A bisindole compound of crotonate and its preparation method and application

Abstract

The invention belongs to the technical field of compounds, and provides a croconoic acid bisindole compound and a preparation method and application thereof. The croconoic acid bisindole compound with a specific structure provided by the invention contains a croconoic acid group, an indole group and a morpholine group in the structure. The croconoic acid group makes the compound structure more stable and also has longer ultraviolet absorption. The indole group gives the compound pH value response, and the morpholine group has a good lysosome targeting mechanism. The croconoic acid bisindole compound provided by the invention has the characteristics of lysosome targeting, pH value responsiveness, near-infrared-photoacoustic dual-modality imaging, and photothermal therapy, and has small toxic and side effects, and can be used for imaging and treatment research of tumors, especially breast cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of compounds, and more specifically to a bisindole crotonate compound and a preparation method and application thereof. Background Art

[0002] Early diagnosis of cancer is the key to improving the cure rate of cancer patients. It is very meaningful to study detection technologies with strong specificity and high sensitivity for early diagnosis of tumors. Optical imaging technology, especially near-infrared fluorescence imaging technology, is favored because its absorption wavelength and emission wavelength are both in the biological optical imaging window, and it has the advantages of strong tissue penetration, small light absorption and autofluorescence interference of biological tissues. At the same time, in the development and optimization of imaging technology, photoacoustic imaging (PAI) has become a powerful tool for preclinical biomedical diagnosis and cancer detection. PAI probes can improve contrast and help identify pathogenic tissues. Therefore, near-infrared and photoacoustic dual-modality imaging technology has more advantages in the field of early diagnostic imaging of tumor cells.

[0003] Cancer has always been a serious threat to human life and health, and traditional treatments, including surgery, chemotherapy, and radiotherapy, all have their shortcomings, so the superiority of new treatments such as photothermal therapy and photodynamic therapy is reflected. Common photothermal agents are divided into inorganic materials and organic materials. Inorganic photothermal agents are difficult to metabolize in the body and have poor biocompatibility. Organic small molecule photothermal agents have good stability, simple structure, adjustable optical properties, and good biocompatibility. They are considered to be ideal probes for treatment. Therefore, it is very meaningful to develop organic small molecules that can achieve near-infrared and photoacoustic dual-modal imaging and photothermal therapy of tumor cells for the preparation of more effective integrated diagnosis and treatment reagents.

[0004] On the other hand, the acidic microenvironment of tumors can be used as a target for targeted drug delivery. Appropriate pH plays an important role in cell growth and metabolism, while abnormal pH can cause cell disorders. The pH of intracellular lysosomes is between 4.6 and 5.5, and its abnormality may affect the normal growth of organisms and cause a series of related diseases. Therefore, real-time monitoring of lysosomal pH is of great significance for the diagnosis of lysosome-related biological processes and diseases. As a near-infrared dye, crotonate dye actually has dual properties. Under physiological conditions, the extracellular pH is about 7.4, which keeps its molecule in an alkaline structure; due to the nonspecific endocytic pathway, the absorption of the molecule is mainly through endocytosis, and the pH value in the lysosome will reach 5.5, which means that the acidic structure molecules under such conditions will have higher wavelength absorption in the cell. In addition, crotonate dye has strong resistance to photobleaching, good chemical stability and thermal stability, and is an ideal photothermal conversion agent. Therefore, it is of great significance to develop a probe that is pH-responsive, lysosome-targeted, and capable of combined near-infrared and photoacoustic imaging and photothermal therapy for tumors for tumor imaging and treatment research.

[0005] The bisindole compounds disclosed in the prior art, such as the compound of formula (IV) (Chinese patent CN105503831A) and the compound of formula (V) (ACS Sens.2021,6,2141-2146), has good pH response and near-infrared imaging efficacy, but there is no report on its lysosome targeting, photoacoustic imaging and photothermal treatment of tumors; Formula (VI) bisindole compound (Chinese patent CN105693590A) is a pH-sensitive photothermal agent, but there is no report on lysosome targeting and near-infrared-photoacoustic dual-modal imaging; Formula (VII) bisindole compound (CN111592482A) is a pH-responsive photothermal / photodynamic / fluorescence integrated reagent, but there is no report on lysosome targeting and photoacoustic imaging; Formula (VIII) bisindole compound (Chinese patent CN111592482A) is a pH-responsive photothermal / photodynamic / fluorescence integrated reagent, but there is no report on lysosome targeting and photoacoustic imaging. The prior art discloses micelles of bisindole oxadiazine compounds, whose structural formula is shown in formula (V) (Adv. Healthcare Mater. 2021, 10, 2002115), which can be used for photoacoustic imaging and photothermal therapy, but there is no report on pH response, lysosomal targeting and near-infrared imaging. The micelles of bisindole oxadiazine compounds, whose structure is shown in formula (IX) (Biomaterials 2021, 267, 120454), can be used for pH response, near-infrared-photoacoustic imaging and photothermal therapy, but have no lysosomal targeting effect. Long-chain polyether compounds are introduced into the above two micelles of bisindole oxadiazine compounds to increase their aggregation concentration in tumor cells, but the large-scale introduction of exogenous substances such as polyethers may cause some unknown toxic side effects.

[0006]

[0007]

[0008] Therefore, there is an urgent need to develop an integrated tumor diagnosis and treatment preparation that has pH responsiveness, lysosome targeting, near-infrared-photoacoustic dual-modality imaging, photothermal therapy, and low toxicity and side effects. Summary of the invention

[0009] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a bisindole compound of crotonate and its preparation method and application. The present invention provides an organic crotonate photothermal small molecule, which is a simple organic small molecule compound with a high aggregation concentration in cells. It has the pH response characteristics of targeting lysosomes, and can recognize tumor cell lysosomes by responding to the cell microenvironment. It can also realize near-infrared-photoacoustic dual-modal imaging. Under near-infrared light, it can also perform photothermal treatment on tumors, especially breast cancer cells, which is of great significance for the research on the development of integrated tumor diagnosis and treatment preparations with less toxic side effects.

[0010] The first aspect of the present invention provides a bisindole compound of crotonate.

[0011] Specifically, a bisindole compound of cremonate has a molecular structure shown in the following formula (I):

[0012]

[0013] The croconoic acid bisindole compound provided by the present invention is a croconoic acid cyanine dye. The compound structure of formula (I) includes a croconoic acid part, an indole part and a morpholine group. The croconoic acid part, as a strong electron-withdrawing group, forms a longer conjugated system with the electron-donating groups on both sides, has longer ultraviolet absorption, and the long-wavelength light has stronger penetration during the treatment process; and the conjugated unit of the central ring system of the croconoic acid small molecule also increases the rigidity and steric hindrance of the molecule, which is not conducive to the attack of singlet oxygen, so that the croconoic acid cyanine dye of the present invention is more Stable; the morpholine group has a photoinduced electron (PET) fluorescence quenching mechanism, and the dye containing the morpholine group can detect the pH value well, and the morpholine group has a good lysosome targeting mechanism, so that the dye can effectively detect the pH value in the cell lysosome; the indole part can be used for pH value response; because the ultraviolet and fluorescence spectra of the bisindole compounds of crotonate are located in the near-infrared region, they can also be used for near-infrared and photoacoustic dual-modal imaging; crotonate bisindole compounds also have a good photothermal conversion effect, which can be used for photothermal therapy research of tumors. The organic small molecule provided by the present invention links the morpholine ring and the indole group on the crotonate dye, and has the characteristics of pH value response, lysosome targeting, combined near-infrared-photoacoustic dual-modal imaging and photothermal therapy of tumors, which is very meaningful for tumor imaging and treatment research.

[0014] The second aspect of the present invention provides a method for preparing bisindole compounds of crotonate.

[0015] A method for preparing a bisindole compound of crotonate comprises the following steps:

[0016] The compound of formula (II) and the compound of formula (III) are mixed and dispersed in a solvent, and a Knoevenagel condensation reaction is carried out in an inert atmosphere to obtain the bisindole compound of crotonate;

[0017] The compound of formula (II) has the following molecular structure:

[0018]

[0019] The compound of formula (III) has the following molecular structure:

[0020]

[0021]

[0022] Preferably, the reaction temperature of the Knoevenagel condensation reaction is 80-130° C., and the reaction time is 6-12 h.

[0023] Preferably, the molar ratio of the compound of formula (II) to the compound of formula (III) is 1:0.5-2.

[0024] Preferably, the solvent is toluene and / or n-butanol.

[0025] More preferably, the solvent is a mixed solution of toluene and n-butanol.

[0026] Preferably, in the mixed solution of toluene and n-butanol, the volume ratio of toluene to n-butanol is 1:1-3.

[0027] Preferably, after the Knoevenagel condensation reaction occurs, a purification step is also included.

[0028] Preferably, the purification step comprises:

[0029] The reaction solution solvent is removed, the solid matter is subjected to silica gel column chromatography, eluted with an organic solvent, the eluate is collected, and the organic solvent in the eluate is removed to obtain the bisindole crotonate compound.

[0030] Preferably, the organic solvent is dichloromethane and / or methanol.

[0031] More preferably, the organic solvent is a mixed solvent of dichloromethane and methanol.

[0032] Further preferably, the organic solvent is a mixed solvent of dichloromethane and methanol in a volume ratio of 200:1-15.

[0033] Preferably, the collected eluent is an eluent having a volume ratio of dichloromethane to methanol of 200:7.

[0034] Preferably, the inert atmosphere is one or more of nitrogen, argon and helium.

[0035] The third aspect of the present invention provides an application of bisindole compounds of crotonate.

[0036] Application of a bisindole compound of crotonate in the preparation of a lysosome preparation targeted to cells.

[0037] A use of a bisindole crotonate compound in the preparation of a preparation responsive to the pH value of tumor cells.

[0038] Application of a bisindole crotonate compound in the preparation of a near-infrared-photoacoustic dual-modality imaging preparation for tumor cells.

[0039] A use of a bisindole crotonate compound in the preparation of a photothermal therapy preparation for tumor cells.

[0040] A pH-responsive lysosome-targeted near-infrared-photoacoustic dual-modality imaging and photothermal therapy integrated diagnostic and therapeutic agent, comprising the crotonate bisindole compound.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) The present invention provides a bis-indole crotonate compound with a specific structure, which contains a crotonate group, an indole group, and a morpholine group. The crotonate group makes the compound structure more stable and has longer ultraviolet absorption. The indole group gives the compound pH response. The morpholine group has a good lysosome targeting mechanism. The crotonate bis-indole compound provided by the present invention has the characteristics of lysosome targeting, pH responsiveness, near-infrared-photoacoustic dual-modality imaging, and photothermal therapy, and has low toxic and side effects. It can be used for imaging and treatment research of tumors, especially breast cancer cells;

[0043] (2) The bisindole compounds of the present invention can be used to prepare pH-responsive, lysosome-targeted, near-infrared-photoacoustic dual-modality imaging and photothermal therapy integrated diagnostic and therapeutic agents, which can be used in the diagnosis and treatment of tumors to improve the cure rate of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The ultraviolet absorption spectra of the bisindole crotonate compound prepared in Example 1 of the present invention at different pH values;

[0045] Figure 2 The fluorescence spectra of the bisindole crotonate compound prepared in Example 1 of the present invention at different pH values;

[0046] Figure 3 This is a photoacoustic intensity variation diagram of the bisindole crotonate compound prepared in Example 1 of the present invention under different pH conditions;

[0047] Figure 4 This is a photothermal performance diagram of the bisindole crotonate compound prepared in Example 1 of the present invention;

[0048] Figure 5 This is a diagram showing the endocytosis and near-infrared imaging of the bisindole crotonate compound prepared in Example 1 of the present invention in 4T1 cells;

[0049] Figure 6 This is an imaging diagram of the targeting of lysosomal cells of 4T1 tumor cells by the bisindole compounds of crezolic acid prepared in Example 1 of the present invention;

[0050] Figure 7 Cell imaging diagrams of 4T1 tumor cells with different pH values ​​at different excitation wavelengths by the bisindole compounds of crotonate prepared in Example 1 of the present invention;

[0051] Figure 8 This is a photoacoustic imaging image of 4T1 tumor cells by the bisindole compounds of crotonate prepared in Example 1 of the present invention;

[0052] Fig. 9 This is a photothermal treatment diagram of 4T1 tumor cells under non-illumination or illumination with the bisindole crotonate compound prepared in Example 1 of the present invention at different concentrations and different pH values. DETAILED DESCRIPTION

[0053] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0054] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0055] Example 1

[0056] A bisindole cremonate compound having a molecular structure shown in the following formula (I):

[0057]

[0058] The preparation method of the above-mentioned bisindole crotonate compound comprises the following steps:

[0059] 1. Add 4-hydrazinobenzoic acid and 3-methyl-2-butanone in a molar ratio of 1:3 into a round-bottom flask, and add glacial acetic acid (AcOH) as a solvent. Heat and reflux at 110°C for 12 hours, then cool to room temperature, and remove the solvent under reduced pressure; the crude product is further purified by column chromatography (volume ratio of dichloromethane: methanol = 40:1) to obtain a pure gray-yellow solid compound 2,3,3-trimethyl-3H-indole-5-carboxylic acid. Under stirring conditions, the compound 2,3,3-trimethyl-3H-indole-5-carboxylic acid (1 eq) was dissolved in cold N,N-dimethylformamide (DMF), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1.08 eq) and N,N-diisopropylethylamine (DIPEA) (2.2 eq) were added in sequence. After stirring for 30 minutes, 4-(2-aminoethyl)morpholine (2.2 eq) was added and stirred for 24 hours. After the reaction was completed, cold distilled water was added, and the mixture was extracted with dichloromethane (DCM). The organic layer was collected and dried with an appropriate amount of anhydrous sodium sulfate. After concentration, the crude product was further purified by column chromatography (volume ratio of dichloromethane: methanol = 50:3) to obtain 2,3,3-trimethyl-5-[(2-morpholinoethyl)carbamoyl]-2H-indole (compound of formula (II));

[0060] 2. Add 1,1,2-trimethyl-1H-benzo[e]indole and ketoic acid in a molar ratio of 1:1 into a round-bottom flask, add a mixed solvent of acetone:water in a volume ratio of 1:1, stir and react at 50°C, and monitor the reaction by thin layer chromatography (TLC); after the reaction is completed, separate through a column to obtain a product in purple color; the elution solvent is dichloromethane:methanol in a volume ratio of 50:2 to obtain the product 5-[(1,1-dimethyl-1H-benzo[e]indole-3-2-)methylene]-2-hydroxy-3,4-dioxolane-1-enolate (compound of formula (III));

[0061] 3. Weigh 76.6 mg of the above-prepared 5-[(1,1-dimethyl-1H-benzo[e]indole-3-2-)methylene]-2-hydroxy-3,4-dioxolane-1-enolate (compound of formula (III), 0.23 mmol) and 84 mg of 2,3,3-Trimethyl-5-[(2-morpholinethyl)carbamoyl]-2H-indole (compound of formula (II), 0.26mmol) was added to a 25mL three-necked flask, 4mL toluene and 8mL n-butanol were added, a condenser was connected, nitrogen was passed, and the mixture was heated under reflux and stirred at 110°C for 5h. The color of the solution changed from yellow to dark brown. After the reaction was completed, the heating was stopped, the solution was cooled to room temperature, and the solvent was dried under reduced pressure. The obtained solid was eluted with a mixed solvent of dichloromethane and methanol (dichloromethane: methanol volume ratio = 200:7) as an eluent and 200-300 mesh silica gel as a stationary phase. After column chromatography elution, separation and purification, a dark brown solid was obtained, and a ketoacid bisindole compound having a molecular structure of formula (I) was obtained, which is a ketoacid cyanine dye and named CR-630 (39mg, 27%).

[0062] The structure of compound CR-630 was identified, and the specific data are as follows:

[0063] H NMR 1 H NMR (DMSO, 400MHz): δ8.37(s,1H),8.23(d,J=7.6Hz,1H),8.08(d,J=6.4Hz,1H),7 .96(s,1H),7.89(br,1H),7.83(d,J=8.4Hz,1H),7.79(d,J=8.0Hz,1H),7.67(t,J =7.2Hz,1H),7.55(t,J=7.2Hz,1H),7.45(dd,J=8.0Hz,6.4Hz,1H),6.28(s,1H),5 .86(s,1H),3.60(br,6H),3.42(br,4H),2.25(br,2H),1.76(s,6H),1.50(s,6H).

[0064] C NMR13 C NMR (DMSO, 100MHz): δ190.8,166.7,166.2,156.3,146.3,131.4,130.7,130.2,128.5 ,128.0,127.5,123.1,122.4,121.2,119.2,66.4,57.7,53.8,53.6,36.8,22.8,15.7.

[0065] Mass spectrum ESI-MS: m / z 631.3 ([M+H] + ).

[0066] The above results prove that the prepared bisindole compounds of crotonate do have the molecular structure shown in formula (I), wherein DMSO is dimethyl sulfoxide.

[0067] Application Examples

[0068] A pH-responsive lysosome-targeted near-infrared-photoacoustic dual-modality imaging and photothermal therapy integrated diagnostic and therapeutic agent is prepared using the bisindole crotonate compound prepared in Example 1 as a raw material.

[0069] Product effect testing

[0070] 1. Responsiveness of CR-630 solution to pH value

[0071] 1. Prepare Britton-Robinson buffers with different pH values ​​(2.0, 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0). CR-630 was prepared into a 100 μM mother solution with DMSO. 100 μL of the mother solution and 900 L of buffers with different pH values ​​were prepared into 1 mL of CR-630 solution. The changes in the UV absorption spectrum when the pH value changed from 2.0 to 9.0 were measured using a UV-visible spectrometer. Figure 1 As shown. Among them, Figure 1 a Ultraviolet absorption spectra of small molecule solutions at different pH values. Figure 1 As can be seen in a, as the pH value increases from 2.0 to 9.0, the absorption spectrum of CR-630 changes, and its peaks at 697nm and 768nm continue to weaken, while a new peak appears at 610-629nm and its intensity continues to increase. Further analysis of the change of the absorbance value ratio A768nm / A610 nm of the small molecule solution with pH value, as shown in Figure 1 As shown in b, Figure 1b is the change of the absorbance ratio A768nm / A610 nm of the small molecule solution with the pH value. It can be seen from the figure that the absorbance ratio changes significantly in the pH range of 4.0-6.5, indicating that the CR-630 solution has good pH response ability in the pH range of 4.0-6.5, and tumor cells have a weak acid environment. The dye CR-630 has good application prospects for pH response in tumor cells. Figure 1 Absorbance is absorbance and Wavelength is wavelength.

[0072] 2. Prepare Britton-Robinson buffers with different pH values ​​(2.0, 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0). Prepare CR-630 with DMSO to a 100 μM compound solution. Take 100 μL of the compound solution and 900L of buffers with different pH values ​​to prepare 1 mL of CR-630 solution. Use a fluorescence spectrometer to measure the fluorescence spectra at excitation wavelengths of 750 nm and 600 nm when the pH value changes from 2.0 to 9.0, as shown in Figure 2. Figure 2 As shown, Figure 2 a is the fluorescence spectrum of CR-630 solution at different pH values ​​when the excitation wavelength is 750nm; Figure 2 b is the fluorescence spectrum of CR-630 solution at different pH values ​​when the excitation wavelength is 600nm; Figure 2 c is the change of the fluorescence intensity ratio I790 nm / I710 nm of CR-630 solution with pH value. Figure 2 a It can be seen that with 750nm as the excitation wavelength, as the pH value continues to increase, the fluorescence intensity continues to weaken; Figure 2 b It can be seen that with 600nm as the excitation wavelength, the fluorescence intensity gradually increases with the increase of pH value. Figure 2 c It can be seen that the fluorescence intensity ratio I790 nm / I710 nm changes significantly in the pH range of 4.0-6.5, indicating that the pH response of the CR-630 solution is in the acidic range of 4.0-6.5. Based on the above results, it can be seen that the UV and fluorescence of CR-630 have obvious changes in the pH range of 4.0-6.5. Figure 2 Where Wavelength is the wavelength and Fluorescence intensity is the fluorescence intensity.

[0073] 2. Photoacoustic intensity and influencing factors of CR-630 solution

[0074] The CR-630 compound was prepared into solutions of different concentrations (0-160 μM) in PBS, and then the photoacoustic imaging effect of the compound was evaluated using a multispectral photoacoustic tomography system, such as Figure 3 As shown in a, Figure 3 a is the photoacoustic spectrum of the small molecule solution at different concentrations and pH value 6.0. It can be seen from the figure that as the concentration of CR-630 increases, its photoacoustic intensity gradually increases. Then, the photoacoustic effect of CR-630 in solutions with different pH values ​​was evaluated. Britton-Robinson buffers with different pH values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) were added to prepare 80μM solutions and tested for their photoacoustic effect. The test range was 680-860nm. The results are shown in the figure. Figure 3 As shown in b. Figure 3 b is a bar graph of the photoacoustic intensity of small molecule solutions at different pH values ​​at 80 μM. It can be seen from the figure that in an acidic environment of 5.0-7.0, CR-630 shows a higher photoacoustic intensity, indicating that CR-630 is also pH-sensitive to the photoacoustic intensity.

[0075] 3. Photothermal performance of CR-630

[0076] The temperature change of the solution was detected by changing the concentration of CR-630 solution, laser power and solution pH. CR-630 was prepared into a 1mM stock solution with DMSO, and the required concentration of CR-630 solution was prepared with Britton–Robinson buffer. The temperature was raised by 808nm laser irradiation, and the real-time temperature change was detected by near-infrared imaging. The results are shown in Figure 2. Figure 4 As shown, Figure 4 a is the pH value of CR-630 solution at different concentrations of 6.0 and 1.0 W / cm 2 The temperature of the solution changes with time under 808nm laser irradiation. Figure 4 b is the change of solution temperature over time of CR-630 solution at 25 μM and pH 6.0 under different laser irradiation powers; Figure 4 c is CR-630 solution at 25 μM and 1.0 W / cm 2 The temperature of solutions with different pH values ​​changes with time under 808nm laser irradiation. Figure 4 As shown in a, when the laser power is 1.0 W / cm 2 As the concentration of the compound in the solution increases from 0 to 50 μM, the temperature that can be reached gradually increases and stabilizes, indicating that CR-630 has a concentration-dependent photothermal conversion effect. At the same time, it can be seen that for a medium concentration of 25 μM and a laser power of 1.0 W / cm 2After 2 minutes of irradiation, the solution temperature can reach 45°C, and after 6 minutes of irradiation, the solution temperature can reach 60°C, showing an excellent photothermal conversion effect. Figure 4 As shown in Figure b, the concentration of CR-630 solution is fixed and the power of the laser is changed. As the power increases, the temperature that the compound can reach also increases. Figure 4 c, when the fixed solution concentration is 25 μM and the power is 1.0 W / cm 2 When the pH value of the solution is changed, it can be seen from the figure that the photothermal conversion efficiency is best when the compound is in acidic conditions. Figure 4 Where Temperature is temperature and Time is time.

[0077] IV. Endocytosis and near-infrared imaging of CR-630 in mouse breast cancer cells (4T1 cells)

[0078] 4T1 cells (8×10 4 4T1 cells were inoculated in a confocal dish and cultured for 24 h. They were incubated with the compound (10 μM) for different time periods (0, 0.5, 1, 3, 6, and 9 h). The medium containing the drug was removed, and PBS was added. Confocal imaging of 4T1 cells was performed on a confocal laser scanning microscope (model: LSM 880 with Airyscan) at an excitation wavelength of 633 nm and an emission wavelength range of 650-800 nm. The results are shown in FIG. Figure 5 As shown in the figure, after 0.5h of incubation, 4T1 cells showed obvious red fluorescence ( Figure 5 CR-630 dyed the cells red), and as the incubation time gradually increased, the fluorescence intensity gradually increased, and when the incubation time reached 6 hours, the fluorescence intensity value reached the maximum. These results show that CR-630 has good cell endocytosis ability and can be used for rapid cell staining and high-quality near-infrared imaging of incubation for 6 hours.

[0079] 5. Targeting of CR-630 to the acidic organelle lysosome in cells

[0080] 4T1 cells (8×10 4 The cells were inoculated in a confocal dish and cultured for 24 h, incubated with the compound (10 μM) for 6 h, and then the targeted nuclei, lysosomes and mitochondria were incubated with different commercial dyes such as Hoechst fluorescent dye 3334 (Hoechst 33342), lysosomal green fluorescent probe (Lyso-Tracker Green), and mitochondrial green fluorescent probe (Mito-Tracker Green) for 20 min, respectively. The cells were imaged using a confocal laser scanning microscope, and the excitation wavelengths were selected as follows: blue light 405 nm, green light 488 nm and red light 633 nm. The experimental results are shown in Figure 6As shown (where the nucleus is stained blue, the lysosomes and mitochondria are stained green, and the CR-630 compound is stained red). As can be seen from the figure, the Pearson coefficient (R) of CR-630 for the nucleus cannot be calculated (No positive correlations found), and the Pearson coefficients for mitochondria and lysosomes are 0.43 and 0.84 respectively. This result shows that CR-630 has obvious targeting recognition for the acidic organelle lysosome in the cell. Among them, Figure 6 In the data, Nucleus refers to the nucleus, Lysosomes refers to the lysosomes, Mitochondria refers to the mitochondria, Tracker refers to tracking, and Merged refers to mixing.

[0081] 6. pH responsiveness of CR-630 to tumor cells

[0082] 4T1 cells were incubated with the compound (10 μM) in culture medium with different pH values ​​(5.5, 6.0, 6.5, 7.0, 7.5, 8.0) for 6 h, and Hoechst 33342 was added for 20 min. After treatment, the cells were imaged under a confocal laser scanning microscope using excitation wavelengths of 561 nm and 633 nm, respectively. The experimental results are shown in Figure 7 shown. Figure 7 a shows the effect of CR-630 on 4T1 tumor cells at different pH values. ex =405nm and λ ex =Cell imaging at 633nm excitation wavelength; Figure 7 b shows the effect of CR-630 on 4T1 tumor cells at different pH values. ex =405nm and λ ex = Cell imaging at 561nm excitation wavelength. Figure 7 It can be seen that in λ ex =633nm, as the pH value gradually increases, the red fluorescence gradually weakens; Figure 7 b can be seen that in λ ex =561nm, as the pH value gradually increases, the red fluorescence gradually increases. These results show that CR-630 is pH-sensitive to 4T1 cells. Figure 7 In the table, Control refers to the control group, Merged refers to the mixed group, and Nucleus refers to the cell nucleus.

[0083] 7. Photoacoustic imaging capability of CR-630 for tumor cells

[0084] 4T1 cells were seeded in a six-well plate and cultured for 24 hours. The culture medium was removed and the cells were incubated with fresh culture medium containing CR-630 (20 μM) for 6 hours. The culture medium was removed, the cells were washed with PBS, digested with trypsin and separated, and then centrifuged. The cells were resuspended in 0.5 mL of hot agarose solution (2%) in a centrifuge tube and subjected to photoacoustic imaging after cooling. The experimental results are shown in Figure 2. Figure 8 As shown in the figure, it can be seen that CR-630 produces stronger photoacoustic intensity (PA Intensity) in 4T1 cells than agarose and cells alone. Figure 8 In the figure, Agarose is agarose, cells is cells, max is maximum, and min is minimum.

[0085] 8. Photothermal therapy effect of CR-630 on tumor cells

[0086] Two groups of 4T1 cells in the exponential growth phase were inoculated into 96-well plates (5000 cells / 100 μL / well), incubated in a constant temperature incubator at 37°C and 5% CO2 for 24 h, the culture medium was discarded, and 100 μL of culture medium containing CR-630 concentration gradient (between 0 and 10 μM) and different pH values ​​(5.5, 6.0, 6.5, 7.0, 7.5, 8.0) was added, respectively, and 3 replicates were made for each concentration. The dark group continued to be incubated in the incubator for 24 h; 12 h after the addition of the drug, the light group was irradiated with 808 nm cell phototoxicity instrument with 1.0 W / cm 2 After irradiation with a power of 10 min, the cells were placed in an incubator and cultured for 12 h. Then, the cell viability of both groups was determined using the standard MTT method. The experimental results are shown in Fig. 9 shown. Fig. 9 a is the photothermal therapy of 4T1 tumor cells by CR-630 at different concentrations and pH values ​​without illumination; Fig. 9 b is the photothermal treatment of 4T1 tumor cells by CR-630 at different concentrations and pH values ​​under illumination. Fig. 9 a It can be seen that in the concentration range of 0-10μM and at different pH values, if there is no laser irradiation, 4T1 still maintains high cell activity, indicating that CR-630 has low dark toxicity. Fig. 9b It can be seen that under laser irradiation, the photothermal killing of tumors by CR-630 has obvious dependence on concentration and pH value. In the pH range of 5.5-6.5, CR-630 shows a higher tumor killing effect. When [CR-630] ≈ 4 μM, 75% of tumor cells are killed. These results show that CR-630 has a certain pH sensitivity while having excellent photothermal killing of tumors. The environment of tumor cells is weakly acidic, while normal cells are weakly alkaline. In vitro solution photothermal experiments also prove that this dye has a better warming effect in an acidic environment than in an alkaline environment, so it has a better killing effect on tumor cells. Among them Fig. 9 In the table, Cell viability refers to cell viability, and [CR-630] refers to the concentration of CR-630.

[0087] In summary, the present invention examines the ultraviolet absorption spectrum, fluorescence spectrum, photoacoustic spectrum and photothermal conversion of CR-630 solution at different pH values, and studies the near-infrared imaging of CR-630 in cells, the lysosome targeting, the near-infrared imaging and photoacoustic imaging at different pH values, and finally performs photothermal therapy by recognizing the pH value of tumor cells. It is found that CR-630 has low dark toxicity and can achieve photothermal therapy under near-infrared light 808nm laser excitation irradiation. It is proved that the CR-630 provided by the present invention is a lysosomal-targeted, pH-responsive, near-infrared-photoacoustic dual-modal imaging, and photothermal therapy-enabled bisindole compounds of crotonate.

Claims

1. A bisindole compound of crotonate, characterized in that: It has the molecular structure shown in the following formula (I):

2. The method for preparing the bisindole ketoic acid compound according to claim 1, characterized in that: The steps include: The compound of formula (II) and the compound of formula (III) are mixed and dispersed in a solvent, and a Knoevenagel condensation reaction is carried out in an inert atmosphere to obtain the bisindole compound of crotonate; The compound of formula (II) has the following molecular structure: The compound of formula (III) has the following molecular structure:

3. The preparation method according to claim 2, characterized in that: The reaction temperature of the Knoevenagel condensation reaction is 80-130° C., and the reaction time is 6-12 hours.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the compound of formula (II) to the compound of formula (III) is 1:0.5-2.

5. The preparation method according to claim 2, characterized in that: After the Knoevenagel condensation reaction occurs, the method further comprises a purification step.

6. Use of the bisindole crezoate compound according to claim 1 in the preparation of a tumor cell lysosome targeting preparation.

7. Use of the bisindole crotonate compound according to claim 1 in the preparation of a preparation responsive to the pH value of tumor cells.

8. Use of the bisindole crotonate compound according to claim 1 in the preparation of a near-infrared-photoacoustic dual-modality imaging preparation for tumor cells.

9. Use of the bisindole crotonate compound according to claim 1 in the preparation of a photothermal therapy preparation for tumor cells.

10. A pH-responsive lysosome-targeted near-infrared-photoacoustic dual-modality imaging and photothermal therapy integrated diagnostic and therapeutic agent, characterized in that: The method is prepared by comprising the bisindole ketoacid compound as described in claim 1.

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