Preparation of molecular probes based on platinum-based chemotherapy drugs and applications thereof

Abstract

The application discloses a platinum-based chemotherapy drug molecular probe preparation and application thereof, and belongs to the technical field of biochemical analysis. The platinum-based chemotherapy drug is cisplatin or oxaliplatin, and the molecular probe is shown in formula (I), formula (II) or formula (III). The simple modification of the molecular probe in the application does not change the activity of the drug, and the antitumor activity of the platinum-based chemotherapy drug itself is retained as much as possible. A crosslinking agent with a photoaffinity group and a click group is used, wherein the bisaziridine photoaffinity group can provide covalent binding of the drug and the target, and the azido group can provide in-situ labeling by using a biological orthogonal reaction. The preparation method of the molecular probe is simple, which is helpful for monitoring the response of tumors to platinum drugs by using clinical tissue or blood samples, and further establishing a tumor chemotherapy monitoring index based on platinum probe information, thereby providing important reference data for clinical treatment.

Description

Technical Field

[0001] This invention relates to the field of biochemical analysis technology, specifically to a method for preparing a divalent platinum compound derivative with antitumor activity and its use in antitumor applications. Background Technology

[0002] Platinum-based chemotherapy drugs are a class of antitumor drugs based on DNase inhibitors and are among the most widely used chemotherapy drugs in clinical practice. After entering cells, they undergo hydrolysis under the influence of 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 the N7 position) on bases such as guanine (G) and adenine (A) in DNA molecules, forming platinum-DNA adducts. This interferes with normal DNA replication and transcription, inducing cells to enter the apoptosis program.

[0003] Platinum-based chemotherapy drugs are used to treat common malignancies 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 anticancer mechanisms and broad anticancer spectrum. However, Pt(II) drugs have drawbacks such as high toxicity, poor efficacy response, and easy development of drug resistance. Therefore, improving the precision of their treatment has become an important research direction in clinical drug use. The key to precision medicine lies in real-time monitoring of the efficacy response of different patients to drugs, but current technology has not yet met this need. Therefore, designing small molecule probes based on clinical platinum-based drugs to study and monitor the mechanism of action between drugs and targets holds promise for providing a method for efficacy detection.

[0004] Existing platinum-based drug probes mostly involve chemically modifying the ligand molecules of platinum-based drugs with fluorescent groups. These probes are used to study drug toxicity or cellular stress responses to drugs via fluorescence imaging or fluorescent gel techniques. However, modifications with larger groups such as fluorescent dyes can affect drug properties, and the reversible coordination bonds of platinum-based drugs may result in signal loss. Therefore, these probes are unsuitable for in-situ analysis of the intracellular action sites of divalent platinum-based chemotherapy drugs and for monitoring drug efficacy. Photoaffinity labeling strategies have long been used to analyze the mechanisms of action of non-covalent drugs. Therefore, a pressing problem needs to be solved to effectively identify the mechanisms of action of platinum-based drugs by appropriately modifying the structure of Pt(II) drugs using minimal chemical tags, labeling the target sites of platinum-based drugs using the photoaffinity reaction of diazide, and then selectively introducing fluorescent reporter groups using the bioorthogonal reaction of azides. This, combined with bioimaging or sensing technologies, will enable rapid detection of clinical efficacy indicators. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide molecular probe preparation and application 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, wherein the platinum-based chemotherapy drug is cisplatin or oxaliplatin, and the molecular probe is as shown in formula (I), formula (II), or formula (III):

[0007]

[0008] Furthermore, the molecular probe is based on a platinum-based chemotherapy drug and incorporates a small molecule chain 69 with azide or a photocrosslinking agent N8 with azide.

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

[0010] According to a second aspect of the present invention, a method for preparing the molecular probe of formula (I) is provided, the preparation method being as follows:

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

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

[0013] A3. Dissolve the above formula (I-2) in tetrahydrofuran, add lithium hydroxide aqueous solution and react at room temperature, then add hydrochloric acid to adjust the pH to 3 to obtain a colorless oily substance; dissolve the colorless oily substance in anhydrous dichloromethane, add EDCI, HOBT, triethylamine and photocrosslinking agent N8 in sequence under ice bath conditions, and then transfer to room temperature to react to obtain formula (I-3);

[0014] A4. Dissolve the formula (Ⅰ-3) in dichloromethane, add a solution of 1,4-dioxane with hydrogen chloride dropwise under ice bath conditions, transfer to room temperature for reaction, and then concentrate under vacuum to obtain a white solid; dissolve the white solid in N,N-dimethylformamide, and then add 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum(Ⅱ) in sequence, react at room temperature, add pre-cooled deionized water to obtain a pale yellow precipitate, wash with water and vacuum dry to obtain the molecular probe of formula (Ⅰ);

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

[0016]

[0017] Further, in A1, the molar ratio of methyl 3-bromo-2-(bromomethyl)propionate to potassium azide is 1:(8-12); in A2, the molar ratio of formula (I-1) to nickel dichloride hexahydrate, ditert-butyl dicarbonate, and sodium borohydride is (0.5-1):(0.003-0.008):(1.5-2):(1.5-2); and in A3, the molar ratio of formula (I-2) to lithium hydroxide aqueous solution 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 and is prepared as follows:

[0019] B1. Under a nitrogen atmosphere, manganese acetate dihydrate and potassium azide are dissolved in acetonitrile, methyl 3-cyclohexene-1-carboxylate is added, and trifluoroacetic acid is slowly added dropwise at -20°C to react. Then the mixture is transferred to room temperature to react fully to obtain formula (Ⅱ-1).

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

[0021] B3. Dissolve the formula (II-2) in a mixed solution of tetrahydrofuran and water, add triethylamine and ditert-butyl dicarbonate, and react at room temperature to obtain formula (II-3);

[0022] B4. Dissolve the above formula (II-3) in tetrahydrofuran, add an aqueous lithium hydroxide solution and react at room temperature to obtain formula (II-4);

[0023] B5. Dissolve the formula (II-4) in anhydrous tetrahydrofuran, add EDCI, HOBT and triethylamine in sequence under ice bath conditions, then add small molecule chain 69, and then transfer to room temperature to react to obtain formula (II-5).

[0024] B6. Dissolve the formula (II-5) in dichloromethane, add a 1,4-dioxane solution of hydrogen chloride dropwise under ice bath conditions, transfer to room temperature for reaction, and then concentrate under vacuum to obtain a white solid; dissolve the obtained white solid in an aqueous sodium hydroxide solution, add an aqueous potassium tetrachloroplatinate solution, react at 60°C, and after restoring to room temperature, obtain a yellow precipitate, wash and then vacuum dry to obtain the molecular probe of formula (II).

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

[0026]

[0027] Further, in B1, the molar ratio of manganese acetate dihydrate to potassium azide and methyl 3-cyclohexene-1-carboxylate is (4-8):(10-15):(2-4); in B2, the molar ratio of formula (II-1) to triphenylphosphine is 1:(1.8-2.5); in B3, the molar ratio of formula (II-2) to triethylamine and di-tert-butyl dicarbonate is (0.5-1):(2-3):(1-1.5); and in B4, the molar ratio of formula (II-3) is... The molar ratio of the substance of the solid in B5 to the aqueous solution of lithium hydroxide is 1:(3-4), the molar ratio of the substance of the substance of formula (II-4) 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 the aqueous solution of sodium hydroxide and the aqueous solution of potassium tetrachloroplatinate is (1-1.5):(2-3):(1-1.5).

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

[0029] C1. Dissolve the formula (Ⅱ-4) in dichloromethane solution, add EDCI, HOBT, and triethylamine sequentially under ice bath conditions, add the photocrosslinking agent N8, and then transfer to room temperature to react, to obtain formula (Ⅲ-1);

[0030] C2. Dissolve the above formula (Ⅲ-1) in dichloromethane, add a solution of 1,4-dioxane hydrogen chloride dropwise under ice bath conditions, react at room temperature and then concentrate under vacuum to obtain formula (Ⅲ-2);

[0031] C3. Dissolve the formula (Ⅲ-2) in an aqueous sodium hydroxide solution, add an aqueous potassium tetrachloroplatinate solution, react at 60°C, and after restoring to room temperature, obtain a brownish-yellow precipitate. Wash and vacuum dry to obtain the molecular probe of the formula (Ⅲ).

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

[0033]

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

[0035] According to a third aspect of the invention, an application of the molecular probe in cell localization imaging is proposed, the application including the molecular probe identifying the interaction and changes between platinum-based chemotherapy drugs and tumor cells, thereby establishing a monitoring system for tumor cells.

[0036] The beneficial effects of this invention are:

[0037] This invention designs and synthesizes molecular probes based on the basic structures of platinum-based chemotherapy drugs, capable of reflecting the intracellular distribution of drugs and in-situ labeling of drug action sites. The molecular probes incorporate photocrosslinking groups and click reaction handles into the drug's intrinsic structure, resulting in a simple preparation method with high stability, good selectivity, and high sensitivity. This invention provides a simple, precise, and efficient method for observing the uptake and accumulation of platinum compounds and their intracellular distribution. Furthermore, it allows for a clearer understanding of drug mechanisms of action and analysis of drug efficacy. Attached Figure Description

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

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

[0040] Figure 3 The proton NMR 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) obtained by in-gel fluorescence imaging and Coomassie brilliant blue staining;

[0042] Figure 5 The in situ protein labeling maps of the molecular probes of formula (II) and formula (III) obtained by in-gel fluorescence imaging and Coomassie brilliant blue staining;

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

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

[0045] Figure 8 Cellular localization imaging of the formula (III) molecular probe in non-small cell lung cancer cells A549, as shown by confocal microscopy. Detailed Implementation

[0046] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0047] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0048] Given the current lack of an efficient and precise method or tool for in situ analysis of the intracellular action sites and drug efficacy of divalent platinum-based chemotherapy drugs in clinical practice, the first objective of this invention is to propose a molecular probe based on platinum-based chemotherapy drugs that can reflect the intracellular distribution of drugs and in situ label the drug action sites.

[0049] The second objective of this invention is to provide a method for preparing the molecular probe, which is simple, highly stable, selective, and sensitive.

[0050] The third objective of this 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] I. Preparation of the molecular probe of formula (I) of this invention

[0053] 1. The molecular probe of formula (Ⅰ) described in this 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 monitored by TCL and the reaction was complete. 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 shown in formula (Ⅰ-1).

[0055] (2) Under a 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 ditert-butyl dicarbonate (1.9 mmol), and 71.9 mg of sodium borohydride (1.9 mmol) at room temperature in the dark for 3 hours. The reaction was monitored by TCL and the reaction was complete. The reaction was quenched with water, extracted twice with ethyl acetate, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by silica gel column chromatography (EtOAc: Hex = 15%) to obtain 127 mg of the compound shown in 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. The reaction was carried out at room temperature for 12 hours under TCL monitoring. The reaction was complete. 1 N hydrochloric acid was added to adjust the pH to 3. 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. Under ice bath conditions, 67.1 mg of EDCI (0.35 mmol) and 47.3 mg of... were added sequentially. HOBT (0.35 mmol), 0.1 mL triethylamine (0.64 mmol), 49 mg of compound N8 (0.32 mmol) were added, and the mixture was then transferred to room temperature and reacted 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 column chromatography (MeOH:DCM = 2%) to obtain 106 mg of the compound shown in formula (Ⅰ-3).

[0057] (4) 106 mg of the compound shown in formula (Ⅰ-3) (0.233 mmol) was dissolved in 3 mL of dichloromethane. 0.35 mL of a 4M hydrogen chloride solution of 1,4-dioxane was added dropwise under ice bath conditions. After reacting at room temperature for 12 hours, the solution was concentrated under vacuum 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. 36 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (0.244 mmol) and 51.5 mg of cis-dichlorobis(dimethyl sulfoxide)platinum(II) (0.122 mmol) were added sequentially. After reacting at room temperature for 72 hours, 2 mL of pre-cooled deionized water was added to obtain a pale yellow precipitate. The precipitate was washed with water and dried under vacuum to obtain the molecular probe of formula (Ⅰ). This molecular probe was a pale yellow solid with a yield of 27%. The 1H NMR spectrum is shown below. 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 molecular probe preparation method of formula (Ⅰ) of the present invention is as follows:

[0060]

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

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

[0063] (1) Under a 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, and 0.41 mL of methyl 3-cyclohexene-1-carboxylate (3 mmol) was added. 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 and monitored by TCL. The reaction was basically complete. Then, saturated sodium bisulfite solution was added, and 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 shown in 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 the mixture was reacted at room temperature for 12 hours. The mixture was concentrated under reduced pressure and separated by silica gel column chromatography (MeOH:DCM=30%+0.1%TFA, ninhydrin staining) to obtain 72 mg of the compound shown in 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), 0.35 mL of triethylamine (2.54 mmol) and 0.24 mL of ditert-butyl dicarbonate (1.06 mmol) were added, and the mixture was reacted at room temperature for 2 hours under TCL monitoring. The reaction was completed 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 shown in formula (Ⅱ-3);

[0066] (4) Dissolve 99 mg of compound (Ⅱ-3) (0.27 mmol) in 5 mL of tetrahydrofuran, add 1 mL of lithium hydroxide (1 mmol) aqueous solution and react at room temperature for 12 hours. The reaction was monitored by TCL and the reaction was complete. Add 1 N hydrochloric acid to adjust the pH to 3, extract with ethyl acetate, dry with anhydrous sodium sulfate, filter, and concentrate to obtain 88 mg of the compound shown in formula (Ⅱ-4).

[0067] (5) 304.7 mg of compound (II-4) (0.85 mmol) was dissolved in 4.5 mL of anhydrous tetrahydrofuran. Under ice bath conditions, 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 sequentially. Then, 128 mg of compound 69 (0.94 mmol) was added. The mixture was then transferred to room temperature and reacted for 2 hours. The mixture was 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 268 mg of the compound shown in formula (II-5).

[0068] (6) 259 mg of compound (II-5) was dissolved in 2 mL of dichloromethane. 0.88 mL of a 4M hydrogen chloride solution of 1,4-dioxane was added dropwise under ice bath conditions. After reacting at room temperature for 12 hours, the solution was 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. The mixture was reacted at 60 °C for 1 hour. After returning to room temperature, a yellow precipitate was obtained. This precipitate was washed successively with deionized water, methanol, and diethyl ether, and then dried under vacuum to obtain the molecular probe of formula (II). This molecular probe was a yellow solid with a yield of 63%. Its 1H NMR spectrum is shown below. 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 for the preparation method of molecular probe of formula (II) described in this 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) described in this 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. Under ice bath conditions, 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 sequentially. Then, 60 mg of compound N8 (0.34 mmol) was added. The mixture was then transferred to room temperature and reacted for 2 hours. The mixture was 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 shown in 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 solution of 1,4-dioxane was added dropwise under ice bath conditions. After reacting at room temperature for 12 hours, the solution was concentrated under vacuum to obtain 90 mg of the compound shown in 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. The mixture was reacted at 60 °C for 1 hour. After returning to room temperature, a brownish-yellow precipitate was obtained. The precipitate was washed successively with deionized water, methanol, and diethyl ether, and then dried under vacuum to obtain the molecular probe of formula (Ⅲ). The molecular probe was a brownish-yellow solid with a yield of 70%. The 1H NMR spectrum is shown in […]. 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 for the preparation method of the molecular probe of formula (III) described in this invention is as follows:

[0079]

[0080] Example 2: Detection of molecular probe labeling of target proteins by in-gel fluorescence imaging and Coomassie brilliant blue staining.

[0081] A549 cells were cultured to approximately 90% confluence and incubated with a 1 μM probe for 2 hours. After UV irradiation / non-irradiation, cells were lysed using IP cell lysis buffer. The protein concentration of the sample was quantified to 1.5 mg / mL using phosphate-buffered saline (PBS). A click chemiluminescent dye was then added and reacted for 1 hour. Acetone precipitation was performed at -20°C to precipitate the protein, and the organic solvent was removed by centrifugation. Loading buffer was added, and separation was performed using polyacrylamide gel electrophoresis. The results were finally obtained by fluorescence imaging, as shown below. Figure 4 and Figure 5 As shown. Figure 4 The in situ protein labeling map of the molecular probe of formula (I) obtained by in-gel fluorescence imaging and Coomassie brilliant blue staining. Figure 5 The images show the in situ protein labeling patterns of molecular probes of formula (II) and formula (III) obtained by in-gel fluorescence imaging and Coomassie brilliant blue staining. Figure 4 and Figure 5 (a) in the images are all intragel fluorescence imaging, and (b) are all Coomassie brilliant blue staining. The molecular probes of formulas (I), (II), and (III) can all label their target proteins, among which the molecular probes of formulas (I) and (III) have higher sensitivity.

[0082] Example 3: Investigating the efficacy of platinum-based chemotherapy drugs before and after treatment using probe cell imaging

[0083] Non-small cell lung cancer (NSCLC) A549 cells were cultured in imaging dishes at 37°C with 5% CO2 and saturated humidity until a density of 50%-60% was reached, followed by probe treatment for 2 hours. For competitive labeling, cells were treated with the corresponding drug for 2 hours before probe addition. After UV irradiation, cells were washed with PBS and then fixed with 4% paraformaldehyde. Cells were incubated in fresh medium containing the corresponding click reporter factor for 1 hour. Nuclear staining and mounting were then performed at room temperature, and images were acquired on different detection channels of a Zeiss LSM980 confocal microscope system. Figure 6 Cellular localization imaging of the molecular probe of formula (I) in non-small cell lung cancer cells A549, as shown by confocal microscopy. Figure 7 Cellular localization imaging of the molecular probe of formula (II) in non-small cell lung cancer cells A549, as shown by confocal microscopy. Figure 8 This is a confocal microscopy image showing the cellular localization of the molecular probe of formula (III) in A549 non-small cell lung cancer cells. Figure 6 , Figure 7 and Figure 8 The first column shows Hoechst nuclear staining images, the second column shows cell images treated with corresponding molecular probes, the third column shows overlay images of cells treated with Hoechst nuclear staining and probes, and the fourth column shows bright-field images related to cell imaging. Additionally, Figure 6 , Figure 7 and Figure 8 The first row consists of blank control group images without drug or molecular probe treatment; the second row consists of experimental group images with molecular probe treatment after drug competition; and the third row consists of experimental group images with molecular probe treatment only. Therefore, it can be seen that the molecular probes of formulas (I), (II), and (III) described in this invention can all identify the interaction between platinum-based drugs and A549 non-small cell lung cancer cells.

[0084] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A molecular probe based on platinum-based chemotherapy drugs, characterized in that, The platinum-based chemotherapy drug is cisplatin or oxaliplatin, and the molecular probe is as shown in formula (I), formula (II), or formula (III): 。 2. A method for preparing a molecular probe as described in claim 1, 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 were reacted at room temperature in a dry N,N-dimethylformamide solution to obtain formula (Ⅰ-1). A2. Under a nitrogen atmosphere, the formula (I-1) is dissolved in anhydrous methanol solution and reacted with nickel dichloride hexahydrate, ditert-butyl dicarbonate and sodium borohydride at room temperature in the dark to obtain formula (I-2). A3. Dissolve the above formula (I-2) in tetrahydrofuran, add lithium hydroxide aqueous solution and react at room temperature, then add hydrochloric acid to adjust the pH to 3 to obtain a colorless oily substance; dissolve the colorless oily substance in anhydrous dichloromethane, add EDCI, HOBT, triethylamine and photocrosslinking agent N8 in sequence under ice bath conditions, and then transfer to room temperature to react to obtain formula (I-3). A4. Dissolve the formula (I-3) in dichloromethane, add a solution of 1,4-dioxane with hydrogen chloride dropwise under ice bath conditions, transfer to room temperature for reaction, and then concentrate under vacuum to obtain a white solid; dissolve the white solid in N,N-dimethylformamide, and then add 1,8-diazabicyclo[5.4.0]undec-7-ene and cis-dichlorobis(dimethyl sulfoxide)platinum (II) in sequence, react at room temperature, add pre-cooled deionized water to obtain a pale yellow precipitate, wash with water and vacuum dry to obtain the molecular probe of formula (I); The structural formula of the photocrosslinking agent N8 is: ; The reaction route of the preparation method is as follows: 。 3. The method for preparing the molecular probe according to claim 2, characterized in that, In A1, the molar ratio of methyl 3-bromo-2-(bromomethyl)propionate to potassium azide is 1:(8~12); in A2, the molar ratio of formula (I-1) to nickel dichloride hexahydrate, di-tert-butyl dicarbonate, and sodium borohydride is (0.5~1):(0.003~0.008):(1.5~2):(1.5~2); and in A3, the molar ratio of formula (I-2) to lithium hydroxide aqueous solution 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).

4. A method for preparing a molecular probe as described in claim 1, characterized in that, The molecular probe of formula (II) is based on oxaliplatin and is prepared as follows: B1. Under a nitrogen atmosphere, manganese acetate dihydrate and potassium azide are dissolved in acetonitrile, methyl 3-cyclohexene-1-carboxylate is added, and trifluoroacetic acid is slowly added dropwise at -20°C to react. Then the mixture is transferred to room temperature to react fully to obtain formula (Ⅱ-1). B2. Under dark conditions, the formula (II-1) was dissolved in a mixed solution of tetrahydrofuran and water, and triphenylphosphine was added and reacted at room temperature to obtain formula (II-2). B3. Dissolve the formula (II-2) in a mixed solution of tetrahydrofuran and water, add triethylamine and ditert-butyl dicarbonate and react at room temperature to obtain formula (II-3). B4. Dissolve the above formula (II-3) in tetrahydrofuran, add lithium hydroxide aqueous solution and react at room temperature to obtain formula (II-4). B5. Dissolve the formula (II-4) in anhydrous tetrahydrofuran, add EDCI, HOBT and triethylamine in sequence under ice bath conditions, then add small molecule chain 69, and then transfer to room temperature to react to obtain formula (II-5). B6. Dissolve the formula (II-5) in dichloromethane, add a 1,4-dioxane solution of hydrogen chloride dropwise under ice bath conditions, transfer to room temperature for reaction, and then concentrate under vacuum to obtain a white solid; dissolve the obtained white solid in an aqueous sodium hydroxide solution, add an aqueous potassium tetrachloroplatinate solution, react at 60°C, and after restoring to room temperature, obtain a yellow precipitate, wash and then vacuum dry to obtain the molecular probe of formula (II). The structural formula of the small molecule chain 69 is as follows: ; The reaction route of the preparation method is as follows: 。 5. The method for preparing the molecular probe according to claim 4, characterized in that, In B1, the molar ratio of manganese acetate dihydrate to potassium azide and methyl 3-cyclohexene-1-carboxylate is (4~8):(10~15):(2~4); in B2, the molar ratio of formula (II-1) to triphenylphosphine is 1:(1.8~2.5); in B3, the molar ratio of formula (II-2) to triethylamine and di-tert-butyl dicarbonate is (0.5~1):(2~3):(1~1.5); in B4, the molar ratio of formula (II-3) to... The molar ratio of the lithium hydroxide aqueous solution is 1:(3~4), the molar ratio of formula (II-4) 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).

6. A method for preparing a molecular probe as described in claim 1, characterized in that, The molecular probe of formula (III) is based on oxaliplatin and is prepared as follows: C1. Dissolve the formula (Ⅱ-4) in dichloromethane solution, add EDCI, HOBT, and triethylamine in sequence under ice bath conditions, add photocrosslinking agent N8, and then transfer to room temperature to react, to obtain formula (Ⅲ-1). C2. Dissolve the above formula (Ⅲ-1) in dichloromethane, add a solution of 1,4-dioxane with hydrogen chloride dropwise under ice bath conditions, react at room temperature and then concentrate under vacuum to obtain formula (Ⅲ-2). C3. Dissolve the formula (Ⅲ-2) in an aqueous sodium hydroxide solution, add an aqueous potassium tetrachloroplatinate solution, react at 60°C, and after restoring to room temperature, obtain a brownish-yellow precipitate. Wash and vacuum dry to obtain the molecular probe of the formula (Ⅲ). The structural formula of the photocrosslinking agent N8 is: ; The reaction route of the preparation method is as follows: 。 7. The method for preparing the molecular probe according to claim 6, characterized in that, The molar ratio of formula (Ⅱ-4) in C1 to EDCI, HOBT, triethylamine, and photocrosslinking agent N8 is (1~1.5):(1~1.5):(1~1.5):(2~3):(0.8~1.2), and the molar ratio of formula (Ⅲ-2) in C3 to aqueous solutions of sodium hydroxide and potassium tetrachloroplatinate is (1~1.5):(2~3):(1~1.5).

8. The application of the molecular probe according to claim 1 in cell localization imaging, characterized in that, The application includes the molecular probe identifying the interaction and changes between platinum-based chemotherapy drugs and tumor cells, thereby establishing a monitoring system for tumor cells. This application is not for disease diagnosis or treatment.

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