Rhodamine-labeled isoquinoline derivative as well as preparation method and application thereof

Abstract

The invention belongs to the field of organic matter preparation, and particularly relates to a rhodamine-labeled isoquinoline derivative as well as a preparation method and application thereof. According to the preparation method, Sigma-2 receptor targeting ligand 6, 7-dimethoxy tetrahydroisoquinoline is covalently linked with rhodamine fluorophore through a specific connecting bridge, so that a derivative containing rhodamine and a compound containing the 6, 7-dimethoxy tetrahydroisoquinoline are prepared. The rhodamine-labeled isoquinoline derivative disclosed by the invention can achieve the diagnosis and treatment effects through one-time administration, and has good biological activity, higher selectivity and lower toxicity.

Description

Technical Field

[0001] This invention belongs to the field of organic compound preparation, specifically relating to a rhodamine-labeled isoquinoline derivative, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human health, and its early and accurate diagnosis, efficient targeted therapy, and real-time monitoring of the treatment process remain prominent challenges in clinical practice. Targeted therapy, by enhancing the specific accumulation of drugs at the lesion site and reducing damage to normal tissues, has become an important direction for the development of precision medicine. Therapeutic technologies integrate diagnostic and therapeutic functions into a single system, providing an innovative strategy for achieving real-time tumor localization and simultaneous intervention. Compared with traditional therapies relying on nanocarriers, small-molecule therapeutic agents have significant advantages such as well-defined chemical structures, ease of synthesis and structural modification, relatively clear in vivo metabolic pathways, and good tissue permeability, thus showing greater potential in translational applications. Summary of the Invention

[0003] To address the technical bottlenecks in existing nanotechnology that hinder the development of precision tumor diagnosis and treatment, this invention provides a small molecule therapeutic candidate compound with a well-defined structure, simple synthesis, strong targeting, and environmentally responsive imaging capabilities, along with its preparation method and applications. The technical solution is as follows: A rhodamine-labeled isoquinoline derivative, with the structural formula shown in X: R1 is selected from H, CH3, or halogen elements; R2 is selected from... .

[0004] Furthermore, the structure can be any of the following structural formulas: , , , , , , , or .

[0005] A method for preparing the above-mentioned rhodamine-labeled isoquinoline derivative includes the following steps: using tert-butyl (2-bromoethyl)carbamate and compound 1: A nucleophilic substitution reaction was carried out to prepare compound 2: ; Hydrolyzing compound 2 yields compound 3: ; Compound 7 was prepared by amidation of compound 3 with 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride. ; Removing the tert-butyl ester group from the outer end of compound 7 yields compound 8. ; Compound 8 was subjected to an amidation reaction with a rhodamine derivative to obtain a rhodamine-labeled isoquinoline derivative.

[0006] Furthermore, the reaction process is as follows: .

[0007] Furthermore, the reaction conditions are as follows: .

[0008] Furthermore, the rhodamine derivative includes one or more of 5-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimide, 6-carboxytetramethylrhodamine, or 6-carboxytetramethylrhodamine succinimide.

[0009] Furthermore, this includes the following steps: a. Compound 1, base and (2-bromoethyl)carbamate tert-butyl ester were dissolved at -5~5℃ and reacted at 55~65℃ for 2~4 days; water was added to the reaction solution, the reaction solution was extracted with ethyl acetate, the organic layers were combined, and then washed successively with water, saturated alkaline solution and saturated brine. The organic layers were dried, the solvent was removed, and compound 2 was obtained by column chromatography. b. At 10~30℃, compound 2 is reacted with an organic solution of base and stirred until the reaction is complete. The solvent is removed, and then water is added. The aqueous layer is extracted with ethyl acetate, and the pH of the aqueous layer is adjusted to 2~3 with an acid solution. Then it is extracted with an organic solvent, the organic layer is dried, and the solvent is removed to obtain compound 3. c. Compound 3 and the condensing agent were dissolved in an organic solvent and activated at -10 to 10 °C; then 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride was added and reacted at room temperature; after the reaction was complete, an organic solvent was added to the reaction solution, and the organic layer was washed successively with acid, alkali and water. The organic phases were combined, the organic layer was dried, and the compound 7 was obtained by purification. d. Compound 7 and its acid solution were reacted in an organic solvent at -10 to 10°C for 1 hour; the temperature was then slowly raised to room temperature, and the reaction was continued for 4 to 6 hours; the solvent was removed to obtain compound 8. e. Dissolve the rhodamine derivative in an organic solvent to prepare a rhodamine derivative solution; activate compound 8 and the base in an organic solvent at room temperature, then add the rhodamine derivative solution and mix and react under a protective atmosphere; after the reaction is complete, remove the solvent and purify to obtain the rhodamine-labeled isoquinoline derivative.

[0010] Further, the molar ratio of compound 1 to (2-bromoethyl)carbamate tert-butyl ester in step a is 1.1~1.5:1; the molar ratio of the base to compound 1 in step a is 1.3~3:1; the molar ratio of compound 2 to base in step b is 1:2~10; the molar ratio of condensing agent to compound 3 in step c is 1:1~3; the molar ratio of compound 7 to acid in step d is 1:10~50; the molar ratio of compound 8 to rhodamine derivative in step e is 1.5~2:1; and the molar ratio of compound 8 to base is 1:20~50.

[0011] Furthermore, the organic solvent includes dichloromethane or N,N-dimethylformamide; the base in step a is an alkaline carbonate; the acid in step d is an organic acid; and the base in step e is an organic base.

[0012] Furthermore, the preparation of 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride includes the following steps: condensing 4-[N-(tert-butoxycarbonyl)amino]butyric acid with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in the presence of a condensing agent to obtain (4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl)carbamate tert-butyl ester; Then, under the action of hydrochloric acid, the reaction was carried out at -10~10℃ for 1~3h to obtain 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride.

[0013] Furthermore, the condensing agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the molar ratio of the condensing agent to 4-[N-(tert-butoxycarbonyl)amino]butyric acid is 1.1 to 1:5; the molar ratio of 4-[N-(tert-butoxycarbonyl)amino]butyric acid to 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is 1:0.8 to 1.2.

[0014] The application of the above-mentioned rhodamine-labeled isoquinoline derivative in the preparation of antitumor drugs.

[0015] By adopting the above scheme, the method of the present invention has the following advantages: 1. This invention covalently links the Sigma-2 receptor targeting ligand (6,7-dimethoxytetrahydroisoquinoline) to the rhodamine fluorophore via a specific linker. Utilizing the high affinity of the 6,7-dimethoxytetrahydroisoquinoline unit for the Sigma-2 receptor, active targeting and precise localization of tumor cells are achieved. Furthermore, by leveraging the fluorescence "off-on" switching characteristics of the rhodamine unit in the acidic tumor microenvironment, high-contrast, specific fluorescence imaging of the tumor site is achieved. Simultaneously, the photodynamic therapy function of rhodamine as a photosensitizer enables simultaneous tumor treatment under imaging guidance, solving problems such as insufficient targeting, low signal-to-noise ratio, and complex preparation in existing technologies.

[0016] 2. The rhodamine-labeled isoquinoline derivative of the present invention exhibits significant antitumor activity against DU145 cells, superior to the positive control drug doxorubicin, and shows lower cytotoxicity than doxorubicin. The fluorescence intensity released by the rhodamine-labeled isoquinoline derivative a of the present invention is inversely proportional to the pH value, indicating that the amide bond breaks under acidic conditions to release rhodamine. This property of being specifically activated under acidic conditions is highly compatible with the acidic microenvironment unique to tumor tissues.

[0017] 3. The rhodamine-labeled isoquinoline derivative of the present invention mainly enters cells via protein receptors, targets tumor cells, and has a certain ability to remain in the tumor area, with a tumor inhibition rate of up to 81.9%. In mice, its metabolism is mainly through the liver, indicating that the rhodamine-labeled isoquinoline derivative of the present invention has a strong tumor-killing effect and good biosafety.

[0018] 4. This invention can achieve both diagnostic and therapeutic effects with a single dose, exhibiting better bioactivity, higher selectivity, and lower toxicity, and has promising application prospects in tumor treatment and fluorescence diagnosis.

[0019] 5. The preparation method of the present invention has strong repeatability, good stability, simple experimental reaction conditions, mild experimental environment, and good yield, and can be used for large-scale production with relatively small investment. Attached Figure Description

[0020] Figure 1 The cell inhibition rates of rhodamine-labeled isoquinoline derivatives in Examples 1-8 on different cell types; Figure 2 The fluorescence intensity of the rhodamine-labeled isoquinoline derivative of Example 1 at different pH gradients; Figure 3 Comparison of laser scanning confocal microscopy images of DU145 cells incubated with 5-carboxytetramethylrhodamine succinimide for 1-4 h in Example 1; Figure 4Laser scanning confocal microscopy comparison of DU145 cells incubated with different concentrations of Example 1 for 6 h and DU145 cells incubated with 10 µM Example 1 for different times; Figure 5 A comparison graph showing the hemolysis rates of different concentrations of rhodamine-labeled isoquinoline derivatives from Example 1; Figure 6 This is a comparison of the tumor inhibition effects of Example 1, 5-carboxytetramethylrhodamine succinimide ester, and the comparative example in tumor-bearing mice. Figure 7 Comparison of tissue sections of major organs of mice (100×). Figure 8 In vitro comparative images of various organs and tumors in mice obtained after 112 h of tail vein injection of Example 1 and Comparative Example 1. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: (1) Methyl salicylate, potassium carbonate and tert-butyl (2-bromoethyl)carbamate were dissolved in DMF in a molar ratio of 1.3:2.5:1 under stirring at 0°C. The temperature was then raised to 65°C and stirred for 3 days. Distilled water was added to the reaction solution and the reaction solution was extracted with ethyl acetate. The organic layers were then combined and washed with distilled water, saturated sodium carbonate solution and saturated brine in sequence. The organic layers were dried overnight and the solvent of the reaction solution was removed by vacuum distillation. The oily product methyl 2-(2-(tert-butyloxycarbonylamino)ethoxy)benzoate was obtained by column chromatography. (2) Compound 2 was added to a flask under stirring at 20°C, followed by a methanol solution of 2.0 mol / L potassium hydroxide, so that the molar ratio of compound 2 to the base was 1:5. The reaction was stirred at room temperature and monitored by TLC. The solvent was removed under reduced pressure, and then distilled water was added. The aqueous layer was extracted with ethyl acetate, and the pH of the aqueous layer was adjusted to 2~3 with 1 mol / L hydrochloric acid. The aqueous layer was then extracted with ethyl acetate, and the organic layer was dried overnight. The solvent was removed under reduced pressure to obtain a white solid 2-(2-(tert-butoxycarbonylamino)ethoxy)benzoic acid. (3) 3 mmol of 4-[N-(tert-butoxycarbonyl)amino]butyric acid was added to a flask containing 10 mL of dichloromethane, and then 2.8 g, 14.6 mmol of EDC·HCl and 0.3 g, 2.2 mmol of 1-hydroxy-benzo-triazole were added. After activation at 0 °C for 1 h, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was added in equimolar amounts to 4-[N-(tert-butoxycarbonyl)amino]butyric acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction was stopped and 80 mL of dichloromethane was added. The organic layer was washed successively with 1 mol / L hydrochloric acid, saturated sodium carbonate solution and distilled water. The organic phases were combined and dried overnight to obtain a white powdery solid (4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl)carbamate tert-butyl ester; White solid, yield 39.1%. 1 H NMR (DMSO- d 6 , 600 MHz); δ 6.83(q, J 1 =J 2 =6.2 Hz, 1H, -CONH), 6.77(d, J =11.1 Hz, 1H, ArH), 6.74 (s, 1H, -ArH), 4.52(d, J =8.3 Hz, 2H, -CH2-), 3.72(q, J 1 =J 2 =1.6 Hz, 6H, -OCH3-), 3.65~3.61(m, 2H, -CH2-), 2.96~2.93(m, 2H, -CH2-), 2.75(t, J =5.9 Hz, 1H, -CH2-), 2.66(t, J =5.8 Hz, 1H, -CH2-),2.39~2.36(m, 2H, -CH2-), 1.65~1.61(m, 2H, -CH2-), 1.38(d, J =2.4 Hz, 9H, -CH3). (4) Under stirring, tert-butyl 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl)carbamate, dichloromethane, and ethyl acetate solution of hydrochloric acid were added sequentially to a flask. The mixture was stirred at 0°C for 2 h, and the solvent was removed under reduced pressure to obtain a white powdery solid 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl-1-ammonium chloride; the molar ratio of the condensing agent in step c to compound 3 was 1:1~3; the molar ratio of compound 7 to acid in step d was 1:10~50. (5) Under stirring, 2-(2-(tert-butoxycarbonylamino)ethoxy)benzoic acid, EDC·HCl was dissolved in dichloromethane at a molar ratio of 1:2. 1-hydroxy-benzo-triazole was added, and the mixture was stirred at 0°C for 1 h to activate the reaction. Then 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the reaction was stopped, and dichloromethane was added to the reaction solution. The organic layer was washed successively with 1 mol / L hydrochloric acid, saturated Na2CO3 solution, and distilled water. The organic phases were combined, and the organic layer was dried overnight to obtain a transparent oily tert-butyl{2-[2-({4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl}carbamoyl)phenoxy]ethyl}carbamate; (6) Under stirring, 1 mmol of tert-butyl{2-[2-({4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl}carbamoyl)phenoxy]ethyl}carbamate, dichloromethane, and 3 mL of trifluoroacetic acid were added to a flask in sequence. The mixture was stirred at 0 °C for 1 h, and the refrigeration power was turned off so that the mixture was slowly raised to room temperature. The reaction was continued for 5 h, and the solvent was removed under reduced pressure to obtain a yellow solid 2-(2-aminoethoxy)-N-[4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl]benzamide; (7) 0.3 mmol of 2-(2-aminoethoxy)-N-[4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl]benzamide, 10 mL of dichloromethane, and 1 mL of triethylamine were added sequentially to a flask and activated at room temperature for 10 min. Then, 4 mL of DMF solution containing 0.17 mmol of 5-carboxytetramethylrhodamine succinimide was added, and the mixture was stirred overnight. The result was analyzed by TLC (Volume, Volume, and Temperature). CH2CL2 :V CH3OH =9:1) TLC to track the reaction; after the reaction was completed, the solvent was removed under vacuum and silica gel column chromatography was used to obtain red solid compound 9A; The proton NMR data shows a yield of 39.1%. 1H NMR (DMSO- d 6 , 400 MHz); δ : 9.15(s, 1H, -NCOH),8.50(s, 1H, -NCOH), 8.27(d, J =7.3 Hz, 2H, ArH), 7.79(d, J =6.9 Hz, 1H, ArH), 7.48 (d, J =6.4 Hz, 1H, ArH), 7.32(d, J =5.8 Hz, 1H, ArH), 7.21(d, J =8.2 Hz, 1H,ArH), 7.05(d, J =6.4 Hz, 1H, ArH), 6.68(s, 2H, ArH), 6.50(t, J =9.6, 4H, ArH), 4.45(d, J =13Hz, 2H, -CH2-), 4.32(s, 2H, -CH2-), 3.81(s, 2H, -CH2-), 3.67(d, J =13.4Hz, 6H, -OCH3-), 3.50(d, J =28.5Hz, 2H, -CH2-), 2.93(s, 12H, -CH3), 2.67 (s,2H, -CH2-), 2.35(d, J =15.6Hz, 4H, -CH2-), 1.74(d, J =18.6Hz, 2H, -CH2-).

[0023] Example 2: The difference from Example 1 is as follows: Methyl salicylate was replaced with methyl 5-bromosalicylate; the final product was red crystal 9B. The proton NMR data shows a yield of 24.5%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.14(s, 1H, -NCOH),8.49(s, 1H, -NCOH), 8.26(d, J =6.7 Hz, 2H, ArH), 7.71(d, J =6.4 Hz, 1H, ArH), 7.48 (d, J=7.2Hz, 1H, ArH), 7.32(d, J =5.3 Hz, 1H, ArH), 7.07 (d, J =7.7 Hz, 1H,ArH), 7.05(d, J =6.4 Hz, 1H, ArH), 6.68(s, 1H, ArH), 6.50(t, J =8.3, 4H, ArH), 4.43(d, J =12.6Hz, 2H, -CH2-), 4.32(s, 2H, -CH2-), 3.80(s, 2H, -CH2-), 3.66(d, J =13.4Hz, 6H, -OCH3-), 3.50(d, J =28.5Hz, 2H, -CH2-), 2.92(s, 12H, -CH3), 2.68 (s,2H, -CH2-), 2.34(d, J =12.7Hz, 4H, -CH2-), 1.74(d, J =15.3Hz, 2H, -CH2-).

[0024] Example 3: The difference from Example 1 is as follows: Methyl salicylate was replaced with methyl 5-iodosalicylate; the final product was red crystal 9C. The proton NMR data shows a yield of 35.1%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.13(s, 1H, -NCOH),8.50(s, 1H, -NCOH), 8.34(d, J =5.4Hz, 2H, ArH), 8.02(d, J =4.7 Hz, 1H, ArH), 7.48(d, J =6.4 Hz, 1H, ArH), 7.32(d, J =5.8 Hz, 1H, ArH), 7.05(d, J =6.4 Hz, 1H, ArH), 6.90(d, J =7.3 Hz, 1H, ArH),6.65(s, 1H, ArH), 6.46(t, J =9.6, 4H, ArH), 4.47(d,J =13Hz, 2H, -CH2-), 4.32(s, 2H, -CH2-), 3.81(s, 2H, -CH2-), 3.67(d, J =14.4Hz, 6H,-OCH3-), 3.50(d, J =25.5Hz, 2H, -CH2-), 2.92(s, 12H, -CH3), 2.67(s, 2H, -CH2-),2.35(d, J =15.6Hz, 4H, -CH2-), 1.73(d, J =18.6Hz, 2H, -CH2-).

[0025] Example 4: The difference from Example 1 is as follows: Methyl salicylate was replaced with methyl 5-methylsalicylate; the final product was red crystal 9D. The proton NMR data shows a yield of 29.8%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.15(s, 1H, -NCOH),8.50(s, 1H, -NCOH), 8.23(d, J =5.5Hz, 2H, ArH), 7.85 (d, J =4.6 Hz, 1H, ArH), 7.23(d, J =5.4 Hz, 1H, ArH), 7.11(d, J =5.7 Hz, 1H, ArH), 7.05(d, J =6.4 Hz, 1H,ArH), 6.90(d, J =6.3 Hz, 1H, ArH),6.68(s, 1H, ArH), 6.46(t, J =9.8, 4H, ArH), 4.47(d, J =13Hz, 2H, -CH2-), 4.32(s, 2H, -CH2-), 3.81(s, 2H, -CH2-), 3.66(d, J =14.4Hz, 6H, -OCH3-), 3.49(d, J =25.5Hz, 2H, -CH2-), 2.91(s, 12H, -CH3), 2.67(s,2H, -CH2-),2.42(d, J=14.3Hz, 3H, -CH3) 2.33(d, J =15.6Hz, 4H, -CH2-), 1.72(d, J =18.6Hz, 2H, -CH2-).

[0026] Example 5: The difference from Example 1 is as follows: Replace 5-carboxytetramethylrhodamine succinimide with 6-carboxytetramethylrhodamine succinimide; the final product is yellow crystal 9E. The proton NMR data shows a yield of 25.6%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.15(s, 1H, -NCOH),8.49(s, 1H, -NCOH), 8.02(d, J =6.3 Hz, 2H, ArH), 7.53(d, J =6.8 Hz, 1H, ArH), 7.25 (d, J =7.8 Hz, 1H, ArH), 7.22(d, J =6.3Hz, 1H, ArH), 7.17(d, J =7.6 Hz, 1H,ArH), 7.05(d, J =6.2Hz, 1H, ArH), 6.69(s, 2H, ArH), 6.50(t, J =9.3 4H, ArH), 4.46(d, J =12.6Hz, 2H, -CH2-), 4.37(s, 2H, -CH2-), 3.81(s, 2H, -CH2-), 3.68(d, J =12.7Hz, 6H, -OCH3-), 3.49(d, J =22.4Hz, 2H, -CH2-), 2.92(s, 12H, -CH3), 2.66 (s,2H, -CH2-), 2.37(d, J =11.6Hz, 4H, -CH2-), 1.74(d, J =15.6Hz, 2H, -CH2-).

[0027] Example 6: The difference from Example 1 is as follows: Methyl salicylate was replaced with methyl 5-bromosalicylate; and 5-carboxytetramethylrhodamine succinimide was replaced with 6-carboxytetramethylrhodamine succinimide; the final product was yellow crystal 9F. The proton NMR data shows a yield of 31.2%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.14(s, 1H, -NCOH),8.48(s, 1H, -NCOH), 8.03(d, J =5.3 Hz, 2H, ArH), 7.44(d, J =6.4 Hz, 1H, ArH), 7.41(d, J =8.2Hz, 1H, ArH), 7.31(d, J =2.3 Hz, 1H, ArH), 7.01 (d, J =7.2 Hz, 1H,ArH), 6.92(d, J =6.4 Hz, 1H, ArH), 6.67(s, 1H, ArH), 6.51(t, J =7.3, 4H, ArH), 4.42(d, J =13.6Hz, 2H, -CH2-), 4.31(s, 2H, -CH2-), 3.89(s, 2H, -CH2-), 3.67(d, J =15.4Hz, 6H, -OCH3-), 3.51(d, J =16.5Hz, 2H, -CH2-), 2.93(s, 12H, -CH3), 2.68 (s,2H, -CH2-), 2.34(d, J =10.7Hz, 4H, -CH2-), 1.74(d, J =15.3Hz, 2H, -CH2-).

[0028] Example 7: The difference from Example 1 is as follows: Methyl salicylate was replaced with methyl 5-iodosalicylate; and 5-carboxytetramethylrhodamine succinimide was replaced with 6-carboxytetramethylrhodamine succinimide; the final product was 9g of red crystals. The proton NMR data shows a yield of 21.3%. 1 H NMR (DMSO- d 6 , 400 MHz); δ: 9.13(s, 1H, -NCOH),8.47(s, 1H, -NCOH), 8.10(d, J =4.7Hz, 2H, ArH), 7.54(d, J =4.9 Hz, 1H, ArH), 7.47(d, J =6.4 Hz, 1H, ArH), 7.22(d, J =5.8 Hz, 1H, ArH), 7.05(d, J =6.4 Hz, 1H, ArH), 6.91(d, J =8.1Hz, 1H, ArH),6.64(s, 1H, ArH), 6.44(t, J =9.6, 4H, ArH), 4.46(d, J =11.9Hz, 2H, -CH2-), 4.31(s, 2H, -CH2-), 3.82(s, 2H, -CH2-), 3.63(d, J =12.4Hz, 6H, -OCH3-), 3.51(d, J =21.3Hz, 2H, -CH2-), 2.91(s, 12H, -CH3), 2.68(s, 2H, -CH2-), 2.33(d, J =14.9Hz, 4H, -CH2-), 1.74(d, J =17.3Hz, 2H, -CH2-).

[0029] Example 8: The difference from Example 1 is as follows: Methyl salicylate was replaced with 5-methylsalicylate; and 5-carboxytetramethylrhodamine succinimide was replaced with 6-carboxytetramethylrhodamine succinimide; the final product was red crystal 9H. The hydrogen spectroscopy data shows a yield of 32.5%. 1 H NMR (DMSO- d 6 , 400 MHz); δ : 9.14(s, 1H, -NCOH),8.50(s, 1H, -NCOH), 8.11(d, J =4.7Hz, 2H, ArH), 7.63 (d, J =4.3 Hz, 1H, ArH), 7.46 (d, J =3.4 Hz, 1H, ArH), 7.07(d,J =5.5Hz, 1H, ArH), 7.02(d, J =5.4 Hz, 1H,ArH), 6.91(d, J =5.3 Hz, 1H, ArH),6.69(s, 1H, ArH), 6.44(t, J =9.8, 4H, ArH), 4.48(d, J =12.7Hz, 2H, -CH2-), 4.31(s, 2H, -CH2-), 3.77(s, 2H, -CH2-), 3.66(d, J =12.6Hz, 6H, -OCH3-), 3.47(d, J =23.6Hz, 2H, -CH2-), 2.89(s, 12H, -CH3), 2.57s,2H, -CH2-),2.36(d, J =13.4Hz, 3H, -CH3) 2.34(d, J =14.3Hz, 4H, -CH2-), 1.72(d, J =17.7Hz, 2H, -CH2-).

[0030] Comparative Example: The difference from Example 1 is that: Only the tert-butyl{2-[2-({4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl}carbamoyl)phenoxy]ethyl}carbamate (7a) obtained in step (5) was prepared. The proton NMR data are as follows: Yield: 68.6% (7a). 1 H NMR (CDCl3, 600 MHz); δ 8.23(t, J =5.3 Hz, 1H, -CONH), 8.14(t, J =8.6 Hz, 1H, -CONH), 7.42~7.36(m, 1H, -ArH), 7.06~6.89(m,2H, -ArH), 6.71~6.52(m, 3H, -ArH), 4.59 (s, 1H, -CH2-), 4.56 (s, 1H, -CH2-),4.22(t, J =4.7 Hz, 1H, -CH2-), 4.19(t, J =4.7 Hz, 1H, -CH2-), 3.86(t, J=8.0 Hz, 6H,-OCH3), 3.74(t, J =5.9 Hz, 1H, -CH2-), 3.67(t, J =5.8 Hz, 1H, -CH2-), 3.61~3.58(m,4H, -CH2-), 2.78(t, J =5.6 Hz, 1H, -CH2-), 2.69(t, J =5.6 Hz, 1H, -CH2-), 2.58(t, J =5.8 Hz, 1H, -CH2-), 2.08~2.04(m, 3H, -CH2-), 1.45 (s, 9H, -C(CH3)3).

[0031] In vitro antitumor activity and cytotoxicity studies: Cell Culture: Cells were cultured in a sterile incubator at a constant temperature of 37°C and a carbon dioxide concentration of 5%. The culture medium preparation method (50 mL version) used in the cell culture process was as follows: Add 5 mL of pre-thawed fetal bovine serum and 1 mL of antibiotic (penicillin) to a 50 mL centrifuge tube, then add DMEM medium to bring the volume to 50 mL. Shake well and seal with film to obtain 50 mL of 10% culture medium. Cell passage frequency was approximately once every two days or once the cell density reached 80%. Specific cell culture procedure: First, pipette the original culture medium and add 1 mL of PBS, washing twice using a cross-shaped vortex method. Then, add 1 mL of trypsin to fully digest the cells. When most cells are observed to be rounded and detached under a microscope, immediately add 1 mL of the prepared fresh culture medium and mix thoroughly by pipetting. Then, transfer the cell suspension to a 15 mL sterile centrifuge tube and centrifuge (900 rpm, 5 min). After centrifugation, remove the supernatant, add 2 mL of culture medium and mix well. Then, pipette 1 mL into each of two new culture dishes containing 4 mL of culture medium.

[0032] Antiproliferative activity test: First, the healthy cells in the culture dish were thoroughly digested with trypsin, the cell suspension was removed, centrifuged and the supernatant was discarded, and the cells were resuspended in culture medium. 100 µL of the cell suspension was added to each well of a 96-well plate, resulting in approximately 5000–10000 cells per well. The outer edge wells were filled with PBS, and the plates were placed in a CO2 incubator for adherent growth for 12 h. When the cells nearly filled the 96-well plate, each well was treated with 100 µL of different concentrations (1 µM, 0.1 µM, 0.01 µM, 0.001 µM) of the isoquinoline derivatives from Examples 1–8, with three parallel groups, a negative control group, and a blank control group. The plates were then incubated in a CO2 incubator for another 12 h, followed by the addition of 10 µL of prepared MTT solution (5 mg / mL) to the 96-well plate for an additional 4 h of incubation. Finally, after aspirating the supernatant from the wells, 100 µL of DMSO was added, and the mixture was shaken for 1 min. The absorbance was measured using a microplate reader at a reference wavelength of 630 nm and a test wavelength of 570 nm. The cell proliferation inhibition rate of the test compound was calculated based on the OD value, and the IC50 value of the test sample was determined using a fitted curve calculation method.

[0033] The cytotoxicity of eight compounds against three cancer cell types: DU145 cells, A549 cells, HeLa cells, and a normal cell type, 293T cells, was analyzed using the standard MTT assay. Figure 1 Examples 1-8 show the cell inhibition rates of different cell types, where... Figure 1 A represents DU145 cells; Figure 1 B is an A549 cell; Figure 1 C represents HeLa cells; Figure 1 D represents 293 T cells. (From...) Figure 1 It is evident that the isoquinoline derivatives in Examples 1-8 do not exhibit high cell inhibition rates against normal cells; even at a concentration of 100 µM, the cell-killing rate against normal cells is below 50%. At the highest concentration of 10 µM, the cell-killing rate of the eight drug molecules against 293T cells is below 35%. Example 1 shows an even lower killing rate, below 30% even at a concentration of 100 µM. In contrast, all eight compounds exhibit significant cytotoxicity against the three types of tumor cell lines. Based on this, we fitted a linear equation using the logarithm of concentration and cell-killing rate to determine the IC50 of the eight compounds against the four cell types. 50 Values. Using the same experimental method, the cytotoxicity and IC50 of doxorubicin against these four cell types were calculated using a commonly used tumor treatment drug (Doxorubicin). 50 value.

[0034] Table 1: Anti-cancer cell proliferation activity and cytotoxicity of Examples 1-8

[0035] The IC50 values ​​in Table 1 clearly show that the anti-cancer cell proliferation activity of Example 1 is generally stronger than that of the other examples and slightly higher than that of doxorubicin, while its cytotoxicity to normal cells is also lower than that of the other examples and doxorubicin. Therefore, Example 1 is the compound with the strongest antitumor activity among all the synthesized products.

[0036] Fluorescence experiments at different pH values: Accurately weigh a certain amount of the 10 mM stock solution prepared by dissolving Example 1 in DMSO. Using a pH meter, adjust the pH of PBS (pH=7.4) to 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, and 8.0 using 1M hydrochloric acid and sodium hydroxide solutions. Dilute the stock solution of Example 1 to 10 µM with solutions of different acidities and measure its fluorescence intensity.

[0037] As Figure 2 As shown, the fluorescence intensity of Example 1 exhibits a significant pH dependence, increasing with decreasing pH. The highest fluorescence intensity at pH=2.0 is approximately four times that at pH=8.0. The pH response of Example 1 is mainly due to the breaking of the amide bond in the molecule under acidic conditions, releasing rhodamine and thus achieving better fluorescence. The fluorescence emission peak of Example 1 reaches its highest intensity at 550 nm, which is basically consistent with the fluorescence emission peak of 5-TAMRASE, proving that the fluorescence generated by 9a is caused by the release of intramolecular 5-TAMRASE. This also confirms that the acidic environment promotes the release of 5-TAMRASE, thereby achieving a targeted enhancement of fluorescence and realizing selective "off-on" diagnosis. The tumor intratumoral environment is acidic; therefore, Example 1 can controllably generate fluorescence in the tumor region, while not generating fluorescence in the normal cellular environment, thus achieving the function of detecting and diagnosing tumors.

[0038] Cell-targeting experiments: To directly observe the targeting effect of Example 1 on tumor cells, DU145 cells were incubated using Example 1 and observed using a confocal laser scanning microscope (CLSM). First, DU145 cells were cultured adherently in 6-well plates for 12 hours. The original culture medium was then aspirated and added to pre-prepared 6-well plates containing coverslips. 2 mL of pre-prepared culture medium containing 2% fetal bovine serum (FBS) was added, followed by 9a (10 µM) and 5-TAMRASE (10 µM). The cells were cultured for 1 hour, 2 hours, 3 hours, and 4 hours. Then, the culture medium was aspirated, DAPI staining solution was added, and the cells were incubated at room temperature in the dark for 5 minutes. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde for 10 minutes, transferred to slides, and observed using CLSM imaging.

[0039] Figure 3 CLSM images of DU145 cells incubated with Example 1 and 5-TAMASE for 1–4 h, respectively. (Source: [Insert Source Here]) Figure 3 As can be seen, for the isoquinoline derivative of the present invention, the fluorescence of rhodamine in the molecule can be observed through the specific optical channel of CLSM. Therefore, the amount of Example 1 entering the cells can be determined based on the intensity of the fluorescence signal. Compared with Example 1, which lacks a sigma-2 receptor response, the number of tumor cells entering the tumor cells by 5-TAMRASE is significantly less than that by Example 1. This indicates that Example 1, which possesses both a sigma-2 receptor and a low pH dual response, can be more effectively taken up by tumor cells, thereby exerting its therapeutic and diagnostic effects.

[0040] Cellular uptake experiment: To observe the uptake of DU145 cells by Example 1 in tumor cells, CLSM was used to observe the uptake by tumor cells after incubation with different concentrations and durations of Example 1. Using the same experimental method as in the cell targeting experiment, the time gradient groups were observed after incubation with 10 µM Example 1 for 1 h, 2 h, 6 h, 8 h, 12 h, and 24 h, respectively, followed by CLSM observation; the corresponding concentration gradient groups were observed after incubation with 1 µM, 5 µM, 10 µM, 20 µM, 30 µM, and 40 µM Example 1 for 6 h, respectively, followed by CLSM observation. Figure 4 This is a comparison graph showing the results of CLSM observations after incubating DU145 cells with different concentrations of Example 1 for 6 hours. As can be seen from the graph, the number of Example 1 cells entering tumor cells initially increases with increasing concentration, then remains stable. This is because the number of sigma-2 receptors in tumors is limited, and the number of Example 1 cells targeting the tumor tends to stabilize after reaching a certain concentration. Figure 4The figures show a comparison of CLSM observations after incubating DU145 cells with 10 µM of Example 1 for 1–24 h. As can be seen from the figures, the fluorescence intensity continuously increases with incubation time, reaching its peak at 6 h. Subsequently, the fluorescence intensity gradually decreases until it approaches 0 at 24 h. This may be related to the metabolism of tumor cells. DU145 cells continuously take up Example 1 over time, reaching a peak at 6 h, and then continuously metabolize Example 1 through some pathway until the metabolism is essentially complete after 24 h.

[0041] The above results confirm that the rhodamine-labeled isoquinoline derivatives of the present invention exhibit low cytotoxicity and good apoptosis and necrosis rates in normal cells.

[0042] Hemolysis test: Blood samples were obtained from female BALB / c mice using an ocular blood sampling method. The blood samples were then centrifuged (4°C, 3000 rpm, 10 min), and the supernatant was collected. 200 µL of PBS was added and thoroughly mixed, and the mixture was centrifuged five more times under the same conditions. The collected red blood cells were then diluted 10-fold with PBS buffer. 500 µL of the diluted red blood cell suspension was taken, and different concentrations of PBS solution (10 µM, 20 µM, 40 µM, 60 µM, 100 µM), deionized water, and PBS solution from Example 1(9a) were added and thoroughly mixed. The mixture was then incubated at 37°C for 3 h. Finally, the OD value at 570 nm was measured using a microplate reader, and the hemolysis rate was calculated using the formula: Hemolysis rate = (Absorbance of BPS group - Absorbance of test sample) / (Absorbance of BPS group - Absorbance of deionized water group) × 100%.

[0043] The results are as follows Figure 5 As shown, the hemolysis rate remained below 5% as the concentration of Example 1 increased from 10 µM to 100 µM. Therefore, when Example 1 is administered intravenously to animals for experiments, it can be ensured that no blood damage is caused to the experimental animals, thus maintaining good biosafety and ensuring that animal experiments can be conducted safely and in a standardized manner within the ethical framework of laboratory animal testing.

[0044] In vivo anti-tumor studies: The mice used in the in vivo antitumor study were all female BALB / c mice (5 weeks old, 18-20 g). First, a mouse tumor model was established by subcutaneously inoculating the right thigh of each mouse with a DU145 cell suspension (10⁵ cells). When the tumor grew to an appropriate size (500 mm³), the mice were randomly divided into four groups: 1) PBS group; 2) 5-TMARSE group; 3) Comparative Example 7a; 4) Example 1, with 5 mice in each group as replicates. Every two days, the corresponding experimental drug was injected into the corresponding group via tail vein injection at a dose of 5 mg / kg. The tumor volume and body weight of the mice were measured every two days. Finally, on day 13, the main organs of the tumor tissue were harvested and preserved in 4% paraformaldehyde for further analysis.

[0045] exist Figure 6 Figure A shows the changes in mouse tumor volume throughout the administration process. Compared with the PBS injection group, all other experimental groups showed significant inhibition of mouse tumor volume. Among the three experimental groups, Example 1 showed the most significant inhibition of mouse tumor volume, with Comparative Example 7a showing better inhibition than 5-TMARSE. Comparative Example 7a contains a 6,7-dimethoxytetrahydroisoquinoline moiety with tumor-killing properties, so its better antitumor activity than 5-TMARSE was predictable. On day 13 of in vivo antitumor treatment, the experimental mice were euthanized according to standard experimental animal requirements, the tumors were dissected, weighed, and further sectioned. Figure 6 As shown in B, the tumor weight of mice treated with Example 1 was significantly reduced compared to other groups, with an inhibition rate as high as 81.9%, indicating that Example 1 had the strongest anti-tumor activity among the groups. Figure 6 As can be seen in E, the tumors in group 9a were the smallest among all treatment groups, demonstrating a significantly stronger therapeutic effect compared to the PBS group. When we observed the histological sections of the tumors ( Figure 6 F), the same conclusion can be drawn above: the tumor tissue cells treated with Example 1 died in large numbers, demonstrating a considerable tumor-killing effect.

[0046] Changes in mouse body weight during treatment ( Figure 6 C) and the weight of the mouse's major organs ( Figure 6 D) was used to test the biosafety of the drug. The figure shows that the weight changes of mice treated with the drug and those injected with PBS were not significantly different; the organ weights of the mice also remained relatively stable. No obvious damage was observed in tissue sections of the main organs of the experimental mice, such as the heart, liver, spleen, and kidneys. Figure 6No obvious toxic symptoms were observed in the mice throughout the experiment, further demonstrating the safety of the entire treatment. This indicates that our designed diagnostic and therapeutic reagent did not cause significant damage to normal organs and tissues during the entire anti-tumor process, demonstrating good biocompatibility.

[0047] To investigate the in vivo targeting effects and metabolic pathways of isoquinoline derivatives, tumor-bearing mice were obtained using the same tumor-bearing mouse model establishment method. In vivo fluorescence detection was performed 12 h after tail vein injection of PBS solutions containing 9a and 5-TMARSE from Example 1. The detection results are as follows: Figure 8 The fluorescence of the injected Example 1 was mainly observed in the tumor area, with a small portion appearing in the liver; while the fluorescence of the injected 5-TMARSE was mostly present in the liver, and the fluorescence intensity retained in the tumor area was much lower than that of the group 9a injected with Example 1. Since fluorescence intensity is directly proportional to drug concentration, we believe that the metabolism of Example 1 is mainly in the liver, and Example 1 also exhibits good tumor targeting and retention capabilities. Therefore, our designed Example 1 has the potential to be developed into a highly targeted, low-toxicity therapeutic diagnostic reagent.

[0048] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. A rhodamine-labeled isoquinoline derivative, characterized in that, The structural formula is shown in X: R1 is selected from H, CH3, or halogen elements; R2 is selected from... .

2. The rhodamine-labeled isoquinoline derivative according to claim 1, characterized in that, The structure can be any of the following structural formulas: 、 、 、 、 、 、 、 or .

3. A method for preparing a rhodamine-labeled isoquinoline derivative as described in claim 1 or 2, characterized in that, Includes the following steps: Using (2-bromoethyl) tert-butyl carbamate and compound 1: A nucleophilic substitution reaction was carried out to prepare compound 2: ; Hydrolyzing compound 2 yields compound 3: ; Compound 7 was prepared by amidation of compound 3 with 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride. ; Removing the tert-butyl ester group from the outer end of compound 7 yields compound 8. ； Compound 8 was subjected to an amidation reaction with a rhodamine derivative to obtain a rhodamine-labeled isoquinoline derivative.

4. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 3, characterized in that, The rhodamine derivatives include one or more of 5-carboxytetramethylrhodamine, 5-carboxytetramethylrhodamine succinimide, 6-carboxytetramethylrhodamine, or 6-carboxytetramethylrhodamine succinimide.

5. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 3, characterized in that, Includes the following steps: a. Compound 1, base and (2-bromoethyl)carbamate tert-butyl ester were dissolved at -5~5℃ and reacted at 55~65℃ for 2~4 days; water was added to the reaction solution, the reaction solution was extracted with ethyl acetate, the organic layers were combined, and then washed successively with water, saturated alkaline solution and saturated brine. The organic layers were dried, the solvent was removed, and compound 2 was obtained by column chromatography. b. At 10~30℃, compound 2 is reacted with an organic solution of base and stirred until the reaction is complete. The solvent is removed, and then water is added. The aqueous layer is extracted with ethyl acetate, and the pH of the aqueous layer is adjusted to 2~3 with an acid solution. Then it is extracted with an organic solvent, the organic layer is dried, and the solvent is removed to obtain compound 3. c. Compound 3 and the condensing agent were dissolved in an organic solvent and activated at -10 to 10 °C; then 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride was added and reacted at room temperature; after the reaction was complete, an organic solvent was added to the reaction solution, and the organic layer was washed successively with acid, alkali and water. The organic phases were combined, the organic layer was dried, and the compound 7 was obtained by purification. d. Compound 7 and its acid solution were reacted in an organic solvent at -10 to 10°C for 1 hour; the temperature was then slowly raised to room temperature, and the reaction continued for 4 to 6 hours; the solvent was removed to obtain compound 8. e. Dissolve the rhodamine derivative in an organic solvent to prepare a rhodamine derivative solution; activate compound 8 and the base in an organic solvent at room temperature, then add the rhodamine derivative solution and mix and react under a protective atmosphere; after the reaction is complete, remove the solvent and purify to obtain the rhodamine-labeled isoquinoline derivative.

6. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 5, characterized in that, The molar ratio of compound 1 to (2-bromoethyl)carbamate tert-butyl ester in step a is 1.1~1.5:1; the molar ratio of the base to compound 1 in step a is 1.3~3:1; the molar ratio of compound 2 to base in step b is 1:2~10; the molar ratio of condensing agent to compound 3 in step c is 1:1~3; the molar ratio of compound 7 to acid in step d is 1:10~50; the molar ratio of compound 8 to rhodamine derivative in step e is 1.5~2:1; and the molar ratio of compound 8 to base is 1:20~50.

7. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 5, characterized in that, The organic solvent includes dichloromethane or N,N-dimethylformamide; the base in step a is an alkaline carbonate; the acid in step d is an organic acid; and the base in step e is an organic base.

8. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 3, characterized in that, The preparation of the 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride includes the following steps: condensing 4-[N-(tert-butoxycarbonyl)amino]butyric acid with 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in the presence of a condensing agent to obtain (4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobutyl)carbamate tert-butyl ester; Then, under the action of hydrochloric acid, the reaction was carried out at -10~10℃ for 1~3h to obtain 4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-4-oxobut-1-ammonium chloride.

9. The method for preparing rhodamine-labeled isoquinoline derivatives according to claim 8, characterized in that, The condensing agent comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the molar ratio of the condensing agent to 4-[N-(tert-butoxycarbonyl)amino]butyric acid is 1.1 to 1:5; the molar ratio of 4-[N-(tert-butoxycarbonyl)amino]butyric acid to 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is 1:0.8 to 1.

2.

10. The use of a rhodamine-labeled isoquinoline derivative as described in claim 1 or 2 in the preparation of an antitumor drug.

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