Synthesis and application of a heptamethine cyanine compound that releases hydrogen sulfide in response to stimulation

Abstract

The present invention provides the synthesis and application of a heptamethine cyanine compound that releases hydrogen sulfide in response to stimulation, and provides a micro-nanostructure formed by self-assembly in an aqueous solution having a structure represented by formula (I) or its isomers, pharmaceutically acceptable salts, hydrates or solvates, as well as the preparation of a pharmaceutical composition and its application in the preparation of fluorescent probes, H2S donors and phototherapy drugs, the preparation of drugs for diagnosing and / or treating cancer, phototherapy combined with gas therapy, and stimulus-responsive release drugs. The compound provided by the present invention can accumulate at the tumor site and can achieve aggregation structure conversion and H2S gas release by responding to the unique redox stimulation of the tumor microenvironment. Under irradiation with a specific excitation wavelength, it can achieve precise phototherapy combined with gas therapy of the tumor in the aggregated state and low-background long-lasting fluorescence imaging at the tumor site after the aggregation disintegration. It has two tumor treatment modes, and the tumor treatment effect is good, the trauma is small, the fluorescence imaging accuracy is high, the time is long, and it has excellent biosafety while achieving integrated diagnosis and treatment of the tumor site.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceuticals and relates to the synthesis and application of a heptamethine cyanine compound that releases hydrogen sulfide in response to stimulation. In response to external environmental stimulation, the compound releases hydrogen sulfide gas and heptamethine cyanine molecules, enabling the compound to combine the effects of gas therapy and photothermal therapy, enabling both high-temperature and low-temperature treatment modes for tumors. Background Art

[0002] Cancer is a disease that seriously threatens human life, and its early diagnosis and treatment are crucial. Photothermal therapy (PTT) is one of the most effective cancer treatment strategies known, but the high temperature of traditional photothermal therapy will inevitably burn the skin, leading to long-term inflammation, permanent tissue damage and tumor regeneration. Low-temperature PTT below 45°C has been shown to reduce the risk of normal tissue damage, but due to the limited penetration of the therapeutic laser and the rapid heat dissipation by blood flow, cancer cells located deep inside and around blood vessels are almost impossible to kill, and thus continue to proliferate, leading to tumor recurrence. Therefore, it is very important to seek other therapies in combination with photothermal therapy to achieve a safe and powerful tumor suppression effect.

[0003] In recent years, gas therapy (GT) has garnered significant attention due to its high efficacy and lack of side effects. Hydrogen sulfide (H2S) is an endogenous biotransmitter with cytoprotective properties. At high concentrations, H2S can disrupt mitochondrial homeostasis and interfere with the respiratory chain by reducing cytochrome c oxidase (COX IV) activation, thereby inhibiting ATP synthesis and downregulating energy-dependent heat shock protein 70 (HSP70). This represents an effective and robust strategy for reversing HSP70-mediated thermal tolerance. H2S can inhibit the NF-κB pathway and, through sulfide, downregulate inducible nitric oxide synthase (iNOS), thereby suppressing inflammatory stimuli. This provides a solid theoretical foundation for H2S to downregulate hyperthermia-induced inflammation in photothermal therapy.

[0004] COS / H2S donors are typically constructed based on the self-elimination reaction of 4-aminobenzyl alcohol. This linker molecule is often used to connect specific substrates to target molecules through stable chemical bonds. External stimulation removes the substrate, forming an aniline derivative. This aniline derivative rapidly undergoes a 1,6-elimination self-elimination reaction to form an azaquinone. This aniline derivative then undergoes decarboxylation, releasing carbonyl sulfide (COS). This is then rapidly converted to H2S by the ubiquitous enzyme carbonic anhydrase (CA). Summary of the Invention

[0005] The present invention aims to address the aforementioned problems existing in the prior art by proposing a supramolecular diagnostic and therapeutic integrated reagent based on heptamethine cyanine and its application. By connecting heptamethine cyanine molecules and H2S donors via a stimuli-responsive linker, the nanoparticles overcome the shortcomings of single-phototherapy approaches and provide new insights into tumor treatment. Furthermore, the nanoparticles can target and accumulate within tumor tissues through the EPR effect, inducing a response within the tumor-specific microenvironment, releasing heptamethine cyanine small molecules and H2S gas. Under irradiation with a specific excitation wavelength, they can achieve low-background, long-lasting fluorescence imaging of the tumor site while simultaneously providing precise photothermal / photodynamic therapy for the tumor, achieving integrated diagnosis and treatment of the tumor site. Furthermore, H2S gas can inhibit mitochondrial respiration, thereby suppressing the expression of heat shock protein 70, thereby achieving low-temperature treatment of tumors and reducing damage to normal tissues. Interestingly, under conventional high-temperature photothermal therapy, H2S gas can reduce the overactive inflammatory response caused by high temperatures, enabling a high-temperature / low-temperature treatment mode that can be selected on demand.

[0006] The technical solution of the present invention is:

[0007] A stimuli-responsive integrated diagnostic and therapeutic compound, wherein the compound has a micro-nanostructure formed by self-assembly in aqueous solution of the structure represented by formula (I) or its isomers, pharmaceutically acceptable salts, hydrates or solvates:

[0008]

[0009] In the above formula (I): W is a stimulus response group, which is selected from

[0010] In the formula (I), X, Y, and Z are independently selected from O or S.

[0011] Furthermore, the compounds are compounds I-1, I-2, I-3, I-4, I-5, and I-6.

[0012] Furthermore, the micro-nano structure of the present invention is a nano-microsphere structure formed by self-assembly of a compound having a structure represented by formula (I), a pharmaceutically acceptable salt, a hydrate or a solvate in an aqueous solution.

[0013] Preferably, the micro-nanostructure of Compound I is formed by self-assembly of Compound I (including Compound I-1 to Compound I-6 in Table 1) in an aqueous solution.

[0014] The final preferred range is 30nm to 200nm.

[0015] The present invention also provides a method for preparing the micro-nano structure, comprising the following steps:

[0016] 1) dissolving the compound having the structure represented by formula (I), its pharmaceutically acceptable salt, hydrate or solvate in an organic solvent;

[0017] The organic solvent selected is a mixture of one or more of alkanes, olefins, aromatic hydrocarbons, alcohols, ketones, aldehydes, carboxylic acids, esters or ethers; specifically, the organic solvent is a mixture of one or more of dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, ethylene glycol, n-propanol, isopropanol, propylene glycol, glycerol, n-butanol, isobutanol, butanediol or polyethylene glycol, acetone, dichloromethane or acetonitrile; preferably dimethyl sulfoxide.

[0018] 2) adding the dissolved solution into water to obtain a compound solution with a final concentration of 1 nM to 1 M;

[0019] The final concentration is preferably 10 nM to 1 mM.

[0020] 3) The compounds self-assemble in aqueous solution to form micro-nanostructures.

[0021] The preparation method is simple, convenient and suitable for large-scale production.

[0022] The present invention also provides a composition comprising:

[0023] 1) A therapeutically effective dose of a compound having a structure represented by formula (I), a pharmaceutically acceptable salt, hydrate or solvate

[0024] 2) Pharmaceutically acceptable carrier.

[0025] Preferably, the pharmaceutically acceptable carrier comprises a diluent, a disintegrant, an excipient, a binder, a stabilizer or a combination thereof.

[0026] The invention provides an application of the compound in the preparation of a drug for treating cancer.

[0027] The cancer is selected from esophageal cancer, non-small cell lung cancer, biliary tract cancer, head and neck cancer, Barrett's esophagus, bladder cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain tumor, breast cancer or skin cancer, including melanoma.

[0028] The present invention provides a use of the micro-nanostructure in preparing a phototherapy combined with gas therapy drug. Preferably, the phototherapy drug is a photothermal therapy drug, a photodynamic therapy drug or a photoacoustic therapy drug; and the H2S donor is a COS / H2S donor.

[0029] The present invention also provides the use of the micro-nanostructure in preparing a drug with stimulus-responsive release, wherein the stimulus-responsive release includes active oxygen response and reduction response.

[0030] The present invention also provides a method for performing phototherapy on a target area of ​​a subject, comprising:

[0031] 1) Providing the micro-nanostructure;

[0032] 2) administering the micro-nanostructure to a subject;

[0033] 3) waiting for the micro-nanostructure to be enriched in the target area;

[0034] 3) Using light in the excitation wavelength band of the micro-nanostructure to irradiate the target area of ​​the subject, preferably, using 808 nm light waves for irradiation.

[0035] Beneficial effects of the present invention:

[0036] (1) The compounds provided by the present invention can self-assemble into micro-nanostructures in aqueous solution. Through the EPR effect, the nanoparticles can be circulated through the blood, stably and efficiently targeting tumor tissues, and stimulated to respond under the unique redox microenvironment of the tumor site, thereby accurately releasing H2S gas and heptamethine cyanine molecules. Under near-infrared light irradiation, low-background enhanced fluorescence imaging of the tumor site is achieved, and it can be used for cancer photothermal / gas combined treatment under the guidance of near-infrared fluorescence imaging, showing higher biosafety, excellent fluorescence imaging accuracy and photothermal / gas combined treatment effect, and has broad prospects in cancer diagnosis and treatment.

[0037] (2) The formed micro-nanostructure has high dimensional stability, high biosafety, excellent photostability and excellent tumor targeting, and can achieve stimulus-responsive breaking of connecting bonds and release of H2S gas under the unique pathological microenvironment of the tumor. It has good targeting and imaging effects and can achieve high-temperature or low-temperature treatment modes. It has great market value and broad commercial prospects.

[0038] (3) The present invention also provides the use of the compound represented by formula (I) for preparing phototherapy combined with gas therapy drugs, and drugs for diagnosing and treating cancer. The compound has good therapeutic effects and minimal trauma, and has great potential in commercialization and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a synthetic route diagram of compound Ⅰ-1 provided by the present invention;

[0040] Figure 2 is the UV absorption spectrum of compound Ⅰ-1 in different polar solvents;

[0041] Figure 3 is the fluorescence emission spectrum of compound Ⅰ-1 in different polar solvents;

[0042] Figure 4This is a transmission electron microscopy image of nanospheres self-assembled by compound Ⅰ-1 in aqueous solution;

[0043] Figure 5 The particle size test results of compound Ⅰ-1 provided by the present invention in aqueous solution;

[0044] Figure 6 This is the cyclic temperature change diagram of compound Ⅰ-1 and control group under 808nm laser irradiation;

[0045] Figure 7 The temperature change diagram of compound Ⅰ-1 with different concentrations under 808nm laser irradiation;

[0046] Figure 8 This is a graph showing the change in ultraviolet absorption of the active oxygen scavenger DPBF when an aqueous solution containing compound I-1 is irradiated with 808 nm laser;

[0047] Figure 9 This is the cell co-localization image after compound Ⅰ-1 was phagocytosed by tumor cells;

[0048] Figure 10 The figure shows the production of reactive oxygen species in 4T1 cells after treatment with compound Ⅰ-1 in different ways;

[0049] Figure 11 This is the dark toxicity and phototoxicity of compound Ⅰ-1 to 4T1 cells;

[0050] Figure 12 Live-dead fluorescence imaging of compound Ⅰ-1 on 4T1 cells;

[0051] Figure 13 The production of H2S by compound Ⅰ-1 in 4T1 cells;

[0052] Figure 14 These are fluorescence images of the tumor site at different time points after compound Ⅰ-1 was intravenously injected into mice;

[0053] Figure 15 Photoacoustic imaging images of mice at different time points after compound Ⅰ-1 was injected into the tail vein of mice;

[0054] Figure 16 This is a thermal imaging image of compound Ⅰ-1 in mice during in vivo treatment;

[0055] Figure 17 This is the image of compound Ⅰ-1 in vivo treatment of mice;

[0056] Figure 18 Images of tumor volume changes after intravenous injection of compound Ⅰ-1 in tumor-bearing mice;

[0057] Figure 19 Images of weight changes in tumor-bearing mice after intravenous injection of compound Ⅰ-1;

[0058] Figure 20 Liver function and blood routine of mice after intravenous injection of compound Ⅰ-1;

[0059] Figure 21 H&E-stained images of heart, liver, spleen, lung, and kidney sections of tumor-bearing mice after intravenous injection of compound Ⅰ-1; DETAILED DESCRIPTION

[0060] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] In order to further understand the present invention, the present invention will be further described with reference to the accompanying drawings and embodiments.

[0062] The present invention provides certain specific examples of compounds, including compounds I-1 to I-6 shown in Table 1 below:

[0063]

[0064] Table 1 Structural formula of compounds I-1 to I-6

[0065]

[0066] The above compounds can be synthesized by the following reaction formula:

[0067]

[0068] The main synthetic steps include:

[0069] (1) providing compounds A, B, C, D and E respectively;

[0070]

[0071] Synthesis of compound A:

[0072] Compound 1' and Compound 2' were dissolved in anhydrous ethanol and stirred under reflux for 12 hours. After the reaction was completed, the purple-red precipitate was filtered and dried under vacuum to obtain Compound A without purification.

[0073] Synthesis of compound B:

[0074]

[0075] Compounds 3' and 4' were added to acetonitrile and heated to 110°C for 24 hours under reflux. The solvent was evaporated under vacuum and the resulting solid was washed three times with ether to obtain a pink solid.

[0076] Compounds D, E, and F were purchased directly.

[0077] (2) Compound C and compound D were dissolved in dichloromethane, EDC·HCl and DMAP were added, and the mixture was allowed to react overnight at room temperature. Compound E was then added to continue the reaction, and the mixture was washed three times with a saturated aqueous ammonium chloride solution and a saturated aqueous sodium chloride solution. After removing water, the resulting solid was purified by column chromatography. The product and compound F were dissolved in dichloromethane, pyridine was added, and the mixture was allowed to react overnight at room temperature. The solid was washed three times with water and purified by column chromatography to obtain the product compound I.

[0078] Example 1 Synthesis of Compound Ⅰ-1 and its Fluorescence Properties

[0079] like Figure 1 As shown, the synthesis of compound I-1 includes the following steps:

[0080] 1) Synthesis of Compound 1': 1.46 g of malononitrile and 0.93 g of magnesium ethoxide were added to 15 mL of ethanol, followed by the addition of 0.75 mL of 3-hydroxy-3-methylbutan-2-one. The mixture was heated to 60°C and reacted for 12 hours. The solvent was removed by rotary evaporation in vacuo, and the resulting solid was purified by column chromatography to yield the desired product 1. 1 HNMR (400MHz, CDCl3): δ (ppm): 2.36 (s, 3H), 1.63 (s, 6H).

[0081] 2) Synthesis of Compound 2': 30 mL of DCM and 30 mL of DMF were added to a reaction flask under ice-cooling and stirred. 26.3 mL of POCl3 was added under constant pressure with stirring. 7.95 mL of cyclohexanone was then added and heated to 80°C for 3 hours. After the reaction, the product was poured into crushed ice to quench the reaction and then refrigerated overnight. The solvent was removed by vacuum rotary evaporation to obtain the crude product, Compound 2', which was directly used for the next synthesis reaction.

[0082] 3) Synthesis of Compound 6: 0.5 g of 5-chloro-2,3,3-trimethylindole and 2.01 g of iodoethanol were added to 50 mL of acetonitrile. The reaction was refluxed at 110°C for 24 hours. After cooling to room temperature, the product was recrystallized from diethyl ether and filtered to obtain the crude product 6, which was used directly in the next step without purification.

[0083] 4) Synthesis of compound A: as described above.

[0084] 5) Synthesis of Compound 7: 0.4 g of Compound A, 0.44 g of Compound 6 and a few drops of pyridine were dissolved in 5 mL of ethanol, heated to 90° C., refluxed for 6 hours, and column chromatography was performed to obtain Compound 7.

[0085] 6) Synthesis of Compound 9: 300 mg of Compound 7 and 50 mg of Compound 8 were dissolved in 3 mL of dichloromethane. 0.28 g of EDC·HCl and 0.05 g of DMAP were added dropwise under ice-cooling. The mixture was allowed to react at room temperature overnight. Subsequently, 40 mL of dichloromethane was added. The mixture was washed three times with 50 mL of saturated aqueous NH4Cl and saturated brine, respectively. After dehydration with anhydrous Na2SO4, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to obtain the title compound 9.

[0086] 7) Synthesis of Compound 11: 368.25 mg of Compound 9 and 68 mg of Compound 10 were dissolved in 5 mL of dichloromethane. 0.28 g of EDC·HCl and 0.05 g of DMAP were added dropwise under ice-cooling. The mixture was allowed to react at room temperature overnight. Subsequently, 40 mL of dichloromethane was added. The mixture was washed three times with 50 mL of saturated aqueous NH4Cl and saturated brine, respectively. After dehydration with anhydrous Na2SO4, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to obtain the target compound 11.

[0087] 1 H NMR (400MHz, DMSO-d6): δ8.39(d,J=14.5Hz,1H),8.11(d,J=13.8Hz,1H),7.54(d,J=7.4Hz,1H), 7.42–7.31(m,4H),7.19(t,J=7.2Hz,1H),7.09–7.02(m,2H),6.24(dd,J=25.1,14.2Hz,2H),4.49 (s,2H),4.44(s,4H),2.68(dt,J=26.9,7.2Hz,9H),2.55(d,J=7.0Hz,2H),2.24(t,J=7.4Hz,2H), 1.95(p,J=7.2Hz,2H), 1.83(p,J=7.5,6.8Hz,2H), 1.69(p,J=7.3Hz,3H), 1.63(d,J=3.9Hz,12H).

[0088] 8) Synthesis of Compound I-1: 120 mg of Compound 11 and 50 mg of Compound 12 were dissolved in 3 mL of dichloromethane in an ice bath. After stirring at 0°C for half an hour, 50 μL of pyridine was added and the mixture was returned to room temperature for 12 hours. The mixture was washed three times with 20 mL of saturated brine and water, respectively. After dehydration with anhydrous Na2SO4, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography to obtain the target compound I-1.

[0089] 1 H NMR (400MHz, DMSO-d6): δ8.39(d,J=14.4Hz,1H),8.10(d,J=13.7Hz,1H),7.65–7.51(m,3H), 7.50–7.40(m,2H),7.34(dd,J=7.9,3.7Hz,3H),7.30–7.10(m,5H),6.24(dd,J=22.2,14.2Hz ,2H),5.59(s,2H),4.44(s,4H),2.77–2.59(m,9H),2.56(d,J=7.0Hz,2H),2.24(t,J=7.4Hz, 2H), 1.96 (p, J = 7.2Hz, 2H), 1.86–1.79 (m, 2H), 1.71 (q, J = 7.2Hz, 3H), 1.63 (d, J = 2.9Hz, 12H).

[0090] The UV absorption spectra and fluorescence emission spectra of compound Ⅰ-1 in water and ethanol are shown as follows: Figure 2 and Figure 3 As shown, the absorption and emission spectra of compound I-1 in the two solvents are significantly different, among which the absorption spectrum of I-1 in aqueous solution is wider and the emission light is less, approaching zero, indicating that the physical properties of compound I-1 in aqueous solution and organic solvent are different.

[0091] Example 2 Synthesis of Compounds I-2 to I-6

[0092] Compounds I-2 to I-6 can be prepared by a method similar to Example 1.

[0093] 1. Synthesis of Compound Ⅰ-2

[0094]

[0095] Compound 12 in Example 1 was replaced by compound 13, and the remaining reagents and preparation methods were the same as steps 1 to 8 of Example 1 to prepare compound I-2.

[0096] 1H NMR (400MHz, DMSO-d6): δ7.65–7.58(m,2H),7.40–7.20(m,7H),7.20–7.13(m,2H),7. 05–6.99(m,3H),6.96(d,J=8.1Hz,1H),6.83(dd,J=6.6,1.5Hz,1H),6.33(dt,J=8.1,0 .9Hz,1H),5.55(t,J=1.0Hz,2H),4.33(t,J=5.8Hz,2H),4.11(t,J=5.8Hz,2H),2.73(d t,J=6.9,5.8Hz,4H),2.67–2.53(m,6H),1.99–1.86(m,4H),1.58(s,8H),1.54(s,6H).

[0097] 2. Synthesis of Compound I-3

[0098]

[0099] Compounds 14 and 15 were used to replace compounds 10 and 12 in Example 1. The remaining reagents and preparation methods were the same as steps 1 to 8 of Example 1 to prepare compound I-3.

[0100] 1 H NMR (400MHz, DMSO-d6): δ7.44–7.36(m,2H),7.34–7.28(m,2H),7.28–7.20(m,4H),7.20–7.14(m, 3H),7.14–7.08(m,2H),7.01(td,J=7.7,1.4Hz,1H),6.96(d,J=8.1Hz,1H),6.83(dd,J=6.6,1.5Hz ,1H),6.33(dt,J=8.1,0.9Hz,1H),4.33(t,J=5.8Hz,2H),4.28(t,J=1.0Hz,2H),4.11(t,J=5.8Hz ,2H),2.73(dt,J=6.9,5.8Hz,4H),2.67–2.53(m,6H),1.99–1.86(m,4H),1.56(d,J=21.4Hz,14H).

[0101] 3. Synthesis of Compound I-4

[0102]

[0103] Compounds 14 and 16 were used to replace compounds 10 and 12 in Example 1, respectively. The remaining reagents and preparation methods were the same as steps 1 to 8 of Example 1 to prepare compound I-4.

[0104] 1 H NMR (400MHz, DMSO-d6): δ7.61–7.55(m,2H),7.40–7.33(m,2H),7.33–7.27(m,3H),7.27–7.20(m ,2H),7.20–7.14(m,2H),7.14–7.08(m,2H),7.01(td,J=7.6,1.4Hz,1H),6.96(d,J=8.1Hz,1H), 6.83(dd,J=6.6,1.5Hz,1H),6.33(dt,J=8.1,0.9Hz,1H),4.40–4.30(m,4H),4.11(t,J=5.8Hz,2 H),2.73(dt,J=6.9,5.8Hz,4H),2.67–2.53(m,6H),1.99–1.86(m,4H),1.56(d,J=21.4Hz,14H).

[0105] 4. Synthesis of Compound I-5

[0106]

[0107] Compound 14 was used to replace compound 10 in Example 1. The remaining reagents and preparation methods were the same as steps 1 to 8 of Example 1 to prepare compound I-5.

[0108] 1 H NMR (400MHz, DMSO-d6): δ7.43–7.35(m,2H),7.34–7.28(m,2H),7.28–7.20(m,4H),7.20–7.0 8(m,5H),7.01(td,J=7.7,1.4Hz,1H),6.96(d,J=8.1Hz,1H),6.83(dd,J=6.6,1.5Hz,1H),6.3 3(dt,J=8.1,0.9Hz,1H),4.57(t,J=0.9Hz,2H),4.33(t,J=5.8Hz,2H),4.11(t,J=5.8Hz,2H) ,2.73(dt,J=6.9,5.8Hz,4H),2.67–2.53(m,6H),1.99–1.86(m,4H),1.56(d,J=21.4Hz,14H).

[0109] 5. Synthesis of Compound I-6

[0110]

[0111] Compounds 14 and 13 were used to replace compounds 10 and 12 in Example 1. The remaining reagents and preparation methods were the same as steps 1 to 8 of Example 1 to prepare compound I-6.

[0112] 1 H NMR (400MHz, DMSO-d6): δ7.70–7.64(m,2H),7.40–7.33(m,2H),7.33–7.08(m,9H),7.05–6.98( m,2H),6.83(dd,J=6.5,1.4Hz,1H),6.37(dt,J=8.1,1.0Hz,1H),4.59(dt,J=13.7,0.9Hz,1H), 4.55–4.49(m,1H),4.36(t,J=5.8Hz,2H),4.09(t,J=5.8Hz,2H),2.74(td,J=5.8,4.7Hz,4H),2 .67–2.56(m,6H),2.48(t,J=8.0Hz,2H),1.94(m,J=16.1,8.0,5.8Hz,4H),1.64–1.54(m,14H).

[0113] Example 3 Preparation method of micro-nanostructure

[0114] Taking the micro-nanostructure self-assembled by compound I-1 as an example, I-1 is dissolved in DMSO (or an organic solvent such as ethanol) to prepare a 2mM storage solution, and 10μL of the storage solution is added to 2mL of deionized water to prepare nanoparticles.

[0115] Example 4 Characterization Method of Micro-Nano Structure

[0116] like Figure 4 As shown, 10 μL of the working solution of compound I-1 was added to a silicon wafer and observed and photographed under a transmission electron microscope (TEM). It was clearly observed that the micro-nanostructure in the form of nanospheres was formed. From the observation results, it was found that the particle size of the micro-nanostructure self-assembled by compound I-1 was about 160nm. At the same time, DLS was used to test the particle size of the nanospheres self-assembled by compound I-1 in aqueous solution. The results were as follows: Figure 5 As shown, the particle size is around 30 nm to 200 nm, and there is no obvious change in the particle size after being placed at room temperature for 24 hours, which proves that the compound has excellent structural stability.

[0117] Test Example 1 Photothermal effect of compound Ⅰ-1 in vitro

[0118] Take 3 mL of each of the 4 groups of samples and add them to the cuvette, then seal it.

[0119] Sample No. 1 was 3 mL of deionized water;

[0120] Sample No. 2 is 10 μM Ⅰ-1, and the specific preparation method is to add 15 μL of Ⅰ-1 stock solution (2 mM, dissolved in DMSO) to 3 mL of deionized water;

[0121] Sample No. 3 is 20 μM Ⅰ-1, and the specific preparation method is to add 30 μL of Ⅰ-1 stock solution (2 mM, dissolved in DMSO) to 3 mL of deionized water;

[0122] Sample No. 4 is 40 μM Ⅰ-1, and the specific preparation method is 3 mL of deionized water plus 60 μL of Ⅰ-1 stock solution (2 mM, dissolved in DMSO).

[0123] Each sample was irradiated with 808 nm laser for 10 minutes, and the temperature data was recorded every 10 seconds using a thermal imager. The temperature corresponding to the time was plotted. Figure 7 The temperature of sample No. 1 remained almost unchanged within 10 minutes, rising only 2.4°C; the temperature of sample No. 2 rose from 21.6°C at room temperature to 40.4°C, an increase of 18.8°C; the temperature of sample No. 3 rose from 23°C at room temperature to 53.6°C, an increase of 30.6°C; the temperature of sample No. 4 rose from 23°C at room temperature to 67.3°C, an increase of 44.3°C.

[0124] Sample No. 2 was selected to test the light and heat stability experiment. Figure 6 As shown, after 10 minutes of 808nm laser irradiation, sample 2 heated from room temperature (21.6°C) to 40.4°C. After allowing the temperature to cool naturally for fifteen minutes, the temperature was irradiated again with an 808nm laser for 10 minutes, followed by natural cooling, and repeated five times. After five repeated irradiations, sample 2 continued to heat from 21.6°C to 28.7°C, demonstrating its ability to maintain a stable photothermal effect during repeated heating and cooling cycles and exhibiting excellent photothermal stability. Furthermore, the photothermal conversion efficiency of compound I-1 was calculated and found to be as high as 46.06%, demonstrating its excellent photothermal effect. The photothermal conversion efficiency of Cy7-TCF-OH, decomposed by I-1 upon stimulation, reached 59.21%, laying the theoretical foundation for combining gas therapy with phototherapy.

[0125] Test Example 2: Photodynamic Effect of Compound Ⅰ-1 in Vitro

[0126] First, 2 ml of a 10 μM aqueous solution of compound I-1 was prepared, and ultrapure water and Cy7-TCF-OH groups were used as controls. The reactive oxygen species indicator probe 1,3-diphenylisobenzofuran (DPBF) was added to each sample to a final concentration of 50 μM. The UV absorption after the addition of DPBF was measured using a UV-visible spectrophotometer and recorded as the initial absorbance. An 808 nm laser was used at 1 W / cm 2The absorbance of the sample was measured every 30 seconds under the laser intensity of 5 min. The absorbance at 420 nm was read from the DPBF UV absorption data and compared with the initial absorbance A0. Figure 8 As shown, under the same excitation light irradiation conditions, the DPBF absorbance value of the blank group showed a certain degree of decrease with the extension of irradiation time, while compound I-1 showed a slight downward trend in absorbance, and Cy7-TCF-OH showed a significant decrease in absorbance. This result indicates that the Cy7-TCF-OH molecules produced by compound I-1 after stimulation have a strong ability to release singlet oxygen under 808nm near-infrared light irradiation. Furthermore, because compound I-1 can self-assemble into nanoparticles in aqueous solution, its photostability and in vivo circulation stability are increased, indicating its potential as a photosensitizer for PDT applications in tumors.

[0127] Experimental Example 3 Cell Imaging Experiment

[0128] The nuclear dye Hoechst33342 (100nM) and I-1 (10μM) were added to the cell culture medium with the lysosomal dye Lyso-Green (75nM) or mitochondrial dye Mito-Green (75nM) respectively, and the cells were stained for 30 minutes. After staining, the cells were rinsed twice with PBS solution and then observed and photographed under a confocal fluorescence microscope. Figure 9 As shown, it shows that compound I-1 is successfully taken up by cells and distributed in lysosomes and mitochondria. The Pearson correlation coefficient of its distribution in mitochondria is 0.79.

[0129] Experimental Example 4: Intracellular Reactive Oxygen Species Generation Detection Experiment

[0130] HeLa cells were plated at 10 per well 5 The cells were initially cultured in a 24-well plate. After 24 hours, Ⅰ-1 was added at a concentration of 10 μM. A blank control (no sample, only DCFH-DA) and a Cy7-TCF-OH group were also set up as controls. After 2 hours of co-incubation, the cells were washed twice with PBS. Serum-free culture medium containing DCFH-DA (5 μM) was added to each well and incubated for 30 minutes. After washing with PBS, the sample groups were divided into groups with or without laser irradiation. The irradiation group used an 808 nm laser with a power of 0.5 W / cm 2 Laser intensity was set at 100 nm and each well was irradiated for 6 min. Then the production of reactive oxygen species in the cells was examined using a laser confocal microscope. Figure 10As shown. It can be seen that due to the high level of reactive oxygen species in tumor cells, a lower intensity of green fluorescence can be observed even with the addition of the DCFH-DA probe group. Regardless of whether there is light exposure, the green fluorescence intensity of the Ⅰ-1 group is almost the same as that of the blank group, while the Cy7-TCF-OH group with laser irradiation has obvious green fluorescence observed over a large range. Combined with the results of in vitro photodynamic therapy, compound Ⅰ-1 itself has no obvious photodynamic therapy effect, but the Cy7-TCF-OH generated after being stimulated by the tumor microenvironment has a strong ability to produce reactive oxygen species. This result shows that Ⅰ-1 can produce reactive oxygen species under near-infrared light irradiation after being stimulated by the tumor cell microenvironment, which provides the possibility for Ⅰ-1 nanoparticles to perform photodynamic therapy.

[0131] Experimental Example 5 Intracellular Therapeutic Effect Detection Experiment

[0132] HeLa cells were plated at 10 per well 4 The cells were initially cultured in a 96-well plate. After 24 hours, different concentrations (0.1 μM to 100 μM) of Ⅰ-1 were added. The dark toxicity test was performed. The plate was placed in an incubator for 24 hours and then observed under a fluorescence microscope. It was found that almost all the cells survived. Figure 11 As shown, it is proved that compound I-1 itself has very low toxicity.

[0133] like Figure 12 As shown, the cell wells tested for phototoxicity were at 0.5 W / cm 2 Each well was irradiated for 6 minutes under an 808 nm laser, and after staining with the live cell dye Calcein-AM and the dead cell dye EthD-I for 20 minutes, photos were taken under a fluorescence microscope and it was found that when the concentration of Ⅰ-1 was greater than 12.5 μM, 95.5% of the cells died, proving that compound Ⅰ-1 has a strong killing effect on cancer cells under laser irradiation and has an excellent phototherapy / gas combined treatment effect.

[0134] Comparison of the phototoxicity and dark toxicity results showed that the compound itself was minimally toxic while exhibiting excellent phototoxicity against cancer cells, suggesting great potential for clinical applications in phototherapy for cancer. Other compounds of the present invention also exhibited similar phototherapy effects.

[0135] Experimental Example 6: Fluorescence imaging experiment in mice

[0136] First, a mouse tumor model was constructed. Female nude mice aged 4 to 5 weeks were selected and 4×10 6 Mouse breast cancer cells 4T1 in the logarithmic growth phase were injected subcutaneously in the chest of the mouse. 3The drug was administered by tail vein injection according to the weight of the mice, and the fluorescence intensity of the chest tumor was continuously monitored using a living fluorescence imager for 120 hours. Figure 14 As shown. Continuous imaging monitoring of fluorescence intensity at the chest tumor site in group I-1 demonstrated that, after tail vein injection, I-1 nanoparticles could precisely target the tumor site through the EPR effect and accumulate there. However, aggregation resulted in fluorescence quenching, resulting in a weak fluorescence signal. As high levels of GSH in the tumor microenvironment continuously stimulated the disulfide bonds of I-1, the fluorescence quenching caused by aggregation was restored after the I-1 nanoparticles responded to GSH. Simultaneously, the binding of the generated Cy7-TCF-OH to serum proteins was enhanced, resulting in continuous enhancement of fluorescence at the tumor site, enabling high-resolution enhanced fluorescence imaging of tumor tissue. Furthermore, fluorescence intensity results at the tumor site after 120 hours demonstrated stable and long-lasting accumulation and fluorescence emission within the tumor tissue, demonstrating its applicability for early detection, contour determination, and fluorescence imaging-guided surgical resection of tumor tissue in vivo, and its potential as an integrated diagnostic and therapeutic reagent.

[0137] Experimental Example 7 Photoacoustic Imaging Test of Mouse Tumors

[0138] First, a mouse tumor model was established. 6-week-old female nude mice were injected intrathoracically with 4×10 6 4T1 cells, and wait until the tumor volume grows to 75 mm 3 200 μg of compound I-1 was injected into mice via tail vein injection. The three-dimensional photoacoustic tomography system was used to monitor the mice at different time points. Figure 15 As shown, after injection, the fluorescence of the mouse tumor site gradually increased over time within 1-12 hours, and there was still a strong photoacoustic signal at 24 hours. This example proves that compound I-1 has an excellent photoacoustic signal. Moreover, the nude mice did not experience spasms, convulsions or other abnormalities within 24 hours, proving that compound I-1 has low toxicity and high safety.

[0139] Experimental Example 8: In vivo phototherapy experiment in mice

[0140] Nude mice were divided into 7 groups. Group 1 was injected with saline without laser irradiation; Group 2 was injected with saline and laser irradiated for 10 minutes; Group 3 was injected with 200 μL of Ⅰ-1 (200 μg) without laser irradiation; Group 4 was injected with 200 μL of Cy7-TCF-OH (200 μg) via the tail vein and laser irradiated with 0.5 W / cm 2 The tumor sites of mice were irradiated with 808 nm laser for 10 minutes; the fifth group was injected with 200 μL Cy7-TCF-OH (200 μg) and the tumor sites were irradiated with 0.25 W / cm 2The 808 nm laser was used to irradiate the tumor site of mice for 10 minutes; in group 6, 200 μL Ⅰ-1 (200 μg) was injected into the mice through the tail vein and the tumor was irradiated with 0.5 W / cm 2 The 808 nm laser was used to irradiate the tumor site of mice for 10 minutes; in group 6, 200 μL Ⅰ-1 (200 μg) was injected into the mice through the tail vein and the tumor was irradiated with 0.25 W / cm 2 The mouse tumor site was irradiated with an 808nm laser for 10 minutes. During the laser irradiation process, the tumor site was continuously photographed using a photothermal imager. The imaging results are shown in Figure 16 After the treatment, in order to accurately monitor the changes in the mouse tumors, the tumor sites were photographed and recorded every day, and the tumor volume and weight changes of each group of mice were measured, and the records were continuously recorded for 15 days.

[0141] like Figure 17 As shown, Ⅰ-1 and Cy7-TCF-OH group 0.5W / cm 2 The mice treated with laser irradiation experienced tumor rupture the next day due to the increase in local temperature and the increase in reactive oxygen species concentration under the light. As time went on, no obvious tumor growth was observed at the primary tumor site in both groups of mice, and the ruptured tumor site began to heal. By the 15th day, the ruptured site had completely healed, leaving only a tiny scar. 2 The Cy7-TCF-OH group experienced relapse on the 4th day, while the 0.25W / cm 2 The Ⅰ-1 group relapsed on the 8th day, and the tumor volume on the 15th day was much smaller than that of the Cy7-TCF-OH group. This shows that Ⅰ-1 has an excellent hypothermia therapeutic effect. There was no abnormal change in the body weight of the mice in the experimental and control groups after phototherapy, and no obvious side effects of Ⅰ-1 were observed, indicating that it is safe and reliable. This result proves that Ⅰ-1 nanoparticles can be stably and passively targeted to the tumor site and accumulate through the blood circulation, and exhibit excellent phototherapy / gas synergistic therapeutic capabilities under near-infrared light irradiation, with few side effects and safe and reliable treatment. Combined with the fluorescence imaging effect shown, it is proved that Ⅰ-1 has the potential to prepare highly safe integrated diagnostic and therapeutic reagents.

[0142] Test Example 9 Safety test of compound Ⅰ-1 in mice

[0143] The nude mice were divided into 6 groups. The first group was injected with normal saline through the tail vein, and the other 5 groups were injected with Ⅰ-1. The blood of the 6 groups of female Kunming mice after injection was collected for blood routine and liver function tests. Figure 20As shown, the blood routine indicators such as lymphocytes, mean corpuscular volume and erythrocyte volume distribution width of the six groups of mice were all within the normal range, and the liver function indicators such as blood protein, alkaline phosphatase and blood glucose were all within the normal range. The results show that compound I-1 has high safety, does not damage the liver in the short term, and is relatively safe and reliable. At the same time, after the end of in vivo phototherapy, the main organs of the mice, including the heart, liver, spleen, lungs, and kidneys, were dissected out and fixed with formalin and then sectioned. The samples were stained with hematoxylin-eosin (H&E) and subjected to pathological histological analysis to evaluate whether the drugs caused damage to the organs after phototherapy. The staining results are shown in Figure 1. Figure 21 As shown, compared with the normal saline group, the Ⅰ-1 injection group did not cause obvious damage to major tissues and organs, showing good biosafety.

[0144] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A diagnostic and therapeutic integrated compound with stimulus responsiveness, characterized in that: The compound has a structure shown in formula (I), and a micro-nano structure formed by self-assembly of a pharmaceutically acceptable salt in an aqueous solution: In the above formula (I): W is a stimulus response group, which is selected from In the formula (I), X, Y, and Z are independently selected from O or S.

2. The compound according to claim 1, characterized in that The compounds are compounds I-1, I-2, I-3, I-4, I-5, and I-6 。 3. The compound according to any one of claims 1 to 2, characterized in that The micro-nano structure of the compound is a nano-microsphere structure formed by self-assembly of a compound having a structure shown in formula (I) and a pharmaceutically acceptable salt in an aqueous solution.

4. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

5. Use of the compound according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of fluorescent probes, preparation of H2S donors and phototherapy drugs, preparation of drugs for diagnosing and / or treating cancer, phototherapy combined with gas therapy, and stimulus-responsive release of drugs.

6. The use according to claim 5, characterized in that The cancer is selected from esophageal cancer, non-small cell lung cancer, biliary tract cancer, head and neck cancer, Barrett's esophagitis, bladder cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, brain tumor, breast cancer or skin cancer; the phototherapy drug is a photodynamic therapy drug, a photothermal therapy drug or a photoacoustic therapy drug; the H2S donor is a COS / H2S donor; and the stimulus-responsive release is selected from reactive oxygen species response and reduction response.

Images