A cyclometalated iridium complex for inhibiting mitochondrial energy metabolism and a preparation method and application thereof

Abstract

This invention discloses a 2,2'-bipyridine compound, a cyclic iridium complex comprising this compound that inhibits mitochondrial energy metabolism, its preparation method, and its applications. The structural formula of the 2,2'-bipyridine compound is shown in Formula (I), and the structural formula of the cyclic iridium complex is shown in Formula (II). The cyclic iridium complex of this invention not only targets mitochondria and induces an increase in intracellular reactive oxygen species levels, but also reduces intracellular ATP levels and affects cellular energy supply processes, exhibiting high antitumor activity and photoresponsive properties. 50 The value is significantly lower than that of existing cisplatin drugs, reduced by approximately 10-100 times; under light conditions, the IC50 value is significantly lower. 50 The value further decreased by 3-5 times. After Ir2 treatment, the intracellular reactive oxygen species level significantly increased to 3.3 times that of the control group, and further increased by 8-9 times under light conditions; the intracellular ATP content decreased by 15-20%, and decreased by about 50% after light treatment; mitochondria showed changes such as swelling, irregular morphology, and reduced or disordered cristae structure. It can be used to prepare anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of chemistry, and specifically relates to a cyclic metal iridium complex that inhibits mitochondrial energy metabolism, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human life and health, characterized by high incidence, high mortality, and difficulty in treatment, and has become one of the most pressing public health problems worldwide. Tumor cells have high energy requirements during rapid proliferation; their growth and survival depend on a continuous intracellular energy supply, especially the maintenance of ATP levels. Therefore, interfering with the energy supply of tumor cells has become one of the important strategies in anti-tumor research.

[0003] Mitochondria are key organelles for energy production within cells, playing a crucial role in maintaining normal cellular physiological functions primarily through related biological processes that contribute to ATP generation. In recent years, targeting mitochondria to regulate their function and thus influence intracellular ATP levels has emerged as a promising anti-tumor therapeutic approach. Directly delivering functional molecules to mitochondria can help improve their efficiency within cells and enhance their inhibitory effect on tumor cells.

[0004] Iridium complexes have attracted widespread attention in the field of antitumor therapy due to their unique antitumor mechanisms and rich structural regulatory properties. Meanwhile, cyclic iridium complexes have shown certain advantages in cellular uptake and subcellular organelle targeting. However, there are still relatively few cyclic iridium complexes that target mitochondria and effectively reduce intracellular ATP levels in current technologies, and their structural design and functional realization require further optimization. Therefore, it is necessary to develop a cyclic iridium complex that can target mitochondria and reduce intracellular ATP levels to expand its application in the antitumor field. Summary of the Invention

[0005] This invention provides a cyclic iridium metal complex that inhibits mitochondrial energy metabolism, its preparation method, and its application, thereby overcoming the shortcomings of existing compounds in regulating intracellular ATP levels.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a 2,2'-bipyridine compound, chemically named (4'-methyl-[2,2'-bipyridine]-4-yl)methyl 6,8-bis(benzylthio)octanoate valerate, with the structural formula shown in Formula (I):

[0007] The preparation method of 2,2'-bipyridine compounds includes the following steps: (a) 6,8-bis(benzylthio)octanoic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, condensing agent and catalyst react in dichloromethane or methanol;

[0008] (b) After the reaction, water was added, the organic phase was extracted, dried and concentrated, and then subjected to column chromatography to obtain the 2,2'-bipyridine compound.

[0009] Preferably, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), or O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU).

[0010] Preferably, the catalyst is 4-dimethylaminopyridine (DMAP) or triethylamine (Et3N).

[0011] More preferably, 6,8-bis(benzylthio)octanoic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, EDC and DMAP in a ratio of 4-7:3-6:4-6:1 are stirred in dichloromethane for 24 hours, the reaction solution is washed with water, the organic phase is concentrated, and column chromatography is performed.

[0012] Another object of the present invention is to provide a cyclic metallic iridium complex [Ir(NC)₂L]. + PF6 - Where L is (4'-methyl-[2,2'-bipyridin]-4-yl)methyl 6,8-bis(benzylthio)octanoate valerate, and NC is 2-phenylpyridine or 2-phenylquinoline, and its general structural formula (the anionic part is omitted) is shown in formula (II):

[0013] In formula (II) for or .

[0014] The above-mentioned cyclic iridium complex [Ir(NC)2L] + PF6 - The preparation method includes the following steps: (a) Under nitrogen protection, the bis(2-phenylpyridine)-dichloroiridium precursor or the bis(2-phenylquinoline)-dichloroiridium precursor and (4'-methyl-[2,2'-bipyridine]-4-yl)methyl 6,8-bis(benzylthio)octanoate valerate were added to a mixed solvent and refluxed at a molar ratio of 1:2-2.5;

[0015] (b) The reaction solution was evaporated to dryness, dissolved in methanol, and then ammonium hexafluorophosphate was added and stirred. The reaction solution was concentrated and recrystallized to obtain the cyclic metal iridium complex that inhibits mitochondrial energy metabolism of the present invention.

[0016] Preferably, the mixed solvent is a dichloromethane-methanol mixed solution with a volume ratio of 1:1.

[0017] Compared with the prior art, the cyclic iridium complex of the present invention has very high antitumor activity and photoresponsive properties. The cyclic iridium complex IC of the present invention... 50 The values ​​are significantly lower than those of existing cisplatin drugs (CDDP), and can be reduced by approximately 10-100 times in some cells. Under light conditions, the Ir1 and Ir2 of this invention have a significantly lower IC50 value for various tumor cells. 50 The value decreased further by 3-5 times.

[0018] The Ir2 of this invention can significantly increase the level of intracellular reactive oxygen species (ROS). After Ir2 treatment, the level of intracellular ROS increased significantly to 3.3 times that of the control group, and further increased by 8-9 times under light conditions. Ir2 can affect the ultrastructure of mitochondria. After treatment, mitochondria showed changes such as swelling, irregular morphology, and reduction or disorder of cristae structure, thereby disrupting the normal physiological function of tumor cells and enhancing the anti-tumor effect.

[0019] The Ir2 of this invention can significantly affect the cellular energy supply process and reduce intracellular ATP levels. After treatment, the ATP content decreases by approximately 15%–20%, and further decreases to approximately 50% under light conditions. This demonstrates that the cyclic iridium complex of this invention can interfere with the energy supply process of tumor cells, thereby inhibiting their growth and survival. Attached Figure Description

[0020] Figure 1 This is a diagram showing the mitochondrial targeting results of the cyclic iridium complex Ir2 prepared in this invention; Figure 2 This invention relates to the effect of the cyclic iridium complex Ir2 prepared in this invention on the reactive oxygen species level in tumor cells. Figure 3 This is a transmission electron microscope image showing the effect of the cyclic iridium complex Ir2 prepared in this invention on the morphology of tumor cells. Figure 4 The figure shows the effect of the cyclic iridium complex Ir2 prepared in this invention on intracellular ATP levels. Detailed Implementation

[0021] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the reagents or testing methods are conventional methods in the art.

[0022] Example 1 (1) Preparation of ligand L: 6,8-Bis(benzylthio)octanoic acid (0.31 g, 0.08 mmol), 4-hydroxymethyl-4'-methyl-2,2'-bipyridine (0.14 g, 0.7 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.19 g, 0.10 mmol), and 4-dimethylaminopyridine (0.01 g, 0.08 mmol) were stirred in dichloromethane for 24 hours. The reaction solution was washed with water, the organic phase was concentrated, and the solution was subjected to column chromatography to give a white solid in 82.5% yield.

[0023] The NMR spectral data are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 8.56 (d, J = 5.0 Hz, 1H), 8.44 (d, J J = 8.5, 4.2 Hz, 2H), 7.04 (d, J = 4.5 Hz, 1H), 5.11 (s, 2H), 3.56 (d, J = 3.2 Hz, 3H), 3.54 (d, J = 4.7 Hz, 2H), 3.38 (q, J = 7.0 Hz, 1H), 2.50 – 2.45 (m, 1H), 2.40 (t, J = 7.3 Hz, 3H),2.34 (s, 3H), 2.28 (t, J = 7.5 Hz, 2H), 1.62 (m, J = 7.3, 3.2 Hz, 3H), 1.47 (m, J = 14.5, 7.4 Hz, 3H), 1.42 – 1.32 (m, 3H), 1.11 (t,J = 7.0 Hz, 1H). 13 C NMR(101 MHz, Chloroform-d) δ 173.16, 156.48, 155.41, 149.43, 148.89, 148.51,146.19, 138.67, 138.53, 128.93, 128.89, 128.53, 128.49, 127.01, 126.97,125.02, 122.20, 121.96, 119.58, 64.43, 44.26, 36.43, 35.20, 34.47, 34.05,28.76, 26.18, 24.73, 21.30. ESI-MS: Theoretical value: m / z 571.24 [M+H] + Actual value: m / z 571.20 [M+H] + .

[0024] (2) Preparation of complex Ir1 Under nitrogen protection, [Ir(ppy)₂Cl]₂ (0.11 g, 0.10 mmol) and ligand L1 (0.12 g, 0.21 mmol) were reacted in a mixed solvent (V... 二氯甲烷 :V 甲醇 The reaction mixture was refluxed in a 1:1 solution for 7 hours, the solution was evaporated to dryness, dissolved in a small amount of methanol, and then ammonium hexafluorophosphate (0.07 g, 0.42 mmol) was added and stirred for 12 hours. The reaction solution was concentrated, dissolved again in dichloromethane, and the organic phase was washed with water. The organic phase was concentrated and added dropwise to diethyl ether, and the precipitate was formed. The precipitate was filtered and dried to give a yellow solid with a yield of 68.2%.

[0025] The NMR spectral data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.78 (d, J = 33.3 Hz, 2H), 8.26 (dd, J = 8.1, 4.3 Hz, 2H), 7.96 – 7.88 (m, 4H), 7.83 (d, J = 5.7 Hz, 1H), 7.70 (d, J = 5.6 Hz, 1H), 7.62 (d, J = 3.9 Hz, 3H), 7.53 (d, J = 5.6 Hz, 1H),7.33 – 7.11 (m, 15H), 7.01 (t, J= 7.5 Hz, 2H), 6.89 (t, J = 7.4 Hz, 2H), 6.19(t, J = 7.1 Hz, 2H), 5.32 (s, 2H), 3.68 (d, J = 8.1 Hz, 3H), 3.63 (s, 2H), 2.53(s, 4H), 2.42 (m, J = 9.2, 7.3, 4.7 Hz, 5H), 1.66 (m, J = 15.0, 7.4 Hz, 3H), 1.43 (m, J = 19.1, 6.8, 6.3 Hz, 5H). 13 C NMR (101 MHz, DMSO-d6) δ 173.06,167.36, 167.32, 155.96, 155.19, 152.09, 151.03, 150.97, 150.31, 149.95,149.61, 149.29, 144.32, 144.27, 139.29, 139.24, 139.21, 131.60, 131.54,130.74, 129.93, 129.29, 128.81, 128.77, 127.23, 127.19, 126.61, 126.22, 125.57, 124.41, 123.16, 122.74, 120.53, 63.63, 44.25, 35.56, 34.38, 34.23, 33.97, 33.60, 28.56, 25.99, 24.51, 21.46. ESI-MS: Theoretical value: m / z 1070.33 [M-PF6] + Actual value: m / z 1070.20 [M-PF6] + .

[0026] (3) Preparation of complex Ir2 Under nitrogen protection, [Ir(mppy)₂Cl]₂ (0.12 g, 0.10 mmol) and ligand L1 (0.12 g, 0.21 mmol) were reacted in a mixed solvent (V2). 二氯甲烷 :V 甲醇The reaction mixture was refluxed in a 1:1 solution for 7 hours. The solution was then evaporated to dryness, dissolved in a small amount of methanol, and then ammonium hexafluorophosphate (0.07 g, 0.38 mmol) was added and stirred for 12 hours. The reaction mixture was concentrated, dissolved again in dichloromethane, and the organic phase was washed with water. The concentrated organic phase was then added dropwise to diethyl ether, and the precipitate was formed. The precipitate was filtered and dried to give an orange solid with a yield of 70.6%.

[0027] The NMR spectral data are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.76 (s,1H), 8.35 – 8.22 (m, 2H), 8.11 – 7.98 (m, 2H), 7.95 – 7.85 (m, 1H), 7.82 –7.74 (m, 1H), 7.70 (s, 2H), 7.65 (d, J = 5.5 Hz, 1H), 7.56 (d, J = 5.6 Hz, 1H), 7.29 (d, J = 2.2 Hz, 2H), 7.28 (s, 2H), 7.26 (d, J = 4.5 Hz, 4H), 7.24 – 7.15(m, 4H), 7.03 – 6.90 (m, 2H), 5.62 (m, J = 9.0, 6.4, 4.5 Hz, 2H), 5.35 (s,2H), 3.67 (s, 2H), 3.64 (s, 2H), 2.55 (s, 4H), 2.42 (t, J = 6.5 Hz, 4H), 1.74– 1.58 (m, 2H), 1.45 (m, J = 19.6, 6.3 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ172.94, 170.25, 155.58, 154.79, 152.20, 151.58, 150.34, 147.79, 147.34,147.20, 146.28, 146.25, 140.91, 139.29, 139.21, 131.51, 131.34, 131.08,129.90, 129.68, 129.28, 128.80, 128.75, 128.20, 127.95, 127.21, 127.17,126.24, 125.44, 124.61, 124.43, 123.18, 122.29, 118.68, 63.44, 44.24, 35.56, 34.38, 34.23, 33.96, 33.54, 31.46, 28.55, 25.95, 24.47, 22.57, 21.24, 14.47. ESI-MS: Theoretical value: m / z 1170.36 [M-PF6] + Actual value: m / z 1171.40 [M-PF6] + .

[0028] Example 2: Cytotoxicity test of cyclic iridium complex Cytotoxicity experiments were performed on the cyclic iridium complexes Ir1 and Ir2 prepared in the examples to test their cytotoxicity in human ovarian cancer cells A2780, human cervical cancer cells HeLa, human lung cancer cells A549, and human embryonic kidney cells 293T, respectively, to evaluate their antitumor activity. The specific CCK8 experimental steps are as follows: (1) The four types of cells required for resuscitation (A2780, Hela, A549 and 293T) were cultured in fresh complete medium (DMEM medium + 10 vol% fetal bovine serum + 1 vol% penicillin-streptomycin) and passaged 3-4 times until the cells reached the logarithmic growth phase to start the experiment. (2) When the cells reach the logarithmic growth phase, seed 5000 cells / well into a 96-well plate and incubate at 37°C in a 5% CO2 incubator; (3) After the cells adhered to the wall, fresh culture medium containing different concentrations of iridium complex or CDDP was added to replace the original culture medium, and the mixture was gently mixed. The dark group was incubated in the dark for 48 hours; the light group was incubated for 12 hours and then treated with a 405 nm laser at 12 mw / cm². 2 Irradiate with high power for 3 minutes, then continue incubation for 36 hours; (4) After incubation for 48 hours, aspirate the original culture medium, add fresh complete culture medium containing 10 vol% CCK8 solution, mix gently, and continue incubation at 37 ℃ for 1-4 hours. Detect A using an enzyme-linked immunosorbent assay (ELISA) reader. 450 nm, calculate the cell proliferation inhibition rate, and determine the IC50. 50 Value (IC) 50 The value is the drug concentration at which the inhibition rate equals 50%, IC50. 50 (The higher the value, the lower the cytotoxicity); the experimental results are shown in Table 1.

[0029] Table 1. Effects of cyclic iridium complexes Ir1 and CDDP on IC50 in different cells. 50 Value (μM)

[0030] As shown in Table 1, the cyclic iridium complexes Ir1 and Ir2 prepared in this invention exhibit good inhibitory activity against various tumor cells, with IC50 values ​​of [missing information]. 50 The values ​​were significantly lower than those of the control drug CDDP, and could be reduced by approximately 10-100 times in some cells. Under light conditions, Ir1 and Ir2 significantly reduced the IC50 of various tumor cells. 50 The value further decreased by 3-5 times, indicating that the complex has very high antitumor activity and photoresponsive properties.

[0031] Correspondingly, the inhibitory effect on normal renal 293T cells was weaker than that on the four types of tumor cells, which may be related to the uptake efficiency of normal cells. Compared with tumor cells, normal cells have lower metabolic and uptake efficiency, meaning they may take up less iridium complexes into the cells, thus reducing the risk of IC50. 50 The value is too high.

[0032] Example 3: Mitochondrial targeting experiment of complex Ir2 The mitochondrial targeting experiments on the cyclic iridium complex Ir2 were performed using the following steps: (1) A549 cells in the logarithmic growth phase were subjected to 2×10⁻⁶ ion exchange rates. 4Cells were evenly seeded in 3 cm confocal culture dishes and cultured for 24 hours until they adhered. Then, 1 μM Ir2 was added and incubated for 1 hour. The culture medium was removed, and the cells were washed once with PBS. Complete culture medium containing 200 nM of commercial mitochondrial green fluorescent dye MitoTracker Deep Green (MTG, 200 nM) and commercial lysosomal green fluorescent dye LysoTracker Deep Green (LTG, 150 nM) was added and incubated for 30 minutes. The culture medium was removed, and fresh culture medium was added. The cells were then photographed under a confocal microscope. The excitation wavelengths were 405 nm for the complex, 490 nm for MTG, and 500 nm for LTG. The emission and receiver wavelengths were 570 ± 30 nm (Ir2), 510 ± 20 nm (MTG), and 510 ± 15 nm (LTG). (2) Organelle colocalization experiments further confirmed this. The experimental results are as follows: Figure 1 visible.

[0033] like Figure 1 As shown, Ir2 has a very high degree of overlap with commercial mitochondrial dyes, with a colocalization coefficient of 0.89. In contrast, Ir2 has a relatively low degree of overlap with commercial lysosomal dyes, with a colocalization coefficient of only 0.21. This indicates that the complex Ir2 has good mitochondrial targeting and can selectively accumulate in mitochondria after entering the cell.

[0034] Example 4: Effect of complex Ir2 on intracellular reactive oxygen species levels The experimental steps for investigating the effect of the cyclic iridium complex Ir2 on intracellular reactive oxygen species levels are as follows: (1) A549 cells were loaded with 3×10 4 Cells were evenly seeded per well in a 6-well plate and cultured for 24 hours until adherence. The original culture medium was then removed, and fresh culture medium containing Ir2 (0.1 μM) was added. The dark group was incubated in the dark for 24 hours, while the light group was incubated for 12 hours before being treated with a 405 nm laser at 12 mw / cm². 2 Irradiate with high power for 3 minutes, then continue incubation for 12 hours; (2) After incubation, cells were collected and tested using a reactive oxygen species detection kit (Beyotime S0033S).

[0035] like Figure 2 As shown, the intracellular reactive oxygen species (ROS) level significantly increased to about 3.3 times that of the control group after treatment with the cyclic iridium complex Ir2 of the present invention, and further increased to 8-9 times under light conditions, indicating that the complex Ir2 can significantly increase the intracellular ROS level.

[0036] Example 5: Effects of complex Ir2 on cell morphology The experimental steps for investigating the effect of the cyclic iridium complex Ir2 on cell morphology are as follows: (1) A549 cells were seeded in 60 mm culture dishes and cultured for 24 hours. After the cells were completely attached, the culture medium was replaced with fresh medium containing Ir2 (0.1 μM) and incubated for another 24 hours. (2) Collect cells and fix them with a solution containing 2.5% paraformaldehyde. Slice the fixed cells, stain them with uranium acetate and lead citrate, and observe and photograph them with a transmission electron microscope (JEM-2100plus).

[0037] like Figure 3 As shown, the control group exhibited intact mitochondrial structure and clearly ordered cristae arrangement. Treatment with the cyclic iridium complex Ir2 of this invention resulted in mitochondria showing changes such as swelling, irregular morphology, and reduced or disordered cristae structure, indicating that the complex Ir2 can affect the ultrastructure of mitochondria.

[0038] Example 6: Effect of complex Ir2 on intracellular ATP levels The experimental steps for investigating the effect of the cyclic iridium complex Ir2 on intracellular ATP levels are as follows: (1) A549 cells were loaded with 3×10 4 Cells were evenly seeded per well in a 6-well plate and cultured for 24 hours until adherence. The original culture medium was then removed, and fresh culture medium containing Ir2 (0.1 μM) was added. The dark group was incubated in the dark for 24 hours, while the light group was incubated for 12 hours before being treated with a 405 nm laser at 12 mw / cm². 2 Irradiate with high power for 3 minutes, then continue incubation for 12 hours; (2) After incubation, cells were collected and tested using an ATP assay kit (Beyotime S0026).

[0039] like Figure 4 As shown, compared with the control group, the intracellular ATP content decreased by 15-20% after treatment with the cyclic iridium complex Ir2 described in this embodiment of the invention, and decreased by about 50% after light treatment, indicating that Ir2 treatment can reduce intracellular ATP levels. These results demonstrate that the complex can reduce intracellular ATP levels, and this effect is significantly enhanced under light irradiation, indicating that it has a significant impact on cellular energy supply processes.

[0040] The cyclic iridium complex of the present invention can accumulate in mitochondria and induce an increase in intracellular ROS levels, reduce intracellular ATP levels and affect cellular energy supply processes, while exhibiting good inhibitory activity and photoresponsive activity against tumor cells.

Claims

1. A 2,2'-bipyridine compound, characterized in that, Its structural formula is shown in equation (Ⅰ): 。 2. A method for preparing the 2,2'-bipyridine compound according to claim 1, comprising: 6,8-bis(benzylthio)octanoic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, condensing agent and catalyst were added to dichloromethane or methanol in a molar ratio of 4-7:3-6:4-6:1 and reacted. The mixture was then washed with water, the organic phase was extracted, dried and concentrated, and column chromatography was performed to obtain the 2,2'-bipyridine compound.

3. A cyclic iridium complex that inhibits mitochondrial energy metabolism, the general structural formula of which is shown in formula (II): In formula (II) for or .

4. A method for preparing the cyclic iridium complex according to claim 3, comprising: adding a bis(2-phenylpyridine)-dichloroiridium precursor or a bis(2-phenylquinoline)-dichloroiridium precursor and the 2,2'-bipyridine compound according to claim 1 to a mixed solvent at a molar ratio of 1:2-2.5 and refluxing; then concentrating under reduced pressure, dissolving, and performing column chromatography.

5. The use of the cyclic iridium complex according to claim 3 in the preparation of antitumor drugs.

6. The application according to claim 5, wherein the tumor includes ovarian cancer, human cervical cancer, and lung cancer.

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