CN111635414B - Rhodol-HBT derivatives and their preparation methods and applications - Google Patents

Abstract

The present invention discloses Rhodol derivatives represented by the general structural formula (I). The present invention introduces benzothiazole into the Rhodol structure, and develops a new type of Rhodol-HBT dye, which has multiple excellent properties of two fluorophores, Rhodol and HBT, and provides two easily modified active sites. The compound of the present invention can be used as a fluorescent probe for the logical detection of thiophenol and hypochlorous acid. With the help of fluorescence confocal microscope, the visualization of the oxidative stress process in cells, zebrafish and nematodes has been successfully realized. Dynamic assessment of senescence at the cellular and nematode levels was also achieved.

Description

Rhodol-HBT derivatives and their preparation methods and applications

technical field

The invention relates to Rhodol-HBT derivatives, a preparation method and application thereof, and belongs to the technical field of chemical biological sensors.

Background technique

Aging is a time-dependent physiological decline that affects most organisms and is the most serious risk factor for many non-communicable diseases, including osteoarthritis, atherosclerosis, Alzheimer's disease, and cancer. Therefore, the detection and evaluation of aging is helpful to the research on the mechanism of aging and aging-related diseases. In 1956, Dr. Harman first proposed the free radical theory, which holds that free radical-induced cellular damage is the cause of aging. Subsequently, the free radical hypothesis was fused with the oxidative stress hypothesis, proposing that oxidative stress damage is the cause of inducing aging, and it is believed that the level of reactive oxygen species (ROS) in the resting state is closely related to the degree of aging. However, recent studies have proposed a new concept "redox stress response capacity (RRC)", which refers to a dynamic response capacity of cells to oxidative stress, that is, the ability to generate reactive oxygen species and how much (Meng J. , Lv Z Y, Qiao X H, et al. Redox Biology, 2017, 11, 365). Simply put, cells respond to the threat of stressors by producing appropriate amounts of reactive oxygen species, which stimulate signaling pathways to ensure cell health. By comparing the ability of young and aged cells and worms to respond to oxidative stress, the study confirms that this dynamic decline is an essential feature of aging, emphasizing that the dynamic is more of a cellular representation of stress than static. and individual vitality. Most of the current research on assessing aging focuses on the detection of aging-related markers in a static state. Therefore, it is very important to develop an efficient and reliable tool based on aging-related RRC, which is very important for dynamic evaluation of aging and then to study the process of aging, but no similar work has been reported so far.

The concept of "oxidative stress" was first proposed by Sohal in 1990. Oxidative stress refers to the process of continuous accumulation of reactive oxygen species in cells of the body when the body is exposed to environmental stress or in a disease state. Under normal circumstances, physiological levels of reactive oxygen species play a positive role in the organism. However, when the organism is stimulated by harmful factors, it will cause a large amount of reactive oxygen species in the body, which will break the balance between the oxidative system and the antioxidant system. This in turn leads to cell damage, resulting in aging. It is well known that many environmental stresses affect cellular redox balance, leading to the overproduction of reactive oxygen species. Thiophenol is a highly reactive aromatic thiol compound that is widely used in the chemical industry such as polymers, pesticides and pharmaceuticals. At the same time, thiophenol is also a highly toxic environmental pollutant. Its odor is very unpleasant and it is very volatile and diffuse. People who have been exposed to it for a long time will not only have serious mental health problems, but also cause shortness of breath, muscle weakness, Liver and kidney dysfunction and central nervous system damage, paralysis, and even death. Studies have shown that trace amounts of thiophenol can cause oxidative stress in cells (Liu Q M, Li A Y, Li X K, et al. Sensors & Actuators B Chemical, 2019, 283, 820), and this stress phenomenon is called Oxidative stress stimulated by poisons induces the production of pathogenic hypochlorous acid during the stress process. Hypochlorous acid is an important reactive oxygen species, and its overexpression can cause oxidative stress damage to cells. However, due to the lack of reliable and effective tools to monitor the process of thiophenol-induced hypochlorous acid production as a chemical stressor, the mechanism remains unclear. Therefore, the visualization of thiophenol-mediated oxidative stress process is particularly important for the exploration and study of stress mechanisms, and further provides a simple and reliable tool for dynamic assessment of aging at the cellular and in vivo levels.

With the development of imaging technology, fluorescence imaging has become a powerful tool for the visualization and real-time detection of target substances due to its advantages of simple technique, fast signal transduction, high sensitivity, high spatiotemporal resolution, strong specificity, and non-invasiveness. In recent years, a series of specific small molecule fluorescent probes have been used for the recognition and detection of thiophenol or hypochlorous acid in organisms, although simply mixing two specific probes together may Signal discrimination detects two analytes, but this strategy is limited by various problems, such as: (1) the possibility of cross-talk between the fluorescence signals of the two probes; (2) the possibility of cross-talk between the two probes; The introduction of these probes may make this strategy have an invasive effect; (3) the two probes may have different localization and metabolism. Therefore, the development of a single-molecule bimodal fluorescent probe, which is ideal for the visualization of thiophenol-induced oxidative stress process and dynamic evaluation of aging, is also very challenging.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a new class of Rhodol derivatives (I) and a preparation method thereof.

Another object of the present invention is to use the ROSH screened from the above Rhodol derivatives (I) as a fluorescent probe for the logical detection of thiophenol and reactive oxygen species, as well as for the oxidative stress process in cells, zebrafish and nematodes Visual Imaging for Dynamic Assessment of Senescence in Cells and C. elegans.

Rhodol derivatives represented by general structural formula (I),

，

，

，

，或

； where R is selected from

,

,

,

,or

;

、

或

。 R' is selected from

,

or

.

The preparation method of the above-mentioned Rhodol derivatives (I), comprising the following steps:

(1) 2-[(2,3,6,7-tetrahydro-10-hydroxy- 1H , 5H -benzo[ij]quinoline-9-)acyl]benzoic acid and 2-(2,4 -Dihydroxyphenyl)-1,3-benzothiazole in the condition of methylsulfonic acid as solvent and catalyst, condensation reaction occurs to obtain compound ROB;

(2) Compound ROB and R-NH ₂ are reacted in alcohol solvent to obtain compound ROR;

，

，

，

，

； R-=

,

,

,

,

;

(3) Compound (I) is obtained by reacting compounds ROR and R'-F in DMF solvent with K ₂ CO ₃ as acid binding agent;

、

或

。 R' is selected from

,

or

.

In the above preparation method, the reaction temperature of step (1) is 80-95°C.

In the above preparation method, the alcohol solvent in step (2) is selected from methanol, ethanol and isopropanol.

In the above preparation method, the reaction temperature of step (2) is 65-85°C.

Use of compound (I) in the preparation of a reagent for the logical detection of thiophenol and hypochlorous acid.

Use of compound (I) in the preparation of imaging reagents for visualization of thiophenol-induced cellular and zebrafish oxidative stress processes.

Use of compound (I) in the preparation of reagents for dynamic assessment of cellular and nematode senescence.

In recent years, hypochlorous acid fluorescent probes based on different reaction types and thiophenol fluorescent probes based on different recognition groups have been widely reported. These reactions include: oxidative deoximation, oxidative unsaturated double bond, oxidative Chalcogenide reaction, oxidative dehydrogenation reaction, oxidative hydrazide reaction, etc. (Wang Yanbao, Zhao Baoxiang. Organic Chemistry, 2016, 36, 1539);

，

，

，

，或

。 like:

,

,

,

,or

.

The thiophenol probes are classified into ether bonds, sulfonamides and sulfonates according to the type of chemical reaction (Ge Wenqi, Sheng Yingying, Qiao Yadong, etc. Chemical Sensors, 2017, 37, 9);

、

或

；like:

,

or

;

;R’为 2, 4-二硝基苯醚基）。ROSH可作为荧光探针用于苯硫酚和次氯酸的逻辑检测，ROSH 检测苯硫酚生成ROCL（R-为

），荧光信号从无转变为绿色，随后ROCL识别次氯酸转化为 ROB，荧光信号从绿色转变为红色，出现两种不同的荧光信号的转变，然而当识别待测物顺 序相反时，ROSH对两种待测物的检测过程仅出现一种荧光信号的变化。探针ROSH借助荧光 共聚焦显微镜，成功实现了ROSH对细胞、斑马鱼、线虫内氧化应激过程的可视化成像。另外， 利用该探针进一步实现了对细胞与线虫水平上衰老的动态评价。 Rhodol fluorophore, due to its mixed structure of half-rhodamine and half-fluorescein, exhibits excellent fluorescence properties such as good photostability, high fluorescence quantum yield and long fluorescence wavelength. It is often used in the field of analysis and detection for "switch" type fluorescence Probe design. In addition, the HBT fluorophore with ESIPT property has a large Stokes shift, so there is no or very little overlap between the excitation and emission spectra, which can greatly improve the sensitivity of fluorescent compounds. The invention combines the ESIPT principle with the switching mechanism, introduces benzothiazole into the Rhodol structure, and develops a new type of Rhodol-HBT dye ROB. The dye has multiple excellent properties of Rhodol and HBT fluorophores, and provides two an easily modified active site. Taking compound ROB as a fluorophore, a hypochlorous acid site based on oxidative hydrazide reaction was introduced into it to synthesize compound ROR, and a thiophenol recognition group of ether bond was further introduced to construct a class of compound (I). In addition, the inventors screened out a highly selective and highly sensitive dual-mode fluorescent probe ROSH (R- is

; R' is 2,4-dinitrophenyl ether group). ROSH can be used as a fluorescent probe for the logical detection of thiophenol and hypochlorous acid, ROSH detects thiophenol to generate ROCL (R- is

), the fluorescence signal changes from none to green, and then ROCL recognizes hypochlorous acid and converts it to ROB, and the fluorescence signal changes from green to red, and two different fluorescence signal transitions appear. During the detection process of the two analytes, only one fluorescence signal change occurred. The probe ROSH successfully visualized the oxidative stress process in cells, zebrafish and nematodes by means of fluorescence confocal microscopy. In addition, the dynamic evaluation of senescence at the cellular and nematode levels was further realized using this probe.

Description of drawings

Fig. 1 is the crystal structure diagram of compound ROSH;

Figure 2 is the fluorescence spectrum of the fluorescent probe ROSH in buffer solution to different concentrations of thiophenol;

Fig. 3 is the working curve diagram of fluorescent probe ROSH in buffer solution to different concentrations of thiophenol;

Fig. 4 is the fluorescence spectrum of the fluorescent probe ROCL to different concentrations of hypochlorous acid in buffer solution;

Fig. 5 is the working curve diagram of fluorescent probe ROCL to different concentrations of hypochlorous acid in buffer solution;

Figure 6 is the selective fluorescence spectrum of the fluorescent probe ROSH in the buffer solution of thiophenol in common anions and cations, biological thiols and reactive oxygen species; Note: 1. Blank, 2 iron ions 3 aluminum ions 4 zinc ions 5 Magnesium ion 6 Bisulfite 7 Bisulfate 8 Thiosulfate 9 Thiocyanate 10 Nitrite 11 Nitrate 12 Bicarbonate 13 Carbonate 14 Fluoride 15 Chloride 16 Bromide 17 Iodide 18 Cysteine Acid 19 Homocysteine 20 Glutathione 21 Hydrogen peroxide 22 Hydroxyl radical 23 Superoxide anion 24 Singlet oxygen 25 Nitric oxide radical 26 Peroxy tert-butanol radical 27 Peroxy tert-butanol 28 Peroxygen Nitrous acid 29 hypochlorous acid 30 thiophenol;

Figure 7 shows the selective fluorescence spectrum of the fluorescent probe ROCL in the buffer solution to hypochlorous acid in common anions and cations, biological thiols, and reactive oxygen species; Note: 1. Blank, 2 iron ions 3 aluminum ions 4 zinc ions 5 Magnesium ion 6 Bisulfite 7 Bisulfate 8 Thiosulfate 9 Thiocyanate 10 Nitrite 11 Nitrate 12 Bicarbonate 13 Carbonate 14 Fluoride 15 Chloride 16 Bromide 17 Iodide 18 Cysteine Acid 19 Homocysteine 20 Glutathione 21 Hydrogen peroxide 22 Hydroxyl radical 23 Superoxide anion 24 Singlet oxygen 25 Nitric oxide radical 26 Peroxy tert-butanol radical 27 Peroxy tert-butanol 28 Peroxygen Nitrous acid 29 hypochlorous acid 30 thiophenol;

Figure 8 is a fluorescence imaging image of the fluorescent probe ROSH in the process of thiophenol-induced oxidative stress in zebrafish;

Figure 9 is a fluorescence imaging diagram of the dynamic evaluation of senescence by the fluorescent probe ROSH in cells;

Figure 10 is a fluorescent imaging image of the dynamic assessment of senescence in C. elegans by the fluorescent probe ROSH.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used can be obtained from commercial sources unless otherwise specified.

Example 1 Preparation and application of fluorescent probe ROSH

(1) Accurately weigh 2.18 g (20 mmol) of the compound m-aminophenol and 4.24 g (40 mmol) of anhydrous sodium carbonate, and accurately weigh 10 mL (100 mmol) of 1-bromo-3 chloropropane into a 50 mL round bottom 15 mL of N,N-dimethylformamide was added to the flask, and under argon protection, the reaction was heated to 80 °C and stirred for 15 h, and the color of the system changed from pale yellow to dark green. After the reaction was completed, the reaction system cooled to room temperature was poured into an appropriate amount of distilled water, the system was extracted several times with dichloromethane as a reagent, the organic phase was recovered and dried by adding anhydrous sodium sulfate for 0.5 h, and the solvent was removed after filtration to obtain a crude product. The compound was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate: 15/1, V/V), and finally 2.62 g of an off-white solid was obtained, which was 8-hydroxyjulonidine, and the yield was 69 %. ¹ H NMR (400 MHz, CDCl ₃ ) δ: 6.67 (d, J = 8.0 Hz, 1H), 6.07 (d, J = 8.0 Hz, 1H), 4.58 (s, 1H), 3.10 (q, J = 5.2 Hz, 4H), 2.69 (dt, J = 15.4, 6.6 Hz, 4H), 2.18 - 1.88 (m, 4H). HRMS (ESI) m/zcalcd for C ₁₂ H ₁₆ NO [M+H] ⁺ : 190.1226, found: 190.1232.

(2) Accurately weigh 2.84 g (15 mmol) of 8-hydroxyjulonidine and 2.96 g (20 mmol) of phthalic anhydride into a 100 mL round-bottomed flask, and then add 30 mL of toluene to the system, under argon The mixture was stirred and refluxed for 24 h under gas protection. After the reaction was completed and cooled to room temperature, the solvent was removed, and the crude product was purified by column chromatography (eluent: methanol/dichloromethane: 1/40, V/V) to finally obtain 2.33 g of a pale yellow solid, which was 2 -[(2,3,6,7-Tetrahydro-10-hydroxy- 1H , 5H -benzo[ij]quinoline-9-)acyl]benzoic acid in 46% yield. ¹ H NMR (600 MHz, CDCl ₃ ) δ: 12.81 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.61 (t, J = 7.0 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 7.4 Hz, 1H), 6.46 (s, 1H), 3.31 - 3.19 (m, 4H), 2.74 (t, J = 6.4 Hz, 2H), 2.48 (t, J = 6.2 Hz, 2H), 2.00 - 1.90 (m, 2H), 1.90 - 1.80 (m, 2H). HRMS(ESI) m/z calcd for C ₂₀ H ₂₀ NO ₄ [M+H] ⁺ : 338.1387, found : 338.1372.

(3) 1.38 g (10 mmol) of 2,4-dihydroxybenzaldehyde, 1.90 g (10 mmol) of sodium metabisulfite, and 1.07 mL (10 mmol) of o-aminothiophenol were added to a 100 mL round-bottomed flask, respectively. 30 mL of DMF was heated to 110 °C and stirred under argon for 2 h. After stopping the reaction and cooling to room temperature, the system was added dropwise to ice water, and a large amount of yellow solid was precipitated. After suction filtration and drying, 1.81 g of solid was obtained, which was 2-(2,4-dihydroxyphenyl)-1, 3-benzothiazole, 74% yield. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ: 11.68 (s, 1H), 10.17 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H) ), 7.91 (d, J = 9.1 Hz, 1H), 7.49(t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 6.46 (s, 2H). HRMS (ESI) m /zcalcd for C ₁₃ H ₁₀ NO ₂ S [M+H] ⁺ : 244.0435, found: 244.0427.

(4) 337 mg (1 mmol) of 2-[(2,3,6,7-tetrahydro-10-hydroxy- 1H , 5H -benzo[ij]quinoline-9-)acyl] Benzoic acid and 365 mg (1.5 mmol) of 2-(2,4-dihydroxyphenyl)-1,3-benzothiazole were added to a 25 mL round bottom flask containing 5 mL of methanesulfonic acid and charged with argon The reaction system was protected, heated to 90 °C for 24 h, and monitored by TLC. After the reaction is completed and cooled, the post-reaction system is poured into ice water, and a large amount of purple-red solid is precipitated, which is filtered with suction and washed with an appropriate amount of brine and ether for three times to obtain a purple-red crude product, which is further separated by column chromatography after drying. Purification (eluent: methanol/dichloromethane: 1/30, V/V), 305 mg of purple-red solid was finally obtained, which was compound ROB, and the yield was 56%. ¹ H NMR (400 MHz, DMSO-d ₆ + drop of CF ₃ COOD-d) δ: 8.30 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.98 - 7.89 ( m, 2H), 7.84 (dd, J = 15.5, 7.7 Hz, 2H), 7.61 (d, J = 7.3 Hz, 1H), 7.48 - 7.35 (m, 2H), 7.28 (s, 1H), 6.46 (s ,1H), 3.72 - 3.51 (m, 4H), 2.95 (s, 2H), 2.48 (s, 2H), 2.16 - 1.93 (m, 2H), 1.87 - 1.65 (m, 2H). HRMS (ESI) m /z calcd for C ₃₃ H ₂₅ N ₂ O ₄ S [M+H] ⁺ : 545.1530, found: 545.1509.

(5) 544 mg (1 mmol) of compound ROB and 20 mL of 80% hydrazine hydrate solution were added to a 100 mL round-bottomed flask containing 30 mL of absolute ethanol. Filled with argon protection, heated to the reaction reflux temperature, continued the reaction for 5 h, and monitored by TLC. The reaction was stopped, the temperature was lowered to room temperature, and the crude product was obtained after removing the solvent, which was further separated and purified by column chromatography (eluent: methanol/dichloromethane: 1/200, V/V) to obtain 236 mg of off-white solid, namely As compound ROCL, the yield was 42%. ¹ HNMR (400 MHz, CDCl ₃ ) δ: 12.73 (s, 1H), 8.11 - 7.94 (m, 1H), 7.89 (d, J = 8.1Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H) , 7.56 - 7.49 (m, 2H), 7.44 (t, J = 7.6 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.19 - 7.05 (m, 1H), 6.93 (d, J = 11.9 Hz,2H), 6.04 (s, 1H), 3.27 - 3.08 (m, 4H), 2.95 (t, J = 6.4 Hz, 2H), 2.65 - 2.36(m, 2H), 2.17 - 1.98 (m, 2H) , 1.97 - 1.74 (m, 2H). ¹³ C NMR (100 MHz, CDCL ₃ ) Δ: 168.41, 166.48, 159.15, 155.79, 151.68, 151.29, 148.29, 144.05, 133.03,132.33, 129.70, 127.85, 125.57, 123.81, 123.81, 123.81, 123.81, 123.81,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23.41,23 121.55, 118.37, 114.10, 111.62, 108.14, 104.92, 104.23, 65.83, 49.99, 49.53, 27.38, 21.90, 21.33, 21.25. HRMS (ESI) m/z calcd for _C33H27N4O3S [ _{M +} H] ⁺ : _559.1798 , found: 559.1781.

(6) 558 mg (1 mmol) of compound ROCL, 10 mL of DMF and 138 mg (1 mmol) of anhydrous potassium carbonate were sequentially added to a 50 mL round-bottomed flask, and finally 279 mg (1.5 mmol) was added to it. 2,4-dinitrofluorobenzene, the reaction was carried out under stirring at room temperature, monitored by TLC, after the reaction was completed, the reaction was stopped, cooled to normal temperature, the system was added dropwise to an appropriate amount of ice water, and a large amount of precipitation was precipitated, Suction filtration, rinsed with ice water several times in small amount, and finally obtained 410 mg of light yellow solid after drying, which is compound ROSH, and the yield is 56%. ¹ H NMR (400 MHz, CDCl ₃ ) δ: 8.92 (s, 1H), 8.05 - 7.96 (m, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.85 (s, 1H), 7.76 (d , J = 7.9Hz, 1H), 7.51 (dd, J = 5.3, 3.0 Hz, 2H), 7.41 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.15 - 7.06 (m, 3H), 6.08 (s, 1H), 3.15 (dd, J = 12.7, 5.7 Hz, 4H), 2.90 (t, J = 6.1 Hz, 2H), 2.67 - 2.41 (m, 2H), 2.03 ( d, J = 4.8 Hz, 2H), 1.90 (d, J = 4.9 Hz, 2H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ: 167.08, 160.02, 155.07,155.03, 152.55, 151.47, 151.14, 147.76, 143.91, 142.39, 139.96, 135.45,135.37, 133.27, 130.75, 129.28, 129.19, 129.00, 126.56, 125.57, 123.79,123.70, 123.41, 122.35, 122.00, 121.42, 119.43, 119.17, 119.13, 118.90,110.09, 65.97, 65.57, 50.04, 49.54, 27.76, 21.24, 15.40. HRMS (ESI) m/zcalcd for C ₃₉ H ₂₈ N ₆ NaO ₇ S [M+Na] ⁺ : 747.1632, found: 747.1629.

The present invention obtains the single crystal structure of compound ROSH (R- is -NH ₂ ; R' is 2, 4-dinitrophenyl ether group) through the ether diffusion method (Fig. 1).

Fluorescence spectrum test of probe ROSH to different concentrations of thiophenol: add 50 μL of ROSH (1 mM) probe stock solution, 0.5 mL DMF, 0.5 mL PBS buffer solution (0.1M, pH = 7.4), and then add 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 μL of thiophenol solution (10 mM) to it, and mix to the volume. The fluorescence spectrum is shown in Figure 2. As the concentration of thiophenol in the system increases, the fluorescence intensity of ROSH increases gradually at the wavelength of 534 nm.

Determination of the working curve of probe ROSH to different concentrations of thiophenol: From the fluorescence spectrum of ROSH to different concentrations of thiophenol, the working curve of the fluorescence intensity at 534 nm with the concentration of thiophenol was obtained. As shown in Figure 3, after thiophenol was continuously added to the system, the fluorescence intensity at 534 nm showed a good linear relationship with the concentration of thiophenol. The linear equation was Y = 21.18182 + 3.1314 X, and the correlation coefficient was 0.9997.

Fluorescence spectrum test of probe ROCL to different concentrations of hypochlorous acid: add 50 μL ROCL (1 mM) probe stock solution, 0.5 mL DMF, 0.5 mL PBS buffer solution (0.1 M, pH = 7.4 to a 5 mL colorimetric tube) ), and then add 0, 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 30, 40 μL of hypochlorous acid solution (1 mM) to it, and mix well after constant volume. The fluorescence spectrum is shown in Fig. 4. As the concentration of hypochlorous acid in the system increases, the fluorescence at 534 nm decreases continuously, and the fluorescence at 602 nm gradually increases.

Determination of the working curve of the probe ROCL to different concentrations of hypochlorous acid: From the fluorescence spectra of ROCL to different concentrations of hypochlorous acid, the working curve of the ratio of the fluorescence intensity at 602 nm to the fluorescence intensity at 534 nm with the concentration of hypochlorous acid was obtained. As shown in Figure 5, after continuously adding hypochlorous acid to the system, the ratio of the fluorescence intensity at 602 nm and the fluorescence intensity at 534 nm showed a good linear relationship with the concentration of hypochlorous acid, and the linear equation was Y = 0.14954 – 0.00761 X , the correlation coefficient is 0.9922.

Selective fluorescence spectrum test of probe ROSH to thiophenol in common anions and cations, biological thiols and reactive oxygen species: add 50 μL of ROSH probe stock solution (1 mM) and 0.5 mL of DMF to a 5 mL colorimetric tube , 0.5 mL PBS buffer solution (0.1M, pH =7.4), and then add 100 µM Fe ³⁺ , Al ³⁺ , Zn ²⁺ , Mg ²⁺ , HSO ₃ ^- , HSO ₄ ^- , S ₂ O ₃ to it respectively ^2- , SCN ^- , NO ₂ ^- , NO ₃ ^- , HCO ₃ ^- , CO ₃ ^2- , F ^- , Cl ^- , Br ^- , I ^- , Cys, Hcy, GSH, H ₂ O ₂ ^, OH, O ₂ ^•− , ¹ O ₂ , NO ^• , TBO ^• , TBHP, ONOO ⁻ , HOCl, PhSH, make up to volume and mix well. The fluorescence spectrum is shown in Figure 6. After mixing ROSH with thiophenol, the fluorescence intensity at the fluorescence wavelength of 534 nm increased significantly, while other reactive oxygen species, biological thiols, and common anions and cations did not cause significant changes in ROSH fluorescence signals. The above results indicated that the fluorescent probe ROSH exhibited good selectivity for the detection of thiophenol.

Selective fluorescence spectrum test of probe ROCL for hypochlorous acid in common anions and cations, biological thiols and reactive oxygen species: add 50 μL ROCL probe stock solution (1 mM), 0.5 mL DMF to a 5 mL colorimetric tube, 0.5 mL PBS buffer solution (0.1M, pH =7.4), and then add 40 µM Fe ³⁺ , Al ³⁺ , Zn ²⁺ , Mg ²⁺ , HSO ₃ ^- , HSO ₄ ^- , S ₂ O ₃ ² to it respectively ^- , SCN ^- , NO ₂ ^- , NO ₃ ^- , HCO ₃ ^- , CO ₃ ^2- , F ^- , Cl ^- , Br ^- , I ^- , Cys, Hcy, GSH, H ₂ O ₂ ^, OH, O ₂ ^•− , ¹ O ₂ , NO ^• , TBO ^• , TBHP, ONOO ⁻ , HOCl, PhSH, make up to volume and mix well. The fluorescence spectrum is shown in Figure 7. After ROCL is mixed with hypochlorous acid, the emission peak at 534 nm disappears, the emission peak appears at 602 nm and the fluorescence intensity increases significantly, while other reactive oxygen species, biological thiols and common anions and cations are No significant change in ROCL fluorescence signal was caused. The above results indicated that the fluorescent probe ROCL showed good selectivity for the detection of hypochlorous acid.

Fluorescence imaging experiment of probe ROSH in thiophenol-induced oxidative stress process in zebrafish: In this experiment, three-day zebrafish were used for stress imaging experiment. Control group: After incubating several zebrafish in 50 µM ROSH probe stock solution for 20 min, the zebrafish were anesthetized and washed with distilled water for several times for fluorescence imaging. Experimental group: Several zebrafish were placed in a solution containing 50 µM thiophenol, and incubated in multiple groups for 10, 30, 60, and 90 min, and then the same zebrafish in each group was placed in 50 µM ROSH probe stock solution After incubation for 20 min, fluorescence imaging was performed after anesthesia. The imaging results are shown in Figure 8. In the control zebrafish, there was no obvious fluorescence signal in the red and green channels, while in the thiophenol-stimulated zebrafish, the red and green channels showed obvious fluorescence signals at 10 min. The fluorescence signal can be clearly observed after 10 min, and the fluorescence in the red channel gradually increases along with the decrease of the fluorescence in the green channel. It can be seen from the superimposed field that with the passage of stress time, the zebrafish changed from reddish whole body and green abdomen to whole body reddening, showing the oxidative stress process induced by thiophenol in zebrafish, indicating that the probe ROSH It can be used to visualize the process of thiophenol-induced oxidative stress in zebrafish.

Probe ROSH in senescence and conventional cell models to evaluate the dynamic visualization of senescence: XL413 is a DNA replication kinase inhibitor, which can induce a variety of cells including HepG2 cells to enter the senescence state with high potency. In this experiment, HepG2 cells were selected as conventional cells, and HepG2 cells after induction with XL413 for five days were used as senescent cells for fluorescence imaging experiments. Control group: 30 µM ROSH probe stock solution was added to both conventional HepG2 cells and senescent HepG2 cells and incubated for 10 min. After washing with PBS buffer solution for several times, fluorescence imaging experiments were performed. Experimental group: In multiple groups of conventional HepG2 cells and senescent HepG2 cells, 30 µM thiophenol was added and incubated for 15, 30, 45, and 60 min, respectively, and then 30 µM ROSH probe stock solution was added to each group for further incubation for 10 minutes. min, rinsed with PBS buffer solution for several times for fluorescence imaging experiments. The imaging results are shown in Figure 9. In the control group, no obvious fluorescent signals were observed in the red and green channels of the cells of the conventional group and the cells of the senescence group. In the experimental group, the normal and senescent HepG2 cells showed the same change trend in the two channels, that is, the fluorescence signal in the green channel gradually weakened, and the fluorescence signal in the red channel gradually increased. However, it can be clearly observed in the superimposed field that the degree of trend change of the two groups of cells is different. Further, by comparing the line graphs of the ratios of the fluorescence quantitative ratios of the red and green channels in the two groups of cells over time, it can be seen that the conventional cell red The change trend of the ratio of green channel is larger than that of senescent cells, that is, the degree of oxidative stress induced by thiophenol in senescent cells is weaker than that of conventional cells, indicating that the probe ROSH can be used for dynamic evaluation of senescence in cells.

The dynamic visualization evaluation experiment of probe ROSH in aging and young nematode models: the life cycle of nematodes is about two weeks, so in this experiment, six-day-old nematodes and twelve-day-old nematodes were selected as the young group nematodes and the aging group, respectively nematodes for research. Control group: 20 μM ROSH probe stock solution was added to both the young group nematodes and the aging group nematodes and incubated for 60 min, and the fluorescence imaging experiments were performed after washing with PBS buffer solution for several times. Experimental group: In multiple groups of young group nematodes and aging group nematodes, 3.33 µM thiophenol was added and incubated for 15, 30, 60, and 90 min, respectively, and then 20 µM ROSH probe stock solution was added to each group for further incubation for 60 minutes. min, rinsed with PBS buffer solution for several times for fluorescence imaging experiments. The imaging results are shown in Figure 10. The fluorescence signals of the green channel of the two groups of nematodes have a tendency to weaken, while the fluorescence signals of the red channel have been significantly enhanced. With the increase of thiophenol stimulation time, the fluorescence color of nematodes in the young group changed from green to red, while the fluorescence color of nematodes in the aging group changed from yellow-green to red. Further quantitative analysis by fluorescence showed that the same as the results of the aging evaluation experiment in cells, the young nematodes The change of the fluorescence ratio of the red and green channels over time is greater than that of the aging nematodes, indicating that the young nematodes are more responsive to oxidative stress stimulated by thiophenol than the aging nematodes. Therefore, the probe ROSH can be used for dynamic evaluation of aging at the in vivo level. .

Claims (7)

1. Rhodol derivatives represented by general structural formula (I),

where R is selected from

,

,

,

,or

; R' is selected from

,

or

. 2. the structural formula of the described Rhodol derivatives of claim 1 is

. 3. the preparation method of the described Rhodol class derivative of claim 1, is characterized in that comprising the following steps: (1) 2-[(2,3,6,7-tetrahydro-10-hydroxy- 1H , 5H -benzo[ij]quinoline-9-)acyl]benzoic acid and 2-(2,4 -Dihydroxyphenyl)-1,3-benzothiazole in the condition of methanesulfonic acid as solvent and catalyst, condensation reaction occurs to obtain compound ROB;

(2) Compound ROB and R-NH ₂ are reacted in alcohol solvent to obtain compound ROR;

R-=

,

,

,

,

; (3) Compound (I) is obtained by reacting compounds ROR and R'-F in DMF solvent with K ₂ CO ₃ as acid binding agent;

R' is selected from

,

or

. 4 . The method for preparing Rhodol derivatives according to claim 3 , wherein the alcohol solvent in step (2) is selected from methanol, ethanol and isopropanol. 5 . 5. The application of the Rhodol derivatives of claim 1 in the preparation of fluorescent probes for the logical detection of thiophenol and hypochlorous acid. 6. The application of the Rhodol derivatives of claim 1 in the preparation of visualization imaging reagents for thiophenol-induced oxidative stress in cells and zebrafish. 7. The application of the Rhodol derivatives according to claim 1 in the preparation of a reagent for dynamic evaluation of cell and nematode senescence.

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