JP7838812B2 - Tetranuclear ligands, nucleic acid cleavage agents, anticancer agents, or tetranuclear metal complexes - Google Patents

Description

This invention relates to a tetranuclear ligand or a tetranuclear metal complex having such a tetranuclear ligand.

Cisplatin is a metal complex used clinically as a chemotherapy agent for cancer. Cisplatin exerts its anti-cancer effect by directly binding to the DNA of cancer cells and distorting the three-dimensional structure of the DNA. However, cisplatin can cause side effects such as vomiting and nephrotoxicity, and in recent years, cisplatin-resistant cancers have also been reported (Non-Patent Literature 1). Therefore, there is a need for chemotherapy drugs that can replace cisplatin.

For example, bleomycin binds to iron in cancer cells, activating oxygen and thereby cleaving DNA strands, thus inhibiting cancer cell proliferation (Non-Patent Literature 2). Bleomycin is widely used in clinical medicine as an excellent chemotherapeutic agent for squamous cell carcinomas of the human skin, head and neck, and cervix, as well as malignant lymphoma. However, bleomycin is a water-soluble glycopeptide antibiotic obtained from the actinomycete Streptomyces verticillus, and there is a need for a chemotherapeutic agent that can be obtained by a simple synthesis method that does not depend on microorganisms.

Furthermore, it has been reported that an iron complex of the N4Py ligand, which mimics _the active site of bleomycin, reacts with hydrogen peroxide ( _H₂O₂ ) to form an active species, which oxidatively cleaves DNA and exhibits anticancer activity (Non-Patent Literature 3). However, this iron complex exhibits high cleavage activity even in the absence _{of H₂O₂} , and therefore lacks selectivity between normal and cancer cells. Thus, there is a need to develop a metal complex that exhibits selectivity between normal and cancer cells.

Y. Kadoya, M. Hata, Y. Tanaka, A. Hirohata, Y. Hitomi and M. Kodera, Inorg. Chem. 2021, 60, 5474-5482. H. Umezawa, K. Maeda, T. Takeuchi, Y. Okami, J. Antibiot., 19 A, 200 (1966). Q. Li, M. G. P. Wijst, H. G. Kazemier, M. G. Rots, G. Roelfes, ACS Chem. Biol., 9, 1044-1051 (2014).

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.

The present invention aims to provide a tetranucleated ligand or tetranuclear metal complex that has minimal impact on normal cells and effectively cleaves DNA in cancer cells.

To improve cytotoxicity against cancer cells, (1) DNA oxidative cleavage activity and (2) improved cell delivery of the complex are necessary. In this invention, we have developed a tetranuclear copper complex with ligands that have been dimerized using various linkers and further substituted with substituents on the pyridyl group, thereby solving the problems. The tetranuclear ligand according to this invention is represented by the following chemical formula (I).

Here, (i) X is H, OMe, Cl, Br, I, Me, _NY2 , _CO2Y , or COYY', and (ii) Y, Y' is H or alkyl.

Furthermore, the tetranuclear metal complex according to the present invention is represented by the following chemical formula (IV).

Here, M is Cu, Fe, Zn, Co, Mn, Re, Ru, Rh, Pd, Pt, or Ce.

According to the present invention, a tetranucleated ligand or tetranuclear metal complex that has minimal impact on normal cells and effectively cleaves the DNA of cancer cells can be easily obtained.

This figure shows the ^1H NMR spectrum of the tetranuclear ligand according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear ligand according to the present invention. This figure shows the ^1H NMR spectrum of the tetranuclear ligand according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear ligand according to the present invention. This figure shows the ^1H NMR spectrum of the tetranuclear ligand according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear ligand according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear complex according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear complex according to the present invention. This figure shows the ESI-MS spectrum of the tetranuclear complex according to the present invention. This figure shows the dependence of the oxidative cleavage reaction of a tetranuclear metal complex according to the present invention on the complex concentration. This figure shows the dependence of the oxidative cleavage reaction of a tetranuclear metal complex according to the present invention on the complex concentration. This figure shows the dependence of the oxidative cleavage reaction of a tetranuclear metal complex according to the present invention on the complex concentration. This figure shows the dependence of the oxidative cleavage reaction of a tetranuclear metal complex according to the present invention on the complex concentration. This figure shows the dependence of the oxidative cleavage reaction of a tetranuclear metal complex according to the present invention on the complex concentration. This figure shows the cytotoxicity of the tetranuclear metal complex according to the present invention.

The embodiments of the present invention will be described below with reference to the attached drawings. However, these embodiments are provided to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which those skilled in the art appropriately substitute the configurations of the embodiments below are also included within the scope of the present invention.

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 possesses high nucleic acid cleavage activity, and have completed the present invention based on this fact.

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.

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

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.

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

Furthermore, the inventors have discovered, as a new finding, that the tetranuclear metal complex shown in the following formula possesses 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.

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

In this invention, tetranuclear metal complexes represented by the following chemical formulas (V) or (VI) are preferred.

In the above, the nucleic acid being cleaved is either DNA or RNA.

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, and therefore _{the H₂O₂} concentration in cancer cells is higher than in normal cells. Furthermore, due to homeostasis, the concentration of reducing substances is also high in cancer cells. 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} and 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. Furthermore, the tetranuclear metal complex according to the present invention has _{high H₂O₂} activation ability because it binds _H₂O₂ at two locations, two of each of the four metal ions (for example, four copper ions ₎ . Therefore, even when used in vivo, it reacts with trace amounts of hydrogen peroxide and exhibits high nucleic acid cleavage activity. Moreover, after the four metal ions (for example, four copper ions) are reduced by reacting with the tetranuclear metal complex reducing substance according to the present invention, reactive oxygen species are generated by activating oxygen molecules. Because it has four metal ions, it can easily provide the three electrons necessary for the activation of oxygen molecules even inside cells, easily generating hydroxyl radicals HO•, which exhibit high nucleic acid cleavage activity.

The tetranucleated ligands and tetranuclear metal complexes according to the present invention can be used for various cancers, and are not particularly limited. For example, they can be used in 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 sarcoma, skin cancer, malignant melanoma, malignant lymphoma, tongue cancer, myeloma, thyroid cancer, metastatic skin cancer, and cutaneous melanoma.

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, for example, as an oral preparation such as injection, capsule, tablet, granule, powder, pill, or fine granule, or as a rectal preparation, oily suppositories, or aqueous suppositories.

Various formulations can be prepared by appropriately adding pharmacologically acceptable additives, such as excipients, binders, lubricants, disintegrants, surfactants, and flow enhancers. 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 esters, and polysorbate 80; and flow enhancers include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

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. 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.

(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 (H ₂ L1)
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 rotor. 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, CDCl ₃ ); 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, CH ₂ ), 4.59 (s, 8H, CH ₂ ), 3.50-3.68 (m, 24H, CH ₂ )

(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 (H ₂ L2)
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 × 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.
¹ H NMR (500 MHz, CDCl ₃ ); 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, CH ₂ ), 4.60 (s, 8H, CH ₂ ), 3.51-3.68 (m, 24H, CH ₂ )

(3) Synthesis of tetranuclear ligands ( _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 a 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, CDCl ₃ ); 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, CH ₂ ), 4.52 (s, 8H, CH ₂ ), 3.42-4.05 (m, 48H, CH _, CH ₂ )

(4) Synthesis of tetranuclear copper complexes
(4-1) Synthesis of tetranuclear copper complex [Cu ₄ (μ-OAc) ₄ (L1)](OAc) ₂ (1)
A rotor was placed in a 100 mL round-bottom flask, and Cu ^II ( _CH3COO ) ₂ (16.5 mg, 90.0 μmol) dissolved in _CH3CN (1 mL) was added. Then, _H2L1 (34.5 mg, 22.0 μmol) dissolved in _CH3CN (0.3 mL) was slowly added using a Pasteur pipette, and the solution turned 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 filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 7.

(4-2) Synthesis of tetranuclear copper complex [Cu ₄ (μ-OAc) ₄ (L2)](OAc) ₂ (2)
A rotor was placed in a 100 mL round-bottom flask, and Cu ^II ( _CH3COO ) ₂ (11.6 mg, 63.5 μmol) dissolved in _CH3CN (1 mL) was added. Then, _H2L2 (25.0 mg, 15.9 μmol) dissolved in _CH3CN (0.3 mL) was slowly added using a Pasteur pipette, and the color of the solution changed 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 filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 8.

(4-3) Synthesis of tetranuclear copper complex [Cu ₄ (μ-OAc) ₄ (L3)](OAc) ₂ (3)
A rotor was placed in a 100 mL round-bottom flask, and Cu ^II ( _CH3COO ) ₂ (8.44 mg, 48.5 μmol) dissolved in _CH3CN (1 mL) was added. Then, _H2L3 (20.0 mg, 11.1 μmol) dissolved in _CH3CN (0.3 mL) was slowly added using a Pasteur pipette, and the color of the solution changed 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 filtered by suction using a Kiriyama funnel to obtain a green solid. The ESI-MS spectrum is shown in Figure 9.

(5) Complex concentration dependence of oxidative cleavage reaction of tetranuclear metal complexes ₁ and 2 in the presence of _H₂O₂ Oxidative cleavage of DNA was performed for complexes 1 and 2 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.

These results are shown in Figures 10 and 11. Our laboratory has found that DNA is not cleaved at all in blank experiments with _{only H₂O₂} , and it was found that the cleavage activity of complexes 1 and 2 greatly increases depending on the concentration of the complex. Because complexes 1 and 2 have two dinuclear structures, two copper ions in each bind to the oxygen atoms _{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.

(6) Complex concentration dependence of oxidative cleavage reaction of tetranuclear copper complexes 1-3 in the presence of AscNa Oxidative cleavage of DNA was performed for complexes 1-3 under the following conditions. For this measurement, a solution was 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 the measurement was performed at 37°C.

These results are shown in Figures 12, 13, and 14. In blank experiments using only AscNa in the absence of complex 1-3, no DNA cleavage occurred. On the other hand, we found that complex 1-3 significantly accelerated DNA cleavage as its concentration increased. The two dinuclear copper atoms in complex 1-3 are reduced by AscNa, then activate oxygen molecules to generate hydroxyl radicals (HO•) as reactive oxygen species. Because complex 1-3 contains two dinuclear copper atoms, it can utilize reducing substances present in cancer cells to easily reduce oxygen molecules by three electrons, even at low concentrations, generating hydroxyl radicals (HO•). This is thought to selectively induce apoptosis in cancer cells through DNA double-strand breaks or endoplasmic reticulum stress responses.

(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 [Cu ₂ (μ-OAc) ₂ (bdpamide)](OAc), and Hbdpamide is represented by the following formula.

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 resulting formalin dye was extracted. After extraction, the absorbance at 570 nm was measured using a spectrophotometer to obtain the data. The 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 within cells, and the generation of the reactive oxygen species hydroxyl radical HO• is thought to be easier than in dinuclear complex 4. This is 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 its uptake into cells. In addition, the increased rate of oxygen molecule activation due to the electronic effect of the 4-position MeO group is thought to have 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. Tetranuclear complex 3 was found to be 6 times and 9 times more cytotoxic to lung and pancreatic cancer cells compared to normal cells, respectively.

It can be used as an anti-cancer drug.

Claims (6)

A tetranuclear ligand characterized by being represented by the following chemical formula (II) or (III).
A nucleic acid cleavage agent characterized by having the tetranucleating ligand described in claim 1 . An anticancer agent characterized by having the tetranucleated ligand described in claim 1 . A tetranuclear metal complex characterized by being represented by the following chemical formula (V) or (VI).
A nucleic acid cleavage agent characterized by having the tetranuclear metal complex described in claim 4 . An anticancer agent characterized by having the tetranuclear metal complex described in claim 4 .