Preparation and application of molecular probe based on platinum chemotherapeutic drugs

Abstract

The invention discloses preparation and application of a molecular probe based on platinum chemotherapeutic drugs, and belongs to the technical field of biochemical analysis. The platinum chemotherapeutic drug is cis-platinum or oxaliplatin, and the molecular probe is shown as a formula (I), a formula (II) or a formula (III): # imgabs0 # imgabs1 #. According to the invention, the molecular probe is simply modified on the basis of not changing the activity of the drug, and the anti-tumor activity of the platinum chemotherapeutic drug itself is retained as far as possible; a cross-linking agent having a photoaffinity group and a click group is also employed, the diaziridine photoaffinity group can provide covalent binding of the drug and the target, and the azide group can provide in-situ labeling by using a bio-orthogonal reaction. The preparation method of the molecular probe is simple and easy, clinical tissues or blood samples can be utilized to monitor the response of tumors to platinum drugs, then tumor chemotherapy monitoring indexes based on platinum probe information are established, and important reference data are provided for clinical treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a method for preparing a divalent platinum compound derivative having anti-tumor activity and its use in anti-tumor aspects. Background Art

[0002] Platinum chemotherapy drugs are a class of anti-tumor drugs based on DNA enzyme inhibitors and are currently one of the most widely used chemotherapy drugs in clinical practice. After entering the cells, they undergo hydrolysis under the influence of the intracellular water environment and chloride ion concentration, producing active platinum ions or platinum complexes. These active substances can covalently bind to nucleophilic groups (such as N7 position) on bases such as guanine (G) and adenine (A) in DNA molecules to form platinum-DNA adducts, interfering with normal DNA replication, transcription and other processes, and inducing cells to enter the apoptosis program.

[0003] Platinum-based chemotherapy drugs are used to treat common malignant tumors such as lung cancer, bladder cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, and head and neck tumors due to their unique anti-cancer mechanisms and broad anti-cancer spectrum. However, Pt(Ⅱ) drugs have the disadvantages of high toxicity and side effects, poor efficacy response, and easy drug resistance during use. Therefore, how to improve the precision of its treatment has become an important research direction in clinical drug use. The key to precision medication is to monitor the efficacy response of different patients to drugs in real time, but current technology has not yet met this demand. Therefore, by designing small molecule probes based on clinical platinum drugs, studying and monitoring the mechanism of action between drugs and targets, it is expected to provide a way to detect efficacy.

[0004] Most of the existing platinum drug probes are chemically modified with fluorescent groups on the ligand molecules of platinum drugs, and the drug toxicity or cell stress response to drugs is studied through fluorescence imaging or fluorescent gel technology. However, the modification of larger groups such as fluorescent dyes will affect the drug properties, and the reversible coordination bonds of platinum drugs may lose some signals. Therefore, such probes are difficult to use for in situ analysis of the action sites of divalent platinum chemotherapy drugs in cells and monitoring of drug effects. For many years, the photoaffinity labeling strategy has been used to analyze the mechanism of action of non-covalent drugs. Therefore, it is urgent to use the smallest chemical label to carry out appropriate structural modification of Pt(Ⅱ) drugs, use the photoaffinity reaction of diazirine to label the target of platinum drugs, and then use the bioorthogonal reaction of azide to selectively introduce fluorescent and other reporter groups, and combine bioimaging or sensing technology to effectively identify the mechanism of action of platinum drugs and realize the rapid detection of clinical efficacy characteristic indicators. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the preparation and application of molecular probes based on platinum-based chemotherapy drugs.

[0006] According to one aspect of the present invention, a molecular probe based on a platinum-based chemotherapy drug is provided. The platinum-based chemotherapy drug is cisplatin or oxaliplatin. The molecular probe is represented by formula (I), formula (II) or formula (III):

[0007]

[0008] Furthermore, the molecular probe uses a platinum-based chemotherapy drug as a main structure, and introduces an azide-containing small molecule chain 69 or an azide-containing photocrosslinker N8.

[0009] Furthermore, the structural formula of the small molecule chain 69 is The structural formula of the photocrosslinking agent N8 is

[0010] According to the second aspect of the present invention, a method for preparing the molecular probe is proposed. The molecular probe of formula (I) is based on cisplatin drug, and the preparation method is as follows:

[0011] A1. Under dark conditions, methyl 3-bromo-2-(bromomethyl)propionate and potassium azide are reacted in a dry N,N-dimethylformamide solution at room temperature to obtain formula (I-1);

[0012] A2. Under a nitrogen atmosphere, the compound of formula (I-1) is dissolved in anhydrous methanol solution and reacted with nickel dichloride hexahydrate, di-tert-butyl dicarbonate and sodium borohydride at room temperature in the dark to obtain the compound of formula (I-2);

[0013] A3, dissolving the compound of formula (I-2) in tetrahydrofuran, adding lithium hydroxide aqueous solution to react at room temperature, and then adding hydrochloric acid to adjust the pH to 3 to obtain a colorless oil; dissolving the colorless oil in anhydrous dichloromethane, adding EDCI, HOBT, triethylamine and photocrosslinking agent N8 in sequence under ice bath conditions, and then transferring to room temperature to react to obtain formula (I-3);

[0014] A4, dissolving the compound of formula (I-3) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, transferring to room temperature for reaction and concentrating in vacuo to obtain a white solid; dissolving the white solid in N,N-dimethylformamide, and then adding 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum(II) in sequence, reacting at room temperature, adding pre-cooled deionized water to obtain a light yellow precipitate, washing with water and vacuum drying to obtain the molecular probe of formula (I);

[0015] The reaction scheme of the preparation method is as follows:

[0016]

[0017] Furthermore, the molar ratio of methyl 3-bromo-2-(bromomethyl)propionate to potassium azide in A1 is 1:(8-12), the molar ratio of the compound of formula (I-1) to nickel dichloride hexahydrate, di-tert-butyl dicarbonate and sodium borohydride in A2 is (0.5-1):(0.003-0.008):(1.5-2):(1.5-2), and the molar ratio of the compound of formula (I-2) to lithium hydroxide aqueous solution in A3 is 1:(3.5-4. 5), the molar ratio of the obtained colorless oil to EDCI, HOBT, triethylamine and the photocrosslinking agent N8 is (1-1.5): (1-1.5): (1-1.5): (1.5-3): (0.8-1.2), and the molar ratio of the white solid in A4 to 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum(II) is (1-1.5): (2-3): (1-1.5).

[0018] Furthermore, the molecular probe of formula (II) is based on oxaliplatin drug, and the preparation method is as follows:

[0019] B1. Under a nitrogen atmosphere, manganese acetate dihydrate and potassium azide were dissolved in acetonitrile, 3-cyclohexene-1-carboxylic acid methyl ester was added, trifluoroacetic acid was slowly added dropwise at -20°C to react, and then the mixture was transferred to room temperature for full reaction to obtain formula (II-1);

[0020] B2. In dark conditions, dissolve the compound of formula (II-1) in a mixed solution of tetrahydrofuran and water, add triphenylphosphine and react at room temperature to obtain the compound of formula (II-2);

[0021] B3, dissolving the compound of formula (II-2) in a mixed solution of tetrahydrofuran and water, adding triethylamine and di-tert-butyl dicarbonate to react at room temperature to obtain the compound of formula (II-3);

[0022] B4, dissolving the compound of formula (II-3) in tetrahydrofuran, adding lithium hydroxide aqueous solution and reacting at room temperature to obtain the compound of formula (II-4);

[0023] B5, dissolving the compound of formula (II-4) in anhydrous tetrahydrofuran, adding EDCI, HOBT and triethylamine in turn under ice bath conditions, and then adding small molecule chain 69, and then transferring to room temperature to react, to obtain the compound of formula (II-5);

[0024] B6, dissolving the compound of formula (II-5) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, transferring to room temperature for reaction and concentrating in vacuo to obtain a white solid; dissolving the obtained white solid in an aqueous sodium hydroxide solution, adding an aqueous potassium tetrachloroplatinate solution, reacting at 60°C, and obtaining a yellow precipitate after returning to room temperature, washing and vacuum drying to obtain the molecular probe of formula (II);

[0025] The reaction scheme of the preparation method is as follows:

[0026]

[0027] Furthermore, the molar ratio of manganese acetate dihydrate to potassium azide and 3-cyclohexene-1-carboxylic acid methyl ester in B1 is (4-8): (10-15): (2-4), the molar ratio of the formula (II-1) to triphenylphosphine in B2 is 1: (1.8-2.5), the molar ratio of the formula (II-2) to triethylamine and di-tert-butyl dicarbonate in B3 is (0.5-1): (2-3): (1-1.5), and the molar ratio of the formula (II-3) in B4 is (0.5-1): (2-3): (1-1.5). ) and the molar ratio of lithium hydroxide aqueous solution is 1:(3~4), the molar ratio of formula (II-4) described in B5 and EDCI, HOBT, triethylamine and small molecule chain 69 is (0.5~1):(0.8~1.5):(0.8~1.5):(2~3):(0.8~1.5), and the molar ratio of the white solid in B6 and the sodium hydroxide aqueous solution and potassium tetrachloroplatinate aqueous solution is (1~1.5):(2~3):(1~1.5).

[0028] Furthermore, the molecular probe of formula (III) is based on oxaliplatin drug, and the preparation method is as follows:

[0029] C1, dissolving the compound of formula (II-4) in a dichloromethane solution, adding EDCI, HOBT, triethylamine in sequence under ice bath conditions, adding the photocrosslinking agent N8, and then transferring to room temperature to react, to obtain the compound of formula (III-1);

[0030] C2, dissolving the compound of formula (III-1) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, reacting at room temperature and concentrating in vacuo to obtain the compound of formula (III-2);

[0031] C3, dissolving the compound of formula (III-2) in an aqueous sodium hydroxide solution, adding an aqueous potassium tetrachloroplatinate solution, reacting at 60° C., and obtaining a brownish yellow precipitate after returning to room temperature. After washing, vacuum drying is performed to obtain the molecular probe of formula (III);

[0032] The reaction scheme of the preparation method is as follows:

[0033]

[0034] Furthermore, the molar ratio of the substance of the formula (II-4) described in C1 to EDCI, HOBT, triethylamine and the photocrosslinker N8 is (1-1.5): (1-1.5): (1-1.5): (2-3): (0.8-1.2), and the molar ratio of the substance of the formula (III-2) described in C3 to aqueous sodium hydroxide solution and aqueous potassium tetrachloroplatinate solution is (1-1.5): (2-3): (1-1.5).

[0035] According to the third aspect of the present invention, an application of the molecular probe in cell localization imaging is proposed, wherein the molecular probe identifies the interaction and changes between platinum chemotherapy drugs and tumor cells, thereby establishing monitoring for tumor cells.

[0036] Beneficial effects of the present invention:

[0037] The present invention designs and synthesizes a molecular probe based on the basic structure of platinum chemotherapy drugs that can reflect the intracellular distribution of drugs and in situ mark the drug action site. The molecular probe uses the drug structure itself as the main structure to introduce a photocrosslinking group and a click reaction handle. The preparation method is simple, and the stability, selectivity and sensitivity are high. The present invention provides a simple, accurate and efficient method, which is beneficial for observing the uptake and accumulation of platinum compounds and their distribution in cells. At the same time, it can further clarify the drug action mechanism and analyze the drug effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The nuclear magnetic hydrogen spectrum of the molecular probe of formula (I) provided by the present invention;

[0039] Figure 2 The nuclear magnetic hydrogen spectrum of the molecular probe of formula (II) provided by the present invention;

[0040] Figure 3 The nuclear magnetic hydrogen spectrum of the molecular probe of formula (III) provided by the present invention;

[0041] Figure 4 The in situ protein labeling map of the molecular probe of formula (I) by in-gel fluorescence imaging and Coomassie Brilliant Blue staining;

[0042] Figure 5 In situ protein labeling maps of the molecular probe of formula (II) and the molecular probe of formula (III) by in-gel fluorescence imaging and Coomassie Brilliant Blue staining;

[0043] Figure 6 Cell localization imaging of the molecular probe of formula (I) in non-small cell lung cancer cells A549 using confocal microscopy;

[0044] Figure 7Cell localization imaging of the molecular probe of formula (II) in non-small cell lung cancer cells A549 displayed by confocal microscopy;

[0045] Figure 8 The cellular localization imaging of the molecular probe of formula (III) in non-small cell lung cancer cells A549 was shown using confocal microscopy. DETAILED DESCRIPTION

[0046] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0047] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0048] In view of the current lack of an efficient and accurate method or tool for monitoring the in situ analysis of the action sites of divalent platinum chemotherapy drugs in cells and the drug effects in the clinic, the first object of the present invention is to propose a molecular probe based on platinum chemotherapy drugs that can reflect the intracellular distribution of drugs and in situ mark the drug action sites.

[0049] The second object of the present invention is to provide a method for preparing the molecular probe, which is simple, has high stability, good selectivity and high sensitivity.

[0050] The third object of the present invention is to propose an application of the molecular probe in cell localization imaging, which is beneficial for observing the uptake and accumulation of platinum compounds and their distribution in cells. At the same time, it can further clarify the drug action mechanism and analyze the drug effect.

[0051] Example 1: Preparation of molecular probes

[0052] 1. Preparation of the molecular probe of formula (I) of the present invention

[0053] 1. The molecular probe of formula (I) of the present invention is based on cisplatin, and its preparation method is as follows:

[0054] (1) Under dark conditions, 0.14 mL of methyl 3-bromo-2-(bromomethyl)propionate (1.0 mmol) and 811.2 mg of potassium azide (10 mmol) were reacted in 10 mL of dry N,N-dimethylformamide solution at room temperature for 24 hours. The reaction was completed by TCL monitoring. The reaction solution was diluted with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (EtOAc: Hex = 5%) to obtain 150 mg of the compound represented by formula (I-1);

[0055] (2) Under nitrogen atmosphere, 140 mg of compound (I-1) (0.76 mmol) was dissolved in anhydrous methanol solution and reacted with 1 mg of nickel dichloride hexahydrate (0.5% mmol), 0.44 mL of di-tert-butyl dicarbonate (1.9 mmol), and 71.9 mg of sodium borohydride (1.9 mmol) at room temperature in the dark for 3 hours. After TCL monitoring, the reaction was complete, and water was added to quench. The mixture was extracted twice with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel chromatography (EtOAc: Hex = 15%) to obtain 127 mg of the compound represented by formula (I-2);

[0056] (3) 127 mg of compound (I-2) (0.38 mmol) was dissolved in 5 mL of tetrahydrofuran, and 1.5 mL of lithium hydroxide (1.5 mmol) aqueous solution was added to react at room temperature for 12 hours. After TLC monitoring, the reaction was complete. 1N hydrochloric acid was added to adjust the pH to 3, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oil. 111.4 mg of the obtained colorless oil (0.35 mmol) was dissolved in 4 mL of anhydrous dichloromethane, and 67.1 mg of EDCI (0.35 mmol), 47.3 mg of HOBT (0.35 mmol), 0.1 mL triethylamine (0.64 mmol), 49 mg compound N8 (0.32 mmol) were added, and then the mixture was transferred to room temperature for reaction for 2 hours. After the reaction was completed, the mixture was diluted with dichloromethane, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel chromatography (MeOH: DCM = 2%) to obtain 106 mg of the compound represented by formula (I-3);

[0057] (4) 106 mg of the compound represented by formula (I-3) (0.233 mmol) was dissolved in 3 mL of dichloromethane, and 0.35 mL of 4M hydrogen chloride in 1,4-dioxane solution was added dropwise under ice bath conditions. The mixture was transferred to room temperature for reaction for 12 hours and then concentrated in vacuo to obtain 68 mg of a white solid. 40 mg of the obtained white solid (0.122 mmol) was dissolved in 0.3 mL of N,N-dimethylformamide, and 36 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (0.244 mmol) and 51.5 mg of cis-dichlorobis(dimethylsulfoxide)platinum(II) (0.122 mmol) were added in sequence. After reaction at room temperature for 72 hours, 2 mL of pre-cooled deionized water was added to obtain a light yellow precipitate, which was washed with water and dried in vacuo to obtain the molecular probe of formula (I). The molecular probe was a light yellow solid with a yield of 27%. The H NMR spectrum showed Figure 1 .

[0058] 1H NMR(500MHz,DMF)δ8.21(s,1H),5.25(s,2H),5.13(s,2H),3.31(t,J=6.6Hz,2H),3.10(dd,J=12.8,6.6Hz,2H), 2.97(d,J=8.4Hz,1H),2.86(dd,J=21.4,9.0Hz,2H),1.74(t,J=6.6Hz,2H),1.65(t,J=7.0Hz,2H),1.28(s,2H).

[0059] 2. The reaction route of the method for preparing the molecular probe of formula (I) of the present invention is as follows:

[0060]

[0061] 2. Preparation of the molecular probe of formula (II) of the present invention

[0062] 1. The molecular probe of formula (II) of the present invention is based on oxaliplatin, and its preparation method is as follows:

[0063] (1) Under nitrogen atmosphere, 1.77 g of manganese acetate dihydrate (6.6 mmol) and 973 mg of potassium azide (12 mmol) were dissolved in 27 mL of acetonitrile, 0.41 mL of 3-cyclohexene-1-carboxylic acid methyl ester (3 mmol) was added, and 2.7 mL of trifluoroacetic acid was slowly added dropwise at -20°C. After reacting for 4 hours, the mixture was transferred to room temperature. TLC monitoring showed that the reaction was almost complete, and then saturated sodium bisulfite solution was added. The mixture was extracted twice with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (EtOAc: Hex = 5%) to obtain 303 mg of the compound represented by formula (II-1);

[0064] (2) Under dark conditions, 150 mg of compound (II-1) (0.67 mmol) was dissolved in a mixed solution of tetrahydrofuran / water (1:1), 351.4 mg of triphenylphosphine (1.34 mmol) was added and reacted at room temperature for 12 hours, concentrated under reduced pressure, and separated by silica gel column chromatography (MeOH:DCM=30%+0.1% TFA, ninhydrin dyeing) to obtain 72 mg of the compound represented by formula (II-2);

[0065] (3) 150 mg of compound (II-2) (0.67 mmol) was dissolved in a mixed solution of tetrahydrofuran / water (1:1), and 0.35 mL of triethylamine (2.54 mmol) and 0.24 mL of di-tert-butyl dicarbonate (1.06 mmol) were added to react at room temperature for 2 hours. After TCL monitoring, the reaction was complete, and the mixture was concentrated under reduced pressure, extracted twice with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (EtOAc: Hex = 20%) to obtain 99 mg of the compound represented by formula (II-3);

[0066] (4) 99 mg of compound (II-3) (0.27 mmol) was dissolved in 5 mL of tetrahydrofuran, and 1 mL of lithium hydroxide (1 mmol) aqueous solution was added to react at room temperature for 12 hours. After TCL monitoring, the reaction was complete, 1N hydrochloric acid was added to adjust the pH to 3, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 88 mg of the compound represented by formula (II-4);

[0067] (5) 304.7 mg of compound (II-4) (0.85 mmol) was dissolved in 4.5 mL of anhydrous tetrahydrofuran, and 180.2 mg of EDCI (0.94 mmol), 127 mg of HOBT (0.94 mmol), and 0.35 mL of triethylamine (2.55 mmol) were added in sequence under ice bath conditions, and 128 mg of compound 69 (0.94 mmol) was added, and then the mixture was transferred to room temperature for reaction for 2 hours, concentrated under reduced pressure, extracted twice with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel chromatography (MeOH:DCM=2%) to obtain 268 mg of the compound represented by formula (II-5);

[0068] (6) 259 mg of compound (II-5) was dissolved in 2 mL of dichloromethane, and 0.88 mL of 4M hydrogen chloride in 1,4-dioxane solution was added dropwise under ice bath conditions. The mixture was transferred to room temperature for reaction for 12 hours and then concentrated under vacuum to obtain 181.7 mg of a white solid; 94 mg of the obtained white solid (0.3 mmol) was dissolved in 1.5 mL of sodium hydroxide aqueous solution (0.6 mmol), and 3 mL of potassium tetrachloroplatinate aqueous solution (0.3 mmol) was added, and the mixture was reacted at 60°C for 1 hour. After returning to room temperature, a yellow precipitate was obtained, which was washed with deionized water, methanol, and ether in turn and then dried under vacuum to obtain the molecular probe of formula (II). The molecular probe was a yellow solid with a yield of 63%. The H NMR spectrum showed Figure 2 .

[0069] 1H NMR (500MHz, DMSO) δ7.76(s,1H),5.50(s,2H),5.10–5.04(m,1H),4.96–4.90(m,1H),3.08(dd,J=12.1,6.0Hz,2H),2.34(s,1H),2.10(d,J=12.3H z,2H),1.72(d,J=13.1Hz,2H),1.65(dd,J=12.7,6.2Hz,3H),1.53(dd,J=24.0,11.8Hz,2H),1.37(td,J=12.7,5.6Hz,2H),1.24(d,J=13.0Hz,1H).

[0070] 2. The reaction route of the method for preparing the molecular probe of formula (II) of the present invention is as follows:

[0071]

[0072] III. Preparation of the molecular probe of formula (III) of the present invention

[0073] 1. The molecular probe of formula (III) of the present invention is based on oxaliplatin, and its preparation method is as follows:

[0074] (1) 139.4 mg of compound (II-4) (0.39 mmol) was dissolved in 8 mL of dichloromethane solution, and 82 mg of EDCI (0.43 mmol), 57.8 mg of HOBT (0.43 mmol), and 0.11 mL of triethylamine (0.78 mmol) were added in sequence under ice bath conditions, and 60 mg of compound N8 (0.34 mmol) was added, and then the mixture was transferred to room temperature for reaction for 2 hours, concentrated under reduced pressure, extracted twice with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (MeOH:DCM=2%) to obtain 124 mg of the compound represented by formula (III-1);

[0075] (2) 124 mg of compound (III-1) was dissolved in 1 mL of dichloromethane, and 0.38 mL of 4 M hydrogen chloride 1,4-dioxane solution was added dropwise under ice bath conditions. The mixture was reacted at room temperature for 12 hours and then concentrated in vacuo to obtain 90 mg of the compound represented by formula (III-2);

[0076] (3) 45 mg of compound (III-2) (0.122 mmol) was dissolved in 0.5 mL of sodium hydroxide aqueous solution (0.244 mmol), and 1.5 mL of potassium tetrachloroplatinate aqueous solution (0.122 mmol) was added, and the mixture was reacted at 60°C for 1 hour. After returning to room temperature, a brownish yellow precipitate was obtained, which was washed with deionized water, methanol, and ether in turn, and then vacuum dried to obtain the molecular probe of formula (III). The molecular probe was a brownish yellow solid with a yield of 70%. The H NMR spectrum showed Figure 3 .

[0077] 1H NMR (500MHz, DMSO) δ7.76(t,J=4.9Hz,1H),5.48(s,2H),5.05(t,J=10.2Hz,1H),4.92(t,J= 10.4Hz,1H),3.21(t,J=6.3Hz,2H),2.94(dt,J=13.1,6.6Hz,1H),2.86(dd,J=12.8,6.1Hz,1 H),2.65(d,J=11.2Hz,1H),2.33(s,1H),2.08(d,J=11.5Hz,2H),1.74(d,J=13.2Hz,1H),1. 64(t,J=6.1Hz,3H),1.53–1.48(m,3H),1.36(td,J=12.4,5.0Hz,1H),1.28–1.21(m,1H).13C NMR(126MHz,DMSO)δ173.59(s),62.85(s),59.53(s),45.74(s),38.14(s), 33.74(s),32.82(s),32.24(s),31.52(s),28.10(s),27.13(s),26.28(s).

[0078] 2. The reaction route of the method for preparing the molecular probe of formula (III) of the present invention is as follows:

[0079]

[0080] Example 2: Detection of the labeling of target protein by molecular probes through in-gel fluorescence imaging and Coomassie Brilliant Blue staining

[0081] A549 cells were cultured to a density of about 90%, and a probe with a concentration of 1uM was added and incubated for 2 hours. After UV irradiation / no irradiation, the cells were lysed with IP cell lysis buffer. The sample protein concentration was quantified to 1.5mg / mL with phosphate buffered saline (PBS). Click chemistry fluorescent dye was then added to react for 1 hour. Pre-cooled acetone at -20℃ was used to precipitate the protein, and the organic solvent was removed by centrifugation. The loading buffer was added and separated by polyacrylamide gel electrophoresis, and the results were finally obtained by fluorescence imaging, such as Figure 4 and Figure 5 shown. Figure 4 The in situ protein labeling map of the molecular probe of formula (I) is obtained by in-gel fluorescence imaging and Coomassie Brilliant Blue staining. Figure 5 The in situ protein labeling patterns of the molecular probe of formula (II) and the molecular probe of formula (III) are obtained by in-gel fluorescence imaging and Coomassie brilliant blue staining. Figure 4 and Figure 5 (a) is in-gel fluorescence imaging, and (b) is Coomassie brilliant blue staining. The molecular probes of formula (I), formula (II) and formula (III) can all label their target proteins, among which the molecular probes of formula (I) and formula (III) have higher sensitivity.

[0082] Example 3: Probe cell imaging to investigate the efficacy of platinum chemotherapy drugs before and after treatment

[0083] Non-small cell lung cancer A549 cells were cultured in a cell imaging glass dish, placed in an incubator at 37°C and 5% CO2 saturated humidity to a density of 50%-60%, and treated with the probe for 2 hours. For competitive labeling, the cells were treated with the corresponding drug for 2 hours before adding the probe. After UV irradiation, the cells were washed with PBS and then fixed with 4% paraformaldehyde. The cells were incubated in fresh culture medium containing the corresponding click reporter factor for 1 hour. The nuclear seal was then stained at room temperature, and images were obtained on different detection channels of the Zeiss LSM980 confocal microscope system. Figure 6 The cellular localization imaging of the molecular probe of formula (I) in non-small cell lung cancer cells A549 was shown by confocal microscopy. Figure 7 The cellular localization imaging of the molecular probe of formula (II) in non-small cell lung cancer cells A549 was shown by confocal microscopy. Figure 8 The cell localization imaging of the molecular probe of formula (III) in non-small cell lung cancer cells A549 is shown by confocal microscopy. Figure 6 , Figure 7 and Figure 8 The first column is all Hoechst nuclear staining, the second column is the cell imaging treated with the corresponding molecular probe, the third column is the overlapping images of cell imaging treated with Hoechst nuclear staining and probe, and the fourth column is the bright field images related to cell imaging. In addition, Figure 6 , Figure 7 and Figure 8 The first row is a blank control group without drug or molecular probe treatment, the second row is an experimental group treated with molecular probe after drug competition, and the third row is an experimental group treated with molecular probe only. It can be seen that the molecular probes of formula (I), formula (II) and formula (III) described in the present invention can identify the interaction between platinum drugs and non-small cell lung cancer cells A549.

[0084] The above describes the specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A molecular probe based on platinum-based chemotherapy drugs, characterized in that: The platinum chemotherapy drug is cisplatin or oxaliplatin, and the molecular probe is represented by formula (I), formula (II) or formula (III):

2. The molecular probe according to claim 1, characterized in that The molecular probe uses a platinum-based chemotherapy drug as the main structure and introduces an azide-containing small molecule chain 69 or an azide-containing photocrosslinker N8.

3. The molecular probe according to claim 2, characterized in that The structural formula of the small molecule chain 69 is The structural formula of the photocrosslinking agent N8 is 4. A method for preparing a molecular probe according to any one of claims 1 to 3, characterized in that: The molecular probe of formula (I) is based on cisplatin and is prepared as follows: A1. Under dark conditions, methyl 3-bromo-2-(bromomethyl)propionate and potassium azide are reacted in a dry N,N-dimethylformamide solution at room temperature to obtain formula (I-1); A2. Under a nitrogen atmosphere, the compound of formula (I-1) is dissolved in anhydrous methanol solution and reacted with nickel dichloride hexahydrate, di-tert-butyl dicarbonate and sodium borohydride at room temperature in the dark to obtain the compound of formula (I-2); A3, dissolving the compound of formula (I-2) in tetrahydrofuran, adding lithium hydroxide aqueous solution to react at room temperature, and then adding hydrochloric acid to adjust the pH to 3 to obtain a colorless oil; dissolving the colorless oil in anhydrous dichloromethane, adding EDCI, HOBT, triethylamine and photocrosslinking agent N8 in sequence under ice bath conditions, and then transferring to room temperature to react to obtain formula (I-3); A4, dissolving the compound of formula (I-3) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, transferring to room temperature for reaction and concentrating in vacuo to obtain a white solid; dissolving the white solid in N,N-dimethylformamide, and then adding 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum(II) in sequence, reacting at room temperature, adding pre-cooled deionized water to obtain a light yellow precipitate, washing with water and vacuum drying to obtain the molecular probe of formula (I); The reaction scheme of the preparation method is as follows:

5. The method for preparing a molecular probe according to claim 4, characterized in that: The molar ratio of 3-bromo-2-(bromomethyl)propionic acid methyl ester to potassium azide in A1 is 1:(8-12), the molar ratio of formula (I-1) to nickel dichloride hexahydrate, di-tert-butyl dicarbonate and sodium borohydride in A2 is (0.5-1):(0.003-0.008):(1.5-2):(1.5-2), and the molar ratio of formula (I-2) to lithium hydroxide aqueous solution in A3 is 1:(3.5-4.5) The molar ratio of the obtained colorless oil to EDCI, HOBT, triethylamine and the photocrosslinker N8 is (1-1.5): (1-1.5): (1-1.5): (1.5-3): (0.8-1.2), and the molar ratio of the white solid in A4 to 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum(II) is (1-1.5): (2-3): (1-1.5).

6. The method for preparing a molecular probe according to claim 4, characterized in that: The molecular probe of formula (II) is based on oxaliplatin, and the preparation method is as follows: B1. Under a nitrogen atmosphere, manganese acetate dihydrate and potassium azide were dissolved in acetonitrile, 3-cyclohexene-1-carboxylic acid methyl ester was added, trifluoroacetic acid was slowly added dropwise at -20°C to react, and then the mixture was transferred to room temperature for full reaction to obtain formula (II-1); B2. In dark conditions, dissolve the compound of formula (II-1) in a mixed solution of tetrahydrofuran and water, add triphenylphosphine and react at room temperature to obtain the compound of formula (II-2); B3, dissolving the compound of formula (II-2) in a mixed solution of tetrahydrofuran and water, adding triethylamine and di-tert-butyl dicarbonate to react at room temperature to obtain the compound of formula (II-3); B4, dissolving the compound of formula (II-3) in tetrahydrofuran, adding lithium hydroxide aqueous solution and reacting at room temperature to obtain the compound of formula (II-4); B5, dissolving the compound of formula (II-4) in anhydrous tetrahydrofuran, adding EDCI, HOBT and triethylamine in turn under ice bath conditions, and then adding small molecule chain 69, and then transferring to room temperature to react, to obtain the compound of formula (II-5); B6, dissolving the compound of formula (II-5) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, transferring to room temperature for reaction and concentrating in vacuo to obtain a white solid; dissolving the obtained white solid in an aqueous sodium hydroxide solution, adding an aqueous potassium tetrachloroplatinate solution, reacting at 60°C, and obtaining a yellow precipitate after returning to room temperature, washing and vacuum drying to obtain the molecular probe of formula (II); The reaction scheme of the preparation method is as follows:

7. The method for preparing a molecular probe according to claim 6, characterized in that: The molar ratio of manganese acetate dihydrate to potassium azide and 3-cyclohexene-1-carboxylic acid methyl ester in B1 is (4-8): (10-15): (2-4), the molar ratio of formula (II-1) to triphenylphosphine in B2 is 1: (1.8-2.5), the molar ratio of formula (II-2) to triethylamine and di-tert-butyl dicarbonate in B3 is (0.5-1): (2-3): (1-1.5), the molar ratio of formula (II-3) to The molar ratio of lithium hydroxide aqueous solution is 1:(3~4), the molar ratio of formula (II-4) described in B5 to EDCI, HOBT, triethylamine and small molecule chain 69 is (0.5~1):(0.8~1.5):(0.8~1.5):(2~3):(0.8~1.5), and the molar ratio of the white solid in B6 to sodium hydroxide aqueous solution and potassium tetrachloroplatinate aqueous solution is (1~1.5):(2~3):(1~1.5).

8. The method for preparing a molecular probe according to claim 4, characterized in that: The molecular probe of formula (III) is based on oxaliplatin drug, and the preparation method is as follows: C1, dissolving the compound of formula (II-4) in a dichloromethane solution, adding EDCI, HOBT, triethylamine in sequence under ice bath conditions, adding the photocrosslinking agent N8, and then transferring to room temperature to react, to obtain the compound of formula (III-1); C2, dissolving the compound of formula (III-1) in dichloromethane, adding dropwise a solution of hydrogen chloride in 1,4-dioxane under ice bath conditions, reacting at room temperature and concentrating in vacuo to obtain the compound of formula (III-2); C3, dissolving the compound of formula (III-2) in an aqueous sodium hydroxide solution, adding an aqueous potassium tetrachloroplatinate solution, reacting at 60° C., and obtaining a brownish yellow precipitate after returning to room temperature. After washing, vacuum drying is performed to obtain the molecular probe of formula (III); The reaction scheme of the preparation method is as follows:

9. The method for preparing a molecular probe according to claim 8, characterized in that: The molar ratio of the substance of the formula (II-4) described in C1 to EDCI, HOBT, triethylamine and the photocrosslinker N8 is (1-1.5): (1-1.5): (1-1.5): (2-3): (0.8-1.2), and the molar ratio of the substance of the formula (III-2) described in C3 to aqueous sodium hydroxide solution and aqueous potassium tetrachloroplatinate solution is (1-1.5): (2-3): (1-1.5).

10. Use of the molecular probe according to any one of claims 1 to 3 in cell localization imaging, characterized in that: The application includes using the molecular probe to identify the interaction and changes between platinum chemotherapy drugs and tumor cells, thereby establishing monitoring for tumor cells.

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