Cysteine-activated fluorescent probe and preparation method and application thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a cysteine-activated fluorescent probe and a preparation method and application thereof. The cysteine-activated fluorescent probe compound provided by the application can be selectively combined with a tumor signal factor Cys, can be excited at a wavelength of 580 nm, and can realize targeted action by 15 times of fluorescence intensity enhancement at a wavelength of 675 nm after combination, and has good selectivity to Cys. Traditional drug treatment is easy to cause drug resistance of cancer cells, and reduces treatment effect. The mechanism of PDT depends on production of active oxygen, direct induction of cell apoptosis or necrosis, and cells are difficult to form effective resistance through gene mutation. The fluorescent probe compound provided by the application can provide a new tool for precise treatment and improvement of treatment effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a cysteine-activated fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) is a non-invasive and highly selective cancer treatment method, which can induce tumor cell apoptosis or necrosis by generating singlet oxygen (O2) or other reactive oxygen species (ROS) under specific wavelength light irradiation through photosensitizers. 3 However, the use of traditional photosensitizers still faces some challenges, including non-specific distribution, phototoxicity, and potential damage to normal tissues. Therefore, developing photosensitizers with targeting and biological responsiveness has become an important research direction in the field of photodynamic therapy.

[0003] Cysteine (Cys) is a key biological small molecule that plays an important role in various physiological and pathological processes, such as antioxidant stress, metabolic regulation, and signal transmission. In some diseases (such as tumors, inflammation, and neurodegenerative diseases), the expression level of Cys is significantly abnormal. Among various methods for detecting Cys, fluorescence probe detection has inherent advantages such as high sensitivity, non-invasiveness, and in-situ detection, and has become one of the most convenient and effective methods. It has long been considered as one of the most powerful tools for detecting Cys in life systems.

[0004] Currently, fluorescence probes for Cys detection can be divided into two categories: reactive fluorescence probes and coordination complex fluorescence probes, which mainly recognize through Michael addition, aldehyde cyclization, disulfide bond cleavage or other recognition mechanisms. However, these probes have detection upper limits, cannot be used in neutral environments, and more importantly, can only be used for detection but not for tumor treatment. SUMMARY

[0005] To solve the technical problems of current Cys-binding fluorescence probes, and more specifically, to target Cys and achieve the effect of activating and eliminating tumor cells, the present application provides a cysteine-activated fluorescent probe and a preparation method and application thereof.

[0006] The specific technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a cysteine-activated fluorescent probe, the structure of which is shown in formula (1):

[0008]

[0009] In a second aspect, the present application provides a preparation method of a cysteine-activated fluorescent probe, characterized by comprising the following steps:

[0010] Step S1: dissolving compound (2) and compound (3) in a solvent, refluxing to obtain compound (4);

[0011] Step S2: dissolving compound (4) and acryloyl chloride in a solvent, and reacting to obtain compound (1);

[0012] wherein:

[0013] The structural formula of compound (2) is:

[0014]

[0015] The structural formula of compound (3) is:

[0016]

[0017] The structural formula of compound (4) is:

[0018]

[0019] The structural formula of compound (1) is:

[0020]

[0021] As a preferred embodiment of the above preparation method, in step S1, the solvent is ethanol.

[0022] As a preferred embodiment of the above preparation method, in step S1, the catalyst for refluxing is piperidine.

[0023] As a preferred embodiment of the above preparation method, in step S1, the temperature for refluxing is 75-80℃.

[0024] As a preferred embodiment of the above preparation method, in step S2, the solvent is dichloromethane.

[0025] As a preferred embodiment of the above preparation method, in step S2, the catalyst for reaction is triethylamine.

[0026] As a preferred embodiment of the above preparation method, in step S2, the temperature for reaction is -5-30℃.

[0027] In a third aspect, the present application provides application of the above fluorescent probe in imaging of cancer biomarkers.

[0028] In a fourth aspect, the present application provides application of the above fluorescent probe in preparation of products based on photodynamic therapy of cancer.

[0029] Compared with the prior art, the present invention has the following technical effects:

[0030] 1. The cysteine-activated fluorescent probe compound (1) provided by the present invention can selectively bind to the tumor signaling factor Cys. After binding, the fluorescence intensity at a wavelength of 675 nm can be enhanced by up to 15 times under excitation at a wavelength of 580 nm, thereby achieving a targeting effect. At the same time, compound (1) has good selectivity for Cys.

[0031] 2. At the same time, the cysteine-activated fluorescent probe compound (1) provided by the present invention can increase the singlet oxygen yield by up to 3 times after reacting with Cys. The compound (1) has a good PDT effect. The probe compound (1) can be successfully applied to the detection of endogenous and exogenous Cys in HeLa cells and successfully used photodynamic therapy to kill HeLa cells.

[0032] 3. Traditional drug treatments can easily induce drug resistance in cancer cells, reducing the effectiveness of treatment. However, the mechanism of PDT relies on the production of reactive oxygen species, which directly induces cell apoptosis or necrosis. It is difficult for cells to develop effective resistance through gene mutation. Fluorescent probe compounds (1) can provide new tools for precise treatment and improving treatment efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The absorption and emission spectra of compound (1) before and after being activated by Cys, wherein -Cys indicates not activated by Cys, +Cys indicates activated by Cys, (a) is the absorption spectrum, and (b) is the emission spectrum;

[0034] Figure 2 The results of the study on the specificity of compound (1) to Cys;

[0035] Figure 3 To study the photodynamic properties of compound (1) after being activated by Cys;

[0036] Figure 4 To study the photodynamic properties of compound (1) that is not activated by Cys;

[0037] Figure 5 The confocal microscopy comparison diagram of compound (1) detecting endogenous and exogenous Cys in Hela cells, wherein the upper diagram is a confocal imaging diagram and the lower diagram is a quantitative result diagram of the fluorescence intensity in the confocal imaging diagram;

[0038] Figure 6 The figure is a comparison of the photodynamic effects of compound (1) on Cys-overexpressing A549 cells before and after illumination;

[0039] Figure 7The results of the Annexin V-PI double staining method to test the photodynamic effect of compound (1) on Cys-overexpressing A549 cells before and after illumination;

[0040] Figure 8 The diagram shows the mechanism of the fluorescence intensity enhancement of compound (1). DETAILED DESCRIPTION

[0041] In a first aspect, the present invention provides a cysteine-activated fluorescent probe, the structure of which is shown in formula (1):

[0042] The above-mentioned cysteine-activated fluorescent probe compound (1) can selectively bind to the tumor signaling factor Cys to achieve a targeting effect. Compound (1) has good selectivity for Cys. Experimental verification shows that after compound (1) is activated by Cys, the fluorescence intensity at a wavelength of 675 nm under excitation at a wavelength of 580 nm can be enhanced by up to 15 times. Figure 8 The diagram shows the mechanism of the fluorescence intensity enhancement of compound (1).

[0043] In one embodiment, the present invention synthesized compound (a) and compound (b) by performing a series of screening on the fluorescent structural changes of fluorescent probe compound (1).

[0044] The structural formula of compound (a) is as follows:

[0045]

[0046] The structural formula of compound (b) is as follows:

[0047]

[0048] Furthermore, in one embodiment, the present invention compares the fluorescence properties of the activated fluorescent probe compound (1) with those of compound (a) and compound (b), and finds that the emission wavelength of compound (1) after reacting with cysteine ​​is 675 nm, while the emission wavelength of compound (a) is only 590 nm and the emission wavelength of compound (b) is 650 nm. The longer the emission wavelength, the better the penetration effect into the biological tissue, which is beneficial for the fluorescence imaging of the organism. It can be seen that the fluorescence properties of the compound after reacting with cysteine ​​of the present invention are more superior. This proves that the fluorescent probe compound (1) of the present invention has a more superior effect in fluorescence imaging effect and PDT performance, which is unexpected.

[0049] In a second aspect, the present invention provides a method for preparing a cysteine-activated fluorescent probe, which is characterized by comprising the following steps:

[0050] Step S1: refluxing compound (2) and compound (3) in a solvent to obtain compound (4);

[0051] Step S2: reacting compound (4) and acryloyl chloride in a solvent to obtain compound (1);

[0052] wherein:

[0053] The structural formula of compound (2) is:

[0054]

[0055] The structural formula of compound (3) is:

[0056]

[0057] The structural formula of compound (4) is:

[0058]

[0059] The structural formula of compound (1) is:

[0060]

[0061] wherein, compound (2) and compound (3) are disclosed compounds, which can be obtained by prior art.

[0062] In an embodiment, in step S1, the solvent is ethanol.

[0063] In an embodiment, in step S1, the catalyst for refluxing reaction is piperidine.

[0064] In an embodiment, in step S1, the molar ratio of compound (2), compound (3) and piperidine is 1:1.5:0.5.

[0065] In an embodiment, in step S1, the temperature for refluxing reaction is 75-80℃.

[0066] In an embodiment, in step S2, the solvent is dichloromethane.

[0067] In an embodiment, in step S2, the catalyst for reaction is triethylamine.

[0068] In an embodiment, in step S2, the molar ratio of compound (4), triethylamine and acryloyl chloride is 1:3:2.

[0069] In an embodiment, in step S2, the temperature for reaction is -5-30℃.

[0070] In another embodiment, in the step S2, the reaction is first carried out at a low temperature of -5°C to 5°C to avoid too violent reaction at the beginning of the reaction stage, and then the reaction is carried out at 10-30°C to promote the reaction and increase the reaction rate.

[0071] In a third aspect, the present application provides use of the above-mentioned fluorescent probe in imaging of a cancer biomarker.

[0072] In a fourth aspect, the present application provides use of the above-mentioned fluorescent probe in preparation of a product based on photodynamic therapy of cancer.

[0073] The present application will be further described below with reference to the embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present application based on the description. In addition, the embodiments of the present application involved in the following description are generally only a part of the embodiments of the present application, not all the embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments in the present application without making creative efforts shall fall within the scope of protection of the present application.

[0074] Preparation of compound (4) in Example 1

[0075] Compound (3) (0.05 g, 0.168 mmol) and compound (2) (4-n-butyl-1,8-naphthalimide, 0.058 g, 0.244 mmol) were dissolved in ethanol, then 2 drops of piperidine (2 μL) were added, and the mixture was uniformly mixed to obtain a mixed solution. The mixed solution was stirred and refluxed at 78°C for 3 hours. Then, the solvent was removed by flash evaporation, and column chromatography was performed to obtain 0.053 g of compound (4) with a yield of 68%.

[0076] The obtained substance was subjected to nuclear magnetic analysis, and the results were as follows:

[0077] The nuclear magnetic hydrogen spectrum data were as follows: 1 H NMR (400 MHz, Chloroform-d): δ 8.63 (d, J = 7.9 Hz, 1H), 8.52 (d, J = 3.4 Hz, 1H), 8.36 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.51 (dd, J = 8.2, 1.8 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 7.10 (d, J = 10.3 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.42 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 10.3 Hz, 1H), 3.31 (ddt, J = 45.6, 14.8, 7.4 Hz, 2H), 1.80 (s, 6H), 1.52 (s, 2H), 1.13 (t, J = 7.1 Hz, 4H), 0.99 (dt, J = 7.1, 3.5 Hz, 6H).

[0078] NMR carbon spectrum data are as follows: 13 C NMR (151 MHz, Chloroform-d): δ 196.42, 165.70, 163.68, 163.02, 134.87, 134.10, 131.80, 130.32, 127.03, 122.89, 122.87, 115.18, 114.93, 40.31, 30.20, 29.70, 20.37, 13.84. HRMS (ESI) calculated for C30H30IN2O3+[M+H]+: 593.1296, found: 593.12957.

[0079] wherein the structural formula of compound (2) is:

[0080]

[0081] The structural formula of compound (3) is:

[0082]

[0083] The structural formula of compound (4) is:

[0084]

[0085] Preparation of compound (1) of Example 2

[0086] Compound (4) (0.04 g, 0.3 mmol) and triethylamine (130 μL, 0.9 mmol) were dissolved in dichloromethane solution (5 mL), and acryloyl chloride (50 μL, 0.6 mmol) was gradually added dropwise in an ice bath. After stirring for 30 min at 0°C, it was warmed to room temperature and stirred for another 30 min to obtain a reaction mixture. The reaction mixture was extracted with DCM, and the organic phase was washed with saturated sodium chloride solution (10 x 3 mL), dried over anhydrous Na2SO4, and purified using silica gel chromatography (E:P = 1:5) to obtain compound (1) (0.020 g, yield 76%).

[0087] The obtained substance was subjected to nuclear magnetic analysis, and the results were as follows:

[0088] NMR hydrogen spectrum data are as follows: 1H NMR (600MHz, Chloroform-d): δ8.61 (dd, J=7.3, 1.2Hz, 1H), 8.45 (s, 1H), 8.28 (dt, J=8.3, 1.4Hz, 1H), 7.89 (s, 1H) ,7.68–7.64(m,1H),7.54–7.50(m,1H),7.05(dd,J=17.0,10.5Hz,1H),6.48(dd,J=22.4,8.2Hz,2H),6.41(dd,J=1 7.0,1.5Hz,1H),5.93(d,J=1.4Hz,1H),4.20–4.17(m,2H),3.79(dd,J=10.2,6.8Hz,1H),3.53(dt,J=14.4,7.3Hz, 1H),3.38–3.33(m,1H),1.70(dd,J=8.1,2.3Hz,2H),1.49–1.45(m,2H),1.28–1.24(m,9H),0.99(d,J=7.2Hz,3H).

[0089] The NMR carbon spectrum data are as follows: 13 C NMR (151MHz, Chloroform-d): δ188.18,164.05,163.51,157.05,146.16,138.15,138.09,1 36.56,133.24,132.89,131.81,130.85,130.53,130.30,130.18,128.68,127.34,126.88, 122.95,121.53,115.18,113.17,111.77,109.06,108.84,79.61,54.87,50.84,40.21,40. 06,39.40,30.27,30.24,26.28,22.52,21.77,20.38,13.84,12.79.HRMS(ESI):Calculated for C33H32IN2O4+:[M+H]+647.1401,found:647.14014.

[0090] Wherein, the structural formula of compound (4) is as follows:

[0091]

[0092] The structural formula of compound (1) is as follows:

[0093]

[0094] Example 3

[0095] Weigh the compound (1) prepared in Example 2 and prepare it into a probe stock solution with a concentration of 1 mM using dimethyl sulfoxide. Draw 2 μL of the probe stock solution and add it to 398 μL of PBS buffer, then add Cys, mix well, transfer to a 96-well plate, and measure its absorbance using an ultraviolet spectrophotometer to determine the fluorescence absorption spectrum of compound (1), where the excitation wavelength is 580 nm and the emission wavelength is 675 nm. The fluorescence spectrum results are shown in Figure 1 .

[0096] The fluorescence absorption spectrum results showed that after incubation with Cys, the fluorescence intensity of compound (1) at 675 nm was enhanced, indicating that the Cys response group of the probe was removed by the reaction and the fluorescence of compound (1) was released.

[0097] Example 4

[0098] 2 μL of the probe stock solution prepared in Example 3 was added to 398 μL PBS buffer, and a total of 16 groups were prepared. Then, 100 μM of Cys, Hcy, Ala, Arg, Asp, Lys, Met, GSH, Phe, Ser, Trp, Val, Tyr, His, Gly, and blank were added respectively. Blank means adding an equal amount of PBS buffer. Then, the fluorescence intensity of each test system was detected by ultraviolet spectrophotometer, wherein the excitation wavelength was 580 nm and the emission wavelength was 675 nm. The results are as follows: Figure 2 Among them, Hcy, Ala, Arg, Asp, Lys, Met, GSH, Phe, Ser, Trp, Val, Tyr, His, and Gly were used as control small molecules, and blank was used as a blank control.

[0099] From the fluorescence detection results and Figure 2 It can be seen that glutathione has a slight fluorescence response, and the probe fluorescence intensity does not fluctuate significantly when reacting with homocysteine. Other amino acids cannot react with compound (1). When Cys is used as a detector, the fluorescence intensity at 675nm increases significantly, and the fluorescence intensity is close to 150, which is 60 times higher than that of the blank control group, indicating that compound (1) has a high selectivity for cysteine.

[0100] Example 5

[0101] (1) After the reaction of compound (1) with cysteine, compound (1) is converted into compound (4). Compound (4) prepared in Example 1 is weighed and prepared into a probe stock solution of compound (4) with a concentration of 1 mM using dimethyl sulfoxide.

[0102] (2) 4 μL of singlet oxygen indicator ABDA stock solution (1 mM, ABDA dissolved in dimethyl sulfoxide) was taken by a pipette and added to 395 μL of PBS (1% DMSO) to obtain an indicator reaction solution; 1 μL of probe stock solution of compound (4) was taken and added to 395 μL of PBS (1% DMSO), and then mixed with the indicator reaction solution. After 0.5 h of reaction at 37 °C, the mixture was added to a 96-well plate, and the UV absorption spectrum of compound (4) was determined by a fluorescence plate reader. Then, the mixture was irradiated by an incandescent lamp (wavelength of 400 nm-780 nm, 250 V, 4 A, 20 mW / cm 2 ) for 1.5 min, and then the UV absorption spectrum of compound (4) was determined. The above process was repeated every 1 min, and the total irradiation time was 450 s. The results are shown in Figure 3 . This step can be used to study the photodynamic properties of activated compound (1).

[0103] As shown in Figure 3 , with the increase of irradiation time, the absorption value gradually decreased, and the absorption value at 378 nm decreased from 0.83 to 0.33, which was relatively obvious. This laid a foundation for photodynamic therapy of tumor cells. (3) The photodynamic properties of compound (1) were measured by the same method as in step (2), which represented the photodynamic properties of unactivated compound (1). The probe stock solution of compound (1) was prepared according to the method of Example 3. The photodynamic properties of compound (1) are shown in Figure 4 . The results showed that the absorption value decreased by 0.4 after 2100 s of irradiation. It can be seen that with the increase of irradiation time, the change of absorption value was not obvious. This indicated that compound (1) did not have photodynamic therapy properties, and only after activation of Cys, the photodynamic therapy properties were turned on.

[0104] Example 6

[0105] Compound (1) was used to study the endogenous and exogenous production of cysteine in HeLa cells (cervical cancer cells), and the specific operation steps were as follows:

[0106] Compound (1) was incubated with HeLa for 30 min, and then confocal imaging experiment was performed. NEM is a thiol blocking agent, which can remove Cys. Compound (1), NEM, and Cys were added to HeLa and incubated for 30 min, and then confocal imaging experiment was performed. The results of the above confocal imaging experiments are shown in Figure 5 .

[0107] From the confocal imaging results, it can be seen that the cell fluorescence intensity of only compound (1) is the strongest. This is because Cys is overexpressed in HeLa cells, and Cys is cyclized back to hydroxyl on the acrylate of compound (1). NEM removes the endogenous cysteine in HeLa cells, and the cell map is basically without red fluorescence after adding NEM. NEM removes the endogenous cysteine in HeLa cells compound (1), but the cell imaging map of the cells incubated with Cys at the same time introduces exogenous Cys, and the fluorescence is obviously increased. Figure 7 The normalized fluorescence map of the lower graph of Figure 7 can visually see the fluorescence change, which fully illustrates that compound (1) can detect the endogenous and exogenous cysteine in HeLa cells.

[0108] Example 7

[0109] (1) About 1 x 10 5 A549 cells were inoculated in a confocal culture dish (20 mm) with 1 mL of culture solution at 37°C. The cells were allowed to adhere to the culture dish for 24 h before adding compound (1). After incubating A549 cells with compound (1) (2.5 μM) at 37°C under light for 30 min, washing with fresh culture solution, and confocal imaging; at the same time, another A549 cells were incubated with compound (1) (2.5 μM) at 37°C in the dark for 30 min, washed with fresh culture solution, and confocal imaging; at the same time, another A549 cells were incubated at 37°C under light for 30 min, washed with fresh culture solution, and confocal imaging. The confocal imaging results are shown in Figure 6 , wherein, Fig. a1-a3 are the imaging results of A549 cells after incubation under light (lighting under white light, wavelength 400 nm-780 nm, 16 mW / cm 2 Fig. b1-b3 are the imaging results of A549 cells after incubation with compound (1) in the dark, and Fig. c1-c3 are the imaging results of A549 cells after incubation with compound (1) under light (lighting under white light, wavelength 400 nm-780 nm, 16 mW / cm 2 .

[0110] As Figure 6 can be seen, Fig. a1-a3 has no fluorescence, Fig. b1-b3 can see the morphology of living cells by fluorescence, and Fig. c1-c3 can see that the morphology of A549 cells has changed significantly from long spindle to round, which fully proves the apoptosis of cells.

[0111] (2) In order to further accurately determine the effect of PDT, the confocal imaging cells of step (1) are imaged again by Annexin V-PI double staining method (Annexin V is used in combination with PI, which can distinguish early apoptotic cells, late apoptotic cells and dead cells), and the results are shown in Figure 7 .

[0112] Annexin V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine (PS). It can specifically recognize the PS on the surface of apoptotic cells, so FITC-conjugated Annexin V can be used to distinguish apoptotic cells and living cells. PI (propidium iodide) dye can bind to DNA in cells. Because the cell membranes of apoptotic cells and living cells are still intact, PI dye cannot freely pass through the cell membrane to bind to DNA in cells. Therefore, PI dye cannot label apoptotic cells and living cells, but only dead cells. Annexin V is used in combination with PI, which can distinguish early apoptotic cells, late apoptotic cells and dead cells. By Figure 7 The change in color can clearly show that the cells have died, further confirming that compound (1) has good photodynamic therapy effect.

[0113] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0114] The above is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment according to the technical essence of the present application still falls within the protection scope of the technical solution of the present application.

Claims

1. A cysteine-activated fluorescent probe, characterized in that: The structure is shown in formula (1): 。 2. A method for preparing a cysteine-activated fluorescent probe, characterized in that: The following steps are involved: Step S1: dissolving compound (2) and compound (3) in a solvent and subjecting the mixture to reflux reaction to obtain compound (4); Step S2: dissolving compound (4) and acryloyl chloride in a solvent and reacting to obtain compound (1); in: The structural formula of compound (2) is: ； The structural formula of compound (3) is: ； The structural formula of compound (4) is: ； The structural formula of compound (1) is: 。 3. The preparation method according to claim 2, wherein: In step S1, the solvent is ethanol.

4. The preparation method according to claim 2, wherein: In step S1, the catalyst for the reflux reaction is piperidine.

5. The preparation method according to claim 2, wherein: In step S1, the temperature of the reflux reaction is 75° C. to 80° C.

6. The preparation method according to claim 2, wherein: In the step S2, the solvent is dichloromethane.

7. The preparation method according to claim 2, wherein: In step S2, the catalyst for the reaction is triethylamine.

8. The preparation method according to claim 2, wherein: In step S2, the reaction temperature is -5°C to 30°C.

9. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 8 in the preparation of a cancer biomarker imaging agent.

10. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 8 in preparing products based on photodynamic therapy for cancer.

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