An organic metal ruthenium macrocyclic complex and its preparation method and application

Abstract

The present invention relates to the field of biomedicine and discloses an organometallic ruthenium macrocyclic complex, its preparation method, and application. The complex has the structure represented by formula (I). Compared with other commercial chemotherapy drugs, the complex provided by the present invention not only has good phototherapy-chemotherapy combination treatment capabilities, but also has fewer toxic side effects and stronger anti-cancer effects. Moreover, the macrocyclic complex can achieve precise guidance of tumor combination therapy using near-infrared second-zone fluorescence imaging technology, and therefore has great application potential in the biomedical field.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an organometallic ruthenium macrocyclic complex and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging technology plays a very wide range of roles in basic research and biomedical fields. Compared with other existing imaging technologies, this technology has the advantages of non-invasiveness and real-time imaging. Compared with traditional near-infrared region 1 (700-900nm) fluorescence imaging, near-infrared region 2 fluorescence imaging technology (1000-1700nm) has deeper tissue penetration depth and higher imaging signal-to-noise ratio. Therefore, using near-infrared region 2 fluorescence imaging technology to apply precise imaging in disease diagnosis and disease monitoring can evaluate the effectiveness of disease treatment in real time.

[0003] Currently, malignant tumors are the second leading cause of death in humans after cardiovascular disease. Chemotherapy, photodynamic therapy (PDT), and photothermal therapy (PTT) have been widely used in basic research and clinical applications in tumor treatment. Although chemotherapy is a conventional method for treating tumors, it has problems such as large toxic side effects and high drug resistance. PDT is a method in which light is irradiated on a photosensitizer to activate oxygen in the surrounding environment, and the reactive oxygen species produced can induce cancer cell death. PTT is a method in which light is irradiated on a photosensitizer to generate heat, and high temperature is used to ablate cancer cells. Both controllable PDT and PTT target local lesions, which can make up for the defect of chemotherapy drugs in producing side effects on healthy tissues, and can also overcome the problem of drug resistance. Therefore, the best therapeutic effect can be achieved through combined chemotherapy / PTT / PDT treatment.

[0004] Platinum complexes such as cisplatin, carboplatin, and oxaliplatin are the most widely used and effective chemotherapy drugs in clinical cancer treatment. It is estimated that these drugs are used in over 50% of cancer treatment regimens. However, platinum-based drugs are associated with significant neurotoxicity, hepatotoxicity, and nephrotoxicity. Ruthenium complexes, however, are considered a promising new class of anticancer agents due to their low cytotoxicity, high antitumor activity, and anti-metastatic properties. To date, two aromatic ruthenium complexes, NAMI-A and KP1019, and their sodium salt, KP1339, have been widely used in clinical chemotherapy trials. The aromatic ruthenium complex TLD1433, a polypyridine ruthenium complex, completed Phase II clinical trials in 2018 and is being used in photodynamic therapy for non-muscle invasive bladder cancer.

[0005] However, most currently reported arene ruthenium complexes have short emission wavelengths, low molar extinction coefficients, and weak tissue penetration, making it difficult to dynamically visualize their delivery, distribution, and metabolism in the body. To further improve tumor treatment efficacy, an arene ruthenium complex with combined therapeutic effects is urgently needed. Summary of the Invention

[0006] The purpose of the present invention is to provide a new organometallic ruthenium macrocyclic complex with near-infrared second-zone luminescence properties.

[0007] In order to achieve the above objectives, the first aspect of the present invention provides an organometallic ruthenium macrocyclic complex having a structure shown in formula (I):

[0008]

[0009] Wherein, in formula (I),

[0010] X1 and X2 are each independently selected from S or Se;

[0011] R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-10 Alkyl, phenyl, halogen, At least one of, and Z is C 1-6 A straight-chain alkoxy group, m is an integer of 1-10;

[0012] (A - ) is (PF6 - ), (CF3SO3 - )、(ClO4 - ) or (BF4 - );

[0013] Y1 and Y2 are each independently selected from at least one of a substituted or unsubstituted phenylene group, a substituted or unsubstituted thienylene group, a group represented by formula (Q1), a group represented by formula (Q2), and a group represented by formula (Q3); and each n is independently selected from an integer of 1 to 18;

[0014] The substituents optionally contained in Y1 and Y2 are independently selected from H, C 1-18 Alkyl, C 1-18 of alkoxy.

[0015] The second aspect of the present invention provides a method for preparing an organometallic ruthenium macrocyclic complex represented by formula (I), comprising: contacting a compound represented by formula (M) with a compound represented by formula (B) in the presence of a solvent,

[0016]

[0017] Wherein, in formula (B), A is PF6, CF3SO3, ClO4 or BF4;

[0018] In formula (M) and formula (B), the definitions of Y1, Y2, X1, X2, R1, R2, R3, R4, R5, and R6 are the same as those described in the first aspect.

[0019] The third aspect of the present invention provides the use of the organometallic ruthenium macrocyclic complex described in the first aspect in anti-tumor drugs and / or antibacterial drugs.

[0020] The fourth aspect of the present invention provides an anti-tumor drug, which contains a therapeutically effective amount of an active ingredient, wherein the active ingredient includes at least one of the organometallic ruthenium macrocyclic complexes described in the first aspect; optionally, the anti-tumor drug further contains an excipient.

[0021] The fifth aspect of the present invention provides the use of the organometallic ruthenium macrocyclic complex described in the first aspect in near-infrared second-zone fluorescence imaging of tumor cells.

[0022] The organometallic ruthenium macrocyclic complex provided by the present invention has near-infrared second-zone luminescence properties, has smaller toxic side effects and stronger anti-cancer effects, and at the same time, the complex has good phototherapy-chemotherapy combined treatment capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the H NMR spectrum of the complex N-1 described in Example 1 according to a preferred embodiment of the present invention.

[0024] Figure 2 The figure shows the NMR fluorine spectrum of the complex N-1 described in Example 1 according to a preferred embodiment of the present invention.

[0025] Figure 3 The figure shows the Roesy spectrum of the complex N-1 described in Example 1 according to a preferred embodiment of the present invention.

[0026] Figure 4 The figure shows the electrospray time-of-flight mass spectrum (ESI-TOF-MS) of the complex N-1 described in Example 1 according to a preferred embodiment of the present invention.

[0027] Figure 5 The diagram shows the ultraviolet absorption spectrum and fluorescence emission spectrum of the complex N-1 described in Test Example 1 according to a preferred embodiment of the present invention.

[0028] Figure 6 Graphs showing the UV absorption intensity of phosphate buffered saline containing 10 μM complex N-1 at different incubation times according to Test Example 2 of a preferred embodiment of the present invention.

[0029] Figure 7 Graph showing the UV absorption intensity of 10 μM complex N-1 under different pH conditions according to Test Example 2 of a preferred embodiment of the present invention.

[0030] Figure 8 Graph showing the UV absorption intensity of the complex N-1 described in Test Example 3 according to a preferred embodiment of the present invention at different irradiation times.

[0031] Figure 9 Graphs showing fluorescence signals of 10 μM complex N-1 in 1% fat emulsions of different thicknesses according to Test Example 4 in a preferred embodiment of the present invention.

[0032] Figure 10 The figures show the test results of photothermal conversion of complex N-1 at different concentrations as described in Test Example 5 according to a preferred embodiment of the present invention.

[0033] Figure 11 Graphs showing the fluorescence intensity of reactive oxygen species (ROS) generated by complex N-1 at different times under DCFH-DA conditions as described in Test Example 6 according to a preferred embodiment of the present invention.

[0034] Figure 12 This figure shows the laser confocal fluorescence imaging of the complex N-1 described in Test Example 7 in Hela cells according to a preferred embodiment of the present invention.

[0035] Figure 13 The figures show the NIR-II fluorescence in vivo imaging of the complex N-1 at different times as described in Test Example 8 according to a preferred embodiment of the present invention.

[0036] Figure 14 The figure shows the growth curve of tumor volume after intravenous injection of PBS, cisplatin and complex N-1 into mice according to Test Example 8 in a preferred embodiment of the present invention.

[0037] Figure 15 The figure shows the weight change curve of mice after intravenous injection of PBS, cisplatin and complex N-1 in Test Example 8 according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0038] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0039] The following terms are explained for the present invention:

[0040] “C1-18 "alkyl" means an alkyl group having a total carbon number of 1 to 18, including C 1-18 Straight chain alkyl, C 1-18 The branched chain alkyl group may be, for example, a straight chain alkyl group having a total number of carbon atoms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, or a branched chain alkyl group having a total number of carbon atoms of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, etc. 1-10 Alkyl", "C 1-6 The "alkyl" has a similar explanation, except that the number of carbon atoms is different.

[0041] “C 1-6 "Straight-chain alkoxy" means a straight-chain alkoxy with a total carbon atom count of 1 to 6, for example, a straight-chain alkoxy with a total carbon atom count of 1, 2, 3, 4, 5 or 6, and may be a methoxy, ethoxy, n-propoxy, n-butoxy, etc. 1-4 The "straight-chain alkoxy" has a similar explanation, except that the number of carbon atoms is different.

[0042] "Phenylene" means that any two H groups on the phenyl group are replaced to form a linking group, for example wait.

[0043] "Thienylene" means that any two H groups on the thienyl group are replaced to form a linking group, for example, wait.

[0044] "Substituted or unsubstituted phenylene" means that the H on the phenylene group may be substituted or unsubstituted with a corresponding group as defined herein. "Substituted or unsubstituted thienylene" has a similar definition, and the H on the thienylene group may be substituted or unsubstituted with a corresponding group as defined herein.

[0045] "Halogen" includes fluorine, chlorine, bromine and iodine.

[0046] In the present invention, m is an integer of 1-10, for example, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0047] In the present invention, each n is independently selected from an integer of 1-18, for example, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0048] As mentioned above, the first aspect of the present invention provides an organometallic ruthenium macrocyclic complex having a structure shown in formula (I), wherein, in formula (I),

[0049] X1 and X2 are each independently selected from S or Se;

[0050] R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-10 Alkyl, phenyl, halogen, At least one of, and Z is C 1-6 A straight-chain alkoxy group, m is an integer of 1-10;

[0051] (A - ) is (PF6 - ), (CF3SO3 - )、(ClO4 - ) or (BF4 - );

[0052] Y1 and Y2 are each independently selected from at least one of a substituted or unsubstituted phenylene group, a substituted or unsubstituted thienylene group, a group represented by formula (Q1), a group represented by formula (Q2), and a group represented by formula (Q3); and each n is independently selected from an integer of 1 to 18;

[0053] The substituents optionally contained in Y1 and Y2 are independently selected from H, C 1-18 Alkyl, C 1-18 of alkoxy.

[0054] Preferably, R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-6 Alkyl, phenyl, halogen, At least one of, and Z is C 1-4 More preferably, R1, R2, R3, R4, R5, and R6 are each independently selected from H, methyl, ethyl, isopropyl, n-propyl, halogen, At least one of , Z is methoxy or ethoxy, and m is an integer of 1-4.

[0055] Preferably, (A - ) is (CF3SO3 - ).

[0056] Preferably, Y1 and Y2 are each independently selected from at least one of a substituted or unsubstituted phenylene group, a substituted or unsubstituted thienylene group, a group represented by formula (Q1), a group represented by formula (Q2), and a group represented by formula (Q3); and each n is independently selected from an integer of 1 to 6; more preferably, Y1 and Y2 are each independently selected from at least one of the groups shown below:

[0057]

[0058] Each n is independently selected from an integer of 1-6.

[0059] In order to make the organometallic ruthenium macrocyclic complex provided by the present invention have a better anti-cancer effect, the present invention provides several preferred specific embodiments hereinafter to illustrate the preferred conditions of the compound represented by formula (I) of the present invention.

[0060] Preferred embodiment 1:

[0061] In formula (I),

[0062] X1 and X2 are each independently selected from S or Se;

[0063] R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-6 Alkyl, phenyl, halogen, At least one of, and Z is C 1-6 A straight chain alkoxy group, m is an integer of 1-6;

[0064] (A - ) is (PF6 - ), (CF3SO3 - )、(ClO4 - ) or (BF4 - );

[0065] Y1 and Y2 are each independently selected from at least one of a substituted or unsubstituted phenylene group, a substituted or unsubstituted thienylene group, a group represented by formula (Q1), a group represented by formula (Q2), and a group represented by formula (Q3); and each n is independently selected from an integer of 1 to 10;

[0066] The substituents optionally contained in Y1 and Y2 are independently selected from H, C 1-10 Alkyl, C 1-10 of alkoxy.

[0067] Preferred embodiment 2:

[0068] In formula (I),

[0069] X1 and X2 are each independently selected from S or Se;

[0070] R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-6 Alkyl, phenyl, fluorine, chlorine, bromine, At least one of, and Z is C 1-6 A straight chain alkoxy group, m is an integer of 1-6;

[0071] (A - ) is (PF6 - ), (CF3SO3 - )、(ClO4 - ) or (BF4 - );

[0072] Y1 and Y2 are each independently selected from at least one of the following groups:

[0073]

[0074] Each n is independently selected from an integer of 1-10.

[0075] Preferred embodiment 3:

[0076] In formula (I),

[0077] X1 and X2 are each independently selected from S or Se;

[0078] R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-4 Alkyl, fluorine, chlorine, bromine, At least one of, and Z is C 1-4 A straight-chain alkoxy group, m is an integer of 1-4;

[0079] (A - ) is (PF6 - ), (CF3SO3 - )、(ClO4 - ) or (BF4 - );

[0080] Y1 and Y2 are each independently selected from at least one of the following groups:

[0081]

[0082] Each n is independently selected from an integer of 1-6.

[0083] Preferred embodiment 4:

[0084] In formula (I),

[0085] X1 and X2 are each independently selected from S or Se;

[0086] R1, R2, R3, R4, R5, R6 are each independently selected from H, methyl, ethyl, isopropyl, n-propyl, fluorine, chlorine, bromine, At least one of, and Z is methoxy, ethoxy, m is an integer of 1-4;

[0087] (A - ) is (CF3SO3 - );

[0088] Y1 and Y2 are each independently selected from at least one of the following groups:

[0089]

[0090] Each n is independently selected from an integer of 1-6.

[0091] Preferred embodiment 5:

[0092] The complex represented by formula (I) of the present invention is any one of the following compounds:

[0093] Compound N-1:

[0094]

[0095] Compound N-2:

[0096]

[0097] Compound N-3:

[0098]

[0099] Compound N-4:

[0100]

[0101] Compound N-5:

[0102]

[0103] Compound N-6:

[0104]

[0105] Compound N-7:

[0106]

[0107] Compound N-8:

[0108]

[0109] As described above, the second aspect of the present invention provides a method for preparing an organometallic ruthenium macrocyclic complex represented by formula (I), the method comprising: contacting a compound represented by formula (M) with a compound represented by formula (B) in the presence of a solvent, wherein, in formula (B), A is PF6, CF3SO3, ClO4 or BF4;

[0110] In formula (M) and formula (B), the definitions of Y1, Y2, X1, X2, R1, R2, R3, R4, R5, and R6 are the same as those described in the first aspect.

[0111] Preferably, the molar ratio of the compound represented by formula (M) to the compound represented by formula (B) is 1:(1-5).

[0112] Preferably, the contacting conditions include: a temperature of 15-35° C. and a time of 12-36 h.

[0113] Preferably, the contacting is performed under light-shielding conditions.

[0114] The aforementioned preparation method of the present invention may also involve various post-processing operations known in the art, such as extraction, washing, filtration, column chromatography, etc. The present invention has no particular limitation thereto, and those skilled in the art should not interpret this as a limitation of the present invention.

[0115] It should be noted that the raw materials involved in the preparation method of the present invention can be synthesized according to the raw material structural formula in combination with organic synthesis methods in the art, or can be obtained commercially. The following text of the present invention exemplifies the preparation methods of several raw materials, which should not be construed as limiting the present invention by those skilled in the art.

[0116] In formula (M), when X1 and X2 are both S, Y1 is Y2 is When , the compound with the structure represented by formula (M) is the compound with the structure represented by formula (M1); in formula (B), when R1 is methyl, R4 is isopropyl, R2, R3, R5, and R6 are all H, and A is CF3SO3, the compound with the structure represented by formula (B) is the compound with the structure represented by formula (B1).

[0117]

[0118] According to a preferred embodiment, the synthetic route for preparing the compound represented by formula (M) is as follows:

[0119]

[0120] Illustratively, a method for preparing a compound represented by formula (M):

[0121] (1) Compound 2, bipinacol borate and potassium acetate (KOAc) were dissolved in 1,4-dioxane, and bis(triphenylphosphine)palladium(II) dichloride [Pd(PPh3)2Cl2] was added under nitrogen protection. The mixture was heated in an oil bath at 80-120°C for 10-16 hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column to obtain intermediate compound 3.

[0122] (2) Argon was introduced into a tetrahydrofuran (THF) solution containing Compound 3 and Compound 4 for 3-8 minutes. Under an argon atmosphere, an aqueous solution of potassium carbonate (K2CO3) and a 1,1-bis(diphenylphosphino)ferrocenepalladium(II) dichloropalladium dichloromethane complex (Pd(dppf)Cl2 CH2Cl2) were added. The mixture was heated in an oil bath at 50-100°C for 22-26 hours, cooled to room temperature, and the solvent was removed in vacuo. The residue was dissolved in dichloromethane, and the resulting organic phase was washed with water and saturated brine, and dried over anhydrous magnesium sulfate. The crude product was purified by silica gel chromatography to obtain the intermediate compound 5.

[0123] (3) Zinc powder and ammonium chloride are added to a mixed solution of methanol (e.g., 90% by volume of methanol) and dichloromethane containing compound 5 at room temperature and protected with argon, and stirred at room temperature for 3-5 hours. The solution is filtered through a celite pad, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain a yellow solid product; then anhydrous pyridine, N-sulfenylanilide and trimethylsilyl chloride are added and heated in an oil bath at 60-100°C for 18-22 hours. After the reaction is completed, it is cooled to room temperature, poured into ice water, extracted with dichloromethane, and the combined organic layer is washed with water and saturated brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The crude product is purified by silica gel chromatography to obtain the compound represented by formula (M).

[0124] Preferably, in step (1), the molar ratio of the compound 2, bipinacol borate, potassium acetate and bis(triphenylphosphine)palladium(II) dichloride is 1:(1.7-1.9):(2.3-2.5):(0.05-0.15); based on 3.5 mmol of compound 2, the amount of 1,4-dioxane used is 10-40 mL.

[0125] Preferably, in step (2), the molar ratio of the compound 3, compound 4, potassium carbonate and 1,1-bis(diphenylphosphino)ferrocenepalladium(II) dichloridepalladium dichloromethane complex is 1:(0.4-0.5):(1.2-1.3):(0.05-0.2); based on 0.85 mmol of compound 3, the amounts of tetrahydrofuran and water dissolved in potassium carbonate are 8-10 mL and 2-4 mL, respectively.

[0126] Preferably, in step (3), the molar ratio of the compound 5, ammonium chloride, zinc powder, N-sulfenylaniline and trimethylchlorosilane is 1: (58-62): (118-122): (50-54): (54-58); based on 0.34 mmol of compound 5, the amounts of methanol and anhydrous pyridine are 10-40 mL and 2-4 mL, respectively.

[0127] According to a preferred embodiment, the synthetic route for preparing the compound represented by formula (B1) is as follows:

[0128]

[0129] Illustratively, the method for preparing the compound represented by formula (B1):

[0130] S1: Dissolve ruthenium (III) chloride hydrate in anhydrous ethanol and add compound 7 under argon. Heat in an oil bath at 60-100°C for 5-7 hours. Concentrate the reaction solution, filter it, and wash it continuously with ethanol to obtain a red solid, compound 8. The molar ratio of compound 7 to ruthenium (III) chloride hydrate is 1:3-4. Based on 7.66 mmol of compound 7, the amount of anhydrous ethanol used is 20-50 mL.

[0131] S2: Under an argon atmosphere, compound 8, compound 9, and sodium acetate were added to anhydrous ethanol. The mixture was heated in an oil bath at 60-100°C for 22-26 hours. The reaction solution was concentrated and filtered, and washed with ethanol, water, acetone, and diethyl ether, respectively, to obtain compound 10 as a black solid. The molar ratio of compound 8, compound 9, and sodium acetate was 1:(1.1-1.3):(2.0-2.2), based on 0.8 mmol of compound 8. The amount of anhydrous ethanol used was 10-40 mL.

[0132] S3: Compound 10 and silver trifluoromethanesulfonate (Ag(CF3SO3), abbreviated as AgOTf) were dissolved in dichloromethane and stirred at room temperature for 2-4 hours. The dark green reaction solution was filtered and washed with methanol to obtain the compound represented by formula (B1). The molar ratio of compound 10 to silver trifluoromethanesulfonate was 1:(2.0-2.2). The amount of dichloromethane used was 1-30 mL based on 0.78 mmol of compound 12.

[0133] It should be noted that the method for preparing the compound represented by formula (I), the compound represented by formula (M) and the compound represented by formula (B) is preferably carried out under stirring conditions. The present invention has no special requirements for the stirring speed and can be carried out using parameters known in the art.

[0134] As mentioned above, the third aspect provides the use of the organometallic ruthenium macrocyclic complex described in the first aspect in anti-tumor drugs and / or antibacterial drugs.

[0135] As mentioned above, the fourth aspect of the present invention provides an anti-tumor drug, which contains a therapeutically effective amount of an active ingredient, wherein the active ingredient includes at least one of the organometallic ruthenium macrocyclic complexes described in the first aspect; optionally, the anti-tumor drug also contains an excipient.

[0136] Preferably, the content of the active ingredient is 1-99.9% by weight.

[0137] More preferably, in the anti-tumor drug, the content of the active ingredient is 5-95 weight%. Exemplarily, the content of the active ingredient is 10 weight%, 15 weight%, 20 weight%, 25 weight%, 30 weight%, 35 weight%, 40 weight%, 45 weight%, 50 weight%, 55 weight%, 60 weight%, 65 weight%, 70 weight%, 75 weight%, 80 weight%, 85 weight%, 90 weight%, etc.

[0138] In the present invention, the auxiliary materials are various auxiliary materials conventionally used in the art, such as solvents, antioxidants, fillers, etc.

[0139] Preferably, the dosage form of the anti-tumor drug is at least one selected from tablets, capsules, and injections.

[0140] As mentioned above, the fifth aspect of the present invention provides the use of the organometallic ruthenium macrocyclic complex described in the first aspect in near-infrared second-zone fluorescence imaging of tumor cells.

[0141] The present invention uses benzothiazole / selenadiazole derivatives as ligand units and half-sandwich ruthenium as a receptor unit, and provides an organometallic ruthenium macrocyclic complex represented by formula (I) with near-infrared second-zone luminescence properties through coordination supramolecular self-assembly. Compared with other commercial chemotherapy drugs, the macrocyclic complex not only has good phototherapy-chemotherapy combined treatment capabilities, but also has smaller toxic side effects and stronger anti-cancer effects. In addition, the macrocyclic complex can achieve tumor combined treatment precisely guided by near-infrared second-zone fluorescence imaging technology. Therefore, as a type of luminescent organometallic ruthenium macrocyclic complex, it has great application potential in the biomedical field.

[0142] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available analytically pure products. Unless otherwise specified, room temperature in the following examples refers to 25±3°C.

[0143] Preparation Example 1

[0144] This preparation example is used to illustrate the preparation of the compound represented by formula (M1).

[0145] (1) Preparation of intermediate 2a

[0146]

[0147] p-Bromoiodobenzene (7.07 mmol) and 4-pyridineboronic acid (5.66 mmol) were dissolved in toluene (40.0 mL). Sodium carbonate (16.94 mmol) was dissolved in 4.0 mL of deionized water. Under an argon atmosphere, the sodium carbonate solution, tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 0.176 mmol), and ethanol (12.0 mL) were added to the toluene solution containing p-bromoiodobenzene and 4-pyridineboronic acid. The reaction was heated in an oil bath at 115°C for 6 h. After completion of the reaction, the mixture was extracted with dichloromethane and water, concentrated under reduced pressure, and the crude product was purified on a silica gel column to obtain the white solid product 2a (yield 72.5%).

[0148] 1 H NMR (400MHz, CDCl3) δ8.68 (d, J = 5.4Hz, 2H), 7.63 (d, J = 8.5Hz, 2H), 7.49 (dd, J = 13.7, 7.3Hz, 4H). 13 C NMR (101MHz, CDCl3) δ150.43,147.12,137.03,132.33,128.57,123.58,121.38.

[0149] (2) Preparation of intermediate 3a

[0150]

[0151] Compound 2a (3.50 mmol), pinacol borate (6.31 mmol), and potassium acetate (KOAc) (8.41 mmol) were dissolved in 1,4-dioxane (25.0 mL). Bis(triphenylphosphine)palladium(II) dichloride (0.35 mmol) was added under nitrogen, and the reaction mixture was heated in an oil bath at 100°C for 12 h. After completion of the reaction, the reaction solution was extracted with ethyl acetate and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column to obtain a colorless solid product 3a (yield 76.2%).

[0152] 1 H NMR (400MHz, CDCl3) δ8.67(d,J=6.0Hz,2H),7.94(d,J=8.1Hz,2H),7.65(d,J=8.1Hz,2H),7.54(dd,J=4.7,1.3Hz,2H),1.38(s,12H). 13 C NMR (151MHz, CDCl3) δ150.05,148.20,140.49,135.53,126.25,121.73,84.01,83.98,24.87.

[0153] (3) Preparation of intermediate 5a

[0154]

[0155] Argon was passed through a tetrahydrofuran solution (7.5 mL) containing compound 3a (0.639 mmol) and compound 4a (0.288 mmol) for 5 min. Potassium carbonate (0.799 mol) was dissolved in water (2 mL) to form an aqueous potassium carbonate solution. Under an argon atmosphere, the aqueous potassium carbonate solution and 1,1-bis(diphenylphosphino)ferrocenepalladium(II) dichloropalladium dichloromethane complex (0.0639 mmol) were added. The mixture was heated in a 75°C oil bath for 24 h, cooled to room temperature, and the solvent was removed in vacuo. The mixture after removal of the solvent was dissolved in dichloromethane, and the resulting organic phase was washed with water and saturated brine, and then dried over anhydrous magnesium sulfate. The crude product was purified by silica gel chromatography to obtain an orange-yellow solid compound 5a (yield: 38.0%).

[0156] 1H NMR (400MHz, CDCl3) δ8.70(dd,J=4.6,1.5Hz,4H),7.75(d,J=8.4Hz,4H),7.66(d,J=8.3Hz,4H),7.57(dd,J=4.6,1 .6Hz,4H),7.44(s,2H),2.81–2.69(m,4H),1.68–1.59(m,4H),1.38(dd,J=14.9,7.4Hz,4H),0.92(t,J=7.3Hz,6H). 13 C NMR (151MHz, CDCl3) δ152.03,150.32,147.46,143.86,141.41,140.36,137.79,134.19,133.50,129.98, 128.03,127.31,121.49,120.74,77.25,77.04,76.83,60.44,32.91,28.42,22.45,21.09,14.19,13.90.

[0157] (4) Preparation of the compound represented by formula (M1)

[0158]

[0159] At room temperature, zinc powder (24.0 mmol) and ammonium chloride (12.0 mmol) were added to a mixed solution of 90% methanol (10.0 mL) and compound 5a (0.20 mmol) in dichloromethane (10.0 mL) under argon protection. The mixture was stirred at room temperature for 4 h, filtered through a pad of celite, dried over anhydrous sodium sulfate, and spin-dried to obtain a yellow solid product. Anhydrous pyridine (2.0 mL), N-sulfenylanilide (10.4 mmol), and trimethylsilyl chloride (11.2 mmol) were then added, and the mixture was heated in an 80°C oil bath for 20 h. After completion of the reaction, the mixture was cooled to room temperature, poured into ice water, and extracted with dichloromethane. The combined organic layers were washed with water and saturated brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The crude product was purified by silica gel chromatography to obtain the compound represented by formula (M1) as a green solid (yield 7.7%).

[0160] 1 H NMR (400MHz, CDCl3) δ8.64(d,J=4.9Hz,4H),7.74(s,8H),7.60(d,J=5.5Hz,4H),7.52(s,2H) ,2.87(d,J=7.7Hz,4H),1.79(d,J=7.7Hz,4H),1.45(d,J=7.5Hz,5H),0.96(d,J=7.3Hz,6H).

[0161] Preparation Example 2

[0162] This preparation example is used to illustrate the preparation of the compound represented by formula (B1).

[0163] 1) Preparation of intermediate 8

[0164]

[0165] Ruthenium (III) trichloride hydrate (7.66 mmol) was dissolved in anhydrous ethanol (35.0 mL). Under the protection of argon, compound 7 (26.8 mmol) was added. The mixture was heated in an oil bath at 80°C for 6 h. The reaction solution was concentrated and filtered, and washed continuously with ethanol to obtain compound 8 as a red solid (yield 88.8%).

[0166] 1 H NMR (400MHz, CDCl3) δ5.49(s,4H),5.34(s,4H),2.92(dt,J=13.9,6.9Hz,2H),2.16(s,6H),1.28(d,J=6.9Hz,12H). 13 C NMR (101MHz, CDCl3) δ101.19,96.73,81.30,80.54,30.61,22.14,18.91.

[0167] 2) Preparation of intermediate 10

[0168]

[0169] Compound 8 (0.800 mmol), compound 9 (0.950 mmol) and sodium acetate (1.71 mmol) were added to anhydrous ethanol (20.0 mL). Under an argon atmosphere, the mixture was heated in an 80°C oil bath for 24 h. After the reaction was completed, the mixture was concentrated and filtered, and washed with ethanol, water, acetone and diethyl ether, respectively, to obtain a black solid product 10 (yield 98.6%).

[0170] 1 H NMR (400MHz, CDCl3) δ6.99 (s, 4H), 5.53 (d, J = 5.7Hz, 4H), 5.27 (d, J = 5.6Hz, 4H), 2.95–2.87 (m, 2H), 2.26 (s, 6H), 1.35 (d, J = 6.9Hz, 12H). 13 C NMR (101MHz, CDCl3) δ171.01,137.07,111.98,100.39,98.00,82.89,79.68,30.79,22.39,17.93.

[0171] 3) Preparation of the compound represented by formula (B1)

[0172]

[0173] Compound 10 (0.78 mmol) and silver trifluoromethanesulfonate (1.60 mmol) were dissolved in dichloromethane (8.0 mL), stirred at room temperature for 3 h, filtered, and washed with methanol to obtain the compound represented by formula (B1) (yield 84.4%).

[0174] 1 H NMR (400MHz, CH3OD) δ7.24 (s, 4H), 5.78 (d, J = 6.2Hz, 4H), 5.53 (d, J = 6.2Hz, 4H), 2.80 (t, J = 6.9Hz, 2H), 2.17 (s, 6H), 1.29 (d, J = 6.9Hz, 12H). 13 C NMR (101MHz, CD3OD) δ171.41,137.24,121.96,118.79,110.31,100.86,98.40,81.38,78.85,30.77,21.00,16.23.

[0175] Example 1

[0176] This example is used to illustrate the preparation of the organometallic ruthenium macrocyclic complex N-1 of the present invention.

[0177] The complex N-1 was prepared using the following reaction formula:

[0178]

[0179] The compound represented by formula (M1) (0.00640 mmol) and the compound represented by formula (B1) (0.00640 mmol) were placed in a brown bottle, followed by the addition of methanol (4.0 mL) and chloroform (2.0 mL). The mixture was stirred at room temperature in the dark for 24 hours. After the reaction was complete, the reaction solution was concentrated, filtered after adding diethyl ether, washed twice with diethyl ether, and dried under vacuum to obtain complex N-1 (yield 75%).

[0180] 1H NMR (400MHz, CD3CN) δ8.19(d,J=6.6Hz,8H),7.74(d,J=8.3Hz,9H),7.45(d,J=8.4Hz,8H),7.42(d,J=6.6Hz,8H),7.10(s,8H) ,5.62(d,J=6.2Hz,8H),5.41(d,J=6.2Hz,8H),4.35(s,8H),4.19(s,8H),2.81(s,4H),2.04(s,12H),1.24(d,J=6.9Hz,24H).

[0181] The complex N-1 was characterized by H NMR spectroscopy (the results are shown in Figure 1 ), NMR fluorine spectrum characterization (results as Figure 2 ), Roesy spectrum characterization (results as Figure 3 ) and electrospray time-of-flight mass spectrometry characterization (results as Figure 4 ).Depend on Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 It can be seen that the present invention successfully prepared complex N-1.

[0182] Test Example 1: Optical Performance Test

[0183] The complex N-1 was dissolved in dichloromethane to a concentration of 10 μM, and the UV absorption spectrum and fluorescence emission spectrum of the complex N-1 were measured. The results are shown in Figure 5 ,from Figure 5 It can be seen that the maximum absorption wavelength is 760 nm and the maximum emission wavelength is 970 nm.

[0184] Test Example 2: Chemical Stability Test

[0185] 10 μM complex N-1 was incubated in 10 mM phosphate buffered saline (PBS, pH = 7.4) for different time periods (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h), and UV-visible absorption spectroscopy was performed. The results are shown in FIG. Figure 6 As shown. Figure 6 It can be seen that the incubation time of complex N-1 in phosphate buffered saline for different times has little effect on the intensity of UV absorption.

[0186] 10 μM complex N-1 was mixed with 10 mM PBS solution, and the pH value was adjusted to 4.5, 5.5, 6.5, and 7.5 respectively with hydrochloric acid and sodium hydroxide. UV-visible absorption spectrum test was performed under different pH conditions (4.5-7.5). The test results are shown in Figure 2. Figure 7 As shown by Figure 7It can be seen that under different pH conditions, the absorption intensity does not change much, indicating that complex N-1 is relatively stable under these conditions.

[0187] Test Example 3: Light Stability Test

[0188] The photostability of complex N-1 was investigated in PBS by continuous laser (808 nm, 0.8 W / cm 2 ) irradiate a 1 cm quartz cuvette containing 200 μL of 10 μM complex N-1 for 60 minutes. The absorbance is recorded every 10 minutes. The test results are as follows Figure 8 As shown by Figure 8 It can be seen that within 60 minutes, complex N-1 has been relatively stable under continuous laser irradiation.

[0189] Test Example 4: Tissue Penetration Depth Test

[0190] 1g agar, 5mL 20% fat emulsion and 100mL water were mixed and the mixture was heated to boiling. Two drops of ink were added and cooled to about 40°C. The solution was poured into a plate to prepare a tissue model (1% fat emulsion). 10μM of complex N-1 was added to each well of a well plate (pore size 0.5mm, depth 10mm) and measured using the Series II 900 / 1700 NIR-II fluorescence imaging system (808nm, 1000LP and 30ms). FL images were analyzed by Image J software, and the test results are shown in Figure 2. Figure 9 As shown by Figure 9 It can be seen that the red light intensity becomes weaker from top to bottom. At 8mm, there is almost no obvious red fluorescence, indicating that the penetration depth of N-1 can reach 7mm.

[0191] Test Example 5: Photothermal Conversion Performance Test

[0192] In a 0.5 mL centrifuge tube, 100 μL of complex N-1 with concentrations of 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM was added. The complex N-1 was irradiated with an 808 nm laser (power density of 0.5 W / cm 2 ) for 5 minutes. The temperature changes were recorded by an infrared thermal imager (Fotric225s). The results are as follows Figure 10 As shown, from Figure 10 It can be seen that the temperature after laser irradiation increases with the concentration of complex N-1, indicating that complex N-1 has a good light-to-heat conversion effect.

[0193] Test Example 6: Photodynamic Performance Test

[0194] In a centrifuge tube, 400 μL of 10 μM complex N-1 was mixed with a working solution prepared according to the instructions of the reactive oxygen species indicator DCFH-DA (purchased from MedChem Express, USA). The working solution was prepared as follows: 25 μL of 10 mM DCFH-DA in DMSO was added to 0.5 μL of 10 M NaOH to generate DCFH. The complex N-1 was illuminated with an 808 nm laser (power density of 0.5 W / cm 2 ) were irradiated for 0s, 60s, 120s, 180s, 240s, and 300s, and the changes in the fluorescence intensity of DCFH at the characteristic peak of 525nm were monitored (e.g. Figure 11 ),Depend on Figure 11 It can be seen that with the extension of irradiation time, the fluorescence emission of DCFH at the characteristic peak of 525 nm increased significantly, indicating that complex N-1 produced reactive oxygen species under light irradiation and had a good ability to produce reactive oxygen species.

[0195] Test Example 7: Detection of Intracellular Reactive Oxygen Species

[0196] Human cervical cancer Hela cells in logarithmic growth phase were seeded in confocal culture dishes at a cell number of 1×10 4 The cells were cultured at 37°C for 6 h, and complex N-1 (600 μL, 10 μM) was added and incubated for 2 h. DCFH-DA (1.2 μL, 20 μM) was added and incubated for 20 min. The cells were illuminated with an 808 nm laser (power density of 0.5 W / cm 2 ) irradiate for 5 minutes, aspirate all the liquid, wash once with PBS buffer, add nuclear stain 4',6-diamidino-2-phenylindole (DAPI, 50μL), stain for 5 minutes at room temperature, aspirate DAPI, and wash three times with PBS buffer. The culture dish was placed under an inverted fluorescence microscope for observation. The results are as follows Figure 12 . Figure 12 The figures show the results of laser confocal fluorescence imaging of the HeLa cells: bright field imaging (Bright Field), complex N-1 (N-1, red fluorescence), the known DCFH-DA fluorescent probe (DCF, green fluorescence), and the overlap (Merge) of the complex N-1 and the DCFH-DA fluorescent probe. The figure shows that the complex N-1 can be used for near-infrared second-zone fluorescence imaging of cells and that reactive oxygen species are generated in the cells under illumination.

[0197] Test Example 8: Anti-tumor application of organometallic ruthenium macrocyclic complexes

[0198] (1) In vitro anti-tumor performance test

[0199] Cytotoxicity tests were performed using the MTT assay. Non-small cell lung cancer A549 cells, human cervical cancer HeLa cells, human liver cancer HepG-2 cells, human lung adenocarcinoma cisplatin-resistant strain A549 / DDP, and human normal bronchial cells 16HBE were cultured in 96-well plates in DMEM medium containing 10% fetal bovine serum (FBS, Ausbian, model WS500T) and 1% penicillin-streptomycin (Solarbio, P7630) at 37°C in a humidified atmosphere of 5% CO2 for 24 hours. 0.5×10 cells were seeded into each well. 4 cells, and then cultured in different concentrations (0 μM, 6 μM, 12 μM, 25 μM, 50 μM, 100 μM, 200 μM) of cisplatin or N-1 culture medium for 12 hours.

[0200] For the A549 hypoxia group, cells in the logarithmic growth phase were trypsinized and seeded into 96-well plates. After 12 hours of quiescence, the 96-well plates were transferred to a hypoxic sealed box containing a fresh hypoxia pack (AnaeroPack-Anaero, Mitsubishi, Japan) (oxygen content <1%). The cells were maintained in a hypoxic environment for more than 6 hours. The original culture medium was then discarded, and the cells were incubated with different concentrations of cisplatin or N-1 before the cytotoxicity test was performed. The cells were kept in a hypoxic environment throughout the incubation process.

[0201] For the dark group, after incubation with N-1 or cisplatin for 12 hours, the culture medium containing N-1 was incubated in the dark for 48 hours; for the light group, after incubation with N-1 or cisplatin for 12 hours, the cells were exposed to 808nm irradiation for 5 minutes and then continued to incubate in the dark for 12 hours. 10μL of 0.5mg / mL MTT (BioFrox, China) solution was added to each well and incubated at 37°C for 4 hours to produce formazan crystals. Then, the supernatant was removed and the product was lysed with 200μL of DMSO. The absorbance value was recorded at 570nm using a microplate reader. The absorbance of cells not treated with drugs was used as the control group, and its absorbance was used as the reference value for calculating 100% cell viability; the blank group was a group in which no cells were added and only culture medium was added for the experiment;

[0202] Inhibition rate % = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%;

[0203] IC 50 Calculation of values: Use the logarithm of compound concentration as the horizontal axis and the inhibition rate as the vertical axis. Fit a nonlinear curve in GraphPadPrism6: log(inhibitor) vs. normalized response. Read IC in the Best-fit values.50 The value is the concentration value at which N-1 or cisplatin induces 50% apoptosis of tumor cells. The results are shown in Table 1;

[0204] Phototoxicity index a =IC 50(黑暗) / IC 50(光照) ;

[0205] Selectivity Index b =IC 50(正常) / IC 50(A549癌细胞) .

[0206] Table 1

[0207]

[0208] As shown in Table 1, compared with the clinical gold standard cisplatin, complex N-1 has better in vitro antitumor activity and lower normal cell toxicity than cisplatin under both normoxic and hypoxic conditions.

[0209] (2) Fluorescence imaging in mice

[0210] A549 tumor-bearing mice were intravenously injected with complex N-1 (200 μL, 1 mg Ru / Kg). NIR-II fluorescence data were collected at 2 h, 4 h, 6 h, 12 h, 24 h, 48 h, and 72 h after injection. Images were taken using a NIR-II fluorescence imaging system (808 nm, 1000 LP, and 30 ms). The NIR-II fluorescence intensity of the tumor was calculated using ImageJ software. The results are shown in Figure 5. Figure 13 As shown. Figure 13 It can be seen that the NIR-II fluorescence intensity of the N-1 tumor reached the maximum accumulation at 12 h.

[0211] (3) In vivo antitumor activity

[0212] The tumor volume and body weight of A549 tumor-bearing mice were measured. The size (length and width) of the tumor was measured with a vernier caliper and the tumor volume was calculated using the formula = (length × width 2 ) / 2. When the average tumor volume reaches about 100mm 3 The mice were randomly divided into five groups, and this day was set as day 1. The mice were intravenously injected with PBS, cisplatin (dosage of 1 mg Pt / kg), and complex N-1 (dosage of 1 mg Ru / kg). For the PBS+Laser and N-1+Laser groups, the mice were injected with PBS or complex N-1 and then laser-excited with 808 nm laser (power density of 1.0 W / cm 2 ) irradiated for 10 min. The tumor volume of A549 tumor-bearing mice was measured every 3 days (the results are shown in Figure 14 ) and weight (results such as Figure 15 ).

[0213] exist Figure 14 and Figure 15 In the table, PBS means that mice were intravenously injected with PBS solution, PBS+Laser means that mice were intravenously injected with PBS and then irradiated with laser, Cisplatin means that mice were intravenously injected with cisplatin, N-1 means that mice were intravenously injected with complex N-1, and N-1+Laser means that mice were intravenously injected with complex N-1 and then irradiated with laser. Figure 14 It can be seen that compared with the injection of PBS, PBS+Laser, and cisplatin, the tumor volume growth rate of mice injected with complex N-1 and N-1+Laser was relatively slow, and the effect of laser irradiation for tumor treatment after the mice were injected with complex N-1 was better than that of the injection of complex N-1 alone. Figure 15 It can be seen that there was almost no significant change in the body weight of mice in each group, indicating that N-1 did not produce systemic toxicity.

[0214] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. An organometallic ruthenium macrocyclic complex, characterized in that The complex has a structure shown in formula (I): Formula (I), Wherein, in formula (I), X1 and X2 are each independently selected from S or Se; R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-6 At least one of the alkyl groups of (A-) is (CF3SO3-); Y1 is , Y2 is , and each n is independently selected from an integer of 1-6.

2. The complex according to claim 1, wherein In formula (I), X1 and X2 are each independently selected from S or Se; R1, R2, R3, R4, R5, and R6 are each independently selected from H, C 1-4 At least one of the alkyl groups of (A-) is (CF3SO3-); Y1 is , Y2 is , and each n is independently selected from an integer of 1-6.

3. The complex according to claim 1 or 2, wherein In formula (I), X1 and X2 are each independently selected from S or Se; R1, R2, R3, R4, R5, and R6 are each independently selected from at least one of H, methyl, ethyl, isopropyl, and n-propyl; (A-) is (CF3SO3-); Y1 is , Y2 is , and each n is independently selected from an integer of 1-6.

4. The complex according to claim 1 or 2, wherein The complex represented by formula (I) is the following compound: Compound N-1: 。 5. A method for preparing the organometallic ruthenium macrocyclic complex of formula (I) according to any one of claims 1 to 4, characterized in that: The method comprises: contacting a compound represented by formula (M) with a compound represented by formula (B) in the presence of a solvent, Formula (M), Formula (B), Wherein, in formula (B), A is CF3SO3; In formula (M) and formula (B), the definitions of Y1, Y2, X1, X2, R1, R2, R3, R4, R5, and R6 are the same as those in any one of claims 1 to 4.

6. The method according to claim 5, wherein: The molar ratio of the compound represented by formula (M) to the compound represented by formula (B) is 1:(1-5).

7. The method according to claim 5 or 6, wherein: The contact conditions include: temperature of 15-35° C. and time of 12-36 hours.

8. Use of the organometallic ruthenium macrocyclic complex according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The anti-tumor drug contains a therapeutically effective amount of active ingredients, wherein the active ingredients include at least one of the organometallic ruthenium macrocyclic complexes according to any one of claims 1 to 4; optionally, the anti-tumor drug further contains excipients.

10. The antitumor drug according to claim 9, wherein The content of the active ingredient is 1-99.9% by weight.

11. The antitumor drug according to claim 9 or 10, wherein The dosage form of the anti-tumor drug is selected from at least one of tablets, capsules, and injections.

12. Use of the organometallic ruthenium macrocyclic complex according to any one of claims 1 to 4 in the preparation of a near-infrared second-region fluorescence imaging agent for tumor cells.

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