A near-infrared light-releasing binuclear ruthenium complex, its preparation, and application in combating drug resistance in non-small cell lung cancer

Abstract

The present invention belongs to the field of pharmaceutical chemistry technology, and specifically relates to a near-infrared light-releasing binuclear ruthenium complex, its preparation, and its application in combating drug resistance in non-small cell lung cancer. The binuclear ruthenium complex disclosed in the present invention has the structural formula shown below. The complex has a strong photochemotherapy effect on cisplatin-resistant human non-small cell lung cancer cell lines. Under light conditions, it has a strong growth and proliferation inhibition ability (IC 50 The phototoxicity of the metallo-photosensitive material was 0.22 μM, while in the dark, its cytotoxicity was only 75.6 μM, and the phototherapy index (PI) was as high as 343, which is of great significance for the study of metallo-medicines for anti-drug-resistant tumors. It is expected to be used to prepare anti-tumor photoactivated drugs or drugs for anti-proliferation of cisplatin-resistant strains of human non-small cell lung cancer, and even anti-tumor metal photosensitizers.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and specifically relates to a near-infrared light-releasing binuclear ruthenium complex, its preparation, and application in combating drug resistance in non-small cell lung cancer. Background Art

[0002] Lung cancer is difficult to detect in its early stages and is also challenging to treat. Consequently, lung cancer is the leading cause of cancer death worldwide, claiming an estimated 1.6 million lives annually. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) being its two most common subtypes. Smoking is the most common cause of lung cancer, accounting for over 80% of cases. Therefore, the development of drugs for NSCLC has long been a hot topic of research.

[0003] Photodynamic therapy (PDT) is a non- or minimally invasive cancer treatment approach that has garnered significant attention in the field of oncology due to its high selectivity, minimal side effects, and low drug resistance. However, many aggressive and drug-resistant cancers are characterized by hypoxia, severely limiting the application of PDT. An alternative PDT approach, photoactivated chemotherapy, has emerged. This approach, through spatially and temporally controlled light irradiation, converts non-toxic prodrugs into active anticancer agents within tumor tissue, ultimately resulting in highly specific therapeutic effects.

[0004] Drug therapy (chemotherapy, targeted therapy, and immunotherapy) is a crucial treatment option for cancer patients. However, nearly all current drugs develop drug resistance over time, leading to a cessation of the drug's effect on cancer cells. Consequently, drug resistance is a major cause of cancer treatment failure, causing rapid recurrence / progression and ultimately death. This presents one of the most challenging challenges facing cancer patients and their families, healthcare professionals, and cancer researchers. Therefore, developing drugs to combat drug-resistant tumors is crucial to extending the survival of cancer patients. Summary of the Invention

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a near-infrared light-releasing binuclear ruthenium complex, which has a strong photochemotherapy effect on cisplatin-resistant strains of human non-small cell lung cancer and is expected to be used in the development of metal drugs against drug-resistant tumors.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides a binuclear ruthenium complex, the structural formula of the binuclear ruthenium complex is as follows:

[0008]

[0009] The second aspect of the present invention provides a method for preparing the binuclear ruthenium complex described in the first aspect, comprising the following steps:

[0010] S1. Under the protection of inert gas, ruthenium (III) chloride hydrate, lithium chloride and 2,9-dimethyl-1,10-phenanthroline are dissolved in an organic solvent, and the mixture is refluxed to generate a RuCl2(2,9-dmp)2 precursor;

[0011] S2, dissolving RuCl2(2,9-dmp)2 precursor and 5-bromo-2,2'-bipyridine in an organic solvent, stirring and reacting, and then adding ammonium hexafluorophosphate for ion replacement to generate a ruthenium complex [Ru(2,9-dmp)2(bpy-Br)](PF6)2;

[0012] S3. Dissolve the ruthenium complex [Ru(2,9-dmp)2(bpy-Br)](PF6)2, 2,5-di(2-ethylhexyl)-3,6-di(5-(trimethyltin)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4-(2H,5H)-dione and tetrakis(triphenylphosphine)palladium in an organic solvent, and stir the mixture under the protection of an inert gas to obtain a binuclear ruthenium complex.

[0013] Preferably, in step S1, the molar ratio of the 2,9-dimethyl-1,10-phenanthroline, ruthenium (III) chloride hydrate and lithium chloride is 2:1:3.

[0014] Preferably, in step S1, the reflux reaction temperature is 150° C. and the time is 8 to 10 hours. More preferably, the reflux reaction temperature is 150° C. and the time is 8 hours.

[0015] Preferably, in step S2, the molar ratio of the precursor RuCl2(2,9-dmp)2 to 5-bromo-2,2'-bipyridine is 1:1.

[0016] Preferably, in step S2, the stirring reaction temperature is 80-100° C. and the time is 8-12 h. More preferably, the stirring reaction temperature is 90° C. and the time is 12 h.

[0017] Preferably, in step S3, the molar ratio of 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethyltin)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4-(2H,5H)-dione, [Ru(2,9-dmp)2(bpy-Br)](PF6)2, and tetrakis(triphenylphosphine)palladium is 1:2:0.1.

[0018] Preferably, in step S3, the stirring reaction temperature is 100-135° C. and the time is 18-24 hours. More preferably, the stirring reaction temperature is 115° C. and the time is 20 hours.

[0019] The third aspect of the present invention provides the use of the binuclear ruthenium complex described in the first aspect in the preparation of anti-tumor photoactivated drugs and / or drugs for preventing the proliferation of cisplatin-resistant strains of human non-small cell lung cancer.

[0020] Preferably, the tumor is human lung cancer,

[0021] Preferably, the human non-small cell lung cancer cisplatin-resistant strain is A549 / DDP cell.

[0022] The fourth aspect of the present invention provides an anti-tumor photoactivated drug or a drug for anti-proliferation of cisplatin-resistant human non-small cell lung cancer strains, wherein the drug has the binuclear ruthenium complex described in the first aspect as the main active ingredient.

[0023] The fifth aspect of the present invention provides an anti-tumor metal photosensitizer, wherein the photosensitizer uses the binuclear ruthenium complex described in the first aspect as a main active ingredient.

[0024] Preferably, the drug or photosensitizer further comprises a pharmaceutically acceptable carrier and / or excipient. That is, the drug or photosensitizer comprises a binuclear metal ruthenium complex as the main active ingredient, mixed with a pharmaceutically acceptable carrier and / or excipient to form a composition, and then prepared into a clinically acceptable dosage form.

[0025] Furthermore, the excipient refers to diluents, adhesives, lubricants, disintegrants, solubilizers, stabilizers and other pharmaceutical matrices that can be used in the pharmaceutical field.

[0026] Furthermore, the carrier is an acceptable functional pharmaceutical excipient in the pharmaceutical field, including surfactants, suspending agents, emulsifiers and some new pharmaceutical polymer materials, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0027] Furthermore, the present invention has no particular limitations on the dosage form of the aforementioned drugs or photosensitizers, and the drugs or photosensitizers can be prepared in the form of tablets, capsules, suppositories, and powder injections, as are known to those skilled in the art. The prepared preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically). If certain drugs are unstable under gastric conditions, they can be prepared as enteric-coated tablets.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention discloses a novel photoactivated binuclear ruthenium complex, which has a strong photochemotherapy effect on cisplatin-resistant human non-small cell lung cancer cell lines. Under light conditions (near infrared light), it has a strong growth and proliferation inhibition ability (IC 50 The phototoxicity of the metallo-photosensitive drug was 0.22 μM, while in the dark, its cytotoxicity was only 75.6 μM, and its phototherapeutic index (PI) was as high as 343, which is of great significance for the study of metallo-medicines for anti-drug-resistant tumors. It is expected to be used to prepare anti-tumor photoactivated drugs or drugs for anti-proliferation of cisplatin-resistant human non-small cell lung cancer strains, and even anti-tumor metal photosensitizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the UV absorption spectrum of the photoactivated binuclear ruthenium complex;

[0031] Figure 2 The UV absorption spectrum changes of the photoactivated binuclear ruthenium complex under light irradiation;

[0032] Figure 3 The ability of photoactivated binuclear ruthenium complexes to photocatalytically generate superoxide anions;

[0033] Figure 4 The ability of photoactivated dinuclear ruthenium complexes to photocatalytically generate singlet oxygen;

[0034] Figure 5 The ability of the photoactivated binuclear ruthenium complex to photocatalytically oxidize NADH;

[0035] Figure 6 The ability of the photoactivated binuclear ruthenium complex to photocatalytically oxidize NADPH;

[0036] Figure 7 To develop a photoactivated binuclear ruthenium complex to counteract the dark toxicity and phototoxicity of cisplatin-resistant human non-small cell lung cancer cell line (A549 / DDP). DETAILED DESCRIPTION

[0037] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0039] Example 1 A novel binuclear ruthenium complex and its preparation

[0040] The structural formula of the binuclear ruthenium complex is shown below:

[0041]

[0042] The synthesis of the binuclear ruthenium complex comprises the following steps:

[0043] (1) Under N2 protection, ruthenium(III) chloride hydrate (0.62 g, 3 mmol), lithium chloride (0.38 g, 8.9 mmol), and 2,9-dimethyl-1,10-phenanthroline (2,9-dmp; 1.25 g, 6 mmol) were added to a round-bottom flask, and 20 mL of N,N-dimethylformamide was added. The mixture was refluxed at 150°C for 8 hours. After cooling to room temperature, 100 mL of acetone was added and the mixture was refrigerated overnight. A purple-black precipitate was obtained by filtration. The precipitate was washed alternately with cold water and acetone to obtain the Ru(dmp)2Cl2 precursor with a yield of 24.3%.

[0044] The chemical reaction equation above is as follows:

[0045]

[0046] (2) The precursor Ru(dmp)2Cl2 (177 mg, 0.3 mmol) and 5-bromo-2,2'-bipyridine (71 mg, 0.3 mmol) were dissolved in 10 mL of ethylene glycol solution, stirred at 90 °C (400 rpm) overnight (12 h), and then cooled to room temperature. 50 mL of saturated NH4PF6 aqueous solution was added and stirred at room temperature for 1 h for ion exchange. The mixture was filtered and the filter cake was washed with water and anhydrous ethanol. After vacuum drying, the ruthenium complex [Ru(2,9-dmp)2(bpy-Br)](PF6)2 was obtained as a yellow powder (91 mg, 40%).

[0047] The chemical reaction equation above is as follows:

[0048]

[0049] (3) [Ru(2,9-dmp)2(bpy-Br)](PF6)2 (150 mg, 0.14 mmol), 2,5-di(2-ethylhexyl)-3,6-di(5-(trimethyltin)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4-(2H,5H)-dione (51 mg, 0.07 mmol), and tetrakis(triphenylphosphine)palladium (7 mg, 0.007 mmol) were dissolved in 16 mL of toluene / N,N-dimethylformamide (V / V=1:1) and stirred at 115°C for 20 h under argon protection. After the reaction, the mixture was cooled to room temperature and 50 mL of saturated brine was added. The precipitate was collected by filtration, washed with water and toluene, and dried in vacuo to obtain 90 mg (69%) of dark green solid powder, i.e., the binuclear ruthenium complex.

[0050] The chemical reaction equation above is as follows:

[0051]

[0052] The mass spectrum of the product is: ESI-MS[CH3OH, m / z]: 467[M−4PF6 - ] 4+ ;

[0053] The H NMR spectrum of the product is: 1 H NMR (500 MHz, DMSO- d 6) δ 9.05 – 9.01 (m, 1H), 8.92(d, J = 8.4 Hz, 1H), 8.70 – 8.64 (m, 2H), 8.62 – 8.48 (m, 4H), 8.43 (s, 2H), 8.28 (dd, J = 16.6, 8.7 Hz, 2H), 8.03 (d, J = 8.1 Hz, 1H), 7.98 (d, J = 8.3Hz, 2H), 7.70 – 7.58 (m, 2H), 7.43 (s, 1H), 7.32 – 7.24 (m, 1H), 7.15 (dd, J = 12.2, 6.6 Hz, 2H), 4.10 (s, 1H), 3.96 (s, 1H), 2.13 – 1.82 (m, 12H), 1.49 –1.01 (m, 9H), 0.82 (dt, J = 57.2, 6.9 Hz, 7H).

[0054] Experimental Example 1 Performance Test of Binuclear Ruthenium Complex

[0055] 1. Absorption spectrum determination of binuclear ruthenium complexes

[0056] The binuclear ruthenium complex of Example 1 was prepared into a 10 μM sample solution using anhydrous ethanol (CH 3 OH) as a solvent, and the UV absorption spectrum of the binuclear ruthenium complex was recorded using a double-beam UV-visible spectrophotometer. Figure 1 Characterization of its absorbance in ethanol showed that it had good light absorption ability in organic solvents.

[0057] 2. Changes in the UV-visible absorption spectrum of binuclear ruthenium complexes after illumination

[0058] A quartz cuvette containing a PBS solution of a binuclear ruthenium complex (10 mM) was placed under a 700 nm light source to detect changes in the solution's UV absorption spectrum at different time intervals. Figure 2 As shown, the ultraviolet absorption spectrum of the binuclear ruthenium complex undergoes a blue shift and an isosbestic point appears after irradiation with a 700nm light source, indicating that the ruthenium complex has the ability to produce new substances after photoactivation.

[0059] 3. Determination of the ability of binuclear ruthenium complexes to generate superoxide anions

[0060] To test the photocatalytic superoxide anion generation ability of the binuclear ruthenium complex synthesized in Example 1, dihydrorhodamine 123 (DHR 123) was used to measure the superoxide anion generation ability of the binuclear ruthenium complex. When superoxide anions are generated in a solution, DHR 123 immediately captures the superoxide anions and is oxidized to form the fluorescent derivative rhodamine 123, which emits bright green fluorescence (Ex / Em = 500 / 536 nm). An increase in fluorescence intensity indicates the generation of superoxide anions in the solution. Monitoring the fluorescence spectrum of a mixed solution of the test sample and DHR 123 under different illumination times using a fluorescence spectrophotometer can reflect the superoxide anion generation ability.

[0061] An aqueous solution containing a binuclear ruthenium complex (5 μM) and DHR 123 reagent (10 μM) was placed in a cuvette and its superoxide anion generation capacity was measured under 700 nm light illumination. Figure 3 As shown, the binuclear ruthenium complex has the ability to generate superoxide anions after light irradiation.

[0062] 3. Determination of the ability of binuclear ruthenium complexes to generate singlet oxygen

[0063] To test the photocatalytic singlet oxygen generation ability of the dinuclear ruthenium complex synthesized in Example 1, the singlet oxygen probe 9,10-anthryl-bis(methylene)dimalonic acid (ABDA) was used to measure the singlet oxygen generation ability of the dinuclear ruthenium complex. When singlet oxygen is generated in solution, ABDA immediately captures the singlet oxygen in the solution, reacting to form an endogenous oxidation product, which causes a decrease in the characteristic absorption peak of ABDA. The rate of decrease in the ABDA absorption peak represents the singlet oxygen generation rate. The singlet oxygen generation ability can be reflected by monitoring the changes in the UV-visible absorption spectrum of the mixed solution of the test sample and ABDA under different illumination times using a UV-visible spectrophotometer.

[0064] Two aqueous solutions containing the same binuclear ruthenium complex (5 μM) and ABDA reagent (200 μM) were placed in a cuvette and their singlet oxygen generation abilities were measured under 700 nm illumination. Figure 4 As shown, the binuclear ruthenium complex has the ability to generate singlet oxygen after light irradiation.

[0065] 4. Determination of the photocatalytic oxidation ability of binuclear ruthenium complexes for NADH / NADPH

[0066] Under light irradiation, the metal complex can oxidize reduced coenzyme I (NADH) and reduced coenzyme II (NADPH) into their oxidized forms NADPH and NADPH. ＋ and NADP ＋ , so the ruthenium complex (5 μM) and NADH or NADPH (A 339nm =1.0) in a cuvette, and its ability to oxidize NADH / NADPH under 700nm light conditions can be measured respectively. Figure 5 、 6 As shown in the figure, the ruthenium complex has obvious photocatalytic oxidation ability for NADH and NADPH.

[0067] 5. Photodynamic therapy effect of binuclear ruthenium complex on cisplatin-resistant human non-small cell lung cancer cell lines

[0068] Resazurin solution is blue and is commonly used as an acid-base indicator (orange at pH 3.8 to deep purple at pH 6.5) and a redox indicator. During cell viability assays, resazurin penetrates cells and is irreversibly reduced by living cells to a pink color, accompanied by the appearance of red fluorescent resorufin. The absorbance or fluorescence intensity of resorufin is positively correlated with cell number and reducing capacity, allowing analysis of cell proliferation using an enzyme-linked immunosorbent assay (ELISA).

[0069] The resazurin experimental steps are as follows:

[0070] (1) First, revive one tube of A549 / DDP tumor cells and culture them with fresh complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin mixture). After passage twice, start the experiment.

[0071] (2) When the cells reach the logarithmic growth phase, they are seeded into two 96-well plates at a density of 5000 cells / well (100 μL of culture medium is used to culture cells in each well, one plate is for the light group and the other plate is for the dark control group), and cultured in a 37°C, 5% CO2 incubator.

[0072] (3) After the cells adhere to the wall, aspirate the original culture medium and add 100 μL of binuclear ruthenium complex at seven concentrations, namely 100, 50, 10, 1, 0.1, 0.01, and 0.001 mM, to each well. Gently shake the cells and incubate them in a carbon dioxide incubator (37 °C, 5% CO2) in the dark.

[0073] (4) After incubation for 8 h, the cell culture plates in the illumination group were placed under a 700 nm light source for 30 min (light dose of 71 J / cm 2 ), and then returned to the incubator for further incubation in the dark for 40 h (cells in the dark control group were kept in the incubator in the dark for incubation).

[0074] (5) After incubation for 40 h, the culture medium was discarded from each well, and 80 μL of resazurin (100 mg / mL) was added to each well. The cells were incubated in a 37°C incubator for another 4 h. EX540 / EM590 was detected using the fluorescence plate of an enzyme-linked immunosorbent assay (ELISA) to calculate the cell proliferation inhibition rate and obtain the IC 50 value (drug concentration at which the inhibition rate is equal to 50%).

[0075] Figure 7 The resazurin method was used to detect the killing effect of different concentrations of binuclear ruthenium complexes on human non-small cell lung cancer cisplatin-resistant cell lines (A549 / DDP cells) under dark and light treatment conditions (near infrared light). As can be seen from the figure, in the absence of light, the IC 50 The IC value for cisplatin-resistant human non-small cell lung cancer cell lines under light conditions was 75.6 μM. 50 The phototherapy index PI is as high as 343, indicating that the binuclear ruthenium complex of the present invention has a strong photodynamic therapy effect.

[0076] In summary, the binuclear ruthenium complex provided by the present invention has a strong photochemotherapy effect on human non-small cell lung cancer cisplatin-resistant cell line (A549 / DDP cells). Under light conditions (near infrared light), it has a strong ability to inhibit the growth and proliferation of human non-small cell lung cancer cisplatin-resistant cells (IC50 The phototherapy index (PI) of the metallo-drug was as high as 343, which is of great significance for the study of metallo-drugs for anti-drug resistant tumors.

[0077] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations of these embodiments may be made without departing from the principles and spirit of the present invention, and the changes still fall within the scope of protection of the present invention.

Claims

1. A binuclear ruthenium complex, characterized in that The structural formula of the binuclear ruthenium complex is shown below:

2. The method for preparing the binuclear ruthenium complex according to claim 1, characterized in that: The following steps are involved: S1. Under the protection of inert gas, ruthenium (III) chloride hydrate, lithium chloride and 2,9-dimethyl-1,10-phenanthroline are dissolved in an organic solvent, and the mixture is refluxed to generate a RuCl2(2,9-dmp)2 precursor; S2, dissolving RuCl2(2,9-dmp)2 precursor and 5-bromo-2,2'-bipyridine in an organic solvent, stirring and reacting, and then adding ammonium hexafluorophosphate for ion replacement to generate a ruthenium complex [Ru(2,9-dmp)2(bpy-Br)](PF6)2; S3. Dissolve the ruthenium complex [Ru(2,9-dmp)2(bpy-Br)](PF6)2, 2,5-di(2-ethylhexyl)-3,6-di(5-(trimethyltin)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4-(2H,5H)-dione and tetrakis(triphenylphosphine)palladium in an organic solvent, and stir the mixture under the protection of an inert gas to obtain a binuclear ruthenium complex.

3. The method for preparing a binuclear ruthenium complex according to claim 2, wherein: In step S1, the molar ratio of the 2,9-dimethyl-1,10-phenanthroline, ruthenium (III) chloride hydrate and lithium chloride is 2:1:

3.

4. The method for preparing a binuclear ruthenium complex according to claim 2, wherein In step S1, the reflux reaction temperature is 150° C. and the time is 8 to 10 hours.

5. The method for preparing a binuclear ruthenium complex according to claim 2, wherein In step S2, the molar ratio of the precursor RuCl2(2,9-dmp)2 to 5-bromo-2,2'-bipyridine is 1:

1.

6. The method for preparing a binuclear ruthenium complex according to claim 2, wherein: In step S2, the stirring reaction temperature is 80-100° C. and the time is 8-12 h.

7. The method for preparing a binuclear ruthenium complex according to claim 2, wherein: In step S3, the molar ratio of 2,5-bis(2-ethylhexyl)-3,6-bis(5-(trimethyltin)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4-(2H,5H)-dione, [Ru(2,9-dmp)2(bpy-Br)](PF6)2, and tetrakis(triphenylphosphine)palladium is 1:2:0.

1.

8. The method for preparing a binuclear ruthenium complex according to claim 2, wherein: In step S3, the stirring reaction temperature is 100-135° C. and the time is 18-24 hours.

9. Use of the binuclear ruthenium complex according to claim 1 in the preparation of anti-tumor photoactivated drugs and / or drugs for inhibiting the proliferation of cisplatin-resistant human non-small cell lung cancer strains, characterized in that: The tumor is human lung cancer.

10. An anti-tumor photoactivated drug or a drug for anti-proliferation of cisplatin-resistant human non-small cell lung cancer strains, characterized in that: The binuclear ruthenium complex according to claim 1 is used as the main active ingredient, and the tumor is human lung cancer.

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