A class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation, their preparation methods, and applications.

Abstract

This invention discloses a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy through azoreductase activation, their preparation method, and their application in selectively killing cancer cells and treating tumors. The first objective is to provide a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy through azoreductase activation. These compounds possess the properties of positively charged cyanine dyes, allowing them to target tumor sites and localize to the mitochondria of cancer cells to a certain extent. Through simultaneous activation of azoreductase, photosensitizers and chemotherapeutic drugs are released, achieving selective killing of cancer cells and targeted tumor therapy. The second objective is to provide a method for preparing the prodrug compound. In an anhydrous system, an indole compound containing an R1 group and modified with different substituents at the benzene ring position is reacted with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde to obtain compound IV. Then, compound IV is reacted with m-nitrophenol and reduced to obtain compound V. Finally, compound V undergoes a diazotization reaction with sodium nitrite, and a compound containing an R2 group is added to continue the reaction to obtain the target prodrug compound.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation, as well as their preparation methods and applications. Background Technology

[0002] Chemotherapy is one of the most common methods of clinical cancer treatment. Alkylating agents, as commonly used chemotherapeutic drugs, inactivate cancer cells by covalently binding to intracellular biomolecules (such as DNA, RNA, and enzymes) or by cutting the DNA double strand. However, conventional chemotherapy methods suffer from low bioavailability and insufficient selectivity. The high concentrations and doses of drugs used during treatment easily lead to drug resistance and severe toxic side effects on normal tissues. Photodynamic therapy (PDT) has advantages such as non-invasiveness, high spatiotemporal selectivity, high treatment efficiency, and low biotoxicity. It can increase the chemosensitivity of cancer cells, reduce the dosage of chemotherapeutic drugs, and decrease toxic side effects, thus compensating for the shortcomings of chemotherapy. Utilizing photosensitive dyes with tumor-targeting and cancer cell subcellular organelle localization capabilities (such as the nucleus, mitochondria, and lysosomes) in its structure, PDT can reduce photosensitizer efflux and kill cancer cells in a more controllable manner, targeting the characteristics and functions of different subcellular organelles, thereby improving the precision of treatment. However, due to the limited penetration depth of light and the short diffusion distance of reactive oxygen species, photodynamic therapy (PDT) has a limited area of ​​effect, only capable of killing cancer cells within a certain range. Chemotherapy, with its wide distribution and range of action, can compensate for the limited area of ​​action of PDT. Therefore, the combined PDT and chemotherapy anti-tumor system overcomes the shortcomings of single treatment modalities and has promising application prospects.

[0003] Most reported photodynamic therapy (PDT) and chemotherapy combination therapies involve encapsulating photosensitizers and chemotherapeutic drugs within nanoparticles. While this design strategy has shown better efficacy than single-modality treatments, the uninhibited activity of the photosensitizers and chemotherapeutic drugs encapsulated in the nanoparticles allows them to act on various cell types upon release, resulting in insufficient tumor targeting and difficulty in achieving highly efficient and selective killing of tumor cells. Furthermore, the synergistic mechanism of the two treatments is unclear, potentially leading to non-specific interactions that trigger a defensive response from the body's immune system, resulting in counterproductive outcomes. Therefore, this strategy has limited efficiency in cancer treatment.

[0004] Preparing drugs into prodrug molecules that can be activated by specific tumor microenvironments (such as low pH, overexpressed enzymes, and oxidative / reducing substances) through chemical modification is a highly effective strategy to improve the selectivity of tumor cell killing and reduce toxic side effects. Azo reductase is a typical overexpressed biomarker in hypoxic cancer cells; it can break azo bonds and is an ideal bioactivator for tumor therapy. Azo reductase linked by azo bonds can activate probes and prodrugs, significantly reducing their toxic side effects and achieving selective killing of tumor cells. Summary of the Invention

[0005] To address the problems of insufficient tumor targeting and difficulty in achieving efficient and selective killing of normal and cancer cells in existing technologies, this invention provides the following technical solution.

[0006] The first objective of this invention is to provide a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation. These prodrug compounds possess the properties of positively charged cyanine dyes, enabling them to target tumor sites and localize to the mitochondria of cancer cells to a certain extent. Through the simultaneous activation of azoreductase, they release photodynamic photosensitizers and chemotherapeutic drugs, thereby achieving selective killing of cancer cells and targeted tumor therapy.

[0007] The second objective of this invention is to provide a method for preparing a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation. This method has the advantages of readily available raw materials, simple preparation, and easy industrialization.

[0008] The third objective of this invention is to provide the application of a class of tumor-targeting prodrugs that synergistically enhance photodynamic / chemotherapy activation in the preparation of tumor therapeutic agents, which simultaneously activates photodynamic photosensitizers and chemotherapeutic drugs, targets tumors, selectively kills cancer cells, and achieves the synergistic promotion of photodynamic therapy and chemotherapy.

[0009] To achieve the first objective mentioned above, the present invention provides the following technical solution: a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation, wherein the prodrug compound has the structure of general formula I:

[0010]

[0011] Where X1 and X3 are H and X2 is I, or X1 and X3 are I and X2 is H;

[0012] R1 is n = 1 - 5;

[0013] R2 is And R2 is a para substituent.

[0014] To achieve the second objective mentioned above, the present invention provides the following technical solution: a method for preparing a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation, wherein the compound of formula I is prepared in an anhydrous system by the following method:

[0015] S1: Compound of formula IV

[0016]

[0017] Indole compound II, which has been modified with different substituents at the benzene ring position and contains an R1 group, reacts with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde III in the presence of a first anhydrous organic solvent and a first catalyst to obtain compound IV.

[0018] S2: Compounds of formula V for preparation

[0019]

[0020] The compound of formula IV reacts with m-nitrophenol in the presence of a second anhydrous organic solvent and a second catalyst to give a first intermediate. This first intermediate then reacts with stannous chloride and concentrated hydrochloric acid in the presence of a third anhydrous organic solvent to give the compound of formula V.

[0021] S3: Preparation of compound of formula I

[0022]

[0023] The compound of formula V reacts with sodium nitrite and aminosulfonic acid in anhydrous mixed organic solvents to give a second intermediate. This second intermediate reacts with a nitrogen mustard compound containing an R2 group in a fourth solvent to give the compound of formula I.

[0024] Further, in step S1, the molar ratio of the indole compound II to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde III is 1:(2-3), the reaction time is 10-12 h, the reaction temperature is 25-40 °C, the first anhydrous organic solvent is selected from acetic anhydride, ethanol or acetonitrile, and the first catalyst is selected from anhydrous sodium acetate or anhydrous potassium carbonate.

[0025] Further, in step S2, the molar ratio of the compound of formula IV to m-nitrophenol is 1:(2-3), the reaction time is 4-5 h, the reaction temperature is 25-40 °C, the second anhydrous organic solvent is selected from anhydrous acetonitrile or anhydrous ethanol, and the second catalyst is selected from anhydrous potassium carbonate or anhydrous sodium carbonate.

[0026] Further, in step S2, the molar ratio of the first intermediate to stannous chloride is 1:(5-6), the reaction time is 10-12 h, the reaction temperature is 60-80 °C, and the third anhydrous organic solvent is selected from anhydrous ethanol or anhydrous DMF.

[0027] Further, in step S3, the molar ratio of the compound of formula V to sodium nitrite is 1:(2-3), the reaction time is 1-2 h, the reaction temperature is 0 °C, and the anhydrous mixed organic solvent is anhydrous acetonitrile:anhydrous dichloromethane = 1:4, v / v.

[0028] Further, in step S3, the reaction molar ratio of the second intermediate to the nitrogen mustard compound containing the R2 group is 1:(5-6), the reaction time is 2-3 h, the reaction temperature is 0 °C, and the fourth solvent is selected from ethanol:water = 4:1, v / v or ethanol:water = 2:1, v / v.

[0029] To achieve the third objective mentioned above, the present invention provides the following technical solution: the application of a class of tumor-targeting prodrug compounds that activate azoreductase and synergistically enhance photodynamic / chemotherapy, wherein the azoreductase-activated prodrug compounds can target tumor sites to a certain extent and be located in the mitochondria of cancer cells, for selective killing of cancer cells and tumor treatment, wherein the tumor is a hypoxic solid tumor overexpressing azoreductase.

[0030] In summary, the present invention has the following beneficial effects:

[0031] First, the tumor-targeting prodrug compound prepared by this invention, which activates azoreductase and synergistically enhances photodynamic therapy / chemotherapy, has absorption and emission wavelengths of around 700 nm and exhibits excellent near-infrared photosensitive dye properties. The prodrug compound has the properties of a positively charged cyanine dye, which can target tumor sites and localize cancer cells to a certain extent. Furthermore, the introduction of specific responsive linkages in the prodrug compound ensures its sensitive responsiveness and high selectivity to azoreductase.

[0032] Second, this invention combines a photodynamic photosensitizer with a chemotherapy drug. Azo reductase is a substance overexpressed by hypoxic cancer cells. After the prodrug compound is activated by azo reductase, it releases a hemicyanine dye photosensitizer and a chemotherapy drug, nitrogen mustard. This can simultaneously activate the photosensitizer and the chemotherapy drug, and the two treatment methods synergistically promote each other.

[0033] Third, after activation, hemicyanine dyes exhibit the ICT effect, restoring fluorescence. The absorbed light energy is released through fluorescence and intersystem crossing, enhancing the fluorescence imaging and photodynamic performance of the photosensitizer. Through specific activation, chemotherapeutic drugs are precisely released into cancer cells, demonstrating excellent cancer cell selectivity. The synergistic mechanism between photodynamic therapy and chemotherapy is discussed in more depth. The photodynamic properties of hemicyanine dyes can improve the release efficiency of chemotherapeutic drugs and enhance the effect of chemotherapy. Similarly, chemotherapeutic drugs can enhance the ability of photodynamic therapy to damage mitochondria and enhance the effect of photodynamic therapy. The two work synergistically to greatly promote the killing effect of cancer cells.

[0034] Fourth, it has good biocompatibility. In mouse tumor suppression experiments, the prodrug was able to image and treat mouse tumors, showing excellent tumor suppression effects. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The following are the absorption and fluorescence spectra of Cy-NM and Cy-NH2 disclosed in the embodiments of the present invention; Figure A is the absorption spectrum; Figure B is the fluorescence spectrum.

[0037] Figure 2 The above are liquid chromatograms of Cy-NH2, Cy-NM, and Cy-NM+SDT disclosed in the embodiments of the present invention.

[0038] Figure 3 This is a fluorescence change diagram of Cy-NM mixed with SDT (0-5mM) disclosed in an embodiment of the present invention;

[0039] Figure 4 This is a selectivity diagram of Cy-NM response to different substances disclosed in the embodiments of the present invention, wherein from left to right are: control; potassium ion; calcium ion; sodium ion; hydrogen peroxide; cysteine; glutathione; glutamate; arginine; gamma-glutamyl transferase; alkaline phosphatase; nitroreductase; sodium dithionite;

[0040] Figure 5Figure A shows the absorption change of DPBF under 660nm light irradiation for Cy-NH2 and Cy-NM disclosed in the embodiments of the present invention; Figure B shows the absorption change of DPBF under 660nm light irradiation for Cy-NH2; Figure C shows the absorption attenuation quantification curves of DPBF at 415nm under 660nm light irradiation for DPBF, Cy-NM, Cy-NH2 and MB respectively.

[0041] Figure 6 The images show confocal imaging cell uptake of Cy-NM on 4T1, HepG2, and BEAS-2B cells as disclosed in this embodiment of the invention; Figure A is a confocal imaging image of Cy-NM; Figure B is a data quantification image of fluorescence imaging.

[0042] Figure 7 The diagrams show the subcellular organelle localization of Cy-NM in 4T1 cells as disclosed in this invention. Figures a, b, c, and d represent Cy-NM staining, commercial mitochondrial dye staining, staining overlay, and overlay localization coefficient diagrams, respectively. Figures e, f, g, and h represent Cy-NM staining, commercial lysosomal dye staining, staining overlay, and overlay localization coefficient diagrams, respectively. Figures i, j, k, and l represent Cy-NM staining, commercial nuclear dye staining, staining overlay, and overlay localization coefficient diagrams, respectively.

[0043] Figure 8 Phototoxicity assays of 4T1 cells after incubation with different concentrations of Cy-NM under normoxic and hypoxic conditions; Figure 8 In the middle section: the selected excitation light was a 660nm near-infrared light source with a light dose of 40mW cm⁻¹. -2 5 minutes;

[0044] Figure 9 The Cy-NM disclosed in the embodiments of this invention was tested in darkness and at a 660nm light source (40mW cm⁻¹). -2 Photo / dark toxicity test of 4T1, HepG2 and BEAS-2B cells during irradiation (5 min);

[0045] Figure 10 Gel electrophoresis images of pBR322 DNA alkylation by Cy-NM, Cy-NH2, and NM, respectively.

[0046] Figure 11 This is a confocal imaging image of γ-H2AX immunofluorescence staining of 4T1 cells by Cy-NM and Cy-NH2 under light and dark conditions, respectively, as disclosed in the embodiments of the present invention.

[0047] Figure 12The diagram shows the mitochondrial membrane potential of 4T1 cells under light and dark conditions, as disclosed in this embodiment of the invention, using Cy-NM and Cy-NH2.

[0048] Figure 13 This is a fluorescence imaging experiment of Cy-NM and Cy-NH2 in a live subcutaneous tumor model as disclosed in an embodiment of the present invention; Figure A is a fluorescence imaging image of Cy-NM and Cy-NH2 in a live subcutaneous tumor model; Figure B is a fluorescence data quantification diagram of Figure A;

[0049] Figure 14 This is a fluorescence imaging experiment of organ distribution of Cy-NM and Cy-NH2 in a live subcutaneous tumor model disclosed in an embodiment of the present invention; Figure A is an organ fluorescence imaging image of Cy-NM; Figure B is an organ fluorescence imaging image of Cy-NH2; Figure C is a data quantization diagram of Figures A and B;

[0050] Figure 15 Figure A shows the tumor suppression experiment of Cy-NM in an in vivo anti-tumor model disclosed in this embodiment of the invention; Figure B shows the change in tumor volume in the in vivo after Cy-NM treatment; Figure B shows the change in body weight in the in vivo after Cy-NM treatment. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the appendices in the embodiments of the present invention. Figure 1-15 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0052] All chemicals involved in this application are from Energie or Aladdin Chemical Reagent Company. The biological consumables, fetal bovine serum, trypsin, and DMEM culture medium were purchased from Giboca. The cells used are from the ATCC cell bank. Other raw materials are commercially available and there are no special requirements.

[0053] Example

[0054] Example 1

[0055] This embodiment discloses that X1 and X3 are H, X2 is I, R1 is CH2CH3, and R2 is... The preparation of a class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation is described below:

[0056]

[0057] (1) Under N2 protection, sodium nitrite (12.6 g, 182.6 mmol) was dissolved in water (45 mL), and 4-iodoaniline (20 g, 91.3 mmol) was dissolved in hydrochloric acid (5.5 M, 15 mL). The solution was cooled to 0 °C and added dropwise to the reaction system. The mixture was stirred at room temperature for 3 h. SnCl2·2H2O (67.99 g, 301.3 mmol) was dissolved in concentrated hydrochloric acid (40 mL) and added dropwise to the reaction system at 0 °C. The mixture was stirred in an ice bath for 2 h and then stirred overnight at room temperature. A light brown precipitate was obtained by filtration, washed three times with water, and dissolved in saturated NaOH solution. The precipitate was extracted with ethyl acetate and Na2S2O3. The organic layer was evaporated to dryness to obtain compound 1, a brown powder, with a yield of 77%.

[0058] (2) Under N2 protection, compound 1 (6 g, 25.6 mmol) was dissolved in glacial acetic acid (100 mL), heated to 118 °C and stirred. 3-methyl-2-butanone (3.68 g, 42.7 mmol) was added dropwise to the reaction system, and the mixture was stirred at 118 °C for 12 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the crude product was extracted with ethyl acetate. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v / v) to obtain compound 2, which is an indole compound with H in X1 and X3 and I in X2, a red oily substance with a yield of 83%.

[0059] (3) Under N2 protection, compound 2 (1.68 g, 5.89 mmol) and iodoethane (4.6 g, 29.5 mmol) were dissolved in 1,2-dichlorobenzene (25 mL), and the mixture was heated to 110 °C and stirred for 12 h. The reaction mixture was cooled to room temperature, the crude product was washed with diethyl ether (150 mL), and then cooled in an ice bath for a few minutes. The solvent was removed by suction filtration, and the residue was collected to give compound 3, which is an indole compound with R1 of CH2CH3, in pink solid, with a yield of 90%.

[0060] (4) Under N2 protection, compound 3 (0.8 g, 1.8 mmol), 2-chloro-3-(hydroxymethylene)cyclohexyl-1-enecarboxaldehyde (0.16 g, 0.9 mmol), and anhydrous sodium acetate (0.15 g, 1.8 mmol) were mixed in acetic anhydride (10 mL) and stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100:3, v / v) to give compound 4, a green solid, in 70% yield;

[0061] (5) Under N2 protection, 3-nitrophenol (0.28 g, 1.98 mmol) and K2CO3 (0.27 g, 1.98 mmol) were dissolved in acetonitrile (10 mL) and stirred at room temperature for 10 min. Compound 4 (0.42 g, 0.66 mmol) was dissolved in acetonitrile (5 mL) and added to the reaction system, and stirred at room temperature for 4 h. After the reaction was complete, the solvent was removed by vacuum distillation, the precipitate was dissolved in dichloromethane, washed three times with water and dried over Na2SO4 to obtain the first intermediate. The first intermediate was placed in methanol (30 mL) for further use.

[0062] Under N2 protection, SnCl2 (2.5 g, 13.2 mmol) was dissolved in concentrated hydrochloric acid (2 mL) and added to the above methanol solution. The mixture was heated to 70 °C and stirred for 12 h. The reaction system was neutralized with saturated Na2CO3, the precipitate was removed by filtration and washed with dichloromethane. The collected filtrate was washed three times with water and dried over Na2SO4. The solvent was removed by vacuum distillation to obtain the crude product, which was purified by silica gel column chromatography (dichloromethane:methanol = 100:7.5, v / v) to give compound Cy-NH2, a green solid, in 70% yield.

[0063] High-resolution mass spectra of compound Cy-NH2: m / z C 27 H 28 I₂N₂O([MI]⁺): Calculated value 523.1241, Test value 523.1257.

[0064] The 1H NMR spectrum analysis of compound Cy-NH2 is as follows: 1 H NMR (400MHz, DMSO-d6) δ8.38(d,J=14.5Hz,1H),8.04(s,1H),7.77(d,J=7.0Hz,2 H),7.46(d,J=8.6Hz,1H),7.27(d,J=8.3Hz,1H),7.09(s,2H),6.80(d,J=8.6Hz, 1H),6.69(s,1H),6.18(d,J=14.5Hz,1H),4.20(q,J=7.1Hz,2H),2.76–2.71(m,2 H),2.67(t,J=5.1Hz,2H),1.85–1.80(m,2H),1.69(s,6H),1.28(t,J=7.1Hz,3H).

[0065] (6) Under N2 protection, compound Cy-NH2 (97.5 mg, 0.15 mmol) was dissolved in an acetonitrile / dichloromethane (1:4, 20 mL) solution containing 1% trifluoroacetic acid, and stirred at 0 °C for 1 h. NaNO2 (20.7 mg, 0.3 mmol) was added to the reaction system, and stirring was continued at 0 °C for 30 min to obtain compound 5. Aminosulfonic acid (29.1 mg, 0.3 mmol) was added to the reaction system, and stirring was carried out at 0 °C for 10 min. N,N-bis(2-chloroethyl)aniline (147.8 mg, 0.9 mmol) was dissolved in ethanol / water (4:1, 2 mL), added to the reaction system, and stirred at 0 °C for 2 h. After the reaction was complete, the product was extracted with dichloromethane, and the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 100:6, v / v) to obtain the target compound Cy-NM as a green solid with a yield of 60%.

[0066] High-resolution mass spectra of the target compound Cy-NM: m / z C 37 H 38 Cl2I2N4O([MI)) + ): Calculated value 751.1462, Test value 751.1498.

[0067] The 1H NMR spectrum analysis of the target compound Cy-NM is as follows: 1 H NMR (400MHz, CD3OD) δ8.52(d,J=15.2Hz,1H),8.08(s,1H),7.81(d,J=8.3Hz,1H),7.76(d,J=8.4Hz,1H), 7.68(d,J=8.6Hz,2H),7.64(d,J=8.3Hz,1H),7.53(s,1H),7.38(s,1H),7.09(d,J=8.4Hz,1H),6.77(d,J =8.6Hz,2H),6.16(d,J=15.6Hz,1H),4.13(dd,J=12.4,6.0Hz,2H),3.95(t,J=6.4Hz,4H),3.82(t,J=6.5 Hz,4H),3.62(d,J=8.1Hz,2H),2.75–2.70(m,2H),2.42–2.37(m,2H),1.85(s,6H),1.33(d,J=6.8Hz,3H).

[0068] The carbon NMR spectrum analysis of the target compound Cy-NM is as follows: 13C NMR (101MHz, CD3OD) δ177.18,154.68,153.56,150.69,146.19,144.55,144.10,140.81,138.16,132.39,132.02,131.09,128.55,125.69,12 3.38,121.65,115.57,114.50,111.94,106.96,104.72,91.87,70.26, 53.04,50.97,40.73,40.50,29.56,29.14,26.96,23.57,20.11,11.68.

[0069] Performance testing

[0070] 1. In the preparation process of the above embodiment, the prepared Cy-NM and Cy-NH2 were respectively prepared into 3mM stock solutions, and their absorption and fluorescence spectra at a concentration of 3μM were tested in dichloromethane.

[0071] The absorbance spectrum was measured using a UV-Vis spectrophotometer, specifically as follows: Figure 1 As shown in A, it can be seen that the absorption spectra of Cy-NM and Cy-NH2 are between 600-700 nm.

[0072] The fluorescence spectrum was tested using a fluorescence spectrometer, specifically as follows: Figure 1 As shown in B, it can be seen that the fluorescence of Cy-NM is significantly quenched relative to Cy-NH2 due to the ICT effect generated by the amino group.

[0073] 2. Simultaneous activation mechanism and post-response fluorescence changes and selectivity testing

[0074] 2.1 During the preparation process of the above examples, the absorption of Cy-NH2 and Cy-NM at 680 nm was monitored by high-performance liquid chromatography (HPLC) within 25 minutes in the mobile phase (methanol:water = 9:1 to pure water). After Cy-NM was incubated with SDT (Na2S2O4) at 37°C for 2 hours, its absorption change at 680 nm was monitored by HPLC in the same manner. The results are shown in [Figure number missing]. Figure 2 Different concentrations of SDT were added to Cy-NM to obtain Cy-NM+SDT, and then incubated at 37℃ for 2 hours. Fluorescence changes were then detected, and the results are shown below. Figure 3 (SDT was used instead of azo reductase for solution testing)

[0075] like Figure 2As shown in the liquid chromatography of Cy-NH2, Cy-NM, and Cy-NM+SDT, after reacting with SDT for 2 hours, the peak intensity of Cy-NM (retention time: 18 min) decreased, and a peak of Cy-NH2 (retention time: 12 min) appeared, indicating that SDT can be converted to Cy-NH2 after reacting with Cy-NM. Figure 3 As shown, the higher the concentration of SDT, the stronger the fluorescence of Cy-NM becomes, indicating that SDT can cleave the azo bonds of Cy-NM to release Cy-NH2 and achieve fluorescence recovery.

[0076] 2.2 In the preparation process of the above embodiments, the prepared Cy-NM was mixed with potassium ions, calcium ions, sodium ions, hydrogen peroxide, cysteine, glutathione, glutamic acid, arginine, gamma-glutamyl transferase, alkaline phosphatase, nitroreductase, and sodium dithionite, respectively. After incubation at 37°C for 2 hours, the fluorescence change at 720 nm was detected. The detection results are shown in [Figure number missing]. Figure 4 .

[0077] like Figure 4 As shown, when other metal ions, amino acids, or enzymes are added, Cy-NM only responds to sodium dithionite, exhibiting good selectivity.

[0078] In conjunction with the embodiments and Figure 2 as well as Figure 3 It can be seen that the mechanism of the response of the target compound Cy-NM prepared in the examples to azo reductase is as follows:

[0079]

[0080] In the embodiments of this application, the hemicyanine dye and the chemotherapeutic drug are linked by an azo bond. The azo reductase overexpressed in hypoxic cancer cells can cleave the azo bond and synchronously activate Cy-NM to release the photodynamic photosensitizer and the chemotherapeutic drug.

[0081] 3. Photodynamic performance test

[0082] In a dichloromethane solvent system, DPBF was added to cuvettes containing Cy-NH2, Cy-NM, and methylene blue solutions, respectively, and a 660 nm light source (3 mW cm⁻¹) was used. -2 The photodynamic properties of each sample were measured under irradiation. Each sample was irradiated for 150 seconds, and its absorption spectrum was measured every 15 seconds. The results are shown below. Figure 5 It can be seen that in the Cy-NH2 system, the absorbance of DPBF decreases significantly at 415 nm, while in the Cy-NM system, the absorbance of DPBF does not change significantly, indicating that Cy-NH2 has higher absorbance than Cy-NM. 1 O2 generation capability.

[0083] 4. Intracellular enzyme activation performance and subcellular organelle localization experiments

[0084] 4.1 4T1 cells were cultured in a 37℃, 5% CO2 incubator using DMEM high-glucose medium containing 10% serum and 1% penicillin antibiotics. The oxygen content was 2% under hypoxic conditions and 21% under normoxic conditions. Cy-NM was added to cell imaging dishes containing 4T1, HepG2, and BEAS-2B cells, respectively, and incubated for 2 hours under normoxic and hypoxic conditions. After washing the cells with PBS, images were collected using confocal fluorescence imaging. The results are shown below. Figure 6 As can be seen, under hypoxic conditions, significant Cy-NM fluorescence signals were observed in 4T1 and HepG2 cells, while almost no fluorescence was observed in BEAS-2B cells. Under normoxic conditions, the fluorescence signals in 4T1, HepG2, and BEAS-2B cells were very weak, and the fluorescence was significantly quenched. This indicates that Cy-NM can only be activated in hypoxic cancer cells and cannot be activated in normoxic and normal cells.

[0085] 4.2. 4T1 cells were seeded in a cell confocal culture dish. Cy-NM (2 μM) was added to the imaging dish and incubated for 2 hours. Commercial dyes Mito (100 nM, mitochondria), Lyso (100 nM, lysosomes), and Hoechst (2 μg / mL) were then added to the dish. -1 Cell nuclei were added to Cy-NM-treated cells and incubated for 30 min. The counterstaining coefficients of Cy-NM and Hoechst staining were determined using laser confocal microscopy. The excitation wavelength of Cy-NM was 640 nm, and the emission wavelength was 660-750 nm; the excitation wavelengths of Mito and Lyso were both 488 nm, and the emission wavelengths were 500-540 nm; the excitation wavelength of Hoechst was 405 nm, and the emission wavelength was 440-480 nm. The results are shown below. Figure 7 As can be seen, Cy-NM can be well localized in the mitochondria of 4T1 cells.

[0086] 5. Evaluation of cell selectivity and killing effect

[0087] 5.1. Under normoxic and hypoxic conditions, 4T1 cells were incubated with Cy-NM at concentrations of 0-16 μM for 2 h, followed by exposure to a 660 nm light source (40 mW cm⁻¹). -2 The cells were exposed to light to compare the cytotoxic effects of Cy-NM on 4T1 cells under normoxic and hypoxic conditions. Results are shown below. Figure 8The results show that the Cy-NM hypoxia group exhibited a stronger killing effect on 4T1 cells than the normoxic group. These differences indicate that after Cy-NM enters hypoxic cancer cells, its azo bonds are cleaved by azoreductase, and the released Cy-NH2, together with NM, enhances cytotoxicity. Under normoxic conditions, Cy-NM cannot be activated and exhibits virtually no cytotoxicity.

[0088] 5.2. Different types of cancer cells (4T1, HepG2) and normal cells (BEAS-2B) were selected to compare the killing effect of Cy-NM on different cell types. The test results are shown in […]. Figure 9 .

[0089] Three cell lines were incubated with 0-16 μM Cy-NM under hypoxic conditions for 2 h, followed by exposure to darkness and 660 nm light source (40 mW cm⁻¹). -2 The sample was subjected to light treatment. After 24 hours, MTT solution (5 mg / mL) was added. -1 After incubation for 4 hours, 100 μL of DMSO was added, and the absorbance at 490 nm was measured. Figure 9 Cy-NM was tested in darkness and at a 660nm light source (40mW cm⁻¹). -2 Cell survival rate graph showing the effects of irradiation on different cell types (5 min) during treatment. Figure 9 It can be seen that Cy-NM exhibits higher dark toxicity to cancer cells than to normal cells, indicating that the chemotherapy drugs released after Cy-NM activation can kill cancer cells. Cy-NM also shows significantly higher phototoxicity to cancer cells than to normal cells, indicating that Cy-NM has good selectivity in killing cancer cells.

[0090] 6. Synergistic enhancement mechanism of photodynamic therapy and chemotherapy

[0091] 6.1. The pBR322 plasmid DNA was first incubated with SDT (5mM) for 4 hours, and then incubated with Cy-NM and Cy-NH2, respectively. The alkylation effect was tested by gel electrophoresis under light / dark conditions. The results are shown in […]. Figure 10 It can be seen that the chemotherapy drug nitrogen mustard can alkylate DNA double strands, thereby killing cancer cells. The degree of alkylation after Cy-NM incubation was significantly higher than that of the Cy-NH2 group. After light irradiation, the alkylation effect was further enhanced, indicating that photodynamic therapy can promote the release of chemotherapy drugs and enhance the alkylation effect of the drugs.

[0092] 6.2. Further validation was performed using γ-H2AX immunofluorescence staining: 4T1 cells were incubated with PBS, Cy-NH2, or Cy-NM for 2 hours, respectively. After washing the cells with PBS, they were treated according to a DNA damage detection kit. Confocal fluorescence imaging was used to collect images. The excitation wavelengths for DAPI and γ-H2AX were 405 nm and 488 nm, respectively, and the emission wavelengths were 415-485 nm and 500-545 nm, respectively. The detection results are shown below. Figure 11 As can be seen, the fluorescence signal of the Cy-NM group was significantly stronger than that of the Cy-NH2 group, indicating that Cy-NM can alkylate the DNA of 4T1 cells. Under irradiation with a 660nm light source, the fluorescence signal of the Cy-NM group was further enhanced, indicating that photodynamic therapy enhances the alkylation ratio of chemotherapeutic drugs.

[0093] 6.3. Verification of changes in mitochondrial membrane potential in 4T1 cells using JC-1 fluorescence imaging: 4T1 cells were incubated with PBS, Cy-NH2, or Cy-NM for 2 h, respectively. After rinsing with PBS, the cells were treated according to the JC-1 mitochondrial membrane potential damage detection kit. Images were collected using confocal fluorescence imaging. The excitation wavelength for both JC-1 monomers and aggregates was 488 nm, and the emission wavelengths were 505-545 nm and 560-590 nm, respectively. The detection results are shown below. Figure 12 It can be seen that the Cy-NH2 and Cy-NM groups exhibited stronger JC-1 monomeric fluorescence signals under 660nm light source irradiation than the dark group, indicating that reactive oxygen species generated by photodynamic therapy can induce mitochondrial membrane potential damage. The Cy-NM irradiation group also showed stronger JC-1 monomeric fluorescence signals than the Cy-NM irradiation group, indicating that chemotherapy enhances the mitochondrial damage caused by photodynamic therapy.

[0094] 7. Mouse tumor targeting and in vivo organ distribution test

[0095] A subcutaneous breast cancer (4T1) tumor model was established in 6-7 week old BALB / c female mice, and the tumor volume was increased to 130 mm. 3 200 μM Cy-NM and Cy-NH2 were intravenously injected into mice, and in vivo fluorescence was monitored in real time using a small animal fluorescence imaging system from 0 to 24 hours post-injection. The excitation wavelength of Cy-NM and Cy-NH2 was 700 nm, and the emission wavelength was 750-770 nm. The detection results are shown below. Figure 13 As can be seen, the fluorescence signal in the Cy-NM group at the mouse tumor site was significantly stronger than that in the Cy-NH2 group, indicating that Cy-NM can respond and accumulate at the tumor site, demonstrating good biocompatibility. Four hours after injection, when the tumor site reached its maximum accumulation level, the major organs were dissected and fluorescence imaging was performed. The detection results are shown below. Figure 14As can be seen, Cy-NM is mainly enriched in tumor sites, with a small amount also distributed in the kidneys and liver. Cy-NH2, on the other hand, is mainly enriched in the kidneys, with lower fluorescence intensity in tumors and the liver, indicating that Cy-NM has better tumor targeting ability than Cy-NH2.

[0096] 8. Tumor inhibition effect test in mice

[0097] Cy-NM (200 μM) was injected intravenously into mice. Four hours after injection, the tumor site was treated with a 660 nm light source (300 mW cm⁻¹). -2 Treatment involved irradiation for 10 minutes, with changes in tumor volume recorded. Results are shown below. Figure 15 As can be seen, throughout the 21-day treatment cycle, the tumor volume in the Cy-NM treatment group gradually decreased and there was no recurrence, while the control group showed a 10-12 fold increase in tumor volume. The experimental results demonstrate that this prodrug has good tumor-suppressive ability. Changes in mouse body weight were recorded throughout the treatment cycle; the results are shown below. Figure 15 As can be seen, the mice's weight did not fluctuate abnormally throughout the entire treatment period, indicating that the prodrug has good biosafety.

[0098] In summary, it can be seen that the selection of photodynamic photosensitizer and chemotherapeutic drugs is key to ensuring the excellent properties of the prodrug. During the incubation process, it is particularly important that the prodrug is cleaved by azoreductase and simultaneously activates the photosensitizer and chemotherapeutic drugs. The two treatment methods can promote each other and work synergistically to ensure that the prodrug has a good tumor treatment effect.

[0099] Compared with the prior art, the prodrug compound provided by this invention has the following inventive features: excellent near-infrared photosensitizing dye properties; simultaneous activation of photosensitizers and chemotherapeutic drugs, with the two treatment methods synergistically promoting each other; specific recognition and selective killing of cancer cells; and certain tumor targeting capabilities.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of tumor-targeting prodrug compounds that synergistically enhance photodynamic / chemotherapy by azoreductase activation, characterized in that, The prodrug compound has the structure of general formula I as follows: Where X1 and X3 are H and X2 is I, or X1 and X3 are I and X2 is H; R1 is n = 1-5; R2 is a phenyl group, and R2 is a para-substituent.

2. The method for preparing a tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation as described in claim 1, characterized in that, Compound I was prepared in an anhydrous system by the following method: S1: Compound of formula IV Indole compound II, which is modified with different substituents X1, X2, and X3 at the benzene ring position and contains an R1 group, reacts with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde III in the presence of a first anhydrous organic solvent and a first catalyst to obtain compound IV. S2: Compounds of formula V for preparation The compound of formula IV reacts with m-nitrophenol in the presence of a second anhydrous organic solvent and a second catalyst to give a first intermediate. The first intermediate reacts with stannous chloride and concentrated hydrochloric acid in the presence of a third anhydrous organic solvent to give a compound of formula V. S3: Preparation of compound of formula I The compound of formula V reacts with sodium nitrite and aminosulfonic acid in anhydrous mixed organic solvent to give a second intermediate. The second intermediate reacts with a nitrogen mustard compound containing an R2 group in the presence of a fourth solvent to give the compound of formula I. Wherein, X1, X2, X3, R1, and R2 are as described in claim 1.

3. The method for preparing a type of tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation according to claim 2, characterized in that, In step S1, the molar ratio of indole compound II to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde III is 1:(2-3), the reaction time is 10-12 h, the reaction temperature is 25-40 °C, the first anhydrous organic solvent is selected from acetic anhydride, ethanol or acetonitrile, and the first catalyst is selected from anhydrous sodium acetate or anhydrous potassium carbonate.

4. The method for preparing a type of tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation according to claim 2, characterized in that, In step S2, the molar ratio of the compound of formula IV to m-nitrophenol is 1:(2-3), the reaction time is 4-5 h, the reaction temperature is 25-40 °C, the second anhydrous organic solvent is selected from anhydrous acetonitrile or anhydrous ethanol, and the second catalyst is selected from anhydrous potassium carbonate or anhydrous sodium carbonate.

5. The method for preparing a type of tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation according to claim 2, characterized in that, In step S2, the molar ratio of the first intermediate to stannous chloride is 1:(5-6), the reaction time is 10-12 h, the reaction temperature is 60-80 °C, and the third anhydrous organic solvent is selected from anhydrous ethanol or anhydrous DMF.

6. The method for preparing a tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation according to claim 2, characterized in that, In step S3, the molar ratio of the compound of formula V to sodium nitrite is 1:(2-3), the reaction time is 1-2 h, the reaction temperature is 0 °C, and the anhydrous mixed organic solvent is anhydrous acetonitrile:anhydrous dichloromethane = 1:4, v / v.

7. The method for preparing a tumor-targeting prodrug compound that synergistically enhances photodynamic / chemotherapy by azoreductase activation according to claim 2, characterized in that, In step S3, the molar ratio of the second intermediate to the nitrogen mustard compound containing the R2 group is 1:(5-6), the reaction time is 2-3 h, the reaction temperature is 0 °C, and the fourth solvent is selected from ethanol:water = 4:1, v / v or ethanol:water = 2:1, v / v.

8. The use of the azoreductase-activated, synergistically enhanced photodynamic / chemotherapy prodrug compound of claim 1 in the preparation of tumor-targeted prodrugs.

9. The use of the azoreductase-activated, synergistically enhanced photodynamic / chemotherapy prodrug compound of claim 1 in the preparation of tumor-targeted prodrugs, characterized in that, The azoreductase-activated prodrug compound that synergistically enhances photodynamic / chemotherapy can target tumor sites and localize to the mitochondria of cancer cells.

10. The use of the azoreductase-activated, synergistically enhanced photodynamic / chemotherapy-targeting prodrug compound of claim 1 in the preparation of tumor-targeting prodrugs, characterized in that, The tumor is a hypoxic solid tumor with overexpression of azoreductase.

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