Tumor-targeted prodrug compound for azo reductase activation synergistic enhancement of photodynamic / chemotherapy and preparation method and application of tumor-targeted prodrug compound for azo reductase activation synergistic enhancement of photodynamic / chemotherapy

Abstract

The invention discloses a tumor targeting prodrug compound for azo reductase activation synergistic enhancement of photodynamic / chemotherapy, a preparation method of the tumor targeting prodrug compound and application of the tumor targeting prodrug compound in cancer cell selective killing and tumor treatment. The first purpose is to provide the azo reductase activated and synergistically enhanced photodynamic / chemotherapy tumor targeting prodrug compound, the compound has the property of a positive charge cyanine dye, can target a tumor site to a certain extent and is positioned in mitochondria of cancer cells, and a photosensitizer and a chemotherapy drug are synchronously activated and released through azo reductase, so that the photodynamic / chemotherapy effect is enhanced. The selective killing and tumor targeting treatment on cancer cells are realized. The second purpose of the invention is to provide a preparation method of the prodrug compound, which comprises the following steps: in an anhydrous system, reacting an indole compound with a benzene ring site modified by different substituent groups and containing an R1 group with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-formaldehyde to obtain a compound IV; reacting the compound IV with m-nitrophenol, and reducing to obtain a compound V; finally, the compound V and sodium nitrite are subjected to a diazotization reaction, a compound containing the R2 group is added, the reaction continues, and the target prodrug compound is obtained.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a class of tumor-targeted prodrug compounds that activate azoreductase to synergistically enhance photodynamic / chemotherapy, and a preparation method and application thereof. Background Art

[0002] Chemotherapy is one of the most common means of clinical tumor treatment. Alkylating agents, as commonly used chemotherapy drugs, inactivate biomacromolecules (such as DNA, RNA, enzymes, etc.) in cells by covalently binding to them or kill cancer cells by cutting DNA double strands. However, conventional chemotherapy methods have problems such as low bioavailability and insufficient killing selectivity. The drug concentration and dose used during treatment are large, which makes the body prone to drug resistance and has serious toxic side effects on normal tissues. Photodynamic therapy (PDT) has the advantages of non-invasiveness, high spatiotemporal selectivity, high therapeutic efficiency, and low biological toxicity. It can increase the chemical sensitivity of cancer cells, reduce the dose of chemotherapy drugs, and reduce toxic side effects, making up for the shortcomings of chemotherapy. Photodynamic therapy using photosensitizers with tumor targeting and cancer cell subcellular organelles (such as nuclei, mitochondria, and lysosomes) can reduce the cellular efflux of photosensitizers and kill cancer cells in a more controllable way according to the characteristics and functions of different subcellular organelles, thereby improving the accuracy of treatment. However, due to the limited penetration depth of light and the short diffusion distance of reactive oxygen species, the area of ​​action of photodynamic therapy is limited, and it can only kill cancer cells within a certain range. Chemotherapy has the characteristics of wide distribution and range of action, which can make up for the limited area of ​​action of photodynamic therapy. Therefore, the anti-tumor system of PDT combined with chemotherapy overcomes the shortcomings of a single treatment mode and has good application prospects.

[0003] Most of the reported strategies for combined photodynamic and chemotherapy treatments are to encapsulate photodynamic photosensitizers and chemotherapy drugs into nanoparticles to achieve the combination of PDT and chemotherapy. Although this design strategy has better effects than a single treatment method, since the activity of the photosensitizers and chemotherapy drugs encapsulated in the nanoparticles is not inhibited, they can act on various cells after release, and the tumor targeting is insufficient, making it difficult to achieve efficient and selective killing of tumor cells. At the same time, the synergistic mechanism of the two treatment methods is unclear, which may cause nonspecific interactions, causing the body's immune system to produce a defensive response, resulting in counterproductive combined treatment results. Therefore, this strategy has limited efficiency in tumor treatment.

[0004] Chemically modifying drugs to prepare prodrug molecules that can be activated by specific tumor microenvironments (such as low pH, overexpressed enzymes, and oxidative / reductive substances) is an extremely effective strategy that can improve the selectivity of tumor cell killing and reduce toxic side effects. Azoreductase is one of the typical overexpressed biomarkers in hypoxic cancer cells. It can break azo bonds and is an ideal activated bioactivator for tumor treatment. Azoreductase-activatable probes and prodrugs connected by azo bonds can greatly reduce their toxic side effects and achieve tumor recognition and selective killing of cancer cells. Summary of the invention

[0005] In view of the problems that the prior art has insufficient tumor targeting and is difficult to achieve efficient and selective killing of normal cells and cancer cells, the present invention provides the following technical solutions.

[0006] The first object of the present invention is to provide a class of tumor-targeted prodrug compounds that activate azoreductase to synergistically enhance photodynamic / chemotherapy. The prodrug compounds have the properties of positively charged cyanine dyes, can target tumor sites to a certain extent and localize in the mitochondria of cancer cells, and release photodynamic photosensitizers and chemotherapeutic drugs through synchronous activation of azoreductase, thereby achieving selective killing of cancer cells and tumor-targeted treatment.

[0007] The second object of the present invention is to provide a method for preparing a class of tumor-targeted prodrug compounds that activate azoreductase to synergistically enhance photodynamic / chemotherapy. The preparation method has the advantages of easy availability of compound raw materials, simple preparation, and easy industrialization.

[0008] The third object of the present invention is to provide a type of azoreductase-activated synergistically enhanced photodynamic / chemotherapy tumor-targeted prodrug for use in the preparation of tumor treatment preparations, which has the effects of synchronously activating photodynamic photosensitizers and chemotherapy drugs, tumor targeting, selectively killing cancer cells, and achieving the synergistic promotion of photodynamic therapy and chemotherapy.

[0009] To achieve the first objective, the present invention provides the following technical solution: a class of tumor-targeted prodrug compounds activated by azoreductase for synergistic enhancement of photodynamic / chemotherapy, wherein the prodrug compound has the following general formula I structure:

[0010]

[0011] wherein 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 above second purpose, the present invention provides the following technical solution: a method for preparing a class of tumor-targeted prodrug compounds that activate azoreductase to synergistically enhance photodynamic / chemotherapy, wherein the compound of formula I is prepared by the following method in an anhydrous system:

[0015] S1: Preparation of the compound of formula IV

[0016]

[0017] An indole compound II modified with different substituents at the benzene ring position and containing 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 a compound of formula IV;

[0018] S2: Preparation of compounds of formula V

[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 obtain a first intermediate, and the first intermediate reacts with stannous chloride and concentrated hydrochloric acid in the presence of a third anhydrous organic solvent to obtain a compound of formula V;

[0021] S3: Preparation of compounds of formula I

[0022]

[0023] The compound of formula V reacts with sodium nitrite and aminosulfonic acid in an anhydrous mixed organic solvent to obtain a second intermediate, and the second intermediate reacts with a nitrogen mustard compound containing an R2 group in the presence of a fourth solvent to obtain a 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-carbaldehyde III is 1:(2-3), the reaction time is 10-12 hours, 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 4-5h, 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 10-12h, 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 1-2h, 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 2-3h, 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 above third purpose, the present invention provides the following technical solution: the use of a class of tumor-targeted prodrug compounds that activate synergistically enhanced photodynamic / chemotherapy by azoreductase, wherein the prodrug compounds activated by azoreductase synergistically enhance photodynamic / chemotherapy can target the tumor site to a certain extent and localize in the mitochondria of cancer cells, and are used for selective killing of cancer cells and tumor treatment, wherein the tumor is a hypoxic solid tumor that overexpresses azoreductase.

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

[0031] First, the azoreductase activated synergistically enhanced photodynamic / chemotherapy tumor-targeted prodrug compound prepared by the present invention has an absorption and emission wavelength of about 700 nm, and has excellent near-infrared photosensitizing dye properties; the prodrug compound has the properties of positively charged cyanine dyes, can target tumor sites to a certain extent and locate in the mitochondria of cancer cells; and the introduction of a specifically responsive linker in the prodrug compound ensures that it has sensitive responsiveness and high selectivity to azoreductase;

[0032] Second, the present invention combines a photodynamic photosensitizer with a chemotherapeutic drug. Azoreductase is a substance overexpressed by hypoxic cancer cells. After the prodrug compound is activated by azoreductase, a semi-cyanine dye photosensitizer and a chemotherapeutic drug nitrogen mustard are released, which can synchronously activate the photosensitizer and the chemotherapeutic drug, and the two treatment methods promote each other synergistically.

[0033] Third, after activation, the semi-cyanine dye has an ICT effect, which restores fluorescence, and the absorbed light energy is released in the form of fluorescence and intersystem crossing, which improves the fluorescence imaging and photodynamic performance of the photosensitizer; through specific activation, the chemotherapy drugs are accurately released in cancer cells, showing excellent cancer cell selectivity; the synergistic mechanism of photodynamic therapy and chemotherapy is discussed in more depth. The photodynamic performance of the semi-cyanine dye can improve the release efficiency of chemotherapy drugs and improve the chemotherapy effect. Similarly, chemotherapy drugs can enhance the ability of photodynamic action to damage mitochondria and enhance the effect of photodynamic therapy. The two work together to greatly promote the killing effect of cancer cells.

[0034] Fourth, it has good biocompatibility. In the mouse tumor inhibition experiment, the prodrug can image and treat mouse tumors, showing excellent tumor inhibition effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0036] Figure 1 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 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 the mixture of Cy-NM and SDT (0-5mM) disclosed in the embodiment of the present invention;

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

[0040] Figure 5The singlet oxygen generation capacity test diagram of Cy-NH2 and Cy-NM disclosed in the embodiment of the present invention; Figure A is a graph showing the absorption change of DPBF of Cy-NH2 under 660nm light irradiation; Figure B is a graph showing the absorption change of DPBF of Cy-NM under 660nm light irradiation; Figure C is a quantitative absorption attenuation curve of DPBF at 415nm of DPBF, Cy-NM, Cy-NH2 and MB under 660nm light irradiation;

[0041] Figure 6 The confocal imaging cell uptake diagram of Cy-NM disclosed in the embodiment of the present invention for 4T1, HepG2 and BEAS-2B cells; Figure A is a confocal imaging diagram of Cy-NM; Figure B is a data quantification diagram of fluorescence imaging;

[0042] Figure 7 It is a subcellular organelle localization map of Cy-NM in 4T1 cells disclosed in an embodiment of the present invention; a, b, c and d respectively represent Cy-NM staining, commercial mitochondrial dye staining, staining superposition, and superposition localization coefficient map; e, f, g and h respectively represent Cy-NM staining, commercial lysosomal dye staining, staining superposition, and superposition localization coefficient map; i, j, k and l respectively represent Cy-NM staining, commercial nuclear dye staining, staining superposition, and superposition localization coefficient map;

[0043] Figure 8 The phototoxicity test diagram of 4T1 cells after incubation with different concentrations of Cy-NM under normoxic and hypoxic conditions; Figure 8 Middle: The selected excitation light is a 660nm near-infrared light source with a light dose of 40mW cm -2 , 5min;

[0044] Fig. 9 The Cy-NM disclosed in the embodiment of the present invention is respectively in the dark and under a 660nm light source (40mW cm -2 , 5min) irradiation for 4T1, HepG2 and BEAS-2B cells light / dark toxicity test diagram;

[0045] Fig.10 The gel electrophoresis images of pBR322 DNA alkylation by Cy-NM, Cy-NH2 and NM respectively;

[0046] Fig.11 These are confocal images of γ-H2AX immunofluorescence staining of 4T1 cells by Cy-NM and Cy-NH2 disclosed in the embodiments of the present invention under light and dark conditions, respectively;

[0047] Fig.12The graphs of the mitochondrial membrane potential test of 4T1 cells by Cy-NM and Cy-NH2 disclosed in the embodiments of the present invention under light and dark conditions, respectively;

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

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

[0050] Fig.15 This is a tumor inhibition experiment of Cy-NM in a living anti-tumor model disclosed in an embodiment of the present invention; Figure A is a graph showing the changes in tumor volume after Cy-NM treatment; Figure B is a graph showing the changes in body weight after Cy-NM treatment. DETAILED DESCRIPTION

[0051] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the appended drawings of the embodiments of the present invention. Figure 1-15 , the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] The chemicals involved in this application are all from Anage or Aladdin Chemical Reagent Company, the biological consumables fetal bovine serum, trypsin, and DMEM culture medium are purchased from Giboca, the cells used are from the ATCC cell bank, and other raw materials are commercially available without 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 Preparation of a class of azoreductase-activated tumor-targeted prodrug compounds that synergistically enhance photodynamic / chemotherapy, the synthesis method is as follows:

[0056]

[0057] (1) Under N2 protection, sodium nitrite (12.6 g, 182.6 mmol) was dissolved in water (45 mL), 4-iodoaniline (20 g, 91.3 mmol) was dissolved in hydrochloric acid (5.5 M, 15 mL), cooled to 0°C, added dropwise to the reaction system, and stirred at room temperature for 3 h. SnCl2·2H2O (67.99 g, 301.3 mmol) was dissolved in concentrated hydrochloric acid (40 mL), added dropwise to the reaction system at 0°C, stirred in an ice bath for 2 h, and stirred at room temperature overnight. A light brown precipitate was filtered, washed three times with water, dissolved in a saturated NaOH solution, extracted with ethyl acetate and Na2S2O3, and the organic layer was spin-dried to obtain compound 1 as 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 stirred at 118°C for 12 h. After the reaction was completed, the solvent was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v / v) to obtain compound 2, which is an indole compound wherein X1 and X3 are H and X2 is I, a red oil, 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), heated to 110°C and stirred for 12 h. The reaction mixture was cooled to room temperature, the crude product was washed with ether (150 mL), and then cooled in an ice bath for a few minutes. The solvent was filtered off and the residue was collected to obtain compound 3, which is an indole compound with R1 being CH2CH3, as a pink solid with a yield of 90%;

[0060] (4) Under N2 protection, compound 3 (0.8 g, 1.8 mmol), 2-chloro-3-(hydroxymethylene)cyclohex-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 completed, the solvent was evaporated under reduced pressure to obtain a crude product which was purified by silica gel column chromatography (dichloromethane: methanol = 100: 3, v / v) to obtain compound 4 as a green solid with a yield of 70%;

[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 completed, the solvent was evaporated under reduced pressure, and the precipitate was dissolved in dichloromethane, washed three times with water and dried with Na2SO4 to obtain the first intermediate. The first intermediate was dissolved in methanol (30 mL) for use in the next step.

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

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

[0064] The H-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 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 stirred 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 stirred 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 completed, the mixture was extracted with dichloromethane and the solvent was evaporated under reduced pressure to obtain a crude product which 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 spectrum of target compound Cy-NM: m / z C 37 H 38 Cl2I2N4O([MI] + ): Calculated value 751.1462, tested value 751.1498.

[0067] The H-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 NMR carbon 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 examples, 3 mM stock solutions of the prepared Cy-NM and Cy-NH2 were prepared respectively, and their absorption and fluorescence spectra at a concentration of 3 μM were tested in dichloromethane.

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

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

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

[0074] 2.1. During the preparation process of the above examples, the absorption of the prepared Cy-NH2 and Cy-NM at 680nm was monitored by 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 2h, the absorption change at 680nm was monitored by HPLC in the same manner. The results are shown in Table 2. Figure 2 Different concentrations of SDT were added to Cy-NM to obtain Cy-NM+SDT, which was then incubated at 37°C and its fluorescence changes were detected after 2 h. The results are shown in Figure 3 (Solution test using SDT instead of azoreductase)

[0075] like Figure 2As shown in the liquid chromatography of Cy-NH2, Cy-NM and Cy-NM+SDT, after reacting with SDT for 2h, the chromatographic peak intensity of Cy-NM (retention time: 18min) decreased, and the chromatographic peak of Cy-NH2 (retention time: 12min) appeared, indicating that SDT can be converted into Cy-NH2 after reacting with Cy-NM. Figure 3 As shown, the higher the concentration of SDT, the more the fluorescence of Cy-NM increases, indicating that SDT can cut the azo bond of Cy-NM to release Cy-NH2 and achieve the effect of fluorescence recovery.

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

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

[0078] Combining the embodiments and Figure 2 as well as Figure 3 It can be seen that the mechanism of the target compound Cy-NM prepared in the example responding to azoreductase is:

[0079]

[0080] In the embodiment of the present application, the semi-cyanine dye and the chemotherapeutic drug are connected by an azo bond, and the azoreductase overexpressed in the hypoxic cancer cells can cut the azo bond and synchronously activate the 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 into cuvettes containing Cy-NH2, Cy-NM and methylene blue solution, respectively, and a 660 nm light source (3 mW cm -2 ) irradiation, and the photodynamic performance under irradiation was tested. Each sample was irradiated for 150s, and its absorption spectrum was measured every 15s. The test results are shown in Figure 5 It can be seen that in the Cy-NH2 system, the absorbance of DPBF at 415nm decreased significantly, while in the Cy-NM system, the absorbance of DPBF did not change significantly, indicating that Cy-NH2 has a higher 1 O2 production capacity.

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

[0084] 4.1. 4T1 cells were cultured in a 37°C, 5% CO2 constant temperature cell culture incubator and cultured in DMEM high glucose medium containing 10% serum and 1% double antibody. The O2 content under hypoxic conditions was 2%, and the O2 content under normoxic conditions was 21%. Cy-NM was added to the cell imaging dishes containing 4T1, HepG2 and BEAS-2B cells, respectively, and incubated for 2 hours under normoxic and hypoxic conditions, respectively. After rinsing the cells with PBS, confocal fluorescence imaging was performed and images were collected. The test results are shown in Figure 6 It can be seen that under hypoxic conditions, obvious Cy-NM fluorescence signals were observed in 4T1 and HepG2 cells, while there was almost no fluorescence 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 shows that Cy-NM can only be activated in hypoxic cancer cells, but not in normoxic and normal cells.

[0085] 4.2. 4T1 cells were seeded in a confocal culture dish, Cy-NM (2 μM) was added to the imaging dish and incubated for 2 h, and commercial dyes Mito (100 nM, mitochondria), Lyso (100 nM, lysosomes), and Hoechst (2 μg mL -1 , cell nuclei) were added to the Cy-NM treated cells and co-incubated for 30 minutes. Laser confocal microscopy was used to image and measure the counterstaining coefficient of Cy-NM and commercial dye. The excitation wavelength of Cy-NM is 640nm, and the emission wavelength is 660-750nm; the excitation wavelength of Mito and Lyso is 488nm, and the emission wavelength is 500-540nm; the excitation wavelength of Hoechst is 405nm, and the emission wavelength is 440-480nm. The test results are shown in Figure 7 , it can be seen that Cy-NM can be well localized in the mitochondria of 4T1 cells.

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

[0087] 5.1. 4T1 cells were incubated with Cy-NM at a concentration of 0-16 μM for 2 h under normoxic and hypoxic conditions, and then illuminated with a 660 nm light source (40 mW cm -2 ) were illuminated to compare the killing effect of Cy-NM on 4T1 cells under normoxic and hypoxic conditions. Figure 8It can be seen that the Cy-NM hypoxia group has a stronger killing effect on 4T1 cells than the normoxic group. These differences indicate that after Cy-NM enters hypoxic cancer cells, the azo bond is cut by azoreductase, and the released Cy-NH2 works together with NM to enhance cytotoxicity. Under normoxic conditions, Cy-NM cannot be activated and has basically 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 cells. The test results are shown in Fig. 9 .

[0089] The three types of cells were incubated under hypoxic conditions with 0-16 μM Cy-NM, and then exposed to darkness and 660 nm light (40 mW cm -2 ) were treated with light. After 24 h, MTT solution (5 mg mL -1 ), after incubation for 4 h, 100 μL DMSO was added and the absorbance at 490 nm was measured. Fig. 9 Cy-NM was observed in the dark and under a 660 nm light source (40 mW cm -2 ,5min) irradiation for different cell killing cell survival rate diagram, from Fig. 9 It can be seen that the dark toxicity of Cy-NM to cancer cells is higher than that of normal cells, indicating that the chemotherapy drugs released after Cy-NM activation can kill cancer cells. The phototoxicity of Cy-NM to cancer cells is significantly higher than that of normal cells, indicating that Cy-NM has good cancer cell killing selectivity.

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

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

[0092] 6.2. Further verification by γ-H2AX immunofluorescence staining experiment: 4T1 cells were incubated with PBS, Cy-NH2 or Cy-NM for 2 hours. After rinsing the cells with PBS, the cells were treated according to the DNA damage detection kit. Confocal fluorescence imaging was performed and images were collected. The excitation wavelengths of DAPI and γ-H2AX were 405nm and 488nm, respectively, and the emission wavelengths were 415-485nm and 500-545nm, respectively. The test results are shown in Fig.11 It can be seen that the fluorescence signal of the Cy-NM group is significantly stronger than that of the Cy-NH2 group, indicating that Cy-NM can alkylate the DNA of 4T1 cells. Under the irradiation of the 660nm light source, the fluorescence signal of the Cy-NM group is further enhanced, indicating that the photodynamic effect enhances the alkylation ratio of chemotherapeutic drugs.

[0093] 6.3. Use JC-1 fluorescence imaging to verify the changes in mitochondrial membrane potential in 4T1 cells: 4T1 cells were incubated with PBS, Cy-NH2 or Cy-NM for 2 hours. After rinsing the cells with PBS, the cells were treated according to the JC-1 mitochondrial membrane potential damage detection kit. Confocal fluorescence imaging was used to collect images. The excitation wavelength of JC-1 monomers and aggregates was 488nm, and the emission wavelengths were 505-545nm and 560-590nm, respectively. The test results are shown in Fig.12 It can be seen that the Cy-NH2 and Cy-NM groups have stronger JC-1 monomer fluorescence signals under 660nm light source than the dark group, indicating that the reactive oxygen species generated by photodynamics can induce mitochondrial membrane potential damage. The Cy-NM illumination group has a stronger JC-1 monomer fluorescence signal than the Cy-NM illumination group, indicating that chemotherapy enhances the mitochondrial damage ability of photodynamics.

[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. 3 200 μM Cy-NM and Cy-NH2 were injected into mice by intravenous injection, and the fluorescence in vivo was monitored in real time by a small animal fluorescence imager 0-24 h after injection. The excitation wavelength of Cy-NM and Cy-NH2 was 700 nm, and the emission wavelength was 750-770 nm. The test results are shown in Fig.13 It can be seen that the fluorescence signal of the Cy-NM group in the mouse tumor site is significantly stronger than that of the Cy-NH2 group, indicating that Cy-NM can respond and enrich in the tumor site and has good biocompatibility. When the tumor site reaches the maximum enrichment 4 hours after injection, the main organs are dissected and fluorescently imaged. The test results are shown in Fig.14It can be seen that Cy-NM is mainly enriched in the tumor site, and is also distributed in the kidney and liver to a small extent. Cy-NH2 is mainly enriched in the kidney, and the fluorescence intensity in the tumor and liver is lower, indicating that Cy-NM has better tumor targeting ability than Cy-NH2.

[0096] 8. Mouse tumor inhibition effect test

[0097] Cy-NM (200 μM) was injected into mice intravenously. 4 h after injection, the tumor was illuminated with a 660 nm light source (300 mW cm -2 ) irradiation for 10 minutes for treatment, and the changes in tumor volume were recorded. The test results are shown in Fig.15 It can be seen that during the entire 21-day treatment cycle, the tumor volume of 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 show that the prodrug has good tumor inhibition ability. The changes in mouse weight during the entire treatment cycle were recorded, and the test results are shown in Fig.15 ,It can be seen that there is no abnormal fluctuation in the weight of mice during 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 the key to ensuring that the prodrug has excellent properties. During the incubation process, it is particularly important that the prodrug is cut by azoreductase and the photosensitizer and chemotherapeutic drugs are activated at the same time. The two treatment methods can promote each other synergistically, ensuring that the prodrug has a good tumor treatment effect.

[0099] Compared with the prior art, the prodrug compound provided by the present invention has the following creativity: excellent near-infrared photosensitizing dye properties; synchronous activation of photosensitizers and chemotherapeutic drugs, and the two treatment methods synergistically promote 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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-targeted prodrug compounds activated by azoreductase for synergistic enhancement of photodynamic / chemotherapy, characterized in that: The prodrug compound has the following structure of general formula I: wherein 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 And R2 is a para substituent.

2. The method for preparing a class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase according to claim 1, characterized in that: In an anhydrous system, the compound of formula I is prepared by the following method: S1: Preparation of the compound of formula IV An indole compound II modified with different substituents X1, X2, X3 at the benzene ring position and containing 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 a compound of formula IV; S2: Preparation of compounds of formula V The compound of formula IV reacts with m-nitrophenol in the presence of a second anhydrous organic solvent and a second catalyst to obtain a first intermediate, and the first intermediate reacts with stannous chloride and concentrated hydrochloric acid in the presence of a third anhydrous organic solvent to obtain a compound of formula V; S3: Preparation of compounds of formula I The compound of formula V reacts with sodium nitrite and aminosulfonic acid in an anhydrous mixed organic solvent to obtain a second intermediate, and the second intermediate reacts with a nitrogen mustard compound containing an R2 group in the presence of a fourth solvent to obtain a compound of formula I.

3. The method for preparing a class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase according to claim 2, characterized in that: In step S1, the molar ratio of the indole compound II to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde III is 1:(2-3), the reaction time is 10-12 hours, 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 class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase 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 4-5h, 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 class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase 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 hours, 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 class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase 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 1-2h, 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 class of tumor-targeted prodrug compounds for synergistically enhancing photodynamic / chemotherapy activated by azoreductase according to claim 2, characterized in that: 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 hours, 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. Use of the tumor-targeted prodrug compound activated by azoreductase for synergistically enhancing photodynamic / chemotherapy according to claim 1 in the preparation of tumor-targeted prodrugs.

9. Use of a class of tumor-targeted prodrug compounds activated by azoreductase for synergistically enhancing photodynamic / chemotherapy according to claim 8 in the preparation of tumor-targeted prodrugs, characterized in that: The azoreductase-activated synergistically enhanced photodynamic / chemotherapy prodrug compound can target tumor sites and localize in the mitochondria of cancer cells.

10. Use of a class of tumor-targeted prodrug compounds activated by azoreductase for synergistically enhancing photodynamic / chemotherapy according to claim 9 in the preparation of tumor-targeted prodrugs, characterized in that: The tumor is a hypoxic solid tumor with overexpression of azoreductase.

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