A fluorescent probe and a preparation method and application thereof

Abstract

This invention belongs to the field of fluorescence technology, and particularly relates to a fluorescent probe, its preparation method, and its application. The fluorescent probe has the structure shown in Formula I. The fluorescent probe provided by this invention can specifically recognize pyruvate kinase M2, exhibiting superior tumor cell imaging characteristics.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence technology, and in particular relates to a fluorescent probe, its preparation method, and its application. Background Technology

[0002] Pyruvate kinase (PK) is a key rate-limiting enzyme in the glycolysis pathway. In the final irreversible step of glycolysis, it catalyzes the phosphorylation of phosphopyruvate (PEP) and adenosine diphosphate (ATP) to produce pyruvate and adenosine triphosphate (ATP). In tumors, PK primarily affects cellular metabolic reprogramming by regulating glycolysis, and the expression of its different isoforms plays a crucial role in tumorigenesis and development. Among the four known PK isoforms, PKM2 is significantly overexpressed in various malignant tumors and is closely related to carcinogenesis. PKM2 exists in two forms with different catalytic activities: tetramer and dimer. The tetramer exhibits high catalytic activity and is mainly responsible for promoting ATP production to maintain energy metabolism; the dimer has lower catalytic activity and is the low-activity state of PKM2, which promotes the entry of glycolytic intermediates into the glycolytic branch pathway. As a key regulatory enzyme in the glycolysis pathway, its abnormal expression has been confirmed to be closely related to metabolic reprogramming in various malignant tumors. Because its expression level is closely related to various cancers (such as colorectal cancer, gastric cancer, hepatocellular carcinoma, lung cancer, and breast cancer), PKM2 has been regarded as a potential tumor biomarker and therapeutic target.

[0003] With the elucidation of the function of PKM2 protein kinase, small molecule agonists targeting its activity regulation (such as TEPP-46 and DASA-58) have become an emerging strategy in anti-tumor research. Molecular fluorescence imaging, due to its advantages such as real-time performance, biocompatibility, low cost, and non-invasiveness, is widely used in early disease diagnosis, intraoperative navigation, and in-situ biochemical process monitoring. However, currently reported fluorescent probes for PKM2 detection still have many inherent defects, making it difficult to meet the practical needs of accurate, stable, and highly specific in vitro detection. Most probes are susceptible to non-specific interference from endogenous biothiols such as glutathione and cysteine ​​in complex biological systems, resulting in high background signals, insufficient signal-to-noise ratio, and significantly reduced reliability of detection results. Although some probes can achieve targeted responses, their fluorescence enhancement is limited, quantum yield is low, and sensitivity in dilute in vitro solutions is insufficient, making accurate quantitative analysis difficult. In addition, some probes have short excitation wavelengths, making them susceptible to interference from the sample's own fluorescence and scattered light. They also exhibit poor selectivity for isoenzymes such as PKM1 and significant cross-response. Furthermore, some probes have poor structural stability and are easily affected by environmental factors such as pH and temperature, which limits their widespread application in actual in vitro samples.

[0004] Early turn-on probes based on nucleophilic substitution (such as the AT-OPD series) could covalently label PKM2, but due to their high reactivity, they readily bound to high concentrations of glutathione (GSH) and cysteine ​​(Cys) in cells and in vitro systems, resulting in extremely strong background fluorescence and a very low signal-to-noise ratio, making it impossible to distinguish the target signal in complex in vitro samples (such as cell lysates and serum). CN115010721A reported a water-soluble fluorescent probe targeting the PKM2 protein, but the cell imaging performance of such probes was generally poor. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a fluorescent probe, its preparation method, and its applications. The probe uses a D-π-A (donor-π-acceptor) structure as its fluorescent backbone, with a strong electron-withdrawing group introduced on one side and an electron-donating group (nucleophilic group) modified on the other side. By conjugating TEPP-46 to this fluorophore, it achieves highly sensitive and selective detection of PKM2, providing a novel molecular tool for cancer diagnosis.

[0006] In a first aspect, the present invention provides a fluorescent probe having the structure shown in Formula I:

[0007] I; Wherein, R is selected from C1-C4 alkyl-substituted or unsubstituted N atoms.

[0008] According to some embodiments of the present invention, the fluorescent probe has any of the following structures: , , , , .

[0009] A second aspect of the present invention provides a method for preparing the above-described fluorescent probe, comprising the following steps: S1. Compound 1 is reacted with N-Boc-1,6-diaminohexane to give compound 2; S2. Reacting compound 2 with quinoline aldehyde to obtain compound 3; or, reacting compound 2 with quinoline aldehyde and then further reacting it with a halo-C1-C4 alkyl group to obtain compound 3; S3. After removing the Boc protecting group under acidic conditions, compound 3 is further reacted with succinic anhydride to obtain compound 4. S4. Compound 4 is reacted with TEPP-46 to obtain the fluorescent probe; Wherein, the structural formula of compound 1 is ; The structural formula of compound 2 is as follows: ; The structural formula of compound 3 is as follows: ; The structural formula of compound 4 is as follows: ; The structural formula of TEPP-46 is as follows: ; R is defined above.

[0010] According to some embodiments of the present invention, in step S1, the reaction is carried out in the presence of an inorganic base; and / or, the temperature of the reaction is 70~90 °C; and / or, the reaction time is 4~8 h.

[0011] According to some embodiments of the present invention, in step S3, trifluoroacetic acid is used to remove the Boc protecting group; and / or, the reaction is carried out in the presence of an organic base; and / or, the temperature of the reaction is 0~40 °C; and / or, the reaction time is 8~24 h.

[0012] According to some embodiments of the present invention, in step S4, the reaction is carried out in a condensing agent, 1 The procedure was carried out in the presence of hydroxybenzotriazole and a catalyst.

[0013] According to some embodiments of the present invention, the condensing agent comprises 1 Ethyl (3 dimethylaminopropyl)carbodiimide hydrochloride, N,N' Dicyclohexylcarbodiimide, hexafluorophosphate O (7 Azabenzotriazole 1 base) N,N,N',N' Tetramethylurea, hexafluorophosphate O benzotriazole 1 base) N,N,N',N' At least one of tetramethylurea; and / or, the catalyst comprises N,N-diisopropylethylamine.

[0014] A third aspect of the present invention provides the use of the above-described fluorescent probe in the preparation of live-cell imaging reagents.

[0015] According to some embodiments of the present invention, the application is the use of fluorescent probes in the preparation of PKM2 protein imaging reagents in live cells.

[0016] A fourth aspect of the present invention provides a method for detecting pyruvate kinase M2, comprising the following steps: S1. Mix the above-mentioned fluorescent probe with the sample to be tested; S2. Measure the fluorescence ratio of the mixed solution at 580~750 nm (e.g. 600~750 nm, 650~750 nm, 650~700 nm, 650~680 nm).

[0017] According to some embodiments of the present invention, at least the following beneficial effects are achieved: The probe of this invention exhibits excellent in vitro fluorescence properties, including emission wavelengths reaching the near-infrared emission region, good viscosity response sensitivity, and stability over a wide pH range; providing an important basis for developing highly sensitive, long-term live-cell targeted imaging tools. Compared with existing probes, the fluorescent probe of this invention possesses near-infrared emission wavelengths and superior signal output capabilities; it is expected to be used for highly selective detection of PKM2 in tumor cells, serving tumor screening and early diagnosis.

[0018] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 It is compound YL1 1 H NMR spectrum (Chloroform-d; 500 MHz) and 13 C NMR spectrum (Chloroform-d; 125 MHz); Figure 2 It is compound YL2 1 H NMR spectrum (Methanol-d4; 500 MHz) and 13 C NMR spectrum (Chloroform-d; 125 MHz); Figure 3 The fluorescence emission spectra of YL1 and YL2 (10 μM in PBS) in different polar solvents and the scatter plot of fluorescence intensity at their respective maximum emission wavelengths are shown. Figure 4 The fluorescence emission spectra of YL1 and YL2 (10 μM in PBS) in different Gly / H2O and the scatter plot of fluorescence intensity at their respective maximum emission wavelengths are shown. Figure 5The fluorescence emission spectra of YL1 and YL2 (10 μM in PBS) at different pH values ​​and the scatter plot of fluorescence intensity at their respective maximum emission wavelengths are shown. Figure 6 The graph shows the fluorescence intensity changes of probes YL1 and YL2 (2 μM) and PKM2, PKM1, pepsin (PEP), human serum albumin (HAS), acetylcholinesterase (AchE), bovine serum albumin (BSA), cellulase, and glutathione (GSH) (20 μM) in PBS buffer. Figure 7 This is a cell imaging image of probes YL1, YL2, and zy-2 (10 µM) after incubation for 4 h in HeLa, A549, and HK2 cells. Detailed Implementation

[0020] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0021] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0022] Unless otherwise specified, "about" in this invention means that the allowable error is within ±2%.

[0023] Unless otherwise specified, "room temperature" in this invention means 25±5 ℃.

[0024] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0025] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] Example 1 In this embodiment, small molecule fluorescent probes YL1 and YL2 were synthesized to specifically detect PKM2 protein.

[0027]

[0028] Intermediate compound 2 was obtained in 58% yield by reacting compound 1 with tert-butyl(6-aminohexyl)carbamate under reflux with ethanol at 80 °C. Intermediate compound 3a was obtained in 86% yield by reacting compound 2 with 4-quinolinecarbaldehyde under basic conditions. Intermediate compound 3b was obtained in 75% yield by reacting compound 3a with iodoethane under neutral conditions (NMR confirmed ethyl selectivity on the five-membered ring). Compounds 3a and 3b were de-BOC-treated under acidic conditions to obtain control compounds Y1 and Y2 in 95% and 90% yields, respectively. Intermediates 4a and 4b were obtained in 66% and 52% yields, respectively, by reacting compound Y1 / Y2 with succinic anhydride under basic conditions. Probe molecules YL1 and YL2 were obtained in 52% and 49% yields, respectively, by reacting compound 4a / 4b with TEPP-46 under basic conditions.

[0029] The specific synthesis steps are as follows: Synthesis of Compound 2: Compound 1 (230 mg, 1.0 mmol) was dissolved in a 25 mL round-bottom flask containing 5 mL of anhydrous ethanol. Then, 470 μL of tert-butyl(6-aminohexyl)carbamate was added, and the mixture was stirred in an oil bath at 80 °C for 5 minutes. Potassium carbonate (207 mg, 1.5 mmol) was then added, and the mixture was heated under reflux for 6 hours. After the reaction was complete as monitored by TLC, the organic phase was extracted with dichloromethane. The solution was purified by silica gel column chromatography to give 248 mg of compound as an orange solid, in 58% yield.

[0030] 1 H NMR (500 MHz, Chloroform-d)) δ 8.12 – 8.01 (m, 2H), 7.08 (s, 1H), 6.72 (dd, J = 9.1, 3.3 Hz, 2H), 3.60 (t, J = 7.4 Hz, 2H), 3.13 (d, J = 7.2Hz, 2H), 3.07 (s, 6H), 2.38 (s, 3H), 1.67 – 1.60 (m, 2H), 1.46 (s, 9H), 1.41– 1.32 (m, 6H). 13 C NMR (125 MHz, Chloroform-d) δ 170.74, 158.94, 156.02,151.51, 134.61, 134.16, 128.87, 122.27, 111.76, 111.32, 40.38, 40.07, 29.92,29.37, 28.44, 26.39, 26.35, 15.69.

[0031] Synthesis of compound 3a: Compound 2 (428.6 mg, 1 mmol) and 4-quinolinecarbaldehyde (157 mg, 1.2 mmol) were placed in a 50 mL round-bottom flask and dissolved in 5 mL of ethanol. Zinc chloride (136.3 mg, 1 mmol) was then added to the system, and the reaction mixture was immediately transferred to an oil bath preheated to 80 °C and stirred for 6 hours. After TLC showed complete reaction, the zinc chloride solid was removed by filtration, and the organic phases were combined after extraction and column chromatography to give 488 mg of a red solid, with a yield of 86%.

[0032] 1 H NMR (500 MHz, Chloroform-d) δ 8.75 (d, J = 15.5 Hz, 1H), 8.33 (s,1H), 8.07 (d, J = 9.1 Hz, 1H), 7.82 (t, J = 7.0 Hz, 1H), 7.74 – 7.64 (m, 3H),7.27 (s, 1H), 7.05 (d, J = 15.4 Hz, 1H), 6.81 – 6.78 (m, 2H), 6.76 (d, J =9.1 Hz, 1H), 4.56 (s, 2H), 3.81 (t, J = 7.3 Hz, 2H), 3.13 (s, 6H), 1.71 (d, J= 14.8 Hz, 2H), 1.50 (q, J = 7.0 Hz, 4H), 1.44 (s, 11H). 13 C NMR (125 MHz, Chloroform-d) δ 171.01, 157.36, 156.02, 151.50, 151.48, 151.25, 140.01,136.01, 134.19, 134.15, 129.47, 127.02, 123.61, 123.32, 111.99, 111.87,111.76, 107.58, 77.32, 77.06, 76.81, 47.46, 42.50, 40.42, 40.37, 40.20,40.12, 40.06, 39.78, 31.52, 29.92, 29.72, 29.37, 28.44, 26.44, 26.38, 26.35, 25.50, 24.50, 16.13.

[0033] Synthesis of compound 3b: Compound 3a (100 mg, 0.176 mmol) and 137 μL of iodoethane were placed in a round-bottom flask, and 1 mL of ethanol was added. The mixture was then transferred to an 80°C oil bath for reaction. After the reaction was complete, the product was purified by silica gel column chromatography to obtain 78 mg of the target product as a red solid, with a yield of 75%.

[0034] 1 H NMR (500 MHz, Chloroform-d) δ 9.01 (d, J = 4.7 Hz, 1H), 8.76 (d, J= 15.5 Hz, 1H), 8.38 – 8.32 (m, 1H), 8.28 (d, J = 8.7 Hz, 1H), 8.23 ​​(d, J =8.5 Hz, 2H), 7.85 (ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.76 – 7.69 (m, 2H), 7.27(s, 1H), 7.08 (d, J = 15.6 Hz, 1H), 6.79 (d, J = 9.0 Hz, 2H), 3.82 (t, J =7.2 Hz, 2H), 3.16 (d, J = 3.3 Hz, 2H), 3.13 (s, 6H), 3.12 – 3.09 (m, 2H), 1.70 (q, J = 7.3 Hz, 2H), 1.49 (d, J = 6.9 Hz, 2H), 1.46 – 1.45 (m, 2H), 1.44 (s, 11H), 1.41 – 1.37 (m, 3H). 13 C NMR (125 MHz, DMSO) δ 169.96, 156.14,156.03, 152.15, 150.81, 148.66, 148.04, 140.61, 135.29, 135.22, 134.49,131.96, 130.27, 130.13, 129.66, 129.25, 127.88, 127.19, 125.97, 124.04,122.37, 121.13, 118.76, 112.39, 112.06, 77.76, 66.99, 40.42, 40.35, 40.25, 40.18, 40.09, 40.01, 39.92, 39.75, 39.59, 39.42, 29.92, 29.81, 29.31, 28.70, 27.31, 26.37, 26.28, 25.86.

[0035] Synthesis of compound Y1: Compound 3a (200 mg, 0.35 mmol) was dissolved in DCM / TFA (3:1, 4 mL) and stirred at room temperature for 30 minutes. After concentration, 156 mg of brown-black solid compound was obtained, with a yield of 95%.

[0036] 1 H NMR (400 MHz, Methanol-d4) δ 9.13 (d, J = 5.8 Hz, 1H), 8.87 (d, J= 15.5 Hz, 1H), 8.73 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 5.8 Hz, 1H), 8.34 –8.23 (m, 3H), 8.19 (ddd, J = 8.5, 6.9, 1.2 Hz, 1H), 8.06 (ddd, J = 8.3, 6.8,1.3 Hz, 1H), 7.72 (d, J = 15.5 Hz, 1H), 7.26 (s, 1H), 6.92 – 6.84 (m, 2H),3.96 (t, J = 7.0 Hz, 2H), 3.16 (s, 6H), 2.92 (t, J = 7.6 Hz, 2H), 1.78 (q, J= 7.0 Hz, 2H), 1.66 (q, J = 7.5 Hz, 2H), 1.48 (s, 2H), 1.45 (d, J = 7.6 Hz, 2H), 1.36 – 1.28 (m, 2H). 13 C NMR (125 MHz, Methanol-d4) δ 170.65, 154.50,152.51, 148.01, 145.52, 143.87, 135.26, 134.42, 131.62, 131.10, 130.73,127.97, 126.63, 126.29, 123.62, 122.05, 120.99, 117.66, 111.57, 39.42, 39.13,38.71, 29.30, 27.02, 25.70, 25.51.

[0037] Synthesis of compound Y2: Compound 3b (200 mg, 0.32 mmol) was dissolved in DCM / TFA (3:1, 4 mL) and stirred at room temperature for 30 minutes. After concentration, the brown-black solid compound Y2 (142 mg) was obtained in 90% yield.

[0038] 1 H NMR (500 MHz, Methanol-d4) δ 9.04 (d, J = 5.3 Hz, 1H), 8.83 (d, J =15.5 Hz, 1H), 8.59 (d, J = 8.5 Hz, 1H), 8.33 – 8.22 (m, 3H), 8.20 (d, J = 8.3Hz, 1H), 8.05 (q, J = 8.1, 6.9 Hz, 1H), 7.93 (t, J = 7.7 Hz, 1H), 7.59 (d, J= 15.5 Hz, 1H), 7.23 (s, 1H), 6.87 (d, J = 8.7 Hz, 2H), 3.95 (t, J = 7.1 Hz,2H), 3.14 (s, 6H), 3.04 (s, 2H), 2.92 (t, J = 7.7 Hz, 2H), 1.77 (d, J = 7.4Hz, 2H), 1.67 (t, J = 7.5 Hz, 2H), 1.47 (s, 2H), 1.38 – 1.32 (m, 2H), 1.34 –1.28 (m, 2H). 13 C NMR (125 MHz, Methanol-d4) δ 170.69, 154.59, 152.49, 148.47,146.18, 143.23, 135.21, 134.42, 131.52, 131.00, 130.76, 127.78, 127.16,126.26, 123.53, 122.05, 120.63, 117.66, 111.56, 39.41, 39.13, 38.70, 29.29,27.02, 25.69, 25.50.

[0039] Synthesis of compound 4a: Compound Y1 (100 mg, 0.214 mmol) was dissolved in a 10 mL reaction flask by sonication with acetonitrile (1 mL). Succinic anhydride (42.9 mg, 0.428 mmol) was dissolved in a 1.5 mL centrifuge tube by sonication with acetonitrile (1 mL). The succinic anhydride solution was poured into a round-bottom flask, and triethylamine (44.8 μL) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was complete as monitored by TLC, excess succinic anhydride was washed away with water, and the organic phase was extracted with dichloromethane. The compound was purified by silica gel column chromatography to obtain 82 mg of black solid, with a yield of 66%.

[0040] 1 H NMR (500 MHz, DMSO-d6) δ 12.40 (s, 1H), 8.98 – 8.78 (m, 2H), 8.11(d, J = 6.8 Hz, 2H), 7.92 (t, J = 14.4 Hz, 1H), 7.61 (d, J = 8.7 Hz, 2H),7.27 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 5.77 (s, 1H), 4.48 (q, J= 7.3 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 2.73 – 2.59 (m, 4H), 2.40 (s, 6H),1.52 (d, J = 7.3 Hz, 2H), 1.49 (d, J = 12.9 Hz, 2H), 1.31 – 1.24 (m, 2H),1.23 (d, J = 8.8 Hz, 2H), 1.20 (d, J = 7.1 Hz, 2H), 1.16 (t, J = 7.3 Hz, 2H). 13 C NMR (125 MHz,Chloroform-d) δ 174.15, 173.88, 173.10, 171.55, 170.37,164.01, 153.95, 152.76, 152.05, 146.36, 143.94, 141.89, 140.29, 138.75,133.01, 132.65, 130.39, 125.15, 123.27, 119.04, 115.41, 113.31, 60.38, 58.72,56.50, 55.31, 52.10, 51.86, 49.35, 47.29, 45.51, 30.16, 29.11, 28.96, 19.01,16.63, 14.61。

[0041] Synthesis of compound 4b: Compound Y2 (300 mg, 0.60 mmol) was dissolved in a 10 mL reaction flask by sonication with 0.5 mL of DMF. Succinic anhydride (42.9 mg, 0.428 mmol) was dissolved in a 1.5 mL centrifuge tube by sonication with 0.5 mL of DMF. The succinic anhydride solution was poured into a round-bottom flask, and triethylamine (100 μL) was added. The mixture was incubated at RT for 12 hours. After the reaction was complete, the mixture was washed with water, and the organic phase was extracted with dichloromethane. The compound was purified by silica gel column chromatography to obtain 191 mg of black solid, with a yield of 52%.

[0042] 1 H NMR (500 MHz, DMSO-d6) δ 12.40 (s, 1H), 8.84 (d, J = 6.6 Hz, 2H), 8.11 (d, J = 6.8 Hz, 2H), 7.94 (d, J = 16.2 Hz, 1H), 7.61 (d, J = 8.7 Hz,2H), 7.27 (d, J = 16.1 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 4.48 (q, J = 7.3Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.59 (s, 2H), 2.65 (d, J = 5.2 Hz, 2H),2.40 (s, 6H), 1.52 (d, J = 7.3 Hz, 2H), 1.49 (d, J = 12.9 Hz, 2H), 1.24 (d, J= 7.9 Hz, 2H), 1.22 (s, 2H), 1.20 (s, 2H), 1.19 – 1.13 (m, 2H). 13C NMR (125MHz, Chloroform-d) δ 173.49, 172.36, 170.53, 155.96, 155.09, 151.93, 149.49,147.56, 144.90, 135.15, 135.04, 132.96, 130.88, 129.90, 127.26, 123.60,122.61, 121.90, 119.76, 111.89, 111.74, 108.39, 100.88, 55.76, 50.42, 50.29,45.30, 42.61, 41.74, 40.08, 39.82, 39.19, 30.02, 29.84, 26.16, 26.10, 22.69, 22.26, 17.17.

[0043] Synthesis of compound YL1: Compound 4a (39.6 mg, 0.070 mol), TEPP46 (20 mg, 0.054 mmol), EDCI (13.4 mg, 0.070 mmol), HOBT (9.43 mg, 0.070 mmol), and DMF (1 mL) were placed in a 10 mL reaction flask, and DIPEA (28 μL) was added. The mixture was stirred at room temperature for 12 hours, and the process was monitored by TLC. After the starting materials were completely consumed, water was added to the reaction solution to remove DMF, and then the mixture was extracted with ethyl acetate. The combined organic phases were purified by silica gel column chromatography to obtain 34 mg of black solid, with a yield of 52% for the target product.

[0044] 1H NMR (500 MHz, Chloroform-d) δ 8.84 (s, 1H), 8.72 (d, J = 15.5 Hz,1H), 8.30 (d, J = 8.0 Hz, 1H), 8.23 – 8.17 (m, 3H), 7.85 – 7.75 (m, 1H), 7.72– 7.65 (m, 1H), 7.64 (d, J = 6.6 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 7.37 (d,J = 2.6 Hz, 1H), 7.23 (s, 1H), 7.22 (s, 1H), 7.05 – 7.00 (m, 1H), 6.76 (d, J= 8.7 Hz, 2H), 6.70 (d, J = 8.8 Hz, 1H), 4.33 (d, J = 7.3 Hz, 2H), 4.26 (d, J= 2.4 Hz, 3H), 3.78 (dt, J = 14.0, 7.3 Hz, 2H), 3.21 (d, J = 6.2 Hz, 2H),3.10 (d, J = 7.1 Hz, 6H), 3.00 (d, J = 2.6 Hz, 3H), 2.67 (dt, J = 8.4, 5.0Hz, 2H), 2.60 (dd, J = 8.0, 4.8 Hz, 2H), 1.73 – 1.61 (m, 4H), 1.50 – 1.46 (m,2H), 1.44 (dd, J = 5.0, 3.0 Hz, 2H). 13C NMR (125 MHz, Chloroform-d) δ 172.45,170.79, 170.65, 155.37, 154.97, 152.01, 151.89, 150.01, 149.21, 148.43,145.97, 144.88, 141.79, 141.33, 138.53, 137.82, 135.12, 135.06, 132.98,131.90, 131.11, 130.99, 129.94, 129.16, 127.28, 126.19, 124.38, 123.73, 123.60, 123.49, 122.54, 120.86, 119.78, 119.38, 119.17, 118.09, 117.83, 116.95, 112.24, 111.90, 77.30, 77.04, 76.79, 54.19, 44.70, 39.51, 39.02, 34.99, 34.45, 34.22, 33.85, 33.17, 31.94, 31.52, 31.45, 30.19, 30.14, 29.71, 29.67, 29.50, 29.38, 29.18, 29.02, 25.72, 25.62, 24.90, 22.71, 14.15. See the spectral results below. Figure 1 .

[0045] Synthesis of compound YL2: Compound 4b (72 mg, 0.121 mmol), TEPP46 (30 mg, 0.081 mmol), EDCI (23 mg, 0.121 mmol), HOBT (16.4 mg, 0.121 mmol), and DMF (1 mL) were placed in a 10 mL reaction flask, and DIPEA (52 μL) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was complete, DMF was washed away with water, and the organic phase was extracted with ethyl acetate. The purified product was 57 mg, a black solid, with a yield of 49%.

[0046] 1H NMR (500 MHz, Methanol-d4) δ 9.14 (d, J = 11.1 Hz, 1H), 8.93 (s,1H), 8.68 (d, J = 15.5 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 9.5Hz, 2H), 8.10 (d, J = 35.6 Hz, 3H), 7.75 (q, J = 12.3, 9.9 Hz, 1H), 7.69 –7.57 (m, 2H), 7.58 – 7.48 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.07 (d, J = 8.0Hz, 1H), 7.00 (d, J = 15.5 Hz, 1H), 6.72 (d, J = 8.7 Hz, 2H), 6.70 – 6.55 (m,2H), 5.32 (s, 2H), 4.18 (s, 3H), 3.74 (q, J = 11.5, 7.5 Hz, 2H), 3.21 – 3.14(m, 2H), 3.06 (d, J = 14.9 Hz, 6H), 2.98 (d, J = 9.3 Hz, 4H), 2.64 (q, J =7.0 Hz, 3H), 2.55 (t, J = 6.9 Hz, 2H), 1.61 (d, J = 13.5 Hz, 2H), 1.42 (d, J= 11.7 Hz, 2H), 1.39 – 1.31 (m, 3H), 1.28 (d, J = 17.7 Hz, 4H). 13C NMR (125MHz, Chloroform-d) δ 172.53, 171.00, 170.59, 155.26, 155.01, 151.96, 151.73,150.03, 148.49, 144.84, 141.25, 138.69, 137.75, 135.08, 134.49, 132.90,131.90, 130.96, 130.85, 129.95, 129.84, 129.08, 127.23, 126.90, 126.15,123.68, 123.55, 122.49, 119.68, 119.46, 119.16, 117.99, 117.81, 116.85, 112.36, 111.88, 111.73, 54.18, 44.56, 40.08, 40.03, 39.65, 39.13, 34.15, 32.94, 31.62, 29.73, 29.70, 29.19, 29.05, 26.05, 25.92, 25.84. The spectral results are shown in [the original text is missing]. Figure 2 .

[0047] Test Example 1 This test example uses solvents of different polarities: H₂O, MeOH, EtOH, n-PrOH, and n-BuOH. 3 µL of the compound's DMSO stock solution (10 mM) was added to 3 mL of each of the different polarity solutions to prepare a 10 µM test solution. This was used to study the effect of polarity on the fluorescence intensity of the compound.

[0048] Test results are as follows Figure 3 As shown, the fluorescence intensity of compounds Y1, Y2, YL1, and YL2 increased with decreasing solvent polarity, exhibiting significant fluorescence quenching with increasing solvent polarity. This is a typical characteristic of intramolecular charge transfer (ICT) fluorescent probes. The main mechanism is the obstruction of intramolecular charge transfer in the excited state. Due to the probe molecule's "push-pull electron structure" (D-π-A), intramolecular charge transfer frequently occurs in its excited state, leading to a significant increase in the excited state dipole moment. Polar solvents stabilize this excited state through solvation relaxation, thereby lowering its energy.

[0049] Test Example 2 To investigate how the fluorescence intensity of the compound changes with pH, ​​the emission spectrum of the probe in PBS buffer in the pH range of 3.0–11.0 was evaluated at room temperature.

[0050] Take 3 µL of the compound and add it to 3 mL of PBS buffer at different pH values ​​to prepare a 10 µM test buffer. Adjust the pH value with 1.0 M NaOH and HCl, and measure the fluorescence spectrum of the compound at different pH values.

[0051] Test results are as follows Figure 4 As shown, when investigating the effect of solvent pH on probe fluorescence intensity, it was found that the fluorescence intensity of compounds YL1 and YL2 increased with increasing pH, and tended to stabilize in the pH range of 8.0 to 11.0.

[0052] Test Example 3 To accurately analyze the effect of viscosity parameters on the fluorescence properties of compounds, this test example constructs a binary solvent system with polarity matching and viscosity gradient control. Water and glycerol with similar polarity parameters but significant viscosity differences are selected to ensure that the polarity deviation is ≤10%, effectively isolating the interference of polarity effect on fluorescence signal. Water-glycerol solutions with different volume ratios are prepared and mixed for later use.

[0053] Specifically, the test procedure is as follows: 3 µL of the compound DMSO stock solution was added to 3 mL of pre-prepared mixed solutions of water and glycerol in different volume ratios and brought to a final volume. The fluorescence spectra of the compound in solutions of different viscosities were obtained by scanning with a fluorescence spectrophotometer at room temperature.

[0054] Test results are as follows Figure 5 As shown, the fluorescence intensity of compounds YL1 and YL2 increased with increasing solvent viscosity, by 46 times and 63 times, respectively.

[0055] Test Example 4 The compound was diluted with PBS to prepare a 2 µM test sample solution, and the 2 µM solution was thoroughly mixed with pyruvate kinase M2 (PKM2) (20 µM), pyruvate kinase M1 (PKM1) (20 µM), bovine serum albumin (BSA) (20 µM), glutathione (GSH) (20 µM), cellulose (20 µM), human serum albumin (HSA) (20 µM), pepsin (PEP) (20 µM), lysozyme (LZ) (20 µM) and acetylcholinesterase (AChE) (20 µM), and incubated at 37 °C for 10 min before testing.

[0056] like Figure 6 As shown, YL1 and YL2 lack selectivity for PKM1 and most kinases, but selectively recognize PKM2 kinase. Furthermore, the fluorescence intensity of the compounds is enhanced compared to their initial values. Experimental results indicate that YL1 and YL2 have a significant effect on PKM2 kinase.

[0057] Test Example 5 After HeLa, A549, and HK2 cell cultures were completed, the cells were washed twice with PBS, digested with trypsin, centrifuged, resuspended in culture medium, and then the diluted cell suspension was counted using a hemocytometer. Based on the hemocytometer results, the cell suspension was diluted to 5 × 10⁻⁶ cells / mL. 4 At a concentration of [number] cells / mL, 1.5 mL of cell suspension was added to a 15 mm confocal culture dish and cultured for 24 h to allow cell adhesion. After cell adhesion and growth, a certain concentration of compound zy-2 (a fluorescent probe reported in CN115010721A) was added. YL1 and YL2 (10 µM) were administered at 2 mL, incubated for 24 h, and then washed more than 3 times with sterile PBS (1 mL) buffer. After washing, 1 mL of PBS solution was added to disperse the cells, and finally, fluorescence cell imaging experiments were performed.

[0058] The results are as follows Figure 7 As shown, at a concentration of 10 µM, zy-2, YL1, and YL2 can all provide clear imaging in tumor cell groups (HeLa, A549), mainly exhibiting significantly enriched fluorescence signals in the cytoplasmic region, consistent with the known subcellular localization of PKM2 (such as the cytoplasm and vicinity of mitochondria). No significant fluorescence signal was detected in the normal cell group (HK2). Furthermore, compared to zy-2, YL1 and YL2 in the embodiments of this invention provide clearer and more distinct imaging of tumor cells, clearly displaying their outlines and demonstrating better imaging performance.

[0059] The above description, in conjunction with specific embodiments, provides a detailed explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A fluorescent probe, characterized in that, It has the structure shown in Equation I: I； Wherein, R is selected from C1-C4 alkyl-substituted or unsubstituted N atoms.

2. The fluorescent probe according to claim 1, characterized in that, The fluorescent probe has any of the following structures: 、 、 、 、 。 3. The method for preparing the fluorescent probe as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Compound 1 is reacted with N-Boc-1,6-diaminohexane to give compound 2; S2. Reacting compound 2 with quinoline aldehyde to obtain compound 3; or, reacting compound 2 with quinoline aldehyde and then further reacting it with a halo-C1-C4 alkyl group to obtain compound 3; S3. After removing the Boc protecting group under acidic conditions, compound 3 is further reacted with succinic anhydride to obtain compound 4. S4. Compound 4 is reacted with TEPP-46 to obtain the fluorescent probe; Wherein, the structural formula of compound 1 is ; The structural formula of compound 2 is as follows: ; The structural formula of compound 3 is as follows: ; The structural formula of compound 4 is as follows: ; The structural formula of TEPP-46 is as follows: ; Wherein, R is as defined in claim 1.

4. The preparation method according to claim 3, characterized in that, In step S1, the reaction is carried out in the presence of an inorganic base; and / or, the temperature of the reaction is 70~90 °C; and / or, the reaction time is 4~8 h.

5. The preparation method according to claim 3, characterized in that, In step S3, trifluoroacetic acid is used to remove the Boc protecting group; and / or, the reaction is carried out in the presence of an organic base; and / or, the reaction temperature is 0~40 °C; and / or, the reaction time is 8~24 h.

6. The preparation method according to claim 3, characterized in that, In step S4, the reaction occurs in the condensing agent, 1 The procedure was carried out in the presence of hydroxybenzotriazole and a catalyst.

7. The preparation method according to claim 6, characterized in that, The condensing agent includes 1 Ethyl (3 dimethylaminopropyl)carbodiimide hydrochloride, N,N' Dicyclohexylcarbodiimide, hexafluorophosphate O (7 Azabenzotriazole 1 base) N,N,N',N' Tetramethylurea, hexafluorophosphate O benzotriazole 1 base) N,N,N',N' At least one of tetramethylurea; and / or, the catalyst comprises N,N-diisopropylethylamine.

8. The use of the fluorescent probe as described in claim 1 or 2 in the preparation of live cell imaging reagents.

9. The application according to claim 8, characterized in that, The application described is the use of fluorescent probes in the preparation of PKM2 protein imaging reagents in live cells.

10. A method for detecting pyruvate kinase M2, characterized in that, Includes the following steps: S1. Mix the fluorescent probe as described in claim 1 or 2 with the sample to be tested; S2. Measure the fluorescence ratio of the mixed solution at 580~750 nm.

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