Preparation method and application of parathyroid-targeted cyanine photosensitizer

Abstract

The application discloses a photosensitizer which can be targeted to parathyroid glands and a preparation method thereof, and the photosensitizer compound structure is shown as formula I. ICG dye is a commercial dye matrix, and a series of screening is conducted on the ICG dye, so that the parathyroid glands are targeted by using the inherent structural targeting characteristics. Meanwhile, the asymmetric F, I and F, Br atoms are introduced into the two side positions of the T800 cyanine dye, the existence of the F atom guarantees the targeting efficiency, meanwhile, the introduction of the heavy atoms provides the photodynamic treatment effect, and the precise targeted imaging and treatment of the parathyroid glands are realized. Furthermore, the contrast molecules with double F, double I and double Br introduced into the symmetrical positions on the two sides are synthesized for comparison. The results show that the T800-FI can realize the precise targeting of the parathyroid glands, and has the optimal photodynamic ablation effect on the parathyroid glands, and can be applied to the photodynamic treatment of parathyroid cancer, parathyroid tumor, hyperparathyroidism and parathyroid cyst and the like diseases, and the precise recognition, positioning and treatment effect can be realized.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of medicine and engineering, specifically relating to a parathyroid-targeting photosensitizer constructed based on heptamethrin dye, which has good phototoxicity to cancer cells and parathyroid cells, and good targeting and phototherapy effects on parathyroid tissue. Background Technology

[0002] The parathyroid glands are one of the endocrine glands in the human body. They consist of two pairs, about the size of mung beans, and are very similar in color and size to the thyroid gland, but their location varies from person to person. They are usually located in the middle and lower part of the back of the left and right lobes of the thyroid gland. Their main function is to secrete parathyroid hormone, which regulates the body's calcium and phosphorus metabolism. Currently, there are two main categories of parathyroid diseases: one is hyperparathyroidism, caused by elevated parathyroid hormone levels, including parathyroid adenomas; the other is hypoparathyroidism, caused by decreased parathyroid hormone levels, with symptoms such as low blood calcium, high blood phosphorus, and seizures. Both hyperparathyroidism and hypoparathyroidism can cause disease, with highly heterogeneous clinical manifestations. Furthermore, because these diseases are relatively rare and clinicians have limited knowledge of them, they are easily missed or misdiagnosed.

[0003] For the treatment of parathyroid diseases, the location of the parathyroid glands varies from person to person, and their size is tiny. Traditional surgical resection is prone to mis-removal or incomplete removal. Chemotherapy and radiotherapy also have limitations such as expensive drugs, significant side effects, and inability to be used long-term. Therefore, new diagnostic and treatment methods with high identification accuracy, fewer toxic side effects, and spatiotemporal tracking of disease progression are urgently needed. Currently, many new treatment methods have been developed, such as radiofrequency ablation and microwave ablation. Radiofrequency ablation is a tumor thermotherapy method. Its basic principle is to use heat energy to kill tumor cells. However, radiofrequency ablation carries risks such as postoperative recurrent laryngeal nerve paralysis and the risk of thyroid autoimmune diseases due to the body's stress response. Microwave ablation is a new treatment method for microadenoma. It is favored due to its advantages of less intraoperative bleeding and less trauma. However, when microwave ablation is performed at high power, it can cause damage to the blood supply to the parathyroid glands or damage to the parathyroid gland itself, leading to symptoms of hypocalcemia such as numbness in the limbs, and even convulsions and muscle spasms.

[0004] Photodynamic therapy boasts a range of advantages, including strong targeting, minimal organ damage, non-invasiveness, low toxicity, and repeatability. It is currently commonly used for skin diseases and cancer treatment, but rarely for treating microglandular diseases. However, it offers significant advantages when targeting microglandular diseases such as parathyroid gland disorders. Photosensitizers often suffer from drawbacks such as complex synthesis, short absorption and emission wavelengths, and poor biocompatibility. In contrast, cyanine dyes are easily modified, have strong tissue penetration, and good biocompatibility, making them particularly advantageous in the treatment of parathyroid tumors.

[0005] The parathyroid glands are extremely small and their color is difficult to distinguish compared to the thyroid gland, making both removal and ablation of the parathyroid glands very challenging. Therefore, accurately identifying the parathyroid glands is a key challenge we need to address. Previous literature has reported that heptamethrin dye modified with element F has inherent structural targeting properties, specifically targeting the parathyroid glands without interference from the thyroid gland. Therefore, we envision using this strategy to develop a photosensitizer that can maintain precise labeling of the parathyroid glands while also possessing photodynamic therapeutic effects. This has significant research implications for the treatment of parathyroid-related diseases. Summary of the Invention

[0006] Purpose of the invention: ICG dye is a commercially available dye matrix. Through a series of screenings, we hope to utilize its inherent structural targeting characteristics to achieve targeting of the parathyroid gland. At the same time, we modify the two sides of the T800 cyanine dye to introduce asymmetric F, Br, and I atoms. While ensuring targeting efficiency, the introduction of heavy atoms provides photodynamic therapy effects, thereby achieving precise targeted imaging and treatment of the parathyroid gland.

[0007] The technical solution of this invention: Compound of Formula I:

[0008]

[0009] Where X1 and X2 are each independently F, Br or I, and X1 and X2 are not simultaneously F or Br;

[0010] Y - The anion is an anion, and the anion has the same charge as the cation in Formula I;

[0011] Y - For F - Cl - ,Br - I - BF4 - NO3 - SO4 2- ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

[0012] Specifically, the compound is selected from the following structures:

[0013]

[0014] Specifically, the compound is selected from the following structures:

[0015]

[0016] Specifically, the compound is selected from the following structures:

[0017]

[0018] Specifically, Y - For F - Cl - Br or I - .

[0019] Application of the above-mentioned compounds in the preparation of photodynamic therapy drugs

[0020] The compounds described above are used to prepare drugs for treating cancer or parathyroid diseases.

[0021] This invention also provides a method for preparing a compound of formula I:

[0022] The synthesis steps are as follows:

[0023]

[0024] Compound A reacts with pentadienal diphenylamine hydrochloride in acetic anhydride to give compound B.

[0025] In an inert gas atmosphere, compound B and compound C react in acetic anhydride in the presence of a catalyst at room temperature to give compound I; the catalyst is one of sodium acetate and pyridine.

[0026] Y- is an anion, and the anion has the same charge as the cation in Formula I.

[0027] Y - For F - Cl - ,Br - I - BF4 - NO3 - SO4 2- ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

[0028] Specifically, the present invention also provides a method for preparing the parathyroid gland-targeting photosensitizer T800-X1X2, the steps of which are as follows:

[0029]

[0030] (1) Compound 1 was dissolved in water and HCl, and sodium nitrite was added dropwise. The mixture was stirred in an ice bath. Stannous chloride was then added dropwise, and the reaction was continued at room temperature. The solvent was removed and the mixture was purified to obtain compound 2.

[0031] (2) Compound 2 and 3-methyl-2-butanone were dissolved in acetic acid, and the reaction mixture was refluxed under N2 atmosphere. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound 3;

[0032] (3) Compound 3 and iodomethane were dissolved in acetonitrile and refluxed under N2 atmosphere. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound 4;

[0033] (4) Compound 4 and pentadienal diphenylamine hydrochloride were added to acetic anhydride in proportion. After the reaction was completed, sodium carbonate was added to adjust to neutrality, the solvent was removed, and compound 5 was obtained and directly added to the next reaction.

[0034] (5) The crude product 5 from the previous step and compound 3 with substituent X2 were dissolved in acetic anhydride in a certain proportion, a catalyst was added, and the mixture was stirred at room temperature under a N2 atmosphere. After the reaction was completed, the mixture was purified to obtain the photosensitizer T800-X1X2.

[0035] Furthermore, in the above technical solution, the combination of halogen atoms X1 and X2 can be one of (F, Br), (F, I), (F, F), (I, I), and (Br, Br).

[0036] Furthermore, in the above technical solution, the catalyst is one of sodium acetate and pyridine.

[0037] Furthermore, in the above technical solution, the method for removing the solvent includes vacuum filtration and vacuum evaporation; the purification method includes extraction and column chromatography.

[0038] Furthermore, in the above technical solution, real-time TLC monitoring is performed during the reaction in steps (2) and (4).

[0039] Furthermore, in the above technical solution, the reaction time of the reactants in step (1) is 2 hours in an ice bath and 2 hours in a room temperature reaction; the reflux time of the reactants in step (3) is 18 hours; and the stirring at room temperature under N2 atmosphere in step (5) is 2 hours.

[0040] Finally, this invention provides an application of a parathyroid gland-targeting photosensitizer. By testing the singlet oxygen yield of T800-X1X2, it is applied to investigate the effect of different halogen substituents on the phototoxicity of cyanine dye photosensitizers. By incubating MCF-7 cells with a medium containing T800-X1X2, cell uptake imaging is used to investigate the differences in photosensitizer uptake efficiency for different halogen substituents. By incubating 4T1 cells, HepG-2 cells, and MCF-7 cells with a medium containing the photosensitizer T800-X1X2 and then irradiating them, it is applied to photodynamic therapy of cancer cells. By injecting the photosensitizer T800-X1X2 intravenously into the body, the fluorescence imaging results at the parathyroid gland site are used to evaluate whether each group of photosensitizers targets the parathyroid gland and its targeting ability. Then, the parathyroid gland sites of rats are irradiated under the same conditions, and the decrease in parathyroid hormone and blood calcium concentration in rats is measured to evaluate the photodynamic therapy effect of each group of photosensitizers on the rat parathyroid gland.

[0041] Furthermore, in the above technical solution, the change in the singlet oxygen content of each group of photosensitizers T800-X1X2 under the same light irradiation is used as an evaluation index for the effect of different halogen atom substituents on the in vitro phototoxicity of the photosensitizers.

[0042] The process of detecting singlet oxygen in solution by the photosensitizer under light irradiation is as follows: each group of T800-X1X2 is dissolved in dimethyl sulfoxide solution to prepare a 10mM T800-X1X2 mother liquor. Take 3 mL of dichloromethane in a quartz dish, add 1,3-diphenylisobenzofuran (DPBF) and T800-X1X2 stock solution until the UV absorption intensity of DPBF is about 1 and the UV absorption intensity of T800-X1X2 is about 0.3. Then, irradiate continuously under an 808 nm, 3 mW laser with 30 s intervals for a total duration of 300 s. Then measure the decrease in UV absorption intensity of DPBF. Then replace T800-X1X2 with ICG and repeat the above steps. Finally, calculate the singlet oxygen yield of T800-X1X2 in each group based on the measured decrease in UV absorption intensity of DPBF, and use this as an evaluation index for the effect of different halogen atom substituents on the in vitro phototoxicity of T800-X1X2.

[0043] Furthermore, in the above technical solution, the cell imaging fluorescence intensity of each group of photosensitizers T800-X1X2 is used as an evaluation index for the effect of different halogen atom substituents on the cell uptake efficiency of photosensitizers.

[0044] The process for detecting the photosensitizer's uptake efficiency in in vitro cells is as follows: For in vitro cells, 2 μL of 10 mM T800-X1X2 stock solution from each group was dissolved in 2 mL of culture medium to prepare a 10 μM incubation solution. The pH of the incubation solution was 7.4–7.5, and the temperature was 37°C. MCF-7 cells were incubated in a culture medium containing the same concentration of T800-X1X2. The change in fluorescence intensity of the cells incubated with each group of T800-X1X2 over time was measured, and this was used as an evaluation index of the effect of different halogen atom substituents on the photosensitizer's uptake efficiency in cells.

[0045] Furthermore, in the above technical solution, the cytotoxicity results of T800-X1X2 are used as an evaluation index to reflect the influence of different halogen atom substituents on the cytotoxicity of photosensitizers.

[0046] The detection process for the photosensitizer's in vitro cytotoxicity is as follows: For in vitro cells, 10 μL of 10 mM T800-X1X2 stock solution from each group was dissolved in 90 μL of dimethyl sulfoxide solution to prepare a 1 mM stock solution for later use. 0 μL, 0.5 μL, 1 μL, 1.5 μL, 2 μL, 3 μL, 4 μL, and 5 μL of the 1 mM stock solution were dissolved in 1 mL of culture medium to prepare incubation solutions of 0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, and 5 μM, respectively, for later use. At this time, the pH of the incubation solution was 7.4-7.5, and the temperature was 37℃. 4T1 cells, HepG-2 cells, and MCF-7 cells were incubated for 1 h in a medium containing T800-X1X2 with the same concentration gradient. Then, they were continuously illuminated at 808 nm and 100 mW for 5 min, followed by incubation at 37℃ for 24 h. The medium was then blotted dry with cotton, and thiazolyl blue was added for incubation for 4 h. After blotting dry with cotton, dimethyl sulfoxide solution was added to test the absorbance of the solution at 490 nm. The cell viability of each photosensitizer group was calculated and used as an evaluation index of the effect of different halogen atom substituents on the cytotoxicity of each photosensitizer group.

[0047] Furthermore, in the above technical solution, the photosensitizer is evaluated for its ability to target the parathyroid gland by measuring the fluorescence imaging results of T800-X1X2 at the parathyroid gland site, and the photodynamic therapy effect of different photosensitizers on the parathyroid gland of rats is evaluated by measuring the decrease in parathyroid hormone and blood calcium concentration in rats.

[0048] The detection process for the photosensitizer's targeting and therapeutic effect on parathyroid tissue is as follows: A PBS solution containing T800-X1X2 is prepared and set aside. The concentrations of T800-X1X2 in the PBS solution are 1 mM and 5 mM. The prepared PBS solution is injected into the body via the tail vein. The change in fluorescence intensity at the parathyroid gland is measured over time, and this is used as an evaluation index of the effect of different halogen atom substituents on the parathyroid targeting of the photosensitizer. An 808 nm laser is used for continuous irradiation at 200 mW for 10 minutes. Blood samples are collected at 0 h, 6 h, 24 h, 36 h, 3 d, 5 d, and 10 d to measure PTH levels and serum calcium concentration, which are used as evaluation indicators of the effect of each group of photosensitizers on the phototherapy effect on parathyroid glands.

[0049] This invention, T800-X1X2, utilizes its inherent structural targeting features to achieve parathyroid gland targeting. The flanking positions of the T800 cyanine dye facilitate modification, and the introduction of F atoms significantly improves targeting efficiency. Simultaneously, the introduction of heavy atoms endows T800 with excellent photodynamic therapy effects. These two factors work together to achieve precise targeted imaging and treatment of the parathyroid gland. The photodynamic therapy effect of T800-X1X2 on the parathyroid gland was evaluated by detecting the fluorescence imaging results of T800-X1X2 in the parathyroid gland region of small animals, as well as the decrease in parathyroid hormone and blood calcium concentrations. T800-FI, while meeting certain photodynamic therapy requirements, exhibits high cellular uptake efficiency and excellent parathyroid gland targeting.

[0050] The T800-X1X2 described in this invention has the following outstanding advantages when used for the treatment of parathyroid tumors:

[0051] (1) Simple synthesis: T800-X1X2 targets the parathyroid glands by means of the inherent targeting properties of its structure, without the need to introduce additional targeting groups, making the synthesis convenient;

[0052] (2) Good targeting: The F atom introduced in T800-X1X2, combined with the inherent targeting of the cyanine dye structure, can ensure its high targeting efficiency for the parathyroid gland;

[0053] (3) Fast targeting speed: Cyanide dyes have a fast metabolic rate and can quickly reach the vicinity of the parathyroid gland after being injected via the tail vein;

[0054] (4) Good phototherapy effect: T800-X1X2 can cause powerful phototoxic killing of both cancer cells and normal parathyroid cells, and has the best photodynamic ablation effect on parathyroid glands, which has broad application prospects. Attached Figure Description

[0055] Figure 1The graph shows the singlet oxygen yield test results of T800-FI, T800-FBr, T800-FF, T800-BrBr and T800-II in solution.

[0056] Figure 2 These are confocal fluorescence imaging results of T800-FI, T800-FBr, T800-FF, T800-BrBr, and T800-II cells.

[0057] Figure 3 These are cytotoxicity test results for T800-FI, T800-FBr, T800-BrBr, and T800-II.

[0058] Figure 4 This is a time-dependent fluorescence imaging test diagram of T800-FBr and T800-FI in the parathyroid gland;

[0059] Figure 5 This is a graph showing the serum calcium concentration in rats after photodynamic therapy with T800-FBr and T800-FI.

[0060] Figure 6 This is a graph showing the PTH level test in rats after photodynamic therapy with T800-FBr and T800-FI;

[0061] Figure 7 This is the T800-FI NMR 1H spectrum.

[0062] Figure 8 It is the 1H NMR spectrum of T800-FBr.

[0063] Figure 9 It is the 1H NMR spectrum of T800-BrBr.

[0064] Figure 10 This is the 1H NMR spectrum of the T800-II.

[0065] Figure 11 This is the 1H NMR spectrum of the T800-FF. Detailed Implementation

[0066] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0067] Example 1:

[0068] This embodiment synthesizes the following five parathyroid-targeting photosensitizers.

[0069]

[0070] The synthetic route for T800-FBr is as follows:

[0071]

[0072] It is prepared in the following manner:

[0073] (1) Compound 1 (6.000 g, 1 eq) was dissolved in 4 mL of water and 20 mL of 37% concentrated HCl (v:v = 1:5). After stirring for 10 min in an ice bath, 10 mL of 20.8% sodium nitrite solution (2.085 g, 1.1 eq) was slowly added dropwise. The reaction was allowed to proceed for 1 h until a clear brownish-yellow solution was obtained. Then, stannous chloride (10.421 g, 2 eq) was dissolved in 7 mL of water and 7 mL of 37% concentrated HCl (v:v = 1:1). The solution was stirred at room temperature for 2 h until a light pinkish-white color appeared. The solution was then filtered to obtain solid compound 2 (4.125 g, 68.7% yield). 1 HNMR (400MHz, DMSO-d6) δ7.45(d,J=8.5Hz,2H),7.04(d,J=16.0Hz,1H),6.92–6.80(m,4H),2.62(s,2H),2.48(s,2H),1.10(s,6H).

[0074] (2) Compound 2 (5.000 g, 1 eq) and 3-methyl-2-butanone (3.678 g, 2 eq) were placed in a 250 mL single-necked flask, dissolved in 50 mL of acetic acid, and refluxed at 120 °C under N2 atmosphere. The reaction was monitored by TLC. After the reaction was completed, saturated sodium carbonate solution was added to remove acetic acid until no more bubbles were produced. The mixture was extracted three times with saturated sodium chloride aqueous solution and ethyl acetate, dried over anhydrous sodium sulfate, and the organic phase was evaporated under reduced pressure. The crude product was purified by column chromatography (EA:PE = 1:1) to give the brown liquid product compound 3 (3.782 g, 43.5% yield). 1 H NMR (500MHz, DMSO-d6) δ8.27 (s, 1H), 7.98 (d, 1H, J = 5.0Hz), 7.70 (d, 1H, J = 5.0Hz), 3.92 (s, 3H), 2.72 (s, 3H), 1.50 (s, 6H); 13 CNMR(125MHz,DMSO-d6)δ196.8,144.9,142.6,138.7,132.9,117.8,96.8,55.0,35.5,22.5,15.3.

[0075] (3) Compound 3 (1.500 g, 1 eq) and iodomethane (2.694 g, 2 eq) were dissolved in 10 mL of acetonitrile and refluxed overnight under N2 atmosphere. The next day, solid precipitation was observed. After the reaction was completed, diethyl ether was added to the reaction mixture, and the mixture was sonicated and filtered to obtain the brownish-yellow solid product compound 4 (1.210 g, 28.6% yield). 1H NMR (500MHz, DMSO-d6) δ8.27 (s, 1H), 7.98 (d, 1H, J = 5.0Hz), 7.70 (d, 1H, J = 5.0Hz), 3.92 (s, 3H), 2.72 (s, 3H), 1.50 (s, 6H); 13 CNMR(125MHz,DMSO-d6)δ196.8,144.9,142.6,138.7,132.9,117.8,96.8,55.0,35.5,22.5,15.3.

[0076] (4) Compound 4 (0.500 g, 1 eq) and pentadienal diphenylamine hydrochloride (0.427 g, 1.1 eq) were placed in a 50 mL round-bottom flask, and 10 mL of acetic anhydride was added. The mixture was stirred at 120 °C for 1 h under N2 atmosphere in the dark. The reaction was detected by TLC. After the reaction was completed, saturated sodium carbonate solution was added to adjust the pH to neutral. The mixture was then extracted three times with dichloromethane and saturated brine, dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness to obtain crude red solid product 5 (0.348 g, 37.5% yield), which was directly added to the next step of the reaction.

[0077] (5) Place the crude product 5 (0.348 g, 1 eq) from the previous step and 3-Br (0.736 g, 1.1 eq) in a 50 mL round-bottom flask, add 10 mL of acetic anhydride and 1 mL of pyridine, stir at 25 °C for 0.5 h in the dark under N2 atmosphere, and detect the reaction by TLC. After the reaction was complete, the pH was adjusted to neutral by adding saturated sodium carbonate solution. The mixture was then extracted three times with dichloromethane and saturated brine, dried over anhydrous sodium sulfate, and the organic phase was evaporated to dryness and purified by column chromatography (DCM:MeOH = 50:1) to obtain a dark green solid T800-FBr (0.214 g, 19.7% yield). ¹H-NMR (400 MHz, DMSO-d6) δ: 1.71 (s, 12H), 3.60 (s, 6H), 6.28 (d, J = 13.6 Hz, 2H), 6.60 (t, J = 12.8 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.53 (s, 2H), 7.67 (t, J = 13.2 Hz, 1H), 7.95 (t, J = 12.8 Hz, 2H). 13 C NMR (100MHz, DMSO-d6): δ29.93,32.60,49.09,104.48,111.47,119.96122.66,128.61,128.71,130.33,130.35,130.37,141.50,142.42.

[0078] The preparation processes of compounds T800-FI, T800-FF, T800-II and T800-BrBr were the same as those of T800-FBr, and the structures of the products were identified.

[0079] Examples 2-7 test the application performance of T800-FBr, T800-FI, T800-FF, T800-II, and T800-BrBr. For ease of description, T800-X1X2 is used as a general term for T800-FBr, T800-FI, T800-II, and T800-BrBr to describe the preparation and testing process of the samples.

[0080] Example 2: Singlet oxygen yield test of T800-FBr, T800-FI, T800-FF, T800-BrBr and T800-II.

[0081] Test system preparation: Prepare 1,3-diphenylisobenzofuran (DPBF) stock solution with dichloromethane solution; dissolve each T800-X1X2 in dimethyl sulfoxide to prepare T800-X1X2 stock solution with a concentration of 10 mM; dissolve ICG in dimethyl sulfoxide to obtain ICG stock solution with a concentration of 10 mM.

[0082] In the singlet oxygen yield test involving T800-X1X2, 3 mL of dichloromethane was placed in a quartz dish, and a certain amount of DPBF mother liquor was added until the UV absorption intensity of DPBF at 415 nm was about 1.0. Then, a certain amount of T800-X1X2 mother liquor was added to the quartz dish until the UV absorption intensity of T800-X1X2 at 760 nm was about 0.3. The quartz dish containing the above solution was then placed under an 808nm laser for intermittent irradiation. The irradiation power was 3mW, the interval irradiation time was 30s, and the total duration was 300s. The decrease in the UV absorption intensity of DPBF at 415nm after each interval irradiation was then measured. Next, 3mL of dichloromethane was added to the quartz dish, and a certain amount of DPBF stock solution was added until the UV absorption intensity at 415nm was about 1.0. Then, a certain amount of ICG stock solution was added to the quartz dish until the UV absorption intensity of ICG at 808nm was about 0.3. The quartz dish containing the above solution was then placed under an 808nm laser for intermittent irradiation. The laser power was 3mW, the interval irradiation time was 30s, and the total irradiation time was 300s. The decrease in the UV absorption intensity of DPBF at 415nm was then measured after each interval irradiation. Finally, the singlet oxygen yield of T800-X1X2 was calculated based on the measured decrease in DPBF UV absorption intensity. Figure 1It can be seen that the singlet oxygen yield of T800-II is the highest at 12.231%, and that of T800-FI is 7.238%. This proves that the introduction of heavy atoms can increase the singlet oxygen yield, and that a certain level of singlet oxygen production can still be maintained when a single heavy atom is present.

[0083] Example 3: Confocal fluorescence imaging assay of T800-FBr, T800-FI, T800-FF, T800-BrBr and T800-II cell uptake

[0084] Test system preparation: Dissolve each T800-X1X2 in dimethyl sulfoxide solution to prepare a 10 mM T800-X1X2 stock solution. Then take 2 μL of the stock solution, dissolve it in 2 mL of culture medium, shake well, and prepare a 10 μM T800-X1X2 incubation solution for later use. At this time, the pH value of the incubation solution is 7.4-7.5, and the temperature is 37℃.

[0085] In the assay of T800-X1X2 uptake efficiency of cells in vitro, T800-X1X2 was added to a confocal dish containing MCF-7 live cells at a growth density of approximately 60% and incubated. The changes in fluorescence intensity of the cells incubated with T800-X1X2 over time were measured. Figure 2 It can be seen that the cell fluorescence reaches its strongest at about 1 hour. Among the five molecules, T800-FI has the strongest fluorescence intensity, indicating that the target molecule T800-FI has the highest cellular uptake efficiency and is easier to achieve fluorescence-guided photodynamic therapy.

[0086] Example 4: Cytotoxicity tests of T800-FBr, T800-FI, T800-BrBr and T800-II

[0087] Test system preparation: Dissolve each T800-X1X2 sample in dimethyl sulfoxide (DMSO) solution to prepare a 10 mM T800-X1X2 stock solution. Then, take 10 μL of the 10 mM stock solution and dissolve it in 90 μL of DMSO solution to prepare a 1 mM stock solution for later use. Take 0 μL, 0.5 μL, 1 μL, 1.5 μL, 2 μL, 3 μL, 4 μL, and 5 μL of the 1 mM stock solution respectively and dissolve them in 1 mL of culture medium to prepare incubation solutions of 0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 3 μM, 4 μM, and 5 μM for later use. At this time, the pH of the incubation solution is 7.4–7.5, and the temperature is 37 °C.

[0088] 4T1, HepG-2, and MCF-7 cells were seeded at specific densities in 96-well plates. When the cell density reached over 80%, 100 μL of T800-X1X2 culture medium at different concentrations was added to each well and incubated for 1 h. Subsequently, the cells were continuously illuminated at 808 nm and 100 mW for 5 min, then transferred to a 37°C incubator for 24 h. After removing residual culture medium, 100 μL of 5 mg / mL thiazolyl blue medium was added to each well, and the cells were incubated at 37°C for 4 h. After removing residual culture medium, 100 μL of dimethyl sulfoxide was added to each well, and the absorbance at 490 nm was immediately measured. The cell viability under different photosensitizers was calculated. Figure 3 It can be seen that although the phototoxicity of the target molecule is lower than that of the control molecule, the target molecule T800-FI can still achieve a similar phototoxicity to the control molecule T800-II due to its high cellular uptake efficiency.

[0089] Example 5: Time-dependent fluorescence imaging of T800-FBr and T800-FI at the parathyroid gland

[0090] Test system preparation: T800-FBr and T800-FI were dissolved in dimethyl sulfoxide to obtain T800-FBr stock solution and T800-FI stock solution with a concentration of 100mM, respectively. Then, 10μL of the 100mM stock solution was dissolved in 990μL of PBS solution to prepare a 1mM stock solution for later use.

[0091] Animal preparation: The experimental animals were approved by the Animal Experiment Ethics Committee of Dalian University of Technology (DUT20210902). The mouse imaging and treatment procedures were carried out in accordance with the "Guidelines for the Protection and Use of Experimental Animals of Dalian Medical University". The 4-5 week old BALB / c female mice used in the experiment were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0092] In the time-dependent fluorescence imaging test of parathyroid tissue by T800-FBr and T800-FI, 100 μL of 1 mM T800-FBr and T800-FI PBS stock solutions were injected into mice via the tail vein, respectively. The change in fluorescence intensity at the parathyroid gland was measured over time. Figure 4 As shown, the T800-FI in the target molecule exhibits longer fluorescence intensity and retention time, which is consistent with the previous cellular uptake experiments and indicates that the target molecule reaches the parathyroid gland.

[0093] Example 6: Test of serum calcium concentration in rats after phototherapy with T800-FBr and T800-FI

[0094] Test system preparation: T800-FBr and T800-FI were dissolved in dimethyl sulfoxide solution to prepare T800-FBr stock solution and T800-FI stock solution with a concentration of 100mM, respectively. 10 μL of T800-X1X2 stock solution with a concentration of 100mM was taken and dissolved in 990 μL of PBS solution to prepare T800-X1X2 stock solution with a concentration of 1mM. 50 μL of T800-X1X2 stock solution with a concentration of 100mM was taken and dissolved in 950 μL of PBS solution to prepare T800-X1X2 stock solution with a concentration of 5mM for later use.

[0095] Animal preparation: The experimental animals were approved by the Animal Experiment Ethics Committee of Dalian University of Technology (DUT20210902). The rat treatment procedures were carried out in accordance with the "Guidelines for the Protection and Use of Experimental Animals of Dalian Medical University". The 250-300g male SD rats used in the experiment were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0096] In the test of serum calcium concentration after phototherapy of rat parathyroid tissue with T800-FBr and T800-FI, 200 μL of prepared T800-X1X2 (T800-FBr, T800-FI) stock solutions with concentrations of 1 mM and 5 mM were injected into rats (approximately 300 g) via the tail vein. After incubation for 12 h, continuous irradiation was performed for 10 min using an 808 nm laser at a light power of 200 mW. Blood samples were collected from each group at 0 h, 6 h, 24 h, 36 h, 3 d, 5 d, and 10 d to measure the serum calcium concentration. Figure 5 It can be seen that the blood calcium concentration of rats in all groups decreased after photodynamic therapy, which indicates that the parathyroid glands of rats were ablated under photodynamic therapy. Moreover, the decrease in blood calcium concentration in the T800-FI group was greater than that in the T800-FBr group, proving that T800-FI has the best photodynamic ablation effect on the parathyroid glands.

[0097] Example 7: Test of PTH Concentration in Rats After Phototherapy with T800-FBr and T800-FI. The test system, phototherapy method, and blood collection protocol were the same as in Example 6. After blood collection from each group, the PTH concentration of the rats was measured. Figure 6 It can be seen that the PTH concentration of rats in all groups decreased after photodynamic therapy, which indicates that the parathyroid glands of rats were ablated under photodynamic therapy. Moreover, the decrease in PTH concentration in the T800-FI group was greater than that in the T800-FBr group, proving that T800-FI has the best photodynamic ablation effect on the parathyroid glands.

Claims

1. The application of the compound in the preparation of photodynamic drugs for treating parathyroid diseases, characterized in that, The compound is selected from the following structures: ； Y - For F - Cl - ,Br - I - BF4 - NO3 - ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

2. The use of the compound according to claim 1 in the preparation of a photodynamic drug for treating parathyroid disease, characterized in that, Y - For F - Cl - Br or I - .

3. The use of the compound according to claim 1 in the preparation of a photodynamic drug for treating parathyroid disease, characterized in that, Y - is I - .

4. A method for preparing the compound as described in claim 1, characterized in that, Using heptamethrin dye as the photosensitizer parent material, the synthesis reaction is as follows: The synthesis steps are as follows: ; Where X1 is F and X2 is I; Compound A reacts with pentadienal diphenylamine hydrochloride in acetic anhydride to give compound B; In an inert gas atmosphere, compound B and compound C react in acetic anhydride in the presence of a catalyst at room temperature to give compound I. The catalyst is one of sodium acetate and pyridine; Y - For F - Cl - ,Br - I - BF4 - NO3 - ClO4 - CH3COO - CH3SO3 - or CF3SO3 - .

5. The preparation method according to claim 4, characterized in that, Y - is F - , Cl - , Br - , I - .

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