A bis-indocyanine green compound with stimuli responsiveness and application thereof

Abstract

The application provides a preparation method and application of a dual-indocyanine green diagnosis and treatment integrated compound with a stimulus response, provides a micro-nano structure formed by self-assembly of a compound with a structure shown in formula (I) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof in an aqueous solution, and preparation of a pharmaceutical composition and application thereof in preparation of a fluorescent probe, a phototherapy drug, a drug for diagnosis and / or treatment of cancer, and stimulus-responsive drug release. The compound provided by the application can accumulate at a tumor site, and can realize aggregation structure conversion and optical performance regulation by responding to a unique redox stimulus of a tumor microenvironment, realize precise photothermal / photodynamic combined treatment of the tumor in an aggregated state under irradiation of a specific excitation wavelength, and realize long-acting fluorescence imaging of a low background at the tumor after disaggregation of the aggregation. The tumor treatment effect is good, the trauma is small, the fluorescence imaging precision is high, the time is long, the biological safety is excellent, and diagnosis and treatment integration at the tumor site is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical pharmacy, and relates to a double-indocyanine green compound with a stimulus response and application thereof. BACKGROUND

[0002] Cancer is a disease that seriously threatens human life, and its early diagnosis and treatment are crucial. At present, surgical resection is still the gold standard for the treatment of many cancers, especially solid tumors. Due to the individual differences of tumors, strong mutation ability, easy metastasis and recurrence and other characteristics, doctors are difficult to accurately distinguish tumor tissues and their boundaries by naked eye and palpation and other methods during surgery, resulting in unclear resection results, which may cause recurrence or metastasis of tumors. Therefore, intraoperative imaging guided therapy is particularly necessary.

[0003] Biological fluorescence imaging technology can detect biological molecules, cells and tissues / organs in real time and visualization in three-dimensional scale, can track various physiological processes of organisms, and has the advantages of non-invasion, visualization, high spatiotemporal resolution, safety and rapidness, and has great advantages in intraoperative imaging of tumors. Among many in vivo imaging technologies, near-infrared (NIR) fluorescence imaging has the advantages of rapid real-time, high sensitivity, economy and convenience, and shows great potential in the fields of early diagnosis of tumors, drug delivery and controlled release, intraoperative boundary positioning and the like.

[0004] Fluorin dyes have high molar absorption coefficient, and are the most commonly used organic dyes for near-infrared fluorescence imaging. Among them, indocyanine green (ICG) is a near-infrared clinical imaging agent approved by the U.S. Food and Drug Administration (FDA), and is a kind of contrast agent commonly used in the clinical diagnosis of cardiovascular system diseases in China at present. It has a fast clearance rate in blood and a high safety factor. At the same time, ICG can be used as a photosensitizer to generate heat and cytotoxic reactive oxygen species (ROS) under near-infrared light irradiation, realizing the combined treatment of photothermal / photodynamic therapy of tumors. Because the normal tissues and organs of the human body hardly absorb near-infrared light, ICG has the potential to become an ideal diagnosis and treatment integrated molecule. However, the biological environment stability of free ICG injected intravenously is poor, it is easy to aggregate in the water medium, and it lacks target specificity, which results in insufficient bioavailability in the tumor site. In order to overcome these shortcomings, ICG is usually encapsulated in the core of nanocarriers by encapsulation or complexation. However, physically encapsulated or adsorbed ICG still has the problems of limited drug loading capacity, non-specific drug release, drug leakage and long-term toxicity of the carrier in the body. Therefore, in view of the excessive expression of ROS in the tumor microenvironment, it is urgent to design a new stimulus-responsive ICG delivery strategy to prolong the in vivo circulation time of ICG, improve the tumor targeting ability of ICG, and ultimately realize the low-background fluorescence imaging of the tumor site while realizing the precise photothermal / photodynamic combined therapy of the tumor site. SUMMARY

[0005] The present application aims to solve the above-mentioned problems existing in the prior art, and proposes an ICG-based supramolecular diagnosis and treatment integrated reagent and its application. The double-ICG molecules are connected through a stimulus-responsive connecting bond, which overcomes the shortcomings of poor anti-photobleaching ability and poor tumor targeting of single ICG molecules. At the same time, the introduction of the stimulus-responsive connecting bond can improve the hydrophilic / hydrophobic performance of ICG, so that the compound self-assembles into stable nanoparticles in water, targets and enriches in tumor tissues through the EPR effect, induces response under the tumor-specific microenvironment, releases ICG small molecules, and under the irradiation of a specific excitation wavelength, can realize low-background long-acting fluorescence imaging at the tumor site while achieving precise photothermal / photodynamic therapy effect on the tumor, realizing diagnosis and treatment integration at the tumor site, with high fluorescence imaging accuracy, long time, good tumor treatment effect, small trauma, and excellent biological safety.

[0006] The technical scheme of the present application is:

[0007] A diagnosis and treatment integrated compound with stimulus responsiveness, the compound has a micro-nano structure formed by self-assembly of a structure represented by formula (I) or its isomer, a pharmaceutically acceptable salt, a hydrate or a solvate in an aqueous solution:

[0008] (I)

[0009] In the above formula (I), B is a stimulus-responsive group, including , , , , , , , , , , , , , , , , , , or .

[0010] In the formula (I), R1, R2 are each independently selected from H, -(CH2) q CH3, -(CH2) q CF3, -(CH2) q CHCH2, -(CH2) q CCH, -(CH2) q OH, -(CH2) q COOH, -(CH2)q NH2, -(CH2) q CHO, -(CH2) q CO(CH2) q 'CH3, -(CH2) q COO(CH2) q 'CH3, -(CH2) q O(CH2) q 'CH3, , , , , , and wherein q, q' are each independently selected from an integer from 0 to 12; preferably, said R1, R2 are -(CH2)5COOH;

[0011] Further, said compound is compound I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23, I-24, I-25, I-26, I-27, I-28.

[0012] Further, the micro-nano structure of the present application is a nano-microsphere structure self-assembled from the compound having the structure shown in formula (I), isomers, pharmaceutically acceptable salts, hydrates or solvates thereof in an aqueous solution.

[0013] Preferably, the micro-nano structure of compound I is self-assembled from compound I (including compound I-1 to compound I-28 in Table 1) in an aqueous solution.

[0014] The particle size of the micro-nano structure is 1 nm to 800 nm, preferably 20 nm to 300 nm, and more preferably 30 nm to 200 nm.

[0015] The micro-nano structure of the present application is a nano-microsphere structure self-assembled from the compound having the structure shown in formula (I), isomers, pharmaceutically acceptable salts, hydrates or solvates thereof in an aqueous solution.

[0016] The present application also provides a preparation method of the micro-nano structure, comprising the following steps:

[0017] 1) dissolving the compound having the structure shown in formula (I), isomers, pharmaceutically acceptable salts, hydrates or solvates thereof with an organic solvent;

[0018] The organic solvent used is one or a mixture of more than one of alkane, alkene, arene, alcohol, ketone, aldehyde, carboxylic acid, ester or ether; specifically, the organic solvent is one or a mixture of more than one 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.

[0019] 2) adding the solution obtained by dissolving into water to obtain a compound solution with a final concentration of 1 nM-1 M of the compound;

[0020] The final concentration is preferably 10 nM-1 mM; more preferably 100 nM-500 μM; most preferably 0.2 μM-300 μM.

[0021] 3) self-assembly of the compound in the aqueous solution to form micro-nano structures.

[0022] The preparation method described above is simple, convenient and suitable for large-scale production.

[0023] The application also provides a pharmaceutical composition comprising:

[0024] 1) a therapeutically effective dose of a compound having the structure shown in formula (I) or an isomer, a pharmaceutically acceptable salt, a hydrate or a solvate thereof, and

[0025] 2) a pharmaceutically acceptable carrier.

[0026] Preferably, the pharmaceutically acceptable carrier comprises a diluent, a disintegrant, an excipient, a binder, a stabilizer or a combination thereof.

[0027] The compound provided by the application is used in the preparation of a stimulus-responsive fluorescent probe, a stimulus-responsive phototherapeutic drug, the preparation of a drug for diagnosing and / or treating cancer. The phototherapeutic drug is a photodynamic therapeutic drug, a photothermal therapeutic drug or a photoacoustic therapeutic drug.

[0028] The cancer includes 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, and the skin cancer includes melanoma.

[0029] The pharmaceutical composition provided by the application can be prepared into an injection, and the injection comprises a therapeutically effective dose of micro-nano structures and an injection solvent or an additional agent or a combination thereof; wherein the injection solvent is one, two or more than two of injection water, ethanol, propylene glycol, glycerol, polyethylene glycol. Preferably, the pharmaceutical composition can be prepared into an injection.

[0030] The micro-nano structure of the present application is a nano-micro sphere structure, and the pharmaceutical composition thereof further comprises an active agent encapsulated in the micro-nano structure, and the active agent is a therapeutic agent or a diagnostic agent, preferably a chemotherapeutic agent or a radiotherapeutic agent, including a small molecule chemotherapeutic drug, a targeted therapeutic drug, a chemotherapeutic drug, an antibody drug, etc.

[0031] The present application also provides the use of the micro-nano structure or the pharmaceutical composition thereof in the preparation of a stimulus-responsive drug release. The stimulus-responsive release includes active oxygen response, reduction response, enzyme response, pH response, hypoxia response, temperature response, light response, sound response and magnetic response.

[0032] In another aspect, the present application also provides the use of the micro-nano structure or the pharmaceutical composition thereof in the preparation of a phototherapy drug, and the use as a photosensitizer. The photosensitizer is used in the preparation of a phototherapy drug. The phototherapy drug is a photodynamic therapy drug, a photothermal therapy drug or a photoacoustic therapy drug.

[0033] The present application also provides the use of the micro-nano structure or the pharmaceutical composition thereof in the preparation of a drug for diagnosing and / or treating cancer. The cancer includes 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.

[0034] The present application also provides a method for phototherapy in a target area of a subject, comprising:

[0035] 1) providing the micro-nano structure;

[0036] 2) administering the micro-nano structure to the subject;

[0037] 3) waiting for the micro-nano structure to accumulate in the target area;

[0038] 3) using the micro-nano structure to irradiate the target area of the subject with light of a wave band, preferably using light waves of 808 nm.

[0039] The present application has the following beneficial effects:

[0040] (1) The compound provided by the present application can self-assemble to form a micro-nano structure in an aqueous solution, and through the EPR effect, the nano-particle can be targeted to tumor tissue stably and efficiently, and can be stimulated to respond under the unique redox microenvironment of the tumor site, and then accurately release small molecule ICG, so as to realize low background enhanced fluorescence imaging at the tumor site under near-infrared light irradiation, and be used for photothermal / photodynamic combined therapy of cancer under the guidance of near-infrared fluorescence imaging, and show higher biological safety, excellent fluorescence imaging accuracy and photothermal / photodynamic combined therapy effect, and have a broad prospect in cancer diagnosis and treatment.

[0041] (2) The micro-nano structure formed has high dimensional stability, high biological safety, excellent anti-photobleaching property and excellent tumor targeting property, and can realize the cleavage of the stimulus-responsive connecting bond and the release of the photosensitizer under the unique pathological microenvironment of the tumor, has good targeting effect and imaging treatment effect, and has great market value and broad commercialization prospect.

[0042] (3) The compound provided by the application contains a stimulus-responsive connecting bond and a high-biological-safety photosensitizer ICG which has been applied in clinic, is passively targeted to tumor tissue through the EPR effect, releases ICG molecules in response to the tumor microenvironment stimulus, produces long-acting low-background fluorescence and releases heat and ROS under near-infrared light irradiation, realizes fluorescence imaging guided photothermal / photodynamic therapy, and has great potential in commercialization and clinicalization. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The synthesis route of the compound I-1 provided by the application is shown in the figure;

[0044] Figure 2 The ultraviolet absorption spectrum of the compound I-1 in different polar solvents is shown in the figure;

[0045] Figure 3 The fluorescence emission spectrum of the compound I-1 in different polar solvents is shown in the figure;

[0046] Figure 4 The transmission electron microscope image of the nanometer microspheres formed by self-assembly of the compound I-1 in an aqueous solution is shown in the figure;

[0047] Figure 5 The particle size test result of the compound I-1 provided by the application in an aqueous solution is shown in the figure, which shows that the compound can self-assemble into a micro-nano structure;

[0048] Figure 6 The cyclic temperature change graph of the compound I-1 under 808 nm laser irradiation is shown in the figure;

[0049] Figure 7 The temperature change graph of the compound I-1 of different concentrations under 808 nm laser irradiation is shown in the figure;

[0050] Figure 8 The ultraviolet absorption change graph of the active oxygen trapping agent DPBF in the aqueous solution containing the compound I-1 under 808 nm laser irradiation is shown in the figure, which proves that the compound I-1 has the ability to produce active oxygen under near-infrared light irradiation;

[0051] Figure 9 The fluorescence emission intensity change of the compound I-1 before and after the active oxygen (ROS) stimulus response is shown in the figure;

[0052] Figure 10The fluorescence emission intensity change of compound I-1 in different concentrations of serum before and after the response to reactive oxygen species (ROS) stimulation;

[0053] Figure 11 The microscopic imaging of the nanoscale microspheres self-assembled by compound I-1 after phagocytosis by cells;

[0054] Figure 12 The colocalization scatter plot of the nanoscale microspheres self-assembled by compound I-1 after phagocytosis by cells and lysosomes and mitochondria;

[0055] Figure 13 The reactive oxygen species production of compound I-1 in HeLa cells after different treatments;

[0056] Figure 14 The dark toxicity and phototoxicity of the nanoscale microspheres self-assembled by compound I-1 to HeLa cells;

[0057] Figure 15 The fluorescence imaging of the dark toxicity and phototoxicity of the nanoscale microspheres self-assembled by compound I-1 to HeLa cells;

[0058] Figure 16 The fluorescence imaging of the tumor site at different time points after intravenous injection of the nanoscale microspheres self-assembled by compound I-1 into tumor-bearing mice;

[0059] Figure 17 The fluorescence intensity change of the tumor site at different time points after intravenous injection of the nanoscale microspheres self-assembled by compound I-1 into tumor-bearing mice;

[0060] Figure 18 The photo-thermal imaging of the phototherapy of compound I-1 in mice;

[0061] Figure 19 The phototherapy image of compound I-1 in mice;

[0062] Figure 20 The tumor volume change image of tumor-bearing mice after intravenous injection of the nanoscale microspheres self-assembled by compound I-1 and phototherapy;

[0063] Figure 21 The body weight change image of mice after intravenous injection of the nanoscale microspheres self-assembled by compound I-1 and phototherapy;

[0064] Figure 22 The liver function and blood routine of mice after intravenous injection of compound I-1;

[0065] Figure 23H&E staining images of heart, liver, spleen, lung and kidney sections of mice after intravenous injection of the nanoscale microspheres assembled from compound I-1 and light treatment; DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present application will be apparently and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without any creative work fall within the protection scope of the present application.

[0067] In order to further understand the present application, the present application will be further described with reference to the drawings and embodiments.

[0068] The present application provides some specific examples of the compounds, including compounds I-1 to I-28 shown in Table 1 below:

[0069]

[0070] Table 1 Structural formulas of compounds I-1 to I-28

[0071]

[0072]

[0073] The above compounds can be synthesized by the following reaction scheme:

[0074]

[0075] The main synthesis steps include:

[0076] (1) providing compounds a, b, c and d, respectively;

[0077] Synthesis of compound a:

[0078]

[0079] Compound 1' and compound 2' are dissolved in toluene, and reacted at 120℃ for 2 h. The solvent is removed by suction filtration, and the obtained solid is washed by acetone to obtain the target compound a.

[0080] Synthesis of compound b:

[0081]

[0082] Compound 3' and compound 4' were dissolved in a mixture of acetic acid and acetic anhydride, and the mixture was reacted at 100°C under nitrogen atmosphere for 2 h. The resulting product was vacuum evaporated to dryness, precipitated with excess ether, suction filtered with a Buchner funnel, dissolved in dichloromethane, re-precipitated with ether, washed with water, and suction filtered with a Buchner funnel again. The resulting solid was purified by column chromatography to obtain product b.

[0083] Synthesis of compound c:

[0084]

[0085] Compound 5' and compound 6' were added to toluene. The mixture was heated to reflux at 110°C under nitrogen atmosphere for 24 h. The resulting solid was washed with water and dichloromethane in turn, suction filtered with a Buchner funnel, and vacuum dried to obtain compound c.

[0086] Compound d was purchased directly.

[0087] (2) Compound a and compound b were dissolved in acetic anhydride, heated to reflux, and then compound c was added. After heating to reflux, the solvent was evaporated under vacuum, and the resulting solid was purified by column chromatography. The obtained product was dissolved in dichloromethane, and EDC•HCl and DMAP were added. After reaction at room temperature overnight, the mixture was washed with saturated aqueous ammonium chloride and saturated aqueous sodium chloride three times, and the resulting solid was purified by column chromatography after drying to obtain product compound I.

[0088] Example 1 Synthesis of compound I-1 and its fluorescent properties

[0089] As shown in Figure 1 , the synthesis of compound I-1 includes the following steps:

[0090] 1) Synthesis of compound 1: Mercaptoacetic acid (9.2 g), acetone (3.54 g), and trifluoroacetic acid (10 μl) were mixed and stirred at room temperature for 6 h, and then the mixture was placed in an ice bath overnight. The resulting solution was centrifuged and filtered, washed with n-hexane and water three times in turn, and dried to obtain compound 1.

[0091] 1 H NMR (400 MHz, CD3OD): δ (ppm): 3.42 (s, 4H), 1.60 (s, 6H).

[0092] 2) Synthesis of compound 2: LiAlH4was transferred into a 25 ml flask, vacuumed and purged with nitrogen 3 times. 2 mL of anhydrous tetrahydrofuran (THF) was added into a constant pressure funnel and slowly dropped into the flask under ice bath condition; similarly, compound 1 (195.87 mg) was dissolved in 2 mL of anhydrous THF and dropped into the flask through the constant pressure funnel under ice bath condition, and the reaction was carried out at room temperature for 10 h. After the reaction was completed, 190.85 mg of deionized water was slowly added into the system under ice bath condition to quench the reaction, and the target compound 2 was obtained in the form of oil after column chromatography purification.

[0093] 1 H NMR (400 MHz, CD3OD): δ (ppm): 3.68 (t, 4H), 2.78(t, 4H), 1.58 (s,6H)。

[0094] 3) Synthesis of compound 3: 1, 1, 2-trimethyl-1H-benzoindole (0.6 g) was mixed with 1, 4-butanesultone (1.17 g) and heated to 120 °C, and stirred for 2 h. The obtained crystalline product was washed with acetone, and then filtered by Buchner funnel, and dried in vacuum to obtain compound 3.

[0095] 1 H NMR (400 MHz, DMSO- d 6): δ(ppm): 8.36 (m, 1H), 8.28 (d, J = 8.9 Hz,1H), 8.21 (m, 2H), 7.75 (d, J = 6.9 Hz, 2H), 4.61 (t, J = 7.8 Hz, 2H), 2.95(s, 3H), 2.54 (d, J = 7.2 Hz, 2H), 2.04 (m, 2H), 1.79 (m, 2H), 1.76 (s, 6H)。

[0096] 4) Synthesis of compound 4: Compound 3 (500 mg) and valerolactam hydrochloride (454 mg) were dissolved in a mixed solution of acetic acid (1.5 mL) and acetic anhydride (6 mL), and then the mixture was stirred at 100 °C under nitrogen atmosphere for 2 h. The obtained product was evaporated in vacuum, precipitated with excess ether, filtered by Buchner funnel, and then the obtained solid was dissolved in dichloromethane, re-precipitated with ether, washed with water, and repeatedly filtered by Buchner funnel. The obtained product was placed in a beaker and purified by column chromatography to obtain the target product compound 4.

[0097] 1 H NMR (400 MHz, DMSO- d 6): δ(ppm): 8.38 (d, J = 8.5 Hz, 1H), 8.23 ​​(d, J = 4.2 Hz, 1H), 8.20 (d, J = 6.6 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 13.6 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.76 (ddd, J = 8.4, 6.9, 1.4 Hz,1H), 7.68 (m, 1H), 7.63 (m, 3H), 7.58 (dd, J = 5.4, 1.8 Hz, 1H), 7.42 (m,2H), 7.08 (d, J = 15.1 Hz, 1H), 6.59 (dd, J = 14.4, 11.1 Hz, 1H), 5.23 (dd, J = 13.6, 11.5 Hz, 1H), 4.50 (t, J = 7.8 Hz, 2H),1.96 (s, 3H), 1.91 (d, J = 1.6Hz, 10H), 1.76 (p, J = 7.4 Hz, 2H).

[0098] 5) Synthesis of Compound 5: 6-Bromhexanoic acid (1.17 g) was completely dissolved in toluene (3 mL). The solution, along with 1,1,2-trimethyl-1H-benzoindole (1.05 g), NaI (0.9 g), and toluene (3 mL), was then added to a 25 mL three-necked flask. The mixture was heated to 110 °C in an oil bath under nitrogen atmosphere and refluxed for 24 h. The resulting solid was washed sequentially with water and dichloromethane, filtered through a Buchner funnel, and dried under vacuum to obtain product compound 5.

[0099] 1 H NMR (400 MHz, CD3OD): δ (ppm): 8.32 (dt, J = 8.5, 1.0 Hz, 1H), 8.24(d, J= 8.9 Hz, 1H), 8.15 (m, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.80 (dd, J =8.4, 6.9 Hz, 1H), 7.71 (dd, J = 8.2, 6.9 Hz, 1H), 4.64 (m, 2H), 2.35 (t, J =7.1 Hz, 2H), 2.05 (m, 2H), 1.84 (s, 6H), 1.71 (m, 2H), 1.59 (m, 2H).

[0100] 6) Synthesis of compound 6: Compound 4 (543 mg) and compound 5 (324 mg) were dissolved in solvent pyridine (6 mL) and refluxed at 70°C under nitrogen atmosphere for 2 h in the dark. After repeated recrystallization from ether, the product was isolated using column chromatography to obtain compound 6.

[0101] 1 H NMR (400 MHz, CD3OD): δ (ppm): 8.20 (d, J = 8.6 Hz, 2H), 7.97 (m,6H), 7.59 (m, 5H), 7.45 (m, 2H), 6.59 (q, J = 12.3 Hz, 2H), 6.34 (dd, J =38.7, 13.6 Hz, 2H), 4.20 (dt, J = 21.0, 7.2 Hz, 4H), 2.91 (t, J = 6.8 Hz,2H), 2.32 (t, J = 7.2 Hz, 2H), 1.98 (m, 16H), 1.85 (m, 2H), 1.70 (p, J = 7.3Hz, 2H), 1.54 (m, 2H).

[0102] 7) Synthesis of compound I-1: 0.8 g of compound 6 and 0.1 g of compound 2 were dissolved in 10 mL of dichloromethane, 0.28 g of EDC-HCl and 0.35 g of DMAP were added dropwise under ice bath, and the reaction was carried out at room temperature overnight, then 40 mL of dichloromethane was added, washed with 50 mL of saturated aqueous NH4Cl and saturated brine for 3 times respectively, dried with anhydrous Na2SO4, and then the solvent was evaporated under vacuum, and the obtained solid was purified by column chromatography to obtain the target compound I-1.

[0103] 1 H NMR (400 MHz, CD3OD): δ (ppm): 8.15 (d, J = 8.6 Hz, 4H), 7.96 (q, J = 8.9 Hz, 12H), 7.53 (m, 14H), 6.59 (s, 4H), 6.26 (dd, J = 73.7, 13.3 Hz,4H), 4.21 (m, 12H), 2.97 (d, J = 7.6 Hz, 4H), 2.86 (t, J = 6.8 Hz, 4H), 2.39(t, J = 7.3 Hz, 4H), 2.01 (m, 32H), 1.89 (t, J = 7.1 Hz, 4H), 1.75 (p, J =7.6 Hz, 4H), 1.57 (d, J = 18.2 Hz, 10H)。

[0104] The UV absorption spectrum and fluorescence emission spectrum of compound I-1 in water and acetonitrile are shown in FIGS. 1 and 2 respectively, and it can be seen that the absorption and emission spectra of compound I-1 in the two solvents are significantly different, wherein the absorption spectrum of I-1 in aqueous solution is wider, and the emission is less, approaching zero, indicating that the physical properties of compound I-1 in aqueous solution and organic solvent are different. Figure 2 and Figure 3

[0105] Example 2 Synthesis of compounds I-2 to I-15

[0106] Compounds I-2 to I-15 can be prepared by using a method similar to that of Example 1.

[0107] 1. Synthesis of compound I-2

[0108] 1. Synthesis of compound I-2

[0109] Compound I-2 was prepared by replacing compounds 5 and 6 in Example 1 with compounds 14 and 16 respectively, and by replacing compound 2 in Example 1 with compound 18, while using the same reagents and preparation methods as in steps 1-7 of Example 1.

[0110] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.81 (m, 3H), 7.74 (m, 2H), 7.62 (m,2H), 7.41 (m, 2H), 7.34 (m, 3H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74 (s, 1H),6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.07 (s,2H), 3.30 (s, 2H), 3.22 (d, J = 12.5 Hz, 1H), 3.15 (m, 3H), 2.70 (d, J = 12.5Hz, 1H), 2.58 (d, J = 12.5 Hz, 1H), 2.05 (s, 2H), 1.89 (s, 2H), 1.82 (s, 2H), 1.74 (d, J = 1.3 Hz, 13H), 1.58 (s, 3H), 1.49 (s, 2H).

[0111] 2. Synthesis of Compound I-3

[0112]

[0113] Compound I-3 was prepared by replacing compound 5 in Example 1 with compound 14 and compound 2 in Example 1 with compound 19, while using the same reagents and preparation methods as in steps 1-7 of Example 1.

[0114] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (dd, J = 8.6, 0.8 Hz, 3H), 7.72(d, J = 13.5 Hz, 2H), 7.63 (m, 2H), 7.41 (m, 2H), 7.36 (d, J= 8.5 Hz, 2H),7.32 (s, 1H), 7.24 (d, J = 0.6 Hz, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.28 (s, 1H), 4.06(d, J = 11.0 Hz, 2H), 3.30 (s, 1H), 3.22 (d, J = 12.4 Hz, 1H), 3.15 (d, J =12.4 Hz, 1H), 3.09 (m, 2H), 2.70 (d, J = 12.5 Hz, 0H), 2.59 (d, J = 12.5 Hz,0H), 2.28 (s, 1H), 2.05 (s, 2H), 1.89 (s, 2H), 1.82 (s, 1H), 1.74 (m, 13H), 1.68 (s, 1H), 1.50 (m, 5H), 1.43 (s, 1H).

[0115] 3. Synthesis of Compound I-4

[0116]

[0117] Compound I-4 was prepared by replacing compounds 5 and 6 in Example 1 with compounds 15 and 17 respectively, and by replacing compound 2 in Example 1 with compound 18, while using the same reagents and preparation methods as steps 1-7 in Example 1.

[0118] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 0.7 Hz, 1H), 7.41 (m, 2H), 7.34 (m, 3H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74(s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.34 (m, 3H), 4.45 (s, 2H), 4.03 (m, 3H), 4.04 (d,J = 12.5 Hz, 1H), 3.31 (d, J = 12.3 Hz, 1H), 3.24 (m, 2H), 3.15 (d, J = 12.3 Hz, 1H), 2.78 (s, 2H), 2.05 (s, 2H), 1.90 (m, 6H),1.74 (d, J = 1.3 Hz, 13H), 1.58 (s, 3H).

[0119] 4. Synthesis of Compound I-5

[0120]

[0121] Compound I-5 was prepared by using compound 15 instead of compound 5 and compound 19 instead of compound 2 in Example 1, and the rest of the reagents and preparation methods were the same as those in steps 1-7) of Example 1.

[0122] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (d, J = 13.5 Hz, 2H), 7.63 (d, J = 0.7 Hz, 1H), 7.41 (m, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.32 (s, 1H), 7.24 (d, J = 0.6 Hz, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.28 (s, 1H), 4.13 (m, 2H), 4.04 (m, 2H), 3.31 (s, 1H), 3.22 (d, J = 12.4 Hz, 1H), 3.14 (m, 2H), 2.78 (s, 1H), 2.28 (s, 1H), 2.05 (s, 2H), 1.93 (s, 1H), 1.88 (d, J = 9.9 Hz, 3H), 1.74 (m, 13H), 1.68 (s, 1H), 1.51 (d, J= 11.3 Hz, 4H), 1.43 (s, 1H).

[0123] 5. Synthesis of compound I-6

[0124]

[0125] Compound 22 is used instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3~7) in Example 1, to prepare compound I-6.

[0126] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 0.8 Hz, 1H), 7.38 (m, 5H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.17 (s, 2H), 4.04 (s, 2H), 3.22 (d, J = 12.5 Hz, 1H), 3.15 (d, J = 12.3 Hz, 1H), 2.91 (s, 2H), 2.26 (s, 2H), 2.05 (s, 2H), 1.89 (s, 2H), 1.74 (d, J = 3.4 Hz, 13H), 1.68 (s, 2H), 1.50 (s, 2H), 1.43 (s, 2H).

[0127] 6. Synthesis of compound I-7

[0128]

[0129] Compound 14 and 16 are used instead of compounds 5 and 6 in Example 1, respectively, compound 22 is used instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3~7) in Example 1, to prepare compound I-7.

[0130] 1H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.61 (m,2H), 7.41 (m, 2H), 7.34 (m, 3H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74 (s, 1H),6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.07 (s,2H), 3.31 (s, 2H), 3.22 (d, J = 12.5 Hz, 1H), 3.14 (m, 3H), 2.74 (s, 2H), 2.05 (s, 2H), 1.89 (s, 2H), 1.82 (s, 2H), 1.74 (d, J = 1.3 Hz, 13H), 1.49 (s, 2H).

[0131] 7. Synthesis of Compound I-8

[0132]

[0133] Compound I-8 was prepared by replacing compound 5 in Example 1 with compound 14 and compound 23 in Example 1, with K selected from S, Se, and Te, and the other required reagents and preparation methods being the same as steps 3-7 in Example 1.

[0134] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (dd, J = 8.7, 0.9 Hz, 3H), 7.72(d, J = 13.5 Hz, 2H), 7.62 (m, 2H), 7.41 (m, 2H), 7.36 (d, J = 8.4 Hz, 2H),7.32 (s, 1H), 7.24 (d, J = 0.6 Hz, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.16 (s, 1H), 4.06(d, J= 11.0 Hz, 2H), 3.32 (s, 1H), 3.22 (d, J = 12.4 Hz, 1H), 3.14 (m, 2H), 2.83 (s, 1H), 2.61 (s, 1H), 2.26 (s, 1H), 2.05 (s, 2H), 1.89 (s, 2H), 1.82 (s, 1H), 1.74 (m, 13H), 1.68 (s, 1H), 1.49 (d, J = 1.4 Hz, 2H), 1.43 (s, 1H).

[0135] 8. Synthesis of compound I-9

[0136]

[0137] Compound I-9 is prepared by using compound 15 and 17 instead of compound 5 and 6 in Example 1, using compound 22 instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3~7) in Example 1.

[0138] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 0.7 Hz, 1H), 7.41 (m, 2H), 7.35 (m, 3H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.13 (m, 3H), 4.04 (d, J = 12.5 Hz, 1H), 3.33 (s, 2H), 3.22 (d, J = 12.5 Hz, 1H), 3.15 (d, J = 12.3 Hz, 1H), 2.77 (s, 2H), 2.05 (s, 2H), 1.90 (m, 6H), 1.74 (d, J = 1.3 Hz, 13H).

[0139] 9. Synthesis of compound I-10

[0140]

[0141] Compound 15 instead of compound 5 in Example 1, compound 23 instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as the steps 3~7) in Example 1, to prepare compound I-10.

[0142] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (dd, J = 8.7, 0.9 Hz, 3H), 7.72(d, J = 13.5 Hz, 2H), 7.63 (d, J = 0.6 Hz, 1H), 7.41 (m, 2H), 7.36 (d, J =8.4 Hz, 2H), 7.32 (s, 1H), 7.24 (d, J = 0.6 Hz, 1H), 7.03 (s, 1H), 6.74 (s,1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.13(m, 3H), 4.04 (m, 2H), 3.33 (s, 1H), 3.22 (d, J = 12.4 Hz, 1H), 3.15 (d, J =12.4 Hz, 1H), 2.83 (s, 1H), 2.78 (s, 1H), 2.26 (s, 1H), 2.05 (s, 2H), 1.93(s, 1H), 1.88 (d, J = 9.9 Hz, 3H), 1.74 (m, 13H), 1.68 (s, 1H), 1.50 (s, 1H),1.43 (s, 1H).

[0143] 10、Synthesis of compound I-11

[0144]

[0145] Compound 24 instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as the steps 3~7) in Example 1, to prepare compound I-11.

[0146] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 0.8 Hz, 1H), 7.35 (m, 5H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74 (s, 1H), 6.67(d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.07 (s, 2H), 3.29 (s,2H), 3.22 (d, J = 12.5 Hz, 1H), 3.15 (d, J = 12.3 Hz, 1H), 2.72 (s,2H), 2.28 (s, 2H), 2.05 (s, 2H), 1.89 (s, 2H), 1.74 (d, J = 3.4 Hz, 13H),1.68 (s, 2H), 1.49 (s, 2H), 1.43 (s, 2H)。

[0147] 11. Synthesis of compound I-12

[0148]

[0149] Compound 14 and 16 are used instead of compound 5 and 6 in Example 1 respectively, compound 25 is used instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3~7) in Example 1, to prepare compound I-12.

[0150] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.07 (s, 2H), 3.29 (s,2H), 3.22 (d, J= 12.5 Hz, 1H), 3.18 - 3.09 (m, 3H), 2.61 (s, 2H), 2.05 (s, 2H), 1.89 (s, 2H), 1.82 (s, 2H), 1.74 (d, J = 1.3 Hz, 13H), 1.49 (s, 2H).

[0151] 12. Synthesis of compound I-13

[0152]

[0153] Compound 14 instead of compound 5 in Example 1, compound 26 instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as the steps 3~7) in Example 1, to prepare compound I-13.

[0154] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (dd, J = 8.7, 0.9 Hz, 6H), 7.72(d, J = 13.5 Hz, 4H), 7.63 (d, J = 0.6 Hz, 2H), 7.58 (s, 1H), 7.41 (m, 4H), 7.34 (m, 6H), 7.24 (d, J = 0.6 Hz, 2H), 7.03 (s, 2H), 6.74 (s, 2H), 6.67 (d, J = 1.0 Hz, 2H), 6.59 (s, 2H), 6.35 (m, 6H), 4.45 (s, 4H), 4.28 (s, 2H), 4.06(d, J = 11.0 Hz, 4H), 3.31 (d, J = 12.3 Hz, 1H), 3.23 (m, 3H), 3.14 (m, 4H), 2.89 (d, J = 12.5 Hz, 1H), 2.81 (d, J= 12.3 Hz, 1H), 2.64 (s, 2H), 2.28 (s,2H), 2.05 (s, 3H), 1.89 (s, 3H), 1.82 (s, 2H), 1.74(m, 25H), 1.68 (s, 2H),1.49 (d, J = 0.6 Hz, 4H), 1.43 (s, 2H).

[0155] 13. Synthesis of compound I-14

[0156]

[0157] Compound 15 and 17 are used instead of compound 5 and 6 in Example 1 respectively, compound 25 is used instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3~7) in Example 1, to prepare compound I-14.

[0158] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (m, 3H), 7.72 (m, 2H), 7.63 (d, J = 0.7 Hz, 1H), 7.38 (m, 4H), 7.32 (s, 1H), 7.24 (s, 1H), 7.03 (s, 1H), 6.74(s, 1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.37 (dd, J = 8.6, 0.9 Hz,2H), 6.32 (d, J = 1.0 Hz, 1H), 4.45 (s, 2H), 4.13 (m, 3H), 4.04 (d, J = 12.5Hz, 1H), 3.22 (d, J = 10.8 Hz, 3H), 3.15 (d, J = 12.3 Hz, 1H), 2.78 (s, 2H),2.05 (s, 2H), 1.90 (m, 5H), 1.74 (d, J = 1.3 Hz, 12H)。

[0159] 14. Synthesis of compound I-15

[0160]

[0161] Compound 15 is used instead of compound 5 in Example 1, compound 26 is used instead of compound 2 in Example 1, K can be selected from S, Se, Te, and the rest of the required reagents, preparation method is the same as steps 3-7) in Example 1, to prepare compound I-15.

[0162] 1 H NMR (400 MHz, CD3OD): δ (ppm): 7.80 (dd, J = 8.7, 0.9 Hz, 3H), 7.72(d, J = 13.5 Hz, 2H), 7.63 (d, J = 0.6 Hz, 1H), 7.41 (m, 2H), 7.36 (d, J =8.5 Hz, 2H), 7.32 (s, 1H), 7.24 (d, J = 0.6 Hz, 1H), 7.03 (s, 1H), 6.74 (s,1H), 6.67 (d, J = 1.0 Hz, 1H), 6.59 (s, 1H), 6.35 (m, 3H), 4.45 (s, 2H), 4.26(s, 1H), 4.13 (m, 2H), 4.04 (m, 2H), 3.30 (d, J = 12.3 Hz, 0H), 3.18 (m, 3H),2.87 (s, 1H), 2.78 (s, 1H), 2.28 (s, 1H), 2.05 (s, 2H), 1.93 (s, 1H), 1.88(d, J = 9.9 Hz, 3H), 1.74 (m, 13H), 1.68 (s, 1H), 1.49 (s, 1H), 1.43 (s, 1H)。

[0163] Example 3 Preparation method of micro-nano structure

[0164] Taking the micro-nano structure self-assembled from compound I-1 as an example, I-1 is dissolved in DMSO (or other organic solvents such as ethanol) to prepare a 2 mM storage solution, 10 μL of the storage solution is added to 2 mL of deionized water to prepare nanoparticles.

[0165] Example 4 Characterization method of micro-nano structure

[0166] As Figure 4As shown, 10 μL of the working solution of compound I-1 was added dropwise to a silicon wafer, and observation and photography were performed under a transmission electron microscope (TEM). It can be obviously observed that the micro-nano structure is in the form of a nanometer microsphere. It is found from the observation result that the particle size of the micro-nano structure self-assembled from compound I-1 is about 150 nm. At the same time, the particle size of the nanometer microsphere self-assembled from compound I-1 in an aqueous solution was tested by DLS, and the result is shown in the following table. Figure 5 As shown, the particle size is about 30 nm to 200 nm, and the particle size does not change obviously after being placed at room temperature for 24 h, proving that the compound has excellent structural stability.

[0167] Example 5 Calculation of the molar extinction coefficient of compound I-1

[0168] Compound I-1 was prepared into a 2 mM DMSO storage solution, and then 10 μL was added into 2 mL of a solution with different polarity (water and acetonitrile). Ultraviolet absorption was tested by using an ultraviolet spectrophotometer to find the maximum absorption wavelength. The molar extinction coefficient of I-1 in different polarity solutions was calculated by the ultraviolet absorption spectrum.

[0169] The characteristic parameters of compound I-1 in different polarity solutions are as follows:

[0170]

[0171] As shown in the above table, the characteristic parameters of compound I-1 in different polarity solutions are different. The maximum absorption wavelength and the maximum excitation wavelength in water are obviously different from those in organic solvents, the molar extinction coefficient is significantly reduced, and the photothermal effect is stronger. The essence of this phenomenon is that compound I-1 self-assembles into a micro-nano structure in an aqueous solution. Due to the change in structural properties, the physical properties and characteristic parameters change, and such a change is beneficial to improve the photothermal effect and light stability. In fact, not only compound I-1 has such characteristics, but also other compounds of the present application have similar properties and can self-assemble into a micro-nano structure in an aqueous solution.

[0172] Test Example 1 In-vitro photothermal effect of compound I-1

[0173] A total of 4 groups of samples were added into a cuvette, and the lid was sealed.

[0174] Sample No. 1 is 3 mL of deionized water;

[0175] Sample No. 2 is 10 μM of I-1, and the specific preparation method is that 3 mL of deionized water is added with 15 μL of I-1 stock solution (2 mM, dissolved in DMSO);

[0176] Sample No. 3 is 20 μM of I-1, and the preparation method is 3 mL of deionized water plus 30 μL of I-1 stock solution (2 mM, dissolved in DMSO);

[0177] Sample No. 4 is 40 μM of I-1, and the preparation method is 3 mL of deionized water plus 60 μL of I-1 stock solution (2 mM, dissolved in DMSO).

[0178] Each sample is irradiated by an 808 nm laser for 10 minutes, and a thermal imager is used to record temperature data every 10 seconds. The temperature corresponding to the time is plotted, as shown in Figure 7 The temperature of Sample No. 1 almost does not change in 10 minutes, and only increases by 2.7℃; the temperature of Sample No. 2 increases from room temperature 12.2℃ to 33.6℃, and the temperature increases by 21.4℃; the temperature of Sample No. 3 increases from room temperature 11.8℃ to 43.6℃, and the temperature increases by 31.8℃; the temperature of Sample No. 4 increases from room temperature 11.6℃ to 50.5℃, and the temperature increases by 38.9℃. At the same time, we calculate the photo-thermal conversion efficiency of compound I-1, and find that the photo-thermal conversion efficiency is as high as 60.3%, which also shows that the compound I-1 has extremely excellent photo-thermal effect.

[0179] The photo-thermal stability experiment is tested by selecting Sample No. 3. As shown in Figure 6 , after irradiation by an 808 nm laser for 10 minutes, Sample No. 3 increases from room temperature 12.8℃ to 43.6℃, and then is naturally cooled for fifteen minutes. Then, Sample No. 3 is irradiated by an 808 nm laser for 10 minutes again, and then is naturally cooled. The above process is repeated for 5 times. The results of 5 repeated experiments show that each time of 808 nm laser irradiation can make Sample No. 3 increase from room temperature to at least 35.6℃, and the temperature increases by 22.8℃, which proves that Sample No. 3 can still release stable photo-thermal effect in the process of repeated temperature rise and fall, and has excellent photo-thermal stability.

[0180] The above experiments prove that the compound I-1 not only has excellent photo-thermal effect, but also improves the photo-thermal stability of small molecule ICG when used as a photosensitizer, and has a broad clinical application prospect. Other compounds of the present application also have similar properties.

[0181] Test Example 2: In vitro photodynamic effect of compound I-1

[0182] First, a 2 ml aqueous solution of compound I-1 with a concentration of 10 μM is prepared, and the ultra-pure water group is used as a control. Active oxygen indicator probe 1, 3-diphenyl isobenzofuran (DPBF) is added to the samples to make the final concentration 50 μM, and the ultraviolet absorption after the addition of DPBF is measured by using an ultraviolet-visible spectrophotometer, which is recorded as the initial absorbance. An 808 nm laser is used to irradiate the samples at a power density of 1 W / cm 2The absorbance was measured every 30 s of irradiation of the sample under the laser intensity, and the total irradiation time was 10 min. The absorbance results at 420 nm in the UV absorption data of DPBF were read respectively, and the initial absorbance A0 was compared and plotted. As shown in Figure 8 , under the same excitation light irradiation conditions, the DPBF absorption value of the blank group showed a certain degree of decline with the extension of irradiation time, which could be used as a control. Compound I-1 showed a significant trend of absorbance decrease. This result shows that under the irradiation of 808 nm near-infrared light, the compound I-1 nanoparticles formed by supramolecular aggregation still have the ability to release singlet oxygen. At the same time, since compound I-1 can form nanoparticles by self-assembly in aqueous solution, the light stability and in vivo circulation stability increase, which has the potential to be used as a photosensitizer for PDT in tumors.

[0183] Test Example 3 Changes in fluorescence intensity of compound I-1 in aqueous solution and different concentrations of FBS before and after ROS response

[0184] First, the working solution of compound I-1 in aqueous solution before and after ROS response was prepared at a concentration of 10 μM, and the fluorescence emission intensity of the two was tested respectively. The results are shown in Figure 9 As shown in the figure, the fluorescence emission after ROS response increased significantly, from almost flat baseline before response to 4.55×10 4 , showing a significant increase in fluorescence emission. The change in optical properties after ROS response enables compound I-1 nanoparticles to stimulate response in a high concentration of ROS environment after targeting tumors, and to exhibit enhanced fluorescence imaging at tumor sites, improving the problem of limited imaging resolution caused by aggregation quenching effect, and effectively realizing high-contrast imaging guided phototherapy, which is conducive to the application in imaging guided cancer therapy.

[0185] In order to further explore the fluorescence emission intensity of compound I-1 after stimulation response in physiological environment, the fluorescence emission spectrum of compound I-1 nanoparticles in different concentrations of FBS cell culture medium before and after ROS response was studied, and the results are shown in Figure 10 The fluorescence spectrum results show that the non-specific binding strength of compound I-1 nanoparticles and serum proteins is low, and before targeting tumors, it will not produce strong biological background signal to mask the signal of cancer lesions, which is theoretically more conducive to low background fluorescence imaging at tumor sites, and it is not easy to be eliminated from the blood, the tumor targeting is improved, and the in vivo circulation time is prolonged; after ROS response, due to the rich blood vessels in tumor tissues, the fluorescence enhancement caused by high protein binding will make the tumor tissue imaging show higher resolution and specificity, so as to realize sustained and long-acting low background fluorescence imaging at the tumor, thus showing great application potential in imaging guided tumor therapy.

[0186] Test Example 4 Cell imaging experiment

[0187] Cell nucleus dye Hoechst33342 (100 nM) and I-1 (10 μM) were added to cell culture medium respectively with lysosome dye Lyso-Green (75 nM) or mitochondrion dye Mito-Green (75 nM) for 30 min cell staining. After staining, the cells were washed twice with PBS solution, and then observed and photographed under a confocal fluorescence microscope. As shown in Figure 10 Figure 11 As shown in

[0188] Test Example 5 Intracellular reactive oxygen species detection experiment

[0189] HeLa cells were initially cultured in a 24-well plate at 10 5 cells per well, and after 24 h, 10 μM I-1 was added, and a blank control (no sample was added, only DCFH-DA was added) and a positive control (40 μM VC was added at the same time as the sample to quench reactive oxygen species) were set up. After 2 h of incubation, the cells were washed twice with PBS, and serum-free medium containing DCFH-DA (5 μM) was added to each well and incubated for 30 min. After washing with PBS, the sample groups were divided into groups with or without laser irradiation, and the light irradiation group used an 808 nm laser with an intensity of 1 W / cm 2 per well for 6 min. At the same time, the blank control group was set as a non-light irradiation group, and the VC group was set as a light irradiation group, and the same laser irradiation treatment was performed. Then, the laser confocal microscope was used to investigate the generation of reactive oxygen species in the cells. The results are shown in Figure 13 As can be seen, because the tumor cells themselves have a high level of reactive oxygen species, a low-intensity green fluorescence can be observed in the group with only DCFH-DA probe added. For the group without laser irradiation, the green fluorescence intensity of the I-1 group is almost the same as that of the blank group, while the group with laser irradiation can observe a large range of obvious green fluorescence. Combined with the results of the in vitro photodynamic test, it is proved that this is because the singlet oxygen produced by the I-1 group after light irradiation further oxidizes the DCFH-DA probe, causing fluorescence enhancement. As a control, after adding the antioxidant VC, the green fluorescence intensity is greatly reduced, because the reactive oxygen species generated after light irradiation is captured and quenched by VC. This result shows that I-1 can produce reactive oxygen species under near-infrared light irradiation after being taken up by cells, which provides the possibility of using I-1 as a photosensitizer for photodynamic therapy. ​

[0190] Test Example 6 In-cell phototherapy effect detection experiment

[0191] HeLa cells were initially cultured in 96-well plates at 10 4 cells per well, and after 24 h, different concentrations (0.1 μM~100 μM) of I-1 were added. The cell plates for dark toxicity detection were placed in the incubator for 24 h, and then observed under a fluorescence microscope. It was found that almost all the cells survived, as shown in FIG. 2, proving that the compound I-1 itself has very little toxicity. Figure 11

[0192] As shown in FIG. 3, the cell plates for phototoxicity detection were irradiated under an 808 nm laser for 6 min per well, and after staining with live cell dye Calcein-AM and dead cell dye EthD-I for 20 min, it was found under a fluorescence microscope that when the concentration of I-1 was greater than 12.5 μM, the cells were 100% dead, proving that the compound I-1 has strong killing power on cancer cells under laser irradiation, and has excellent photothermal / photodynamic combined therapy effect. Figure 10

[0193] Comparing the results of the phototoxicity group and the dark toxicity group, it is shown that the compound itself has very little toxicity, which continues the advantage of high safety of small molecule ICG, and at the same time, it shows excellent optical killing effect on cancer cells, and has great potential in the clinical application of phototherapy of cancer. Other compounds of the present application also have similar phototherapy effect.

[0194] Test Example 7 In vivo fluorescence imaging experiment of mice

[0195] In order to explore whether the self-assembly strategy of introducing a stimulus-responsive linkage is more advantageous than the strategy of polymer micelles loaded with ICG delivery, the present application uses ICG wrapped by polyether F-127 as a control to perform an in vivo fluorescence imaging experiment of mice. First, a tumor-bearing mouse model was constructed. 4~5-week-old female BALB / c mice were selected and randomly divided into two groups, namely the I-1 group and the ICG@F127 group. 4×10 6 mouse mammary cancer cells in the logarithmic growth phase were injected subcutaneously in the chest of the mice. When the tumor volume grew to 80 mm 3 , according to the body weight of the mice, the drug was injected through the tail vein, and the fluorescence intensity at the chest tumor was continuously imaged and monitored using a live fluorescence imager, for 8 days, and the results are shown in FIG. 6. The fluorescence intensity at the chest tumor was quantitatively analyzed by ROI, and the results are shown in FIG. 7. Figure 16 Figure 17 ​​​The results of continuous imaging monitoring of the fluorescence intensity at the tumor site in the I-1 group showed that after the injection of I-1 nanoparticles via the tail vein, I-1 nanoparticles could precisely target the tumor site and accumulate at the tumor site through the EPR effect within 6 h. With the continuous stimulation of the high level of ROS in the tumor microenvironment to the TK bond of I-1, the fluorescence quenching caused by aggregation of I-1 nanoparticles was restored after the response of I-1 nanoparticles to ROS, and the binding with serum proteins was enhanced, so the fluorescence at the tumor site was continuously enhanced, realizing high-resolution enhanced fluorescence imaging at the tumor tissue. At the same time, the fluorescence intensity results at the tumor site after 8 days showed that the in vivo circulation time of I-1 was effectively prolonged compared with that of the ICG@F127 group, and stable and long-acting accumulation and fluorescence emission were also exhibited in the tumor tissue, which had applicability in the early detection, contour determination and fluorescence imaging guided surgical resection at the tumor tissue in vivo, and had potential as a diagnosis and treatment integrated reagent.

[0196] Test Example 8 Optical treatment experiment in vivo of mice

[0197] The tumor-bearing nude mice were divided into 6 groups. The first group was injected with normal saline without laser irradiation; the second group was injected with normal saline and laser irradiation for 10 minutes; the third group was injected with 200 μL I-1 (200 μg) without laser irradiation; the fourth group was injected with 200 μL I-1 (200 μg) into the mouse body through the tail vein, and the mouse tumor site was irradiated with an 808 nm laser for 10 minutes; the fifth group was injected with 200 μL ICG@F127 (200 μg) without laser irradiation; and the sixth group was injected with 200 μL ICG@F127 (200 μg) into the mouse body through the tail vein, and the mouse tumor site was irradiated with an 808 nm laser for 10 minutes. During the laser irradiation process, a photothermal imager was used to continuously take pictures of the tumor site, and the imaging results are shown in Figure 18 After the end of the phototherapy, in order to accurately monitor the changes in the tumor site of the mice, the tumor site of the mice was photographed and recorded every day, and the tumor volume and body weight changes of the mice in each group were measured, and the recording was continued for 20 days.

[0198] As shown in Figure 19As shown, mice treated with laser irradiation in the I-1 and ICG@F127 groups experienced tumor ulceration the following day due to increased local temperature and elevated reactive oxygen species concentration under light irradiation. Over time, no significant tumor growth was observed at the primary tumor site in the I-1 treatment group, and the ulcerated tumors began to heal, completely healing by day 18, leaving only a small scar. Tumors in the ICG@F127 treatment group recurred on day 8. No abnormal changes in body weight were observed in either the experimental or control groups after phototherapy, and no significant side effects of I-1 were observed, indicating its safety and reliability. These results demonstrate that I-1 nanoparticles can be stably and passively targeted to and accumulated at the tumor site via blood circulation, exhibiting excellent photothermal / photodynamic synergistic therapeutic capabilities under near-infrared light irradiation, with minimal side effects and high safety. Combined with the observed fluorescence imaging effects, this demonstrates that I-1 can be used as a photosensitizer for in vivo imaging-guided therapy, showing potential for the preparation of highly safe therapeutic reagents.

[0199] Experimental Example 9: Safety test of compound I-1 in mice

[0200] Nude mice were divided into 6 groups. Group 1 received a tail vein injection of physiological saline, while the other 5 groups received injections of I-1. Blood samples were collected from female Kunming mice from all 6 groups after the injections for complete blood count and liver function tests. Figure 14 As shown, the complete blood count (CBC), mean corpuscular volume (MCV), and red blood cell distribution width (RDV) of the six groups of mice were all within the normal range, as were liver function indicators (ALB), alkaline phosphatase (ALP), and blood glucose. These results indicate that compound I-1 has high safety, does not damage the liver in the short term, and is relatively safe and reliable. Simultaneously, after in vivo phototherapy, the mice's major organs, including the heart, liver, spleen, lungs, and kidneys, were dissected, fixed in formalin, sectioned, stained with hematoxylin and eosin (H&E), and subjected to histopathological analysis to evaluate whether the drug caused organ damage after phototherapy. The staining results are shown below. Figure 23 As shown in the figure, compared with the saline group, no significant damage to major tissues and organs was found in the I-1 injection group, demonstrating good biocompatibility.

[0201] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A therapeutic compound with stimulus-responsive properties, characterized in that, The compound has the structure shown in formula (I-1), or formula (I-4), or formula (I-5), or a pharmaceutically acceptable salt thereof: 、 （Ⅰ-1） 、 （Ⅰ-4） ， （Ⅰ-5）。 2. Micro / nano structures formed by the self-assembly of the compound of claim 1 and its pharmaceutically acceptable salt in aqueous solution.

3. The micro / nano structure according to claim 2, characterized in that, The micro / nano structure of the compound is a nanosphere structure formed by the self-assembly in an aqueous solution of a compound having the structure shown in formula (I-1), or formula (I-4), or formula (I-5), or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition, characterized in that, It includes the compound of claim 1 or the micro / nano structure of any one of claims 2-3, and a pharmaceutically acceptable carrier.

5. The use of the compound of any one of claims 1 to 3 or the pharmaceutical composition of claim 4 in the preparation of fluorescent probes for the detection of cervical cancer or breast cancer, the preparation of phototherapy drugs for cervical cancer or breast cancer, the preparation of drugs for the diagnosis and / or treatment of cervical cancer or breast cancer, and the preparation of stimuli-responsive release drugs; wherein the stimuli-responsive release is selected as reactive oxygen species response.

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