An N^N^N type terpyridine platinum complex and its preparation method and application

Abstract

The present invention discloses an N^N^N type terpyridine platinum complex, its preparation method, and application. The present invention relates to the technical field of fluorescent probe material preparation, and specifically to an N^N^N type terpyridine platinum complex, its preparation method, and application. The present invention mixes 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) with K2PtCl4, adds a halogenated hydrocarbon solvent and an alcohol solvent, and conducts a heating reaction. After the reaction, the solvent is removed by reduced pressure distillation, and column chromatography is used for purification to obtain an N^N^N type terpyridine platinum complex. The preparation method of the N^N^N type terpyridine platinum complex of the present invention has readily available raw materials, a simple reaction process, and can be industrially produced. It has specific targeting to lysosomes, low cytotoxicity, and good two-photon absorption activity, and has good application prospects in lysosomal analysis and imaging products.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probe material preparation, and in particular to an N^N^N type terpyridine platinum complex, a preparation method and application thereof. Background Art

[0002] Globally, the incidence and mortality rates of malignant tumors, with the exception of cervical and esophageal cancers, are increasing year by year. Lysosomes, as one of the most important functional organs in cells, play a crucial role in human health. Lysosomes significantly influence the survival and metastasis of cancer cells. Therefore, the use of fluorescent probes targeting lysosomes can enable precise tumor tracking and treatment, resulting in better therapeutic outcomes.

[0003] Currently, the lysosomal fluorescent probes that are widely studied are mostly organic small molecules. This type of probe has problems such as high toxicity and poor photostability. In addition, common imaging technologies are mainly limited to short-wave excitation, and have disadvantages such as weak tissue penetration and large photodamage.

[0004] Chinese patent publication number CN105062463A discloses binuclear ruthenium complexes with varying alkyl chain lengths as lysosomal probes in cells. These complexes can easily penetrate cell membranes and target lysosomes for lysosomal staining. Due to the correlation between DNA binding and cytotoxicity, currently reported ruthenium compounds generally suffer from high dark toxicity, resulting in high cytotoxicity.

[0005] Chinese patent document publication number CN114437010A discloses a lysosome-localized viscosity-responsive two-photon fluorescent probe, its preparation method, and application. The lysosome-localized viscosity-responsive two-photon fluorescent probe of this invention can achieve lysosome localization and viscosity response, but the two-photon absorption band and cross-section are small, which limits the practical application of two-photon absorption. Summary of the Invention

[0006] The present invention aims to provide an N^N^N type terpyridine platinum complex and its preparation method and application. The raw materials are readily available, the reaction process is a one-step synthesis, the synthesis is simple, and industrial production can be achieved. It has specific targeting to lysosomes and has the advantages of low cytotoxicity and good two-photon absorption activity.

[0007] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0008] An N^N^N type terpyridine platinum complex, which can be abbreviated as Pt-S1, has the following structural formula:

[0009] .

[0010] The present invention also provides a technical solution for the preparation of the above-mentioned N^N^N type terpyridine platinum complex:

[0011] First, 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) was prepared. Then, 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) was mixed with K2PtCl4. A halogenated hydrocarbon solvent and an alcohol solvent were added thereto, and the mixture was heated to react. After the reaction, the solvent was removed by distillation under reduced pressure. After purification using a chromatography column, a yellow solid product was obtained, which was an N^N^N-type terpyridine platinum complex.

[0012] The synthetic route is as follows:

[0013] .

[0014] 2-Acetylpyridine and KOH were weighed, water was added, and the mixture was stirred at room temperature until the solution turned yellow. 3,4-Diethoxybenzaldehyde and ethanol were then added, followed by dropwise addition of ammonia to allow the reaction to proceed. After the reaction, the precipitated solid was recrystallized from ethanol to yield yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0015] Furthermore, the mass ratio of 2-acetylpyridine to KOH is 1:0.47-1.16; the mass ratio of KOH to water is 1:2-4; the mass ratio of 3,4-diethoxybenzaldehyde, 2-acetylpyridine and ammonia water is 1:1.2-2.5:8.7-17.5; the mass ratio of 3,4-diethoxybenzaldehyde to ethanol is 1:5-6.3; the heating reaction temperature is 60-80°C, and the reaction time is 5-8h; the mass ratio of the precipitated solid to ethanol is 1:3-6.5, and the recrystallization heating temperature is 80-100°C, and the reaction time is 30min-60min.

[0016] Furthermore, the mass ratio of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) to K2PtCl4 is 1.5-4.8:1; the reaction temperature is 60-80°C for 8-15 hours; and the vacuum distillation pressure is -0.05-0.1 MPa, the temperature is 50-65°C, and the time is 30-65 minutes.

[0017] Furthermore, the halogenated hydrocarbon solvent is dichloromethane or chloroform, and the alcohol solvent is methanol or ethanol.

[0018] Furthermore, the mass ratio of the halogenated hydrocarbon solvent, the alcohol solvent and K2PtCl4 is 8-16:9-31:1.

[0019] Furthermore, in the chromatography column purification, the ratio of non-polar solvent I to polar solvent I is first controlled to be 5-30:1 to separate 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine, and the ratio of non-polar solvent II to polar solvent II is controlled to be 10-30:1 to separate the N^N^N type terpyridine platinum complex.

[0020] Furthermore, the non-polar solvent I is petroleum ether, the polar solvent I is ethyl acetate or acetone; the non-polar solvent II is dichloromethane, and the polar solvent II is methanol or propanol.

[0021] The present invention also provides a technical solution for the application of N^N^N type terpyridine platinum complexes, and applies the N^N^N type terpyridine platinum complexes to lysosome analysis and imaging products.

[0022] Beneficial effects of the present invention:

[0023] 1. The present invention utilizes readily available raw materials. 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) is prepared using the pharmaceutical intermediate 3,4-diethoxybenzaldehyde. The electron-rich nitrogen in ligand S1 coordinates with platinum, resulting in a one-step synthesis. The N^N^N-type terpyridine platinum complex incorporates a flexible chain, enhancing its biocompatibility. The inclusion of platinum atoms further enhances intramolecular electron mobility and two-photon absorption activity. The simple synthesis process allows for industrial production.

[0024] 2. The N^N^N-type terpyridine platinum complex obtained in the present invention has low toxicity. In the cell survival rate experiment, it was found that at a high concentration of 25 μM of the N^N^N-type terpyridine platinum complex, the survival rate could still reach more than 80%.

[0025] 3. The introduction of platinum atoms in this invention not only imparts a certain anti-tumor effect but also enhances electron mobility within the molecule, significantly improving its two-photon absorption activity. The two-photon absorption band reaches the second near-infrared region (960 nm), with a two-photon absorption cross section of 875-1780 GM. Due to the unique molecular structure and excellent assembly properties of platinum compounds, their particle size distribution, as measured by a Malvern laser particle size analyzer, ranges from 150-350 nm, facilitating cellular absorption.

[0026] 4. The N^N^N-type terpyridine platinum complex prepared by the present invention was subjected to cell confocal imaging experiments, and it was found that the staining location and phenomenon were consistent with lysosomal commercial staining, showing specific targeting of lysosomes, which has great practical significance for the development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is the mass spectrum of the N^N^N type terpyridine platinum complex in Example 1 of the present invention, where Relative Abundance is the relative abundance;

[0028] Figure 2 This is the H NMR spectrum of the N^N^N type terpyridine platinum complex in Example 1 of the present invention;

[0029] Figure 3 This is a two-photon Z-scan test image of the N^N^N type terpyridine platinum complex in Example 1 of the present invention under a 960 nm laser in the near-infrared region II;

[0030] Figure 4 This is a two-photon absorption cross-section diagram of N3 in Example 1 of the present invention under a laser with a wavelength of 680-1000 nm;

[0031] Figure 5 This is a Malvern particle size distribution diagram of the N^N^N type terpyridine platinum complex in Example 1 of the present invention when the PBS content is 60%;

[0032] Figure 6 is a scanning electron microscope (SEM) image of the N^N^N type terpyridine platinum complex in Example 1 of the present invention;

[0033] Figure 7 This is a diagram showing the cytotoxicity experiment of the N^N^N type terpyridine platinum complex in Example 1 of the present invention;

[0034] Figure 8 This is a co-localization experiment diagram of the N^N^N type terpyridine platinum complex stained in Hela cells in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. Example 1

[0036] 7.26 g of 2-acetylpyridine and 3.36 g of KOH were weighed, 6.72 g of water was added, and the mixture was stirred at room temperature until the solution turned yellow. 5.82 g of 3,4-diethoxybenzaldehyde and 29.1 g of ethanol were then added dropwise, followed by 51 g of aqueous ammonia. The reaction temperature was controlled at 60°C and the reaction was allowed to proceed for 8 hours. After the reaction, 5.14 g of the precipitated solid was recrystallized from 15.42 g of ethanol at 80°C for 60 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0037] 1.9 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.25 g of K2PtCl4 were dissolved in a mixture of 10.08 g of chloroform and 11.52 g of methanol. The reaction temperature was controlled at 60°C and the reaction was allowed to proceed for 15 h. After the reaction, the solvent was removed by vacuum distillation at -0.05 MPa and 50°C for 30 min. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-ethyl acetate (v / v = 30:1), and dichloromethane-methanol (v / v = 30:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0038] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1780 GM.

[0039] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 280 nm. SEM showed that the platinum complex had a spherical structure.

[0040] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 2

[0041] 11.13 g of 2-acetylpyridine and 6.18 g of KOH were weighed, 14.21 g of water was added, and the mixture was stirred at room temperature until the solution turned yellow. 7.77 g of 3,4-diethoxybenzaldehyde and 40.4 g of ethanol were then added dropwise, followed by 81.6 g of aqueous ammonia. The reaction temperature was controlled at 60°C and the reaction was allowed to proceed for 8 hours. After the reaction, 8.01 g of the precipitated solid was recrystallized from 28.04 g of ethanol at 80°C for 60 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0042] 2.62 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.31 g of K2PtCl4 were dissolved in a mixture of 11.79 g of chloroform and 17.03 g of methanol. The reaction temperature was controlled at 60°C and the reaction was allowed to proceed for 14 hours. After the reaction, the solvent was removed by vacuum distillation at -0.05 MPa and 50°C for 35 minutes. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-ethyl acetate (v / v = 20:1), and dichloromethane-methanol (v / v = 25:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0043] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1635 GM.

[0044] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 305 nm. SEM showed that the platinum complex had a spherical structure.

[0045] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 3

[0046] Weigh 15.73 g of 2-acetylpyridine and 10.19 g of KOH, add 26.49 g of water, and stir at room temperature until the solution turns yellow. Then, add 9.7 g of 3,4-diethoxybenzaldehyde and 52.38 g of ethanol, followed by 110.5 g of aqueous ammonia dropwise. The reaction temperature is controlled at 65°C and the reaction is allowed to proceed for 7 hours. After the reaction, 9.53 g of the precipitated solid is recrystallized from 38.12 g of ethanol at 85°C for 50 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0047] 3.38 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.35 g of K2PtCl4 were dissolved in a mixture of 13.5 g of chloroform and 21.6 g of ethanol. The reaction temperature was controlled at 65°C and the reaction was allowed to proceed for 13 h. After completion of the reaction, the solvent was removed by vacuum distillation at -0.07 MPa and 55°C for 40 min. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-ethyl acetate (v / v = 10:1), and dichloromethane-methanol (v / v = 20:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0048] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1568 GM.

[0049] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 324 nm. SEM showed that the platinum complex had a spherical structure.

[0050] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 4

[0051] Weigh 21.05 g of 2-acetylpyridine and 15.59 g of KOH, add 42.21 g of water, and stir at room temperature until the solution turns yellow. Then, add 11.64 g of 3,4-diethoxybenzaldehyde and 65.18 g of ethanol, followed by 142.5 g of aqueous ammonia dropwise. The reaction temperature is controlled at 65°C and the reaction is allowed to proceed for 7 hours. After the reaction, 11.25 g of the precipitated solid is recrystallized from 50.63 g of ethanol at 85°C for 45 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0052] 4.17 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.39 g of K2PtCl4 were dissolved in a mixture of 15.29 g of chloroform and 27.8 g of ethanol. The reaction temperature was controlled at 65°C and the reaction was allowed to proceed for 12 h. After completion of the reaction, the solvent was removed by vacuum distillation at -0.07 MPa and 55°C for 45 min. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-ethyl acetate (v / v = 5:1), and dichloromethane-propanol (v / v = 10:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0053] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1439 GM.

[0054] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 350 nm. SEM showed that the platinum complex had a spherical structure.

[0055] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 5

[0056] Weigh 27.1 g of 2-acetylpyridine and 22.6 g of KOH, add 72.32 g of water, and stir at room temperature until the solution turns yellow. Then, add 13.85 g of 3,4-diethoxybenzaldehyde and 80.33 g of ethanol, followed by 178.5 g of aqueous ammonia dropwise. The reaction temperature is controlled at 73°C and the reaction is allowed to proceed for 6 h. After the reaction, 12.4 g of the precipitated solid is recrystallized from 62.4 g of ethanol at 90°C for 40 min to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0057] 5.08 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.45 g of K2PtCl4 were dissolved in a mixture of 17.4 g of dichloromethane and 33.35 g of methanol. The reaction temperature was controlled at 70°C and the reaction was allowed to proceed for 11 hours. After the reaction, the solvent was removed by vacuum distillation at -0.09 MPa and 60°C for 50 minutes. The product was purified by column chromatography (using Al2O3 as a packing material) in the following order: petroleum ether, petroleum ether-acetone (v / v = 30:1), and dichloromethane-propanol (v / v = 30:1), yielding a yellow solid. This was the target N^N^N-type terpyridine platinum complex.

[0058] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1382 GM.

[0059] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 240 nm. SEM showed that the platinum complex had a spherical structure.

[0060] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 6

[0061] 33.88 g of 2-acetylpyridine and 31.36 g of KOH were weighed, 109.76 g of water was added, and the mixture was stirred at room temperature until the solution turned yellow. 15.52 g of 3,4-diethoxybenzaldehyde and 93.12 g of ethanol were then added dropwise, followed by 231.2 g of aqueous ammonia. The reaction temperature was controlled at 73°C and the reaction was allowed to proceed for 6 h. After the reaction, 14.29 g of the precipitated solid was recrystallized from 78.6 g of ethanol at 90°C for 40 min to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0062] 6 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.5 g of K2PtCl4 were dissolved in a mixture of 19.5 g of dichloromethane and 39 g of methanol. The reaction temperature was controlled at 70°C and the reaction was allowed to proceed for 10 h. After completion of the reaction, the solvent was removed by vacuum distillation at -0.09 MPa and 60°C for 55 min. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-acetone (V / V = 20:1), and dichloromethane-propanol (V / V = 25:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0063] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1246 GM.

[0064] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 215 nm. SEM showed that the platinum complex had a spherical structure.

[0065] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 7

[0066] 41.38 g of 2-acetylpyridine and 42.13 g of KOH were weighed, 160.09 g of water was added, and the mixture was stirred at room temperature until the solution turned yellow. 17.46 g of 3,4-diethoxybenzaldehyde and 108.25 g of ethanol were then added dropwise, followed by 290.7 g of aqueous ammonia. The reaction temperature was controlled at 80°C and the reaction was allowed to proceed for 5 hours. After the reaction, 16.63 g of the precipitated solid was recrystallized from 99.78 g of ethanol at 100°C for 30 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0067] 6.98 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.55 g of K2PtCl4 were dissolved in a mixture of 21.7 g of dichloromethane and 44.95 g of methanol. The reaction temperature was controlled at 80°C and the reaction was allowed to proceed for 9 hours. After the reaction, the solvent was removed by vacuum distillation at -0.1 MPa and 65°C for 60 minutes. The product was purified by column chromatography (using Al2O3 as a packing material) in the following order: petroleum ether, petroleum ether-acetone (V / V = 10:1), and dichloromethane-propanol (V / V = 20:1), yielding a yellow solid. This was the target N^N^N-type terpyridine platinum complex.

[0068] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 1054 GM.

[0069] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 173 nm. SEM showed that the platinum complex had a spherical structure.

[0070] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects. Example 8

[0071] Weigh 48.4 g of 2-acetylpyridine and 56 g of KOH, add 224 g of water, and stir at room temperature until the solution turns yellow. Then, add 19.4 g of 3,4-diethoxybenzaldehyde and 122.22 g of ethanol, followed by the dropwise addition of 340 g of aqueous ammonia. The reaction temperature was controlled at 80°C and the reaction was allowed to proceed for 5 hours. After the reaction, 18.52 g of the precipitated solid was recrystallized from 120.38 g of ethanol at 100°C for 30 minutes to obtain yellow needle-like crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1).

[0072] 7.68 g of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and 1.6 g of K2PtCl4 were dissolved in a mixture of 25.6 g of dichloromethane and 49.6 g of methanol. The reaction temperature was controlled at 80°C and the reaction was allowed to proceed for 8 h. After the reaction, the solvent was removed by vacuum distillation at -0.1 MPa and 65°C for 65 min. Purification by column chromatography (using Al2O3 as a packing material) in the order of petroleum ether, petroleum ether-acetone (V / V = 5:1), and dichloromethane-propanol (V / V = 10:1) afforded a yellow solid product. This was the target N^N^N-type terpyridine platinum complex.

[0073] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, and a two-photon Z-scan test was performed under the excitation of a laser with a wavelength of 680-1000 nm. The two-photon absorption cross section at 960 nm was 875 GM.

[0074] Preparation of platinum complex Pt-S1 in DMSO (1×10 -3 mol / L) solution, to which a 60% volume fraction PBS solution was added. The particle size was measured by a Malvern laser particle size analyzer and found to be uniformly distributed at 150 nm. SEM showed that the platinum complex had a spherical structure.

[0075] The dark toxicity of Pt-S1 (25 μM) on normal human liver cells HL-7702 was detected under dark conditions, and the cell survival rate reached more than 80%, indicating that Pt-S1 has good biocompatibility and can meet the requirements of cell imaging and other aspects.

[0076] In order to illustrate the use of the N^N^N type terpyridine platinum complex Pt-S1 in Example 1 of the present invention in anti-tumor drugs, proliferation inhibition activity experiments on various human tumor cell lines were carried out.

[0077] 1. This experiment used four human cell lines: human liver cancer HepG2 cells, human cervical cancer HeLa cells, human gastric cancer MGC80-3 cells, and human normal liver HL-7702 cells. All cell lines were cultured in RPMI-1640 medium supplemented with 100 U / mL penicillin, 10 wt% calf blood, and 100 U / mL streptomycin at 37°C in a 5% CO2 incubator.

[0078] Preparation of test compounds: The purity of the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) and the complex Pt-S1 used must be ≥95%. Their DMSO solutions are diluted with physiological buffer to a final solution of 20 μmol / L (final DMSO concentration ≤1%). The degree of inhibition of each compound on the growth of normal cells or selected tumor cells at this concentration is tested.

[0079] 2. Cell growth inhibition assay (MTT assay)

[0080] (1) Take tumor cells in the logarithmic growth phase, digest them with trypsin, and prepare a cell suspension with a concentration of 20,000 cells / ml in a culture medium containing 10% calf serum. Inoculate 190 μL per well in a 96-well culture plate to a cell density of 100 to 10,000 cells / well (fill the edge wells with sterile PBS);

[0081] (2) Incubate at 5% CO2 and 37°C for 24 h until the cell monolayer covers the bottom of the well. Add 10 μL of a certain concentration gradient of drug to each well, and set up 4 replicate wells for each concentration gradient.

[0082] (3) Incubate at 37°C in 5% CO2 for 48 h and observe under an inverted microscope.

[0083] (4) Add 10 μL of MTT solution (5 mg / mL PBS, i.e., 0.5% MTT) to each well and continue incubation for 4 h.

[0084] (5) Terminate the culture, carefully remove the culture medium from the wells, add 150 μL of DMSO to each well to fully dissolve the formazan precipitate, mix on an oscillator, and measure the optical density of each well using a microplate reader at a wavelength of 570 nm and a reference wavelength of 450 nm.

[0085] (6) Set up zero adjustment wells (culture medium, MTT, DMSO) and control wells (cells, culture medium, MTT, drug dissolution medium of the same concentration, DMSO) at the same time.

[0086] (7) The number of living cells can be determined based on the measured optical density (OD value). The larger the OD value, the stronger the cell activity. Use the formula:

[0087] .

[0088] Calculate the inhibition rate of each compound on the selected cell growth, and then calculate the IC of each test compound on each selected cell line using the Bliss method. 50 The results are shown in Table 1 below.

[0089] Table 1 IC values ​​of compounds against various cell lines50 Value (μM)

[0090] .

[0091] From IC 50 The results of the activity screening showed that the complex Pt-S1 had a generally higher proliferation inhibitory activity against the three tested human tumor cell lines (HepG2, Hela, and MGC80-3 cells) than the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1), indicating that the complex Pt-S1 formed a significant positive synergistic effect by organically coordinating the ligand 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine (S1) with the platinum central atom. Among them, the complex Pt-S1 had a better inhibitory effect on human cervical cancer Hela cells, with an IC 50 The value was 1.31±0.16 μM, which is approximately 19.7-fold and 10.8-fold higher than that of the ligand and cisplatin, respectively. Furthermore, the complex Pt-S1 exhibited less cytotoxicity against normal human liver cells HL-7702 than cisplatin, demonstrating good selectivity. In summary, the complex Pt-S1 exhibited superior in vitro antitumor activity and has potential pharmaceutical value.

[0092] like Figure 3 、 Figure 4 The N^N^N type terpyridine platinum complex Pt-S1 of the present invention is excited by a 960 nm laser in the second near-infrared region. After the ligand is coordinated with platinum, the electron mobility in the molecule is further improved, and its two-photon absorption cross section is 875-1780 GM. It is a multi-photon absorption material with excellent nonlinear optical properties.

[0093] like Figure 5 、 Figure 6 The N^N^N-type terpyridine platinum complex Pt-S1 of the present invention has a planar structure and abundant intermolecular forces, resulting in self-assembly behavior of the platinum complex. When PBS is added to its DMSO solution, where the PBS content is 60%, it can be seen by SEM that the self-assembled structure morphology is spherical, and its particle size distribution is relatively uniform between 150 and 350 nm.

[0094] like Figure 7 Cytotoxicity experiments with the N^N^N terpyridine platinum complex Pt-S1, presented in this paper, examined its dark toxicity against normal human hepatocytes, HL-7702 cells. Under dark conditions, even at a high concentration of 25 μM, the N^N^N terpyridine platinum complex maintained a survival rate exceeding 80%. This demonstrates that Pt-S1 exhibits low cytotoxicity and meets requirements for cell imaging and other applications.

[0095] like Figure 8This study used confocal imaging of cells using the N^N^N-type terpyridine platinum complex Pt-S1. Hela cells were stained with 5 μM Pt-S1 and incubated for 40 minutes. The culture dish was then gently washed 3-4 times with PBS buffer. The green channel (Pt-S1) was excited at 473 nm, and fluorescence was collected at wavelengths between 480 and 580 nm. The red channel (lysosome stain Lyso-tracker deep red) was excited at 577 nm, and fluorescence was collected at wavelengths between 580 and 680 nm. The fluorescence signal at 480-580 nm was consistent with the staining location and appearance of the lysosome stain, demonstrating that Pt-S1 can target lysosomes within cells.

Claims

1. An N^N^N type terpyridine platinum complex, characterized in that: The general formula of the N^N^N type terpyridine platinum complex is as follows: 。 2. The method for preparing the N^N^N type terpyridine platinum complex according to claim 1, characterized in that: 2-acetylpyridine and KOH are added with water and stirred at room temperature until the solution turns yellow, 3,4-diethoxybenzaldehyde and ethanol are added, and then ammonia water is added dropwise to carry out heating reaction. After the reaction is completed, the precipitated solid is recrystallized from ethanol to obtain yellow needle-shaped crystals of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine; 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine is mixed with K2PtCl4, and a halogenated hydrocarbon solvent and an alcohol solvent are added thereto, and the mixture is heated to react. After the reaction is completed, the solvent is removed by distillation under reduced pressure, and a yellow solid product is obtained after purification using a chromatography column, which is an N^N^N type terpyridine platinum complex.

3. The method for preparing the N^N^N type terpyridine platinum complex according to claim 2, characterized in that: The mass ratio of 2-acetylpyridine to KOH is 1:0.47-1.16; the mass ratio of KOH to water is 1:2-4; the mass ratio of 3,4-diethoxybenzaldehyde, 2-acetylpyridine and ammonia water is 1:1.2-2.5:8.7-17.5; the mass ratio of 3,4-diethoxybenzaldehyde to ethanol is 1:5-6.3; the heating reaction temperature is 60-80°C, and the reaction time is 5-8 hours; the mass ratio of the precipitated solid to ethanol is 1:3-6.5, and the recrystallization heating temperature is 80-100°C, and the reaction time is 30 minutes to 60 minutes.

4. The method for preparing the N^N^N type terpyridine platinum complex according to claim 2, wherein: The mass ratio of 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine to K2PtCl4 is 1.5-4.8:1; the heating reaction temperature is 60-80°C, and the time is 8-15 hours; the reduced pressure distillation pressure is -0.05~-0.1 MPa, the temperature is 50-65°C, and the time is 30-65 minutes.

5. The method for preparing the N^N^N type terpyridine platinum complex according to claim 2, characterized in that: The halogenated hydrocarbon solvent is dichloromethane or chloroform, and the alcohol solvent is methanol or ethanol.

6. The method for preparing the N^N^N type terpyridine platinum complex according to claim 2, characterized in that: The mass ratio of the halogenated hydrocarbon solvent, the alcohol solvent and K2PtCl4 is 8-16:9-31:

1.

7. The method for preparing the N^N^N type terpyridine platinum complex according to claim 2, characterized in that: The chromatography column purification is to purify the product by controlling the ratio of the polar solvent to the non-polar solvent in the eluent. First, the ratio of the non-polar solvent I to the polar solvent I is controlled to be 5-30:1 to separate 4'-(3,4-bisethoxyphenyl)-2,2':6',2''-terpyridine, and the ratio of the non-polar solvent II to the polar solvent II is controlled to be 10-30:1 to separate the N^N^N type terpyridine platinum complex.

8. The method for preparing the N^N^N type terpyridine platinum complex according to claim 7, characterized in that: The non-polar solvent I is petroleum ether, and the polar solvent I is ethyl acetate or acetone; the non-polar solvent II is dichloromethane, and the polar solvent II is methanol or propanol.

9. Use of the N^N^N type terpyridine platinum complex according to claim 1, characterized in that: The N^N^N type terpyridine platinum complex is used in the preparation of lysosome analysis and imaging products.

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