2-pyridylmethyl)carbamoyl)-1-hydroxy-4-benzamide (187.3 mg, 261 μmol) dissolved in DMSO (2 mL) was added dropwise, and the mixture was stirred overnight. After stirring, the mixture was separated with H2O (30 mL) and CH2Cl2 (50 mL x 3). The organic layer was removed, dehydrated with Na2SO4, and filtered through a Nutsche filter. After removing the filtrate under reduced pressure and vacuum drying, it was dissolved in the minimum amount of CHCl3 and purified by alumina column chromatography (gradient CHCl3 / MeOH from 1 / 30 to 1 / 5) and HPLC to obtain a colorless oily substance (34.5 mg, yield 34%). The NMR and ESI MS spectra are shown in Figures 1 and 2. 1 H NMR (500 MHz, CDCl3); δ / ppm: 8.44-8.54 (m, 8H, CH), 8.26 (s, 1H, CH), 7.96 (s, 4H, CH), 7.86 (d, J = 5.7 Hz, 2H, CH), 7.71 (t, J = 7.5 Hz, 4H, CH), 7.61 (t, J = 6.8 Hz, 4H, CH), 7.48 (d, J = 7.5 Hz, 4H, CH), 7.28 (s, 1H, CH), 7.19-7.26 (m, 8H, CH), 7.10-7.19 (m, 8H, CH, NH), 4.90 (s, 8H, CH2), 4.59 (s, 8H, CH2), 3.50-3.68 (m, 24H, CH2)

[0040] [ka]

[0041] (2) Synthesis of 1,4-bis(N-(N-(8-(2,6-bis(N,N-bis(2-pyridylmethyl)carbamoyl)-1-hydroxy)-4-benzamide)-3,6-dioxaoctyl))benzamide (H2L2) A 100 mL two-necked round-bottom flask containing N-(8-amino-3,6-dioxaoctyl)-2,6-bis(N,N-bis(2-pyridylmethyl)carbamoyl)-1-hydroxy-4-benzamide (90.8 mg, 126 μmol) was added to HATU (88.0 mg, 231.5 μmol) and DIPEA (30 μL), and then DMSO (8 mL) was added. Terephthalic acid (10.0 mg, 60.1 μmol) dissolved in DMSO (7 mL) was slowly added dropwise, and the mixture was stirred overnight. After monitoring the reaction by ESI-MS, the mixture was separated with H2O (30 mL) and CH2Cl2 (50 mL x 3). The organic layer was removed, dehydrated with Na2SO4, filtered through a Nutsche filter, and concentrated using a rotary evaporator. After vacuum drying, the solution was dissolved in the minimum amount of CHCl3 and purified by alumina column chromatography (gradient CHCl3 / MeOH from 1 / 30 to 1 / 5) and HPLC to obtain a colorless oily substance (47.0 mg, yield 24%). The NMR and ESI MS spectra are shown in Figures 3 and 4. 1H NMR (500 MHz, CDCl3); δ / ppm:8.44-8.54 (m, 8H, CH), 7.92 (s, 4H, CH), 7.80 (t, J = 5.7 Hz, 2H, NH), 7.75 (t, J = 7.4 Hz, 4H, CH), 7.65 (s, 4H, CH), 7.61 (t, J = 7.4 Hz, 4H, CH), 7.50 (d, J = 7.4 Hz, 4H, CH), 7.23-7.26 (m, 4H, CH), 7.12-7.22 (m, 8H, CH), 7.03 (t, J = 5.7 Hz, 2H, NH), 4.92 (s, 8H, CH2), 4.60 (s, 8H, CH2), 3.51-3.68 (m, 24H, CH2)

[0042] [ka]

[0043] (3) Synthesis of tetranuclear ligand (H2L3) HATU (31.1 mg, 82.0 μmol) dissolved in DMSO was reacted with isophthalic acid (6.20 mg, 37.0 μmol) for 30 minutes. Then, N-(8-Amino-3,6-dioxaoctyl)-2,6-di(N,N-bis(2-(4-methoxypyridyl)methyl)carbamoyl)-1-hydoroxy-4-benzamide (62.4 mg, 74.0 μmol) and DIPEA (78 μL) were added. After stirring overnight, the mixture was separated with H2O (30 mL) and CH2Cl2 (50 mL x 3). The organic layer was removed, dehydrated with Na2SO4, filtered through a Nutsche filter, and concentrated using a rotary evaporator. After vacuum drying, the substance was dissolved in the minimum amount of CHCl3 and purified using alumina column chromatography (gradient CHCl3 / MeOH from 1 / 30 to 1 / 5) and HPLC to obtain a colorless oily substance (21.0 mg, yield 16%). The NMR and ESI MS spectra are shown in Figures 5 and 6. 1 H NMR (500 MHz, CDCl3); δ / ppm: 8.20-8.56 (m, 8H, CH), 7.90 (bs, 4H, CH), 7.86 (d, J = 7.5 Hz, 2H, CH), 7.75 (s, 1H, CH), 7.28 (s, 1H, CH), 7.01 (bs, 4H, CH), 6.72 (bs, 4H, CH), 6.51-6.69 (m, 8H, CH), 4.84 (s, 8H, CH2), 4.52 (s, 8H, CH2), 3.42-4.05 (m, 48H, CH , CH2)

[0044] [ka]

[0045] (4) Synthesis of tetranuclear copper complexes (4-1) Synthesis of tetranuclear copper complex [Cu4(μ-OAc)4(L1)](OAc)2(1) Place the rotor in a 100 mL round-bottom flask and add Cu dissolved in CH3CN (1 mL). II (CH3COO)2 (16.5 mg, 90.0 μmol) was added, and then H2L1 (34.5 mg, 22.0 μmol) dissolved in CH3CN (0.3 mL) was slowly added using a Pasteur pipette, causing the solution to change color to green. After tracking the reaction by ESI-MS, the solution was concentrated using a rotary evaporator, and a small amount of Et2O was added, causing a green solid to precipitate. This was then filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 7.

[0046] (4-2) Synthesis of tetranuclear copper complex [Cu4(μ-OAc)4(L2)](OAc)2(2) Place the rotor in a 100 mL round-bottom flask and add Cu dissolved in CH3CN (1 mL). II(CH3COO)2 (11.6 mg, 63.5 μmol) was added, and then H2L2 (25.0 mg, 15.9 μmol) dissolved in CH3CN (0.3 mL) was slowly added using a Pasteur pipette, causing the solution to change color to green. After tracking the reaction by ESI-MS, the solution was concentrated using a rotary evaporator, and a small amount of Et2O was added, causing a green solid to precipitate. This was then filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 8.

[0047] (4-3) Synthesis of tetranuclear copper complex [Cu4(μ-OAc)4(L3)](OAc)2(3) Place the rotor in a 100 mL round-bottom flask and add Cu dissolved in CH3CN (1 mL). II (CH3COO)2 (8.44 mg, 48.5 μmol) was added, and then H2L3 (20.0 mg, 11.1 μmol) dissolved in CH3CN (0.3 mL) was slowly added using a Pasteur pipette, causing the solution to change color to green. After tracking the reaction by ESI-MS, the solution was concentrated using a rotary evaporator, and a small amount of Et2O was added, causing a green solid to precipitate. This was then filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 9.

[0048] (5) Complex concentration dependence of the oxidative cleavage reaction of tetranuclear metal complexes 1 and 2 in the presence of H2O2 For complexes 1 and 2, oxidative cleavage of DNA was performed under the following conditions. For this measurement, a solution was prepared with [NaCl] = 10 mM, [buffer] = 10 mM (pH 6.0 (MES)), [complex] = 0-30 μM, [pUC19 DNA] = 50 μM bp, and [H2O2] = 50 μM, and the measurement was performed at 37°C.

[0049] These results are shown in Figures 10 and 11. Our laboratory has found that DNA is not cleaved at all in blank experiments with H2O2 alone, and it was found that the cleavage activity of complexes 1 and 2 greatly increases depending on the concentration of the complex. Since complexes 1 and 2 have two dinuclear structures, two copper ions in each bind to the oxygen atom of H2O2, easily forming two dinuclear copper hydroperoxo complexes. Therefore, it is thought that the complexes can easily cleave double strands of DNA even at low concentrations.

[0050] (6) Complex concentration dependence of the oxidative cleavage reaction of tetranuclear copper complexes 1-3 in the presence of AscNa For complexes 1-3, oxidative cleavage of DNA was performed under the following conditions. For this measurement, solutions were prepared with [NaCl] = 10 mM, [buffer] = 10 mM (pH 6.0 (MES)), [complex] = 0-10 μM, [pUC19 DNA] = 50 μM bp, and [AscNa] = 150 μM, and measurements were performed at 37°C.

[0051] These results are shown in Figures 12, 13, and 14. In blank experiments using only AscNa in the absence of complexes 1-3, no DNA was cleaved at all. On the other hand, we found that complexes 1-3 significantly accelerated DNA cleavage as their concentration increased. The two dinuclear copper atoms in complexes 1-3 are reduced by AscNa, and then activate oxygen molecules to generate hydroxyl radicals HO· as reactive oxygen species. Because complexes 1-3 have two dinuclear copper atoms in their molecule, they can utilize reducing substances present in cancer cells to easily reduce oxygen molecules by three electrons even at low concentrations to generate hydroxyl radicals HO·, which is thought to selectively induce apoptosis in cancer cells through DNA double-strand breaks or endoplasmic reticulum stress responses.

[0052] (7) Cytotoxicity of tetranuclear complexes 1 and 3 The cytotoxicity of conventional dinuclear complex 4 and tetranuclear complexes 1 and 3 was evaluated by MTT assay. Conventional dinuclear complex 4 is [Cu2(μ-OAc)2(bdpamide)](OAc), and Hbdpamide is represented by the following formula.

[0053] [ka]

[0054] MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) taken up into cells is reduced by mitochondrial dehydrogenases to produce formazan dye. Since the amount of dye correlates with the number of metabolically active cells, the number of viable cells was measured by quantifying the dye using a colorimetric method (absorbance at 570 nm). HeLa cells treated with MTT / culture medium (0.25 mg / 0.5 mL) at 37°C for 180 minutes were placed in microtubes, and the generated formalin dye was extracted. After extraction, the absorbance at 570 nm was measured using a spectrophotometer to obtain data. These results are shown in Figure 15. The cytotoxicity of HeLa cells 24 hours after exposure to each complex was in the order of 3 > 1 >> 4. Because complexes 1 and 3 have a tetranuclear structure, the reaction in which oxygen molecules are reduced by three electrons after being reduced by a reducing substance proceeds smoothly even within cells, and it is thought that the generation of the reactive oxygen species hydroxyl radical HO· is easier than in dinuclear complex 4. This is the reason why the cytotoxicity of complex 1 is improved compared to complex 4. Furthermore, complex 3 showed higher cytotoxicity than complex 1. This is thought to be because complex 3 has a MeO group at the 4-position of the pyridyl group in its ligand, making it highly hydrophobic and increasing the amount taken up by cells. In addition, it is thought that the increased rate of oxygen molecule activation due to the electronic effect of the 4-position MeO group achieved high cytotoxicity. Regarding cancer cell selective toxicity, tetranuclear complex 1 was similar to dinuclear complex 4, but tetranuclear complex 3, which has a MeO group at the pyridyl group, showed a significant improvement, and it was found that tetranuclear complex 3 was 6 times and 9 times more cytotoxic to lung and pancreatic cancer cells compared to normal cells, respectively. [Industrial applicability]

[0055] It can be used as an anticancer drug.

Claims

1. A tetranuclear ligand characterized by being represented by the following chemical formula (II) or (III). 【Chemistry 1】 【Chemistry 2】

2. A nucleic acid cleavage agent characterized by having the tetranucleating ligand described in claim 1.

3. An anticancer agent characterized by having the tetranucleated ligand described in claim 1.

4. A tetranuclear metal complex characterized by being represented by the following chemical formula (V) or (VI). 【Transformation 3】 【Chemistry 4】

5. A nucleic acid cleavage agent characterized by having the tetranuclear metal complex described in claim 4.

6. An anticancer agent characterized by having the tetranuclear metal complex described in claim 4.

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