A mitochondrial DNA-targeted cyclometallated iridium complex and its preparation method and application

Abstract

The present invention discloses a mitochondrial DNA-targeted cyclometallated iridium complex, a preparation method, and an application thereof. The structural formula of the cyclometallated iridium complex is: #imgabs0#. Compared with the prior art, the cyclometallated iridium complex of the present invention has a high quantum yield, a large Stokes shift, a long fluorescence lifetime, and good photostability; has high selectivity for H2S, a fast response speed, and can be used as a fluorescent probe for H2S; can target mitochondrial DNA and simultaneously induce mitochondrial DNA damage; and can be used as a drug for photodynamic therapy of tumors.

Description

Technical Field

[0001] The invention belongs to the field of chemistry, and particularly relates to a preparation method of a mitochondrial DNA-targeted iridium complex and its anti-tumor application. Background Art

[0002] Mitochondria are important organelles in biological cells that are responsible for energy supply and regulating cell survival and death. They are also the only organelles besides the nucleus that have DNA. The main target of traditional platinum drugs is nuclear DNA. After entering the cell, they covalently bind to guanine on DNA, causing DNA damage and ultimately killing cancer cells. Since nuclear DNA has a powerful damage repair mechanism, it will greatly reduce the anti-tumor effect of the drug, but the mitochondrial DNA damage mechanism is relatively scarce and single. Therefore, drugs targeting mitochondrial DNA can, on the one hand, effectively circumvent the reduced efficacy caused by the nuclear DNA damage repair mechanism by inducing mitochondrial DNA damage. On the other hand, they can directly destroy the energy system of cancer cell mitochondria, generate reactive oxygen species and activate mitochondria-dependent cell death signaling pathways, thereby overcoming the drug resistance of traditional drugs.

[0003] H2S plays a crucial physiological role in the body, but its real-time quantitative detection remains a challenge. Developing biocompatible, highly targeted, and sensitive H2S fluorescent probes is crucial. Furthermore, H2S is a key species in maintaining intracellular redox homeostasis and can regulate intracellular GSH levels, with GSH depletion being a key trigger of ferroptosis. Furthermore, H2S is highly expressed in some tumor cells, offering a new avenue for designing and synthesizing tumor-specific drugs.

[0004] Iridium complexes are widely studied for their anti-tumor and bioimaging properties. Most iridium complexes have been shown to increase intracellular ROS levels, another key trigger of ferroptosis. Currently, a few iridium complexes have been shown to induce mitochondrial DNA damage and ferroptosis, but the relationship between mitochondrial DNA damage and ferroptosis remains unclear. Therefore, addressing the shortcomings of existing technologies, it is crucial to provide a tumor-specific mitochondrial DNA-targeting cyclometallated iridium complex to address these shortcomings. Summary of the Invention

[0005] The main purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a tumor-specific mitochondrial DNA-targeted iridium complex and its application as an anti-tumor drug.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The present invention provides a 2,2'-bipyridine compound, which serves as a ligand L of a cyclometallated iridium complex. The chemical name of the compound is (4'-methyl-[2,2'-bipyridine]-4-yl)methyl 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionate. The general structural formula of the ligand L is shown in formula (I):

[0008]

[0009] The preparation method of ligand L comprises the following steps:

[0010] (a) reacting 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, a condensing agent and a catalyst in a first solvent;

[0011]

[0012] (b) After the reaction, a second solvent is added for washing, and the organic phase is extracted, dried, concentrated, and subjected to column chromatography to obtain an off-white solid, which is ligand L.

[0013] Preferably, the first solvent is dichloromethane or chloroform; and the second solvent is water.

[0014] Preferably, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or dicyclohexylcarbodiimide (DCC) or 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU).

[0015] Preferably, the catalyst is 4-dimethylaminopyridine (DMAP) or triethylamine (Et3N).

[0016] More preferably, 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, EDC and DMAP in a stoichiometric ratio of 6:5:6:1 are stirred in dichloromethane for 24 hours, the reaction solution is washed with water, the organic phase is concentrated, and chromatographed to obtain an off-white solid, which is ligand L.

[0017] Another object of the present invention is to provide a cyclometalated iridium complex [Ir(N–C)2L]PF6, wherein L is (4'-methyl-[2,2'-bipyridyl]-4-yl)methyl 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionate, N–C=2-phenylpyridine (Ir1), N–C=4-(pyridin-2-yl)benzaldehyde (Ir2), and its general structural formula (the anion part is omitted) is shown in formula (II):

[0018]

[0019] In formula (II),

[0020] The preparation method of the cyclometalated iridium complex [Ir(N–C)2L]PF6 comprises the following steps:

[0021] (a) reacting a bis(2-phenylpyridine)-dichloroiridium precursor ([Ir(ppy)2]2Cl2) or a bis(2-phenylquinoline)-dichloroiridium precursor ([Ir(pba)2]2Cl2) and a ligand L of formula (I) in a mixed solvent under reflux;

[0022]

[0023] (b) After the reaction, the mixture was concentrated under reduced pressure, dissolved in methanol, and then recrystallized by adding ammonium hexafluorophosphate to obtain a yellow solid, which was the target complex.

[0024] Preferably, the mixed solvent is a dichloromethane-methanol solution, and the volume ratio of dichloromethane to methanol is 1:1.

[0025] Preferably, in step (b), after adding ammonium hexafluorophosphate, the mixture is reacted at room temperature overnight, concentrated, and redissolved in dichloromethane. The organic phase is washed with water, concentrated, and then added dropwise to ether to precipitate. The precipitate is filtered and dried to obtain a yellow solid, which is the target complex probe.

[0026] More preferably, a bis(2-phenylpyridine)-iridium dichloride or bis(4-(pyridin-2-yl)benzaldehyde)-iridium dichloride precursor and ligand L in a stoichiometric ratio of 1:2.2 are refluxed in a 1:1 dichloromethane-methanol mixed solvent, concentrated under reduced pressure after the reaction, dissolved in methanol, and then ammonium hexafluorophosphate is added. After the reaction, the mixture is concentrated and recrystallized to obtain a yellow solid, which is the target complex.

[0027] Another object of the present invention is to provide a cyclometallated iridium complex for use in mitochondrial DNA-targeted anti-tumor and photodynamic therapy. The cyclometallated iridium complex has a high phosphorescence quantum yield and strong resistance to photobleaching, enabling real-time monitoring of H2S changes within living cells. It exhibits high H2S sensitivity, enabling differentiation of tumor cells based on their intracellular H2S content, further enabling selective tumor treatment. Furthermore, it exhibits high mitochondrial DNA targeting and anti-tumor activity, enabling selective and efficient killing of target tumor cells.

[0028] Beneficial effects of the present invention:

[0029] The cyclometallated iridium complex of the present invention has the following advantages over conventional organic small molecules in the prior art in terms of H2S imaging and anti-tumor capabilities:

[0030] 1. The cyclometallated iridium complex of the present invention has diverse coordination configurations, is easy to synthesize, has high quantum yield, large Stokes shift and good photostability;

[0031] 2. After the cyclometalated iridium complex of the present invention reacts with H2S, the fluorescence at 540nm is significantly enhanced. Under the same conditions, there is almost no fluorescence response to other molecular ions, and the selectivity for H2S is high;

[0032] 3. The cyclometallated iridium complexes of the present invention can recognize different types of tumor cells and tumors;

[0033] 4. The cyclometallated iridium complex of the present invention can selectively bind to mitochondrial DNA;

[0034] 5. The cyclometallated iridium complex of the present invention can induce a significant downregulation of the ferroptosis marker protein GPX4, thereby triggering ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a graph showing the change in fluorescence intensity of the reaction between the complex Ir1 and H2S in Example 2;

[0036] Figure 2 is a graph showing the change in fluorescence intensity of the reaction between the complex Ir2 and H2S in Example 2;

[0037] Figure 3 is the fluorescence change after the complex Ir1 reacts with H2S and other molecular ions in Example 2;

[0038] Figure 4 is the fluorescence change after the complex Ir2 reacts with H2S and other molecular ions in Example 2;

[0039] Figure 5 This is the imaging of the complex Ir2 in Example 2 and H2S in different types of cancer cells;

[0040] Figure 6 This is the imaging of the complex Ir2 in Example 2 and H2S in different types of tumors;

[0041] Figure 7 This is the imaging of the complex Ir2 and intracellular DNA in Example 2;

[0042] Figure 8 This is the effect of the complex Ir2 in Example 2 on the expression level of cellular GPX4 protein. DETAILED DESCRIPTION

[0043] Example 1

[0044] (1) Preparation of ligand L:

[0045] 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionic acid (0.56 g, 1.8 mmol), 4-hydroxymethyl-4'-methyl-2,2'-bipyridine (0.3 g, 1.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.34 g, 1.8 mmol) and 4-dimethylaminopyridine (0.037 g, 0.3 mmol) were stirred in dichloromethane for 24 h. The reaction solution was washed with water, the organic phase was concentrated, and the product was purified by column chromatography to give an off-white solid with a yield of 83.8%.

[0046] 1 H NMR (500MHz, CDCl3) δ8.58(d,J=7.3Hz,1H),8.54(d,J=4.9Hz,1H),8.51(d,J=8. 0Hz,1H),8.48(d,J=4.9Hz,1H),8.38(d,J=8.4Hz,1H),8.24(s,1H),8.19(s,1H), 7.69(t,J=7.8Hz,1H),7.37(d,J=7.9Hz,1H),7.22(d,J=4.8Hz,1H),7.14(d,J=4. 8Hz,1H),5.19(s,2H),4.54(t,J=7.1Hz,2H),2.90(t,J=7.1Hz,2H),2.45(s,3H). 13C NMR (126MHz, CDCl3) δ170.98,163.83,163.37,156.37,155.36,149.19,148.92,148.16,145.58,143.56,132.28,131.82, 129.08,128.93,126.77,124.80,124.27,122.27,121.89,119.44,118.52,114.62,64.86,36.15,32.82,21.19.NMR(500M Hz, CDCl3),8.81(d,J=2.4Hz,1H),8.59(d,J=4.8Hz,1H),8.48(d,J=4.8Hz,1H),8.20(s,1H),8.16(s,1H),8.10-8.08(m,1 H),7.98-7.96(dd,J=2.4,2.4Hz,1H),7.71-7.66(m,3H),7.21-7.15(m,2H),6.97(d,J=9Hz,1H),5.27(s,2H),2.46(s,3H).

[0047] (2) Preparation of complex Ir1:

[0048] Under nitrogen protection, [Ir(ppy)2Cl]2 (0.11 g, 0.1 mmol) and ligand L (0.11 g, 0.22 mmol) were mixed in a solvent (V DCM :V MeOH =1:1) and refluxed for 5 h. The reaction solution was spin-dried and dissolved in methanol. Ammonium hexafluorophosphate (0.0732 g, 0.4 mmol) was then added and stirred at room temperature overnight. The reaction solution was concentrated and purified by column chromatography to obtain a yellow solid in a yield of 68.2%.

[0049] 1 H NMR(500MHz, CDCl3)δ9.73(s,2H),8.63(s,1H),8.60–8.51(m,3H),8.43(d,J=8.4Hz,1 H),8.06(t,J=8.4Hz,2H),7.88(dt,J=21.9,6.5Hz,5H),7.77–7.66(m,4H),7.55(t,J=7 .0Hz,2H),7.45(d,J=8.0Hz,1H),7.39(d,J=5.7Hz,1H),7.24(d,J=5.4Hz,3H),6.73(d ,J=8.2Hz,2H),5.36(s,2H),4.48(t,J=7.1Hz,2H),2.86(t,J=7.2Hz,2H),2.60(s,3H). 13C NMR (126MHz, CDCl3) δ192.61,170.82,165.75,163.89,163.43,155.97,155.07,15 2.95,150.17,149.74,149.51,149.38,143.90,138.90,136.88,132.69,132.42,1 32.03,129.29,129.14,126.92,126.57,126.09,125.42,125.01,124.68,124.35, 123.58,122.16,121.20,118.36,114.82,63.94,35.98,32.63,21.50.ESI–MS:m / z 1049.23[M-PF6] + (calcd:1049.24).(500MHz,CDCl3),8.92(d,J=2.4Hz,1H),8.52(s,1H),8.42(s,1H) ,8.20(d,J=7.5Hz,1H),8.12-8.11(dd,J=2.4,2.4Hz,1H),7.91-7.84(m,3H),7.76-7. 66(m,8H),7.53(t,J=10Hz,2H),7.32(d,J=5.6Hz,1H),7.19(d,J=5.6Hz,1H),7.08-6. 97(m,5H),6.91-6.88(m,2H),6.30-6.27(t,J=13.4Hz,2H),5.45(s,2H),2.57(s,3H).

[0050] (3) Preparation of complex Ir2:

[0051] Under nitrogen protection, [Ir(pba)2Cl]2 (0.12 g, 0.1 mmol) and ligand L (0.11 g, 0.22 mmol) were mixed in a solvent (V DCM :V MeOH =1:1) and refluxed for 5 h. The reaction solution was spin-dried and dissolved in methanol. Ammonium hexafluorophosphate (0.0732 g, 0.4 mmol) was then added and stirred at room temperature overnight. The reaction solution was concentrated and purified by column chromatography to obtain a yellow solid in a yield of 66.3%.

[0052] 1H NMR (500MHz, CDCl3) δ8.61(d,J=7.2Hz,2H),8.56(d,J=7.8Hz,2H),8.46(d,J=8.3Hz,1H),7.91(d,J =5.6Hz,3H),7.77(dd,J=13.4,6.6Hz,4H),7.70(dd,J=7.5,3.5Hz,2H),7.59(s,2H),7.47(d,J=7.9H z,1H),7.36(d,J=5.3Hz,1H),7.23(d,J=5.2Hz,1H),7.05(dt,J=11.7,6.1Hz,4H),6.93(q,J=7.2Hz ,2H),6.33(t,J=7.2Hz,2H),5.39(s,2H),4.52(t,J=6.9Hz,2H),2.89(t,J=7.0Hz,2H),2.61(s,3H). 13 C NMR (126MHz, CDCl3) δ170.86,167.68,163.88,163.42,156.12,155.23,152.42, 150.39,150.16,149.69,149.49,148.93,143.84,143.53,137.98,132.43,132. 01,131.72,130.76,129.12,126.91,126.47,125.82,124.74,124.40,123.47,1 22.56,122.26,119.55,118.48,114.80,64.12,36.04,32.70,21.54.ESI–MS:m / z 993.25[M-PF6] + (calcd:993.25).

[0053] Example 2

[0054] Fluorescence response experiment of complex Ir2 to H2S: During the experiment, PBS buffer was prepared by ultrapure water and corresponding salts. Before the test, PBS buffer (pH=7.2) was deoxygenated by blowing nitrogen. The solution used in the test was V DMSO :V PBS =1:9, the complex used in the test process was first prepared with DMSO to a 20mM stock solution. Other ions (1) HS - ,(2)Hcy,(3)Cys,(4)GSH,(5)SO3 2- ,(6)HSO3 - ,(7)SCN - ,(8)S2O3 - ,(9)CO3 2-,(10)I - ,(11)HCO3 - ,(12)K + ,(13)NO3 - ,(14)Mg 2+ ,(15)SO4 2- ,(16)CH3COO - ,(17)Cl - ,(18)C2O4 2- ,(19)NO2 - ,(20)Br - .Use ultrapure water to prepare, and the ion concentration is 20mM, all ion solutions are prepared fresh. In order to study the responsiveness of iridium complex to H2S, 20μM iridium complex solution is used as the test solution, and 200μM HS - The fluorescence spectrum of the solution was measured at 298K over time. Figure 1 This is the time-dependent fluorescence spectrum of the reaction between complex Ir1 and H2S; Figure 1 It can be seen that with the increase of time, the fluorescence at 535nm is significantly enhanced. Figure 2 This is the time-dependent fluorescence spectrum of the reaction between the complex Ir2 and H2S; Figure 2 It can be seen that the fluorescence at 540nm increases significantly with time. In order to detect H2S in complex living systems, selectivity is an important criterion. A 20μM iridium complex solution was used as the test solution, and the fluorescence intensity was tested after reacting with 1mM solutions of different molecular ions for 30 minutes. Figure 3 and Figure 4 It is clear from the results that the iridium complex has a high selectivity for H2S, but under the same conditions, Ir2 has a higher selectivity and shows almost no fluorescence response to other molecular ions. Therefore, subsequent experiments will use Ir2 as an example for further property testing.

[0055] Example 3

[0056] Cellular and in vivo imaging of H2S by the complex Ir2.

[0057] Cell imaging: A375 cells and HepG2 cells were seeded in 35 mm confocal culture dishes for 24 hours. Some A375 cells were pretreated with aminooxyacetic acid (AOAA) and S-adenosyl-L-methionine (SAM), respectively, and then incubated with iridium complex Ir2 for 1 hour. The cells were washed three times with PBS and observed and photographed using a confocal microscope.

[0058] In vivo imaging: 5- to 6-week-old female Balb / c nude mice were used to establish A375 and HepG2 tumor models in the flank. During the experiments, the mice were anesthetized with isoflurane in an oxygen stream. For exogenous hydrogen sulfide imaging in live mice, Ir2 (polyoxyethylene castor oil:ethanol = 1:1, 1.0 mM, 50 μL) was injected subcutaneously into the tumor and normal tissues of the mice, and the mice were photographed.

[0059] In order to study the selectivity of Ir2 for different tumor cells, HepG2 cells lacking H2S and A375 cells rich in H2S were used for imaging comparison. Figure 5 As shown, Ir2 exhibited bright fluorescence after entering A375 cells, but only weak fluorescence after entering HepG2 cells, indicating that Ir2 can specifically identify tumor cells based on the level of intracellular H2S. Furthermore, the intracellular fluorescence was significantly reduced in cells treated with the H2S inhibitor AAOA. Meanwhile, in the SAM group, which increases intracellular H2S, the fluorescence of the complex was significantly enhanced, indicating that the complex selectively reacts with H2S within the cell to emit fluorescence.

[0060] In order to study the selectivity of Ir2 for different tumors, Figure 6 As shown in the figure, in the A375 tumor model with a high H2S content, Ir2 can produce a strong fluorescence signal, and the fluorescence signal is the strongest 2 hours after injection. In the HepG2 tumor model with a low H2S content, Ir2 only produces a weak fluorescence signal. In addition, Ir2 does not produce a fluorescence signal for normal tissues, indicating that Ir2 can distinguish normal tissues from different tumor tissues based on the level of H2S content.

[0061] Example 4

[0062] Imaging of mitochondrial DNA by the complex Ir2. A375 cells were seeded in a 35 mm confocal culture dish for 24 hours, then incubated with the iridium complex Ir2 and ethidium bromide EB for 1 hour, respectively. The cells were then stained with the nucleic acid dye Pico-green, washed three times with PBS, and observed and photographed using a confocal microscope.

[0063] In order to study the selectivity of Ir2 for mitochondrial DNA, Pico-green is a fluorescent nucleic acid dye. Pico-green was incubated with A375 cells for a period of time. Figure 7As shown, the entire cell exhibited green fluorescence. Addition of Ir2 quenched the green fluorescence of Pico-green in the cytoplasm. Addition of ethidium bromide (EB) also quenched the fluorescence of the entire cell. This is because EB is nuclear permeable and can enter and quench the green fluorescence of Pico-green in the cell nucleus. This result indicates that Ir2 can target mitochondrial DNA after entering A375 cells.

[0064] Example 5

[0065] The complex Ir2 induces ferroptosis in cells. The complex affects the expression level of GPX4, a key protein in the ferroptosis process:

[0066] A375 cells were seeded in culture dishes and incubated with 2.0 μM Ir2 for 12 hours (light group, incubated for 6 hours, 50 mW·cm -2 Cells were lysed in RIPA buffer (irradiated with 425 nm light for 3 minutes and incubated for an additional 6 hours). Proteins were then separated on SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with primary antibodies overnight at 4°C. Following washes, the membranes were blocked with secondary antibodies for 30 minutes. The membranes were developed using an ultrasensitive ECL chemiluminescence kit. Images were captured using a CLINX 6300 imaging station and analyzed manually using Alpha Innotech software.

[0067] Glutathione peroxidase 4 (GPX4) is a key factor in ferroptosis. Down-regulation of GPX4 expression can lead to the accumulation of lipid peroxides, thereby inducing ferroptosis. The results show that the complex Ir2 obtained in the present invention can significantly down-regulate the expression level of GPX4 under light conditions, indicating that the complex can induce ferroptosis. The results are shown in the attached figure. Figure 8 shown.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A 2,2'-bipyridine compound, whose chemical name is (4'-methyl-[2,2'-bipyridine]-4-yl)methyl 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionate, and whose general structural formula is shown in Formula (I):

2. A method for preparing the 2,2'-bipyridine compound according to claim 1, comprising the following steps: (a) reacting 3-(6-azido-1,3-diketo-1H-benzo[de]isoquinolin-2(3H)-yl)propionic acid, 4-hydroxymethyl-4'-methyl-2,2'-bipyridine, a condensing agent and a catalyst in a first solvent; (b) After the reaction, water is added for washing, the organic phase is extracted, dried, concentrated, and subjected to column chromatography to obtain an off-white solid, which is a 2,2'-bipyridine compound; The first solvent is dichloromethane or chloroform.

3. The method for preparing a 2,2'-bipyridine compound according to claim 2, characterized in that: The condensing agent is EDC, DCC, HATU or HBTU.

4. The method for preparing a 2,2'-bipyridine compound according to claim 2, characterized in that: The catalyst is DMAP or Et3N.

5. A cyclometalated iridium complex comprising a compound of formula (I) as claimed in claim 1 as a ligand, wherein the general structural formula thereof is shown in formula (II): In formula (II), for 6. A method for preparing the cyclometalated iridium complex according to claim 5, comprising the following steps: (a) reacting a bis(2-phenylpyridine)-dichloroiridium precursor or a bis(4-(pyridin-2-yl)benzaldehyde)-dichloroiridium precursor and a compound of formula (I) under reflux in a mixed solvent; (b) After the reaction, the mixture was concentrated under reduced pressure, dissolved in methanol, and then ammonium hexafluorophosphate was added. The mixture was purified by column chromatography to obtain a yellow solid, i.e., a cyclometalated iridium complex.

7. The method for preparing a cyclometalated iridium complex according to claim 6, wherein: The mixed solvent is a dichloromethane-methanol solution with a volume ratio of 1:

1.

8. Use of the cyclometallated iridium complex according to claim 5 in the preparation of reagents for H2S detection and imaging.

9. Use of the cyclometallated iridium complex according to claim 5 in preparing reagents for mitochondrial DNA detection and imaging.

10. Use of the cyclometallated iridium complex according to claim 5 in the preparation of anti-tumor drugs.

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