Unconventional dye for tumor microenvironment regulation photosensitive Type I / II process switching as well as synthesis method and application of unconventional dye

Abstract

The invention discloses nitroreductase activated selenium-substituted hemicyanine dye as well as a synthesis method and application thereof, and belongs to the field of fine chemical engineering. The structure of the nitroreductase activated selenium-substituted hemicyanine dye is as shown in a general formula I. In the general formula I, R1 is one of H, phenyl, p-phenyl benzyl chloride, p-phenyl benzyl bromide, SO3R4, COOR4 or C1-C12 alkyl; r2 is C (CH3) 2, S or Se; r3 is one of H, a phenyl group or a C1-C12 alkyl group; r4 is one of H or alkyl with 1 to 12 carbon atoms; y is Cl, Br or I. The nitroreductase activated selenium-substituted hemicyanine dye provided by the invention has spectral properties of near-infrared absorption and activatable near-infrared emission and high active oxygen generation capacity, and provides an ideal platform for cancer cell diagnosis and treatment. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of fine chemical engineering, and relates to an activatable selenium-substituted semi-cyanine dye, a synthesis method thereof, and an application thereof. Background Art

[0002] In recent years, the photodynamic therapy (PDT) method has attracted the research interest of scientific researchers as a non-invasive and real-time operable tumor treatment means, and it is an effective means for treating diseases by using the photodynamic effect of photosensitizer dyes. Especially in the treatment of tumors, it has received extensive attention due to its superior spatial specificity, non-invasiveness, good treatment effect, and low drug resistance. In current clinical tumor photodynamic therapy: the photosensitizer dye is initially in the ground state (S 0 ), and after being excited by incident light of a specific wavelength, the photosensitizer molecule absorbs photon energy and jumps to the excited singlet state (S 1 ). Due to the short lifetime of S 1 , the photosensitizer molecule undergoes intersystem crossing (ISC) to reach the relatively stable excited triplet state (T 1 ). According to different reaction processes, T 1 can trigger two different reaction mechanisms, namely Type I and Type II reactions. In the process of Type I reaction, electron transfer occurs to generate free radicals, mainly superoxide anions (O 2·- ) and hydroxyl radicals (·OH). In the process of Type II reaction, energy transfer occurs to generate cytotoxic 1 O 2 . Thus, it can be seen that the performance of the photosensitizer dye largely determines the tumor PDT effect. However, most of the reported photosensitive dyes can only generate a single type of reactive oxygen species of Type I or Type II during photodynamic therapy. Therefore, it is necessary to develop an unconventional dye for regulating the switching of the photosensitive Type I / II process in the tumor microenvironment.

[0003] Fluorescence imaging is an emerging imaging technology with advantages such as simple operation, high resolution, and low harm to organisms. It provides a powerful tool for biological research and biomedical applications and is widely used in research fields such as oncology, mechanism of drug action, and in vivo fluorescence imaging. Currently, the organic fluorescent probes used in fluorescence imaging can be divided into: "always-on" and "activatable / responsive" probes. Most of the fluorescent probes in current research are the former, and the fluorescent signal always exists, which leads to a low signal-to-noise ratio (S / N) when the probe is used for in vivo imaging and cannot distinguish normal tissues from cancer cells; while the "activatable / responsive" fluorescent probe remains silent before being activated by specific cell markers, which enables the fluorescent probe to have a high recognition ability for cancer cells. In particular, near-infrared fluorescence (NIR) imaging, due to its use of excitation light and emission light with longer wavelengths and lower energies, in the near-infrared region Fluorescence imaging can effectively avoid the interference of self-absorption and autofluorescence of biological tissues and become the "optical window" of in vivo imaging. Hemicyanine dyes, due to their good biocompatibility, excellent photophysical properties, and highly diverse structural tunability, are often used as near-infrared fluorescent reporter molecules and are widely used in biological imaging and bio-labeling. Therefore, it is necessary to develop hemicyanine dyes that have the function of monitoring the switching of photosensitizing Type I / II processes in tumor microenvironment regulation with near-infrared fluorescence. Summary of the Invention

[0004] To solve the problems existing in the prior art, the present invention provides a nitroreductase-activated selenium-substituted hemicyanine dye and its synthesis method and application. The present invention regulates the photodynamic therapy effect of the dye molecule through the tumor marker nitroreductase, and regulates the types of reactive oxygen species through the specifically expressed nitroreductase in tumors during photodynamic therapy, greatly enhancing the photosensitive property while maintaining low dark toxicity.

[0005] In the first aspect, the present invention provides a nitroreductase-activated selenium-substituted hemicyanine dye, the structure of which is shown in General Formula I:

[0006]

[0007] In General Formula I:

[0008] R 1 is one of H, phenyl, p-phenylbenzyl chloride, p-phenylbenzyl bromide, SO 3 R 4 、COOR 4 or C 1 -C 12 alkyl (such as methyl);

[0009] R 2 is C(CH 3 ) 2 、S or Se;

[0010] R 3 is one of H, phenyl or C 1 -C 12 alkyl;

[0011] R 4 is H or C 1 -C 12 alkyl.

[0012] Y is Cl, Br or I.

[0013] Preferably, R 2 is Se.

[0014] Preferably, R 3 is H or phenyl.

[0015] Preferably, Y is Br or I.

[0016] In a second aspect, the present invention provides a method for synthesizing the above-mentioned nitroreductase-activated selenium-substituted hemicyanine dye, comprising the following steps:

[0017] (1) Compound 1 and Compound 2 are reacted in a molar ratio of 1:3.0 - 5.0 to prepare Compound 3;

[0018]

[0019] (2) Compound 3 and Compound 4 are reacted in a molar ratio of 2.0 - 2.5:1 to prepare Compound 5;

[0020]

[0021] (3) Compound 6 and Compound 7 are reacted in a molar ratio of 1:2.0 - 3.0 to prepare Compound 8;

[0022]

[0023] (4) Compound 5 and Compound 8 are reacted in a molar ratio of 1:1.2 - 1.5 to prepare the compound of general formula I.

[0024]

[0025] (5) Compound 9 and Compound 10 are reacted in a molar ratio of 1:4.0 - 5.0 to prepare the compound of general formula I.

[0026]

[0027] Furthermore, in the above technical solution, in step (1), the reaction solvent of Compound 1 and Compound 2 is anhydrous acetonitrile, and the reaction condition is to react at 85 - 90 °C for 4 - 5 h.

[0028] Further, in the above technical solution, in step (2), the reaction solvent of compound 3 and compound 4 is a mixed solution of acetic acid, acetic anhydride and triethylamine, and the reaction condition is to react at 65-70 °C for 1-3 h.

[0029] Further, in the above technical solution, in the mixed solution, the volume ratio of acetic acid, acetic anhydride and triethylamine is 2-3:1:1.

[0030] Further, in the above technical solution, in step (3), the reaction solvent of compound 6 and compound 7 is dimethyl sulfoxide, using AgNO 3 as a catalyst, and the reaction condition is to react at 120-130 °C for 2-3 h. Among them, the molar ratio of compound 6 to AgNO 3 is 1:3.0-5.0.

[0031] Further, in the above technical solution, in step (4), before compound 8 reacts with compound 5, the selenol intermediate is first reduced with a sodium borohydride and citric acid system; the reaction solvent of the selenol intermediate and compound 5 is anhydrous acetonitrile, using cesium carbonate as a catalyst, and under N 2 protection, react at 65-70 °C for 2-3 h. Among them, the molar ratio of compound 5 to cesium carbonate is 1:5.0-7.0.

[0032] Further, in the above technical solution, the preparation method of the selenol intermediate is: compound 8 is first reacted in N 2 protection and at 0±1 °C for 10-20 min in the presence of NaBH 4 using anhydrous ethanol as a solvent, and then citric acid is added and the reaction continues for 5-8 min. The reaction solution is extracted with ether and water (for example, the volume ratio of ether to water is 1-2:1), and the organic layer is washed with a mixed aqueous solution of saturated NH 4 Cl and saturated NaCl (for example, the volume ratio of saturated NH 4 Cl solution to saturated NaCl aqueous solution is 1:1), dried with Na 2 SO 4 , and the ether solvent is removed under reduced pressure to obtain the selenol intermediate. Among them, the molar ratio of compound 8 to NaBH 4 is 1:2.0-4.0, preferably 1:3.0; the molar ratio of compound 8 to citric acid is 1:4.0-7.0, preferably 1:4.0-6.0, more preferably 1:5.0.

[0033] Further, in the above technical solution, in step (5), compound 9 is first reacted under potassium carbonate conditions for 30 - 40 min, and then reacted with compound 10. The reaction solvent is anhydrous acetonitrile, and the reaction is carried out at 60 - 65 °C for 5 - 6 h. Among them, the molar ratio of compound 9 to potassium carbonate is: 1 - 4.0 - 5.0.

[0034] In the third aspect, the present invention provides an application of the above nitroreductase-activated selenium-substituted hemicyanine dye in the preparation of fluorescent dyes or tumor diagnosis and treatment drugs, especially in cell imaging and photodynamic killing of cancer cells.

[0035] Further, in the above technical solution, the nitroreductase-activated selenium-substituted hemicyanine dye is used to regulate the switching of the photosensitive Type I / II process in the tumor microenvironment.

[0036] The nitroreductase-activated selenium-substituted hemicyanine photosensitizer dye of the present invention can regulate the types of reactive oxygen species through nitroreductase in tumors, greatly reducing the dependence on oxygen to a large extent. The fluorescence of the nitroreductase-activated selenium-substituted hemicyanine photosensitizer dye of the present invention can be changed from "off" to "on" through the tumor marker nitroreductase, greatly improving the detection sensitivity, having a high signal-to-noise ratio, and enabling precise identification and specific photodynamic killing of cancer cells. The nitroreductase-activated selenium-substituted hemicyanine dye provided by the present invention has spectral properties of near-infrared absorption and activatable near-infrared emission and a high reactive oxygen species generation ability, providing an ideal platform for cancer cell diagnosis and treatment.

[0037] Beneficial effects:

[0038] The present invention utilizes the differences in the tumor microenvironment to construct a nitroreductase fluorescence-activatable photosensitizer dye, and can regulate the types of reactive oxygen species of the photosensitizer dye during photodynamic therapy through nitroreductase, greatly reducing the dependence of the photosensitizer on oxygen compared with simple Type II photosensitizers. This type of dye has good biocompatibility, can penetrate cell membranes efficiently and rapidly, has a good nitroreductase response in tumor cells, and has been successfully applied to the imaging and treatment of tumor cells. Description of the drawings

[0039] Figure 1 It is the ultraviolet-visible absorption spectrum a) and fluorescence emission spectrum b) of the dye (10 μM) in Example 1 of the present invention in a PBS solution (containing 10% DMSO). Wherein a) the abscissa is the wavelength (nm), and the ordinate is the absorption intensity; b) the abscissa is the wavelength (nm), and the ordinate is the fluorescence intensity, and the excitation light wavelength is 760 nm.

[0040] Figure 2It is the evaluation of the response of the dye in Example 1 of the present invention to nitroreductase in vitro. Among them, a) is the fluorescence response of the dye to nitroreductase. b) is the specific response of the dye fluorescence to nitroreductase (where 1. ammonium chloride, 2. sodium sulfate, 3. zinc chloride, 4. ferric trichloride hexahydrate, 5. L-ascorbic acid, 6. L-glutamic acid, 7. L-cystine, 8. L-cysteine, 9. glutathione, 10. sodium acetate, 11. sodium carbonate, 12. sodium chloride, 13. potassium chloride, 14. glucose, 15. citric acid, 16. L-isoleucine, 17. L-histidine, 18. L-arginine, 19. L-phenylalanine, 20. L-threonine, 21. L-aspartic acid, 22. nitroreductase and NADH).

[0041] Figure 3 It is the evaluation of the ability of the dye in Example 1 of the present invention to generate reactive oxygen species. Among them, a) is the change in the ultraviolet-visible absorption spectrum of the DPBF singlet oxygen scavenger and HCySe-O-NO 2 under irradiation by a 760 nm laser for different times; b) is the change in the fluorescence spectrum of the HPF hydroxyl radical scavenger and the dye HCySe-O-NO 2 under irradiation by a 760 nm laser for different times; c) is the change in the fluorescence spectrum of the DHR123 superoxide anion radical scavenger and HCySe-O-NO 2 under irradiation by a 760 nm laser for different times. At the same time, the ability of the fluorophore parent compound 9HCySe-OH to generate reactive oxygen species was increased, and the test types and methods were the same as above. d) is the change in the ultraviolet-visible absorption spectrum of the DPBF singlet oxygen scavenger and HCySe-OH under irradiation by a 760 nm laser for different times; e) is the change in the fluorescence spectrum of the HPF hydroxyl radical scavenger and the dye HCySe-OH under irradiation by a 760 nm laser for different times; f) is the change in the fluorescence spectrum of the DHR123 superoxide anion radical scavenger and HCySe-OH under irradiation by a 760 nm laser for different times.

[0042] Figure 4 It is the test of the cell viability of the dye in Example 1 of the present invention. Among them, a) is HCySe-O-NO 2 The dark / light toxicity test of the dye on breast cancer cells under normoxic culture conditions; b) is HCySe-O-NO 2 The dark / light toxicity test of the dye on breast cancer cells under hypoxic culture conditions.

[0043] Figure 5Cell imaging characterization of the dye of Example 1 of the present invention. Among them, a) is the normal oxygen culture time of cells for 20 min; b) is the normal oxygen culture time of cells for 40 min; c) is the normal oxygen culture time of cells for 60 min; d) is the hypoxic culture time of cells for 20 min; e) is the hypoxic culture time of cells for 40 min; f) is the hypoxic culture time of cells for 60 min;

[0044] Figure 6 Verification of the production of reactive oxygen species by the dye of Example 1 of the present invention in cells. Among them, a) is the normoxic group, and b) is the hypoxic group.

[0045] Figure 7 Live / dead cell staining of the dye of Example 1 of the present invention. Among them, a) is the normoxic dark treatment group; b) is the normoxic light treatment group; c) is the hypoxic dark treatment group; d) is the hypoxic light treatment group. Detailed implementation manners

[0046] For better explaining the present invention and facilitating understanding, the present invention will be described in detail below with reference to the accompanying drawings through specific implementation manners.

[0047] To better understand the above technical solutions, the exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more clear and thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0048] The present invention first provides a nitroreductase-activated selenium-substituted hemicyanine dye having the structure of General Formula I:

[0049] In a first aspect, the present invention provides a nitroreductase-activated selenium-substituted hemicyanine dye, the structure of which is shown in General Formula I:

[0050]

[0051] In General Formula I:

[0052] R 1 is one of H, phenyl, p-phenylbenzyl chloride, p-phenylbenzyl bromide, SO 3 R 4 , COOR 4 or C 1 -C 12 alkyl;

[0053] R 2 is C(CH 3 ) 2 , S or Se;

[0054] R 3is H, phenyl or C 1 -C 12 is one of the following alkyl groups;

[0055] R 4 is H or C 1 -C 12 is one of the following alkyl groups

[0056] Y is Cl, Br or I.

[0057] Preferably, R 2 is Se.

[0058] Preferably, R 3 is H or phenyl.

[0059] Preferably, Y is Br or I.

[0060] On the other hand, the present invention provides a method for synthesizing an activation-type selenium-substituted hemicyanine dye of nitroreductase, comprising the following steps:

[0061] (1) Reacting compound 1 and compound 2 in a molar ratio of 1:3.0 to 5.0 to prepare compound 3;

[0062]

[0063] (2) Reacting compound 3 and compound 4 in a molar ratio of 2.0 to 2.5:1 to prepare compound 5;

[0064]

[0065] (3) Reacting compound 6 and compound 7 in a molar ratio of 1:2.0 to 3.0 to prepare compound 8;

[0066]

[0067] (4) Reacting compound 5 and compound 8 in a molar ratio of 1:1.2 to 1.5 to prepare a compound of general formula I.

[0068]

[0069] (5) Reacting compound 9 and compound 10 in a molar ratio of 1:4.0 to 5.0 to prepare a compound of general formula I.

[0070]

[0071] In a specific embodiment, the method for preparing the selenium-substituted hemicyanine dye mother body comprises the following steps:

[0072] (1) Compounds 1 and 2 were added to anhydrous acetonitrile solvent in a molar ratio of 1:3.0 - 5.0, and the mixture was refluxed at 85 - 90 °C for 4 - 5 h. After the reaction ended, the system was cooled to obtain a solid. The obtained crude product was added to acetonitrile to form a supersaturated hot solution, which was left standing in a 4 °C refrigerator for 12 - 14 h to obtain pure compound 3.

[0073] (2) Compounds 3 and 4 were added to a mixed solution of acetic acid, acetic anhydride and triethylamine (the volume ratio of acetic acid:acetic anhydride:triethylamine was 2 - 3:1:1) in a molar ratio of 2.0 - 2.5:1, and the reaction was carried out at 65 - 70 °C for 1 - 3 h. Subsequently, the solvent was removed under reduced pressure, and then dichloromethane and saturated brine were added successively for extraction 3 times. The organic phase was collected, dried with anhydrous sodium sulfate, and the solvent was removed to obtain a crude product. After purification by column separation technology (the volume ratio of the eluent system was dichloromethane:methanol = 100:2 - 100:5), compound 5 was obtained.

[0074] (3) Compounds 6 and 7 were added to dimethyl sulfoxide solvent in a molar ratio of 1:2.0 - 3.0 and stirred, and then an appropriate amount of AgNO 3 (the molar ratio of compound 6 to AgNO 3 was 1:3.0 - 5.0) was added, and the reaction was carried out at 120 - 130 °C for 2 - 3 h. After removing the solvent under reduced pressure, dichloromethane and saturated brine were added successively for extraction 3 times. The organic phase was collected, dried with anhydrous sodium sulfate, and the solvent was removed to obtain a crude product. After purification by column separation technology (the volume ratio of the eluent system was petroleum ether:ethyl acetate = 5:1 - 2:1), compound 8 was obtained.

[0075] (4) Compound 8 was first reacted in anhydrous ethanol as a solvent with NaBH 2 at N 4 (the molar ratio of compound 8 to NaBH 4 was 1:4.0 - 5.0) at 0 °C for 10 - 20 min, and then citric acid (the molar ratio of compound 8 to citric acid was 1:4.0 - 7.0) was added to continue the reaction for 5 - 8 min to obtain a selenol intermediate. The reaction solution was diluted with ether and deionized water was added (the volume ratio of ether to deionized water was 1 - 2:1). The organic layer was washed with a mixed aqueous solution of saturated NH 4 Cl and saturated NaCl (the volume ratio of saturated NH 4 Cl solution to saturated NaCl aqueous solution was 1:1), and Na 2 SO 4Dry, remove the ethyl ether solvent under reduced pressure. Without further purification, it can be directly used for subsequent reactions. Subsequently, compound 5 (the molar ratio of compound 5 to compound 8 is 1:1.2 - 1.5), cesium carbonate (as a catalyst, the molar ratio of compound 5 to cesium carbonate is 1:5.0 - 7.0), and anhydrous acetonitrile (as a solvent) are successively added. Under N 2 protection, react at 65 - 70 °C for 2 - 3 h. After the reaction is completed, remove the solvent under reduced pressure, then dissolve it with dichloromethane, add acidic saturated brine (5 mL of 10 M hydrochloric acid is added to 100 mL of saturated NaCl solution) for extraction, collect the organic phase, and remove the solvent to obtain the crude product. Purify it by column separation technology (the volume ratio of the eluent system is dichloromethane:methanol = 100:2 - 100:7) to obtain compound 9. (5) Compound 9 is first reacted under the condition of potassium carbonate (the molar ratio of compound 9 to potassium carbonate is 1 - 4.0 - 5.0) for 30 - 40 min, then compound 10 (the molar ratio of compound 9 to compound 10 is 1:4.0 - 5.0) is added for reaction. The reaction solvent is anhydrous acetonitrile, and react at 60 - 65 °C for 5 - 6 h. After removing the solvent under reduced pressure, dichloromethane and saturated sodium chloride aqueous solution are successively added for extraction 3 times, collect the organic phase, and dry it with anhydrous sodium sulfate. Remove the solvent to obtain the crude product. Purify it by column separation technology (the volume ratio of the eluent system is dichloromethane:methanol = 100:3) to obtain HCySe - O - NO 2 molecule.

[0076] The following examples can enable those of ordinary skill in the art to more comprehensively understand the content of the present invention, but do not limit the present invention in any way.

[0077] Example 1

[0078] Synthesis of dye molecule HCySe - O - NO 2 The synthesis steps of the dye molecule HCySe - O - NO

[0079] Synthesis of dye molecule HCySe - O - NO 2 are as follows:

[0080]

[0081] Synthesis of compound 3

[0082] Compound 1 (5 g) and 2 (19.59 g) are added to anhydrous acetonitrile solvent (5 ml), heated to reflux at 84 °C and monitor the reaction progress. The reaction takes about 4 h. After the reaction is completed, the system is cooled to obtain a solid. Recrystallize with acetonitrile to obtain pure compound 3. The structure of compound 3 is characterized by NMR and high - resolution mass spectrometry.

[0083] 1 H NMR(500MHz,DMSO - d 6)δ: 8.08 - 7.91 (m, 1H), 7.86 - 7.81 (m, 1H), 7.68 - 7.57 (m, 2H), 4.49 (q, J = 7.3 Hz, 2H), 2.83 (s, 3H), 1.53 (s, 6H), 1.44 (t, J = 7.3 Hz, 3H). ESI - HRMS: m / z theoretical value C 13 H 18 N + [M - I] - 188.1434; measured value: 188.1438.

[0084] Synthesis of Compound 5

[0085] Compound 3 (2 g) and Compound 4 (553 mg) were added to a mixed solution of acetic acid (14 mL), acetic anhydride (7 mL) and triethylamine (7 mL), and the reaction was carried out at 65 °C for 2 h. Subsequently, the solvent was removed under reduced pressure, and then dichloromethane and saturated brine were added successively for extraction 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed to obtain the crude product. Purification by column separation technology (the volume ratio of the eluent system was dichloromethane: methanol = 100:5) gave pure Compound 5. The structure of Compound 5 was characterized by NMR and high - resolution mass spectrometry.

[0086] 1 H NMR (500 MHz, DMSO - d 6 )δ: 8.37 (d, J = 8.4 Hz, 2H), 8.30 (d, J = 8.9 Hz, 2H), 8.22 (d, J = 8.2 Hz, 2H), 8.16 (d, J = 8.9 Hz, 2H), 7.79 (t, J = 7.6 Hz, 2H), 7.73 (t, J = 7.5 Hz, 2H), 4.62 (q, J = 7.4 Hz, 4H), 2.94 (s, 6H), 1.76 (s, 12H), 1.50 (t, J = 7.3 Hz, 6H). ESI - HRMS: m / z theoretical value C 34 H 40 ClN 2 + [M - I] - 511.2875; measured value: 511.2876.

[0087] Synthesis of Compound 8

[0088] Compound 6 (1 g) and Compound 7 (603 mg) were added to dimethyl sulfoxide solvent (5 ml) and stirred, and then AgNO 3(3g) React at 120 °C for 2 h. After removing the solvent under reduced pressure, dichloromethane and saturated brine were successively added and extracted 3 times. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed to obtain the crude product. It was purified by column separation technology (the volume ratio of the eluent system was petroleum ether: ethyl acetate = 2:1) to obtain Compound 8. The structure of Compound 8 was characterized by NMR and high-resolution mass spectrometry.

[0089] 1 H-NMR(400MHz,DMSO-d 6 )δ:7.51(dd,J=1.7,1.0Hz,1H),7.50(dd,J=1.7,0.9Hz,1H),7.42(t,J=2.0Hz,2H),7.38(t,J=7.9Hz,2H),7.08(dd,J=2.3,1.0Hz,1H),7.06(dd,J=2.3,1.0Hz,1H),2.26(s,6H).ESI-HRMS:m / z theoretical value C 16 H 14 NaO 4 Se 2 + [M+Na] + 452.9115; measured value: 452.9128.

[0090] Synthesis of Compound 9

[0091] Compound 8 (96 mg) was dissolved in anhydrous ethanol (2 mL) under N 2 and at 0 °C. Sodium borohydride (23 mg) was first added and reacted for 15 min, then citric acid (192 mg) was added and the reaction was continued for 5 min to reduce Compound 8 to the selenol intermediate. The obtained selenol intermediate was extracted with an ether (5 mL) and deionized water (3 mL) system. The organic phase was collected and washed with a mixed aqueous solution of saturated NH 4 Cl and saturated NaCl (volume ratio 1:1), Na 2 SO 4 dried, and the ether solvent was removed under reduced pressure. Without further purification, it was directly used for the subsequent reaction. Subsequently, Compound 5 (100 mg), cesium carbonate (203 mg), and anhydrous acetonitrile (2 ml, as the solvent) were successively added, and the reaction was carried out at 65 °C for 2 h under N 2 protection conditions. After the reaction was completed, the solvent was removed under reduced pressure, then dissolved in dichloromethane, and extracted with acidic saturated brine (5 mL of 10 M hydrochloric acid was added to 100 mL of saturated NaCl solution). The organic phase was collected, and the solvent was removed to obtain the crude product. It was purified by column separation technology to obtain the compound of general formula I. (The volume ratio of the eluent system was dichloromethane: methanol = 100:7) The structure of Compound 9 was characterized by NMR and high-resolution mass spectrometry.

[0092] 1 H NMR(400MHz, Methylene Chloride-d 2 ) δ: 8.12 (d, J=13.8Hz, 1H), 7.72 (s, 1H), 7.56 - 7.44 (m, 3H), 7.39 (t, J=7.4Hz, 1H), 7.26 (d, J=7.8Hz, 1H), 7.22 (s, 1H), 7.13 - 7.04 (m, 1H), 6.29 (d, J=13.8Hz, 1H), 4.17 (q, J=7.3Hz, 2H), 2.78 (t, J=6.0Hz, 2H), 2.60 (t, J=6.2Hz, 2H), 2.01 - 1.86 (m, 2H), 1.77 (s, 6H), 1.47 (t, J=7.1Hz, 3H). ESI-HRMS: m / z calculated for C 27 H 28 NOSe + [M + Na] + 462.1331; found 462.1330.

[0093] Synthesis of compound HCySe-O-NO 2 Compound 9 (100 mg) and potassium carbonate (100 mg) were added to anhydrous acetonitrile (2 ml) and stirred. The mixture was stirred at 60 °C for 30 min, then compound 10 (182.47 mg) was added and the reaction continued for 5 h. After removing the solvent under reduced pressure, dichloromethane and saturated aqueous sodium chloride solution were added successively for extraction three times. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed to obtain the crude product. It was purified by column chromatography (the volume ratio of the eluent system was dichloromethane: methanol = 100:3). The structure of compound HCySe-O-NO

[0094] was characterized by NMR and high-resolution mass spectrometry. 2 The structure of compound HCySe-O-NO

[0095] 1 H NMR(600MHz, Methanol-d 4 ) δ 8.28 (d, J=8.6Hz, 3H), 7.73 (d, J=8.8Hz, 5H), 7.61 - 7.50 (m, 3H), 7.47 (d, J=2.4Hz, 1H), 7.14 (s, 2H), 6.83 (d, J=14.6Hz, 1H), 5.34 (s, 2H), 4.49 (q, J=7.3Hz, 2H), 2.85 - 2.81 (m, 2H), 2.71 (t, J=6.2Hz, 2H), 1.82 (s, 6H), 1.52 (t, J=7.3Hz, 3H).

[0096] ESI-HRMS: Theoretical m / z for C 34 H 33 N 2 O 3 Se + [M] + is 597.1651; measured value is 597.1660.

[0097] Example 2

[0098] Dissolve the dye HCySe-O-NO 2 molecule synthesized in Example 1 in dimethyl sulfoxide solvent to prepare a mother liquor of the dye (10 mM). Use a pipette to take a certain volume of the mother liquor of the dye and dissolve it in PBS (containing 10% dimethyl sulfoxide) to prepare a 10 μM dye solution. Then, collect the ultraviolet-visible absorption and fluorescence spectra on an Agilent ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer respectively. It is found through testing that the ultraviolet absorption of the dye in PBS (containing 10% dimethyl sulfoxide) is the largest at 650 nm in the range of 500 - 900 nm, and the fluorescence is quenched ( Figure 1 ).

[0099] Example 3

[0100] Add the mother liquor of the dye prepared in Example 2 to a certain volume of PBS (containing 10% dimethyl sulfoxide) to prepare a dye solution with a final concentration of 10 μM. Add a certain amount of nitroreductase (NTR, Adamas) with a final concentration of 10 μg / ml, and add a certain amount of reduced coenzyme I (NADH, Sigma) with a final concentration of 500 μM / L to prepare three solutions, namely: a mixed solution containing the dye and nitroreductase (HCySe-O-NO 2 +NTR), a mixed solution containing the dye and reduced coenzyme I (HCySe-O-NO 2 +NADH), and a mixed solution containing the dye, nitroreductase and reduced coenzyme I (HCySe-O-NO 2 +NADH). Then, use an Agilent fluorescence spectrophotometer to collect the fluorescence spectra of the mixed solution containing the dye and nitroreductase, the mixed solution containing the dye and reduced coenzyme I, and the mixed solution containing the dye, nitroreductase and reduced coenzyme I respectively ( Figure 2 a). Prepare a dye solution with a final concentration of 10 μM and add different endogenous substances with a final concentration of 500 μM / L, test the fluorescence spectra of the mixed solutions, and draw a bar chart of the fluorescence intensity at 780 nm ( Figure 2b) It was found that the dye was specifically activated by nitroreductase only in the presence of reduced coenzyme I (wherein 1. ammonium chloride, 2. sodium sulfate, 3. zinc chloride, 4. hexahydrate and ferric chloride, 5. L-ascorbic acid, 6. L-glutamic acid, 7. L-cystine, 8. L-cysteine, 9. glutathione, 10. sodium acetate, 11. sodium carbonate, 12. sodium chloride, 13. potassium chloride, 14. glucose, 15. citric acid, 16. L-isoleucine, 17. L-histidine, 18. L-arginine, 19. L-phenylalanine, 20. L-threonine, 21. L-aspartic acid, 22. nitroreductase (10 μg / ml) and reduced coenzyme I (500 μm / L)).

[0101] Example 4

[0102] This example uses four methods to determine the dye HCySe-O-NO in Example 1. 2 The commercial probe DPBF detects singlet oxygen, the commercial probe HPF detects hydroxyl radicals, and the commercial probe DHR123 detects superoxide anions. Under 760nm laser irradiation, the absorbance of DPBF did not decrease significantly, indicating that the dye HCySe-O-NO 2 No singlet oxygen was generated. After being irradiated at 760nm, HPF and DHR123 were oxidized by the generated hydroxyl radicals and superoxide anions, respectively, which significantly increased their fluorescence intensity. Figure 3 (ac) It can be seen that after 760nm laser irradiation, the dye HCySe-O-NO 2 The ROS generated are mainly hydroxyl radicals and superoxide anion radicals, and no singlet oxygen is produced. Figure 3 (df) It can be seen that under 760nm laser irradiation, the absorbance of DPBF has a very obvious decrease, indicating that the dye HCySe-OH produces singlet oxygen. However, the fluorescence intensity of HPF and DHR123 did not increase significantly after irradiation at 760nm, indicating that no hydroxyl radicals and superoxide anions were generated.

[0103] Example 5

[0104] This example uses the MTT detection method to study the dye HCySe-O-NO in Example 1 under different treatment conditions. 2 Dark toxicity and phototoxicity to human breast cancer cells MCF-7. MCF-7 cells were seeded into 96-well plates at a seeding density of approximately 1×10 5 After the cells adhered to the wall, they were placed in a normoxic incubator (37°C, 5% CO 2 , 95% air) and hypoxic incubator (37°C, 5% CO 2 , 2% O 2) Incubate for 8 h, wash each well with 100 μL of PBS, and then add DMEM complete medium (containing 10% fetal bovine serum and 1% antibiotics) with 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5 μM of the dye HCySe-O-NO 2 into the wells and continue to incubate for 1 h. Cells cultured under normoxia and hypoxia are respectively subjected to dark treatment (without light treatment) and light treatment (Laser, 760 nm, 50 mw / cm 2 , 10 min). Each group of experiments is performed in parallel 6 times. After 24 h, remove the medium in each well, and then add medium containing 5 mg / ml MTT (100 μL) and incubate for 3 h. A large amount of blue-violet crystals are formed. After removing the medium, add 100 μl of dimethyl sulfoxide to each well, wrap it with tin foil and gently shake for 15 min to fully dissolve the formazan, and then use an enzyme-linked immunosorbent assay reader to test. As Figure 4 shown, whether cultured under normoxia or hypoxia, the dye HCySe-O-NO 2 has good biocompatibility without light treatment, while under light conditions, the cell survival rate is significantly reduced.

[0105] Example 6

[0106] Seed human breast cancer cells MCF-7 into a confocal dish. After the cells adhere to the wall and reach an appropriate cell density, place them in a normoxia incubator (37 °C, 5% CO 2 , 95% air) and a hypoxia incubator (37 °C, 5% CO 2 , 2% O 2 ) and incubate for 8 h. Add the dye HCySe-O-NO 2 synthesized in Example 1 (5 μM) and continue to incubate for different times (20 min, 40 min, 60 min), and then use a laser confocal microscope to perform fluorescence imaging on the cells. The excitation wavelength is 639 nm, and the receiving band is 700 - 730 nm. The imaging results are as Figure 5 described. Under normoxia culture conditions, the fluorescence brightness does not change significantly with the extension of time, while under hypoxia culture conditions, with the extension of the dye incubation time, the fluorescence brightness becomes stronger and stronger, indicating that the dye is relatively easy to enter the cells, and under hypoxia culture conditions, the dye fluorescence is activated by intracellular nitroreductase to produce fluorescence, while cells under normoxia culture conditions lack nitroreductase and cannot activate the dye fluorescence.

[0107] Example 7

[0108] Seed human breast cancer cells MCF-7 into a confocal dish and place it in a cell culture incubator. When the cell density is appropriate, place it in a normoxia incubator (37 °C, 5% CO 2 , 95% air) and a hypoxia incubator (37 °C, 5% CO 2 , 2% O2 ) Incubate for 8 h. First, add the DCFH-DA commercial reactive oxygen species probe to the confocal cell culture dish to incubate the cells for 2 h, and then add the dye HCySe-O-NO synthesized in Example 1 2 (5 μM) and continue to culture the cells for 1 h. Divide the confocal dish containing the cells into four groups: normoxia dark treatment group, normoxia light treatment group (760 nm laser irradiation for 10 min); hypoxia dark treatment group, hypoxia light treatment group (760 nm laser irradiation for 10 min). Finally, the imaging results of cells in different groups by laser confocal microscopy show that: there is no obvious green fluorescence in the cells treated in the dark under either normoxia culture or hypoxia culture; there is obvious green fluorescence in the cells of the light irradiation group ( Figure 6 ). The imaging results prove that the dye HCySe-O-NO synthesized in Example 1 2 can generate reactive oxygen species after light treatment in cells. The excitation wavelength is 488 nm, and the reception band is 510 - 530 nm.

[0109] Example 7

[0110] Inoculate human breast cancer cells MCF-7 into a confocal dish and place it in a cell culture incubator. When the cell density is appropriate, place it in a normoxia incubator (37 °C, 5% CO 2 , 95% air) and a hypoxia incubator (37 °C, 5% CO 2 , 2% O 2 ) and culture for 8 h. Add the dye HCySe-O-NO synthesized in Example 1 2 (5 μM) and continue to culture the cells for 1 h. Divide the confocal dish containing the cells into four groups: normoxia dark treatment group, normoxia light treatment group (760 nm laser irradiation for 10 min); hypoxia dark treatment group, hypoxia light treatment group (760 nm laser irradiation for 10 min). Add the live / dead cell kit (Calcein-AM (calcein) and Propidium Iodide (PI, propidium iodide)) to the cells treated as above. Finally, the imaging results of cells in different groups by laser confocal microscopy show that: there is obvious fluorescence in the green channel and no fluorescence in the red channel in the cells without light treatment ( Figure 7 a and 7c); there is no obvious fluorescence observed in the green channel and obvious fluorescence in the red channel in the cells with light treatment ( Figure 7 b and 7d). The Calcein-AM / PI cell double-staining imaging results show that the reactive oxygen species generated by the dye HCySe-O-NO 2 under light treatment can effectively kill MCF-7 cells. Green channel: excitation wavelength 488 nm, reception band 510 - 530 nm; red channel: excitation wavelength 543 nm, reception band 569 - 700 nm.

[0111] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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 nitroreductase-activated selenium-substituted hemicyanine dye, characterized in that: The structure is shown in general formula I: In the general formula I: R1 is H, phenyl, p-phenylbenzyl chloride, p-phenylbenzyl bromide, SO3R4, COOR4 or C1-C 12 One of the alkyl groups; R2 is C(CH3)2, S or Se; R3 is H, phenyl or C1-C 12 One of the alkyl groups; R4 is H or C1-C 12 One of the alkyl groups; Y is Cl, Br or I.

2. The nitroreductase-activated selenium-substituted hemicyanine dye according to claim 1, characterized in that R2 is Se; R3 is H or phenyl; Y is Br or I.

3. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 1 or 2, characterized in that: The steps include: (1) Compound 1 and Compound 2 are reacted at a molar ratio of 1:3.0-5.0 to prepare Compound 3; (2) Compound 3 and Compound 4 are reacted at a molar ratio of 2.0 to 2.5:1 to prepare Compound 5; (3) Compound 6 and Compound 7 are reacted at a molar ratio of 1:2.0-3.0 to prepare Compound 8; (4) Compound 5 and Compound 8 are reacted at a molar ratio of 1:1.2-1.5 to prepare Compound 9; (5) Compound 9 and compound 10 are reacted at a molar ratio of 1:4.0-5.0 to prepare a compound of formula I.

4. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 3, characterized in that: In step (1), the reaction solvent of the compound 3 and the compound 4 is anhydrous acetonitrile, and the reaction conditions are 85-90° C. for 4-5 hours; In step (2), the reaction solvent of the compound 3 and the compound 4 is a mixed solution of acetic acid, acetic anhydride and triethylamine, and the reaction conditions are 65-70° C. for 1-3 hours.

5. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 3, characterized in that: In step (3), the reaction solvent of the compound 6 and the compound 7 is dimethyl sulfoxide, AgNO3 is used as a catalyst, and the reaction conditions are 120-130°C for 2-3h.

6. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 3, characterized in that: In step (4), before the compound 8 reacts with the compound 5, the selenol intermediate is first reduced with a sodium borohydride and citric acid system; the reaction solvent of the selenol intermediate and compound 5 is anhydrous acetonitrile, cesium carbonate is used as a catalyst, and the reaction is carried out at 65-70°C for 2-3h under N2 protection.

7. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 6, characterized in that: The preparation method of the selenol intermediate is as follows: the compound 8 is first reacted in the presence of NaBH4 with anhydrous ethanol as solvent under N2 protection and 0±1°C for 10 to 20 minutes, and then citric acid is added to continue the reaction for 5 to 8 minutes, the reaction solution is extracted with ether and water, the organic layer is washed with saturated NH4Cl aqueous solution and saturated NaCl, dried with Na2SO4, and the ether solvent is removed under reduced pressure to obtain the selenol intermediate.

8. The method for synthesizing the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 3, characterized in that: In step (5), the compound 9 is first reacted under potassium carbonate for 30 to 40 minutes, and then reacted with the compound 10. The reaction solvent is anhydrous acetonitrile, and the reaction is carried out at 60 to 65° C. for 5 to 6 hours.

9. Use of the nitroreductase-activated selenium-substituted hemicyanine dye according to claim 1 or 2 in the preparation of fluorescent dyes or tumor diagnosis and treatment drugs.

10. The use according to claim 9, characterized in that: The application of the fluorescent dyes shown in this figure in cell imaging and photodynamic cancer cell killing.

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