Porfin-BSH complex and its use in boron neutron capture therapy

Abstract

This invention relates to a porfin-BSH complex and its use in boron neutron capture therapy, and belongs to the pharmaceutical technology field. The porfin-BSH complex of the present invention is synthesized by coupling porfin to BSH using an amino-substituted maleimide compound as a linker, the method is simple, the porfin compound has the properties of tumor cell targeting, good uptake by tumor cells, safety and low toxicity, and fluorescence phenomenon of the photosensitizer itself, enabling targeted delivery of mercaptododecaborane disodium salt (BSH), a second-generation boron carrier, to tumor cells, and compared to BSH, intracellular 10 The B atom content can be increased to different degrees. Furthermore, by utilizing the fluorescence properties of porfin itself to monitor the distribution of the complex within the body in real time, optimal neutron irradiation conditions can be determined. This complex is not only safe and low-toxicity, but also... 10 This significantly increases the content of B. Furthermore, since this type of complex yields good results in in vitro activity tests of BNCT, it can be studied in the future as a potential boron carrier for BNCT.

Description

[Technical Field]

[0001] This invention belongs to the field of pharmaceutical technology and, more specifically, relates to porfin-BSH complexes and their use in boron neutron capture therapy. [Background technology]

[0002] Gliomas are the most common primary brain tumors, arising from the cancerous transformation of glial cells in the brain and spinal cord. Their high recurrence and mortality rates are due to their strong metastatic, highly invasive, and drug-resistant characteristics. In China, the annual incidence of gliomas is reported to be 5-8 cases per 100,000 people, and the 5-year mortality rate is the third highest among systemic tumors, after pancreatic and lung cancer. Conventional treatments mainly include surgical resection, radiotherapy, and chemotherapy. However, complete surgical resection is difficult due to problems such as the diffusion and infiltration of tumor cells and unclear infiltration boundaries during the progression of gliomas. Radiotherapy is less effective because the dose delivered to the brain is limited and resistance is easily developed. Furthermore, the blood-brain barrier (BBB) ​​in the brain restricts the accumulation of over 95% of small molecule drugs and biomolecules in brain tissue, making drug therapy less effective. Therefore, the development of highly efficient and low-toxicity glioma treatment strategies is crucial.

[0003] Boron neutron capture therapy (BNCT), a two-dimensional tumor radiotherapy developed based on the principles of precision medicine, is a non-invasive tumor treatment at the cellular scale. It offers potential for treating deep tumors due to its higher tumor targeting and stronger neutron penetration capabilities, as well as its shorter treatment time and cycle, and lower side effects. Its mechanism of action involves the isotope boron-10 ( 10 When B) is irradiated with low-energy (0.025 eV) thermal neutrons or epithermal neutrons (10,000 eV), a neutron capture-fission reaction occurs, and the fission reaction produces two high-energy-density radiation particles called alpha particles (4He). 2+ ) and 7Li 3+Particles are generated, which further play a role in killing tumor cells. The killing range of these two types of lethal radiation is only 5-8 microns, limited to the diameter of a single cell, so it kills tumor cells without significantly affecting surrounding normal cells.

[0004] The advantages of BNCT therapy include the following: (1) The alpha rays generated can simultaneously kill tumor cells in both the mitotic and quiescent phases (conventional radiotherapy and chemotherapy primarily act on rapidly dividing cells and are insensitive to quiescent tumor cells). (2) It can simultaneously kill hypoxic tumor cells without requiring additional oxygen supply (conventional radiotherapy is not highly sensitive to hypoxic cell therapy). (3) The resulting lethal and potentially lethal damage cannot be repaired by DNA, resulting in a remarkable therapeutic effect on refractory tumors that can repair DNA damage after chemotherapy and radiotherapy, and effectively suppressing tumor recurrence.

[0005] Currently, BNCT is not widely used in tumor treatment. This is because it takes a certain amount of time to build BNCT treatment equipment, and there are not many boron carriers currently available for clinical use. 10 Targeting and accumulating boron atoms in tumor cells is a major challenge for current boron carriers. Porfins offer several advantages as carriers for targeted delivery of boron. On the one hand, porfins are safe and low-toxicity molecules that can target tumor cells and accumulate in them in large quantities. On the other hand, this type of molecule belongs to the category of photosensitizers with good fluorescence properties, and this property can be used to track the fluorescence of compounds in the body and observe the distribution of drugs within the body.

[0006] Currently, there are no reports, either in China or internationally, of using porfin as a carrier to load BSH, a second-generation boron carrier, and targeting its delivery to gliomas by crossing the blood-brain barrier. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The objective of the present invention is, 10 The objective is to provide a tumor-targeted amphipathic porfin-BSH complex that enables efficient delivery of B atoms and tracking of compounds within the body, and is applicable to BNCT therapy. A compound obtained by linking a porfin core compound to a linker is coupled to BSH to form a water-soluble target molecule, and this type of complex is suitable for many 10 In addition to efficiently and safely transporting B to tumor cells, the distribution of the drug can also be observed through fluorescence tracking. [Means for solving the problem]

[0008] The technical solution of the present invention is as follows: The porfin-BSH complex used in boron neutron capture therapy has the following structure: TIFF2026514905000002.tif119130 Here, R is independent of each other, TIFF2026514905000003.tif810 or The filename is TIFF2026514905000004.tif1553, where n1 is an integer between 1 and 10, and n2 is an integer between 1 and 10.

[0009] The aforementioned X + M is a monovalent cation, and M is a metal or metallic nuclide.

[0010] Specifically, R is independent of each other. TIFF2026514905000005.tif810 or The filename is TIFF2026514905000006.tif1553, where n1 is an integer between 1 and 5, and n2 is an integer between 1 and 5.

[0011] Specifically, X + These are sodium ions, quaternary ammonium cations, or potassium ions.

[0012] Specifically, each M is independently Cu, 64 Cu, 68 Ga, Fe, Zn, Mg, Ni, Co, Pt, Pd, Sn, or Ti.

[0013] Specifically, the quaternary ammonium cation is selected from a tetramethyl quaternary ammonium cation, a tetraethyl quaternary ammonium cation, a tetrapropyl quaternary ammonium cation, and a tetrabutyl quaternary ammonium cation.

[0014] Specifically, the present invention provides the complex having a structure shown by the following formula. This structure is prepared using chlorophyll a extracted from a natural compound as a starting material and retains the chiral positions of chlorophyll a. Also, in Specific Examples 1 and 2 of the present invention, two specific complexes having the chiral structure shown by the following formula were prepared. They have excellent boron delivery ability, cause a strong killing effect on tumor cells, and demonstrate low toxicity to normal cells. TIFF2026514905000007.tif102111

[0015] In some of the specific complexes shown by the above formula, n1 is an integer from 1 to 5, and n2 is an integer from 1 to 5.

[0016] In some of the specific complexes shown by the above formula, each R is independently TIFF2026514905000008.tif810, and n1 is an integer from 1 to 10.

[0017] In some of the specific complexes shown by the above formula, each R is independently TIFF2026514905000009.tif810, and n1 is an integer from 1 to 5.

[0018] In some of the specific complexes shown by the above formula, X + is a sodium ion, a quaternary ammonium cation, or a potassium ion.

[0019] In some specific composites shown in the above equation, M is independently Cu, 64 Cu, 68 These are Ga, Fe, Zn, Mg, Ni, Co, Pt, Pd, Sn, or Ti.

[0020] The porphine nuclei are as follows: TIFF2026514905000010.tif4587

[0021] The linking groups are as follows: TIFF2026514905000011.tif1628

[0022] The boron agent is either mercaptododecaborane disodium salt, mercaptododecaborane quaternary ammonium salt, or mercaptododecaborane dipotassium salt, both second-generation boron carriers.

[0023] Specifically, the boron compound is mercaptododecaborane disodium salt, and its structure is as follows. TIFF2026514905000012.tif1536

[0024] The porphine core of the aforementioned complex is reacted with a metal salt (for example, a metal chloride or metal acetate complex) to obtain a complex chelated with a divalent or tetravalent metal.

[0025] A method for preparing a porfin-BSH complex in boron neutron capture therapy, wherein the structure is as follows: Compound 1 is obtained by esterifying and hydrolyzing compound pheophorbide a, and then compound 1 is subjected to amidation and thiol coupling reactions with compound A and compound BSH in order to obtain target compound I, or compound pheophorbide a is subjected to a degreasing reaction to obtain compound 2, and then compound 2 is subjected to amidation and thiol coupling reactions with compound A and compound BSH in order to obtain target compound II.

[0026] Here, R and X + The definition is the same as the definition in the structural formula.

[0027] Compounds I and II are reacted with salts containing metal M in a solvent to obtain compounds III and IV, respectively.

[0028] Here, R and X + The definition of M is the same as the definition in the structural formula.

[0029] The specific preparation methods for the porfin-BSH complex in the two types of boron neutron capture therapy include the following steps.

[0030] (1) Using compound pheophorbide a as a starting material, dissolve it in a 5% methanol sulfate solution to a concentration of 0.01 M and react at 25°C for 4 hours to obtain a methyl esterification product. Then, dissolve the obtained methyl esterification product in methanol, add 2 equivalents of sodium methoxide, and react at 25°C for 12 hours to obtain compound 11. Dissolve compound 11 in a 5% KOH:THF = 1:1 mixed solution, sonicate for 5 minutes, and react at 40°C for 12 hours to obtain compound 1. Dissolve compound 1 in DMF and sequentially add EDCI, N-(2-amino)maleimide, and triethylamine, where the molar ratio of compound 1:EDCI:N-(2-amino)maleimide is 1:1.1:1.1, and react at room temperature for 12-24 hours to obtain compound 4. Compound 4 is dissolved in a mixed solution of DCM:EtOH=1:1, and mercaptododecaborane disodium salt and triethanolamine are added sequentially, where compound 4:BSH=1:1.2. The reaction is carried out at room temperature for 4-8 hours, and the reaction equation is as follows. TIFF2026514905000015.tif35167

[0031] (2) Using compound pheophorbide a as a starting material, dissolve it in pyridine at a concentration of 0.01 M and react it at 115°C for 12 to 18 hours to obtain compound 2. Dissolve compound 2 in DMF solution and sequentially add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBT), N-(2-amino)maleimide and triethylamine, where the molar ratio of compound 2:EDCI:1-hydroxybenzotriazole (HOBT):N-(2-amino)maleimide is 1:2:2:2, and react at room temperature for 12 to 24 hours to obtain compound 5. Dissolve compound 5 in a mixed solution of DCM:EtOH=1:1 and sequentially add mercaptododecaborane disodium salt and triethanolamine, where the ratio of compound 5:BSH=1:1.2, and react at room temperature for 4 to 8 hours, with the reaction equation being as follows. TIFF2026514905000016.tif45157

[0032] Examples 1 and 2 of the present invention illustrate the detailed preparation steps of compound 7 and compound 8.

[0033] The reaction equations for the other two porfin-BSH complexes in boron neutron capture therapy are as follows: TIFF2026514905000017.tif66155TIFF2026514905000018.tif65150

[0034] The specific reaction steps are the same as those for the preparation of compounds 4 and 7.

[0035] Specifically, the reaction equation for chelating metal ions into the porphyrin nucleus is as follows: TIFF2026514905000019.tif4799

[0036] Compound 8 is added to a mixed solvent of dichloromethane and methanol in a 1:1 ratio, copper acetate solid is added, and the mixture is reacted at room temperature for 1.5 hours. The mixture is then passed through a silica gel column to obtain compound 10.

[0037] The above-mentioned porfin-BSH complex is used to prepare targeted antitumor drugs with the BNCT-targeted boron agent as the active site. The tumors include head and neck tumors (including pharyngeal cancer, thyroid cancer, rhinitis cancer, tonsil cancer, lymphoma, and sarcoma), intracranial tumors (including glioma, medulloblastoma, ependymoma, and metastatic brain tumor), breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, gastric cancer, and rectal cancer. [Effects of the Invention]

[0038] The beneficial effects of the present invention are as follows: The porfin-BSH complex molecule described in the present invention is in salt form, which effectively improves the water solubility of the entire molecule. By using the porfin nucleus as a carrier, the porfin compound has the properties of tumor cell targeting, good uptake by tumor cells, safety and low toxicity, and fluorescence of the photosensitizer itself, enabling targeted delivery of mercaptododecaborane disodium salt (BSH), a second-generation boron carrier, to tumor cells. This effectively solves the problems of insufficient tumor targeting and low intratumor cell accumulation capacity of BSH in clinical use, and the introduction of the porfin nucleus allows for intratumor cell uptake. 10 The content of B atoms increased significantly. Furthermore, the fluorescence properties of porfin itself were utilized to monitor the distribution of the complex in the body in real time, enabling the formulation of optimal neutron irradiation conditions. Combining the results of dark toxicity and in vitro activity tests of BNCT, the porfin-BSH complex exhibited strong killing activity against tumor cells while showing low toxicity to normal cells. This type of molecule can be used in the preparation of third-generation boron carriers for BNCT therapy. [Brief explanation of the drawing]

[0039] [Figure 1] This is a characterization diagram of the compounds. Here, (a) is the high-performance liquid chromatogram of compound 8, and (b) is the high-performance liquid chromatogram of compound 7. [Figure 2] This is a spectral data diagram of the porfin-BSH complex. Here, (a) is the ultraviolet-visible absorption spectrum of porfin-BSH, and (b) is the fluorescence spectrum of porfin-BSH. [Figure 3] This figure shows the uptake of the porfin-BSH complex by GL261 cells, as monitored by a fluorescence microscope. [Figure 4] This figure shows the uptake of the porfin-BSH complex by bEND.3 cells, as monitored by a fluorescence microscope. [Figure 5] This figure shows the uptake of porfin-BSH by GL261 cells, analyzed using flow cytometry. [Figure 6] This figure shows the toxicity of porfin-BSH complex formulations to bEND.3 cells and GL261 cells, as evaluated using the MTT method. [Figure 7] This figure shows the accumulation of 10B in GL261 cells after incubation of the complex for different time periods, as detected by ICP-OES. [Figure 8] This is an actual diagram of the liquid level in the Transwell chamber on day 5, and diagrams showing the change in liquid level height at 1, 4, and 9 hours on days 1, 3, and 5 after chamber construction. [Figure 9] This is a diagram illustrating the permeation experiment of fluorescein sodium in an in vitro blood-brain barrier model. [Figure 10] This figure evaluates the ability of porfin-BSH complex formulations to cross the blood-brain barrier in vitro. [Figure 11] This figure shows the activity data of the porfin-BSH complex in GL261 cells after BNCT irradiation. Here, (a) is the total neutron fluence of 5.54 × 10⁶ n / cm² after 16 minutes of BNCT irradiation, and (b) is the total neutron fluence of 8.31 × 10⁶ n / cm² after 24 minutes of BNCT irradiation. [Modes for carrying out the invention]

[0040] The extraction and preparation steps for pheophorbide a in Example 1 were prepared with reference to the following document (Guan Lei. Study on the analysis and preparation process of CHP in Spirulina platensis. Master's degree, Dalian University of Technology, 2020).

[0041] Example 1 Extraction and synthesis of the compound pheophorbide a. 100g of Spirulina platensis powder from Chenghai Lake was accurately weighed, immersed in 500mL of acetone, and transferred to a 1L three-necked flask. The three-necked flask was placed in an oil bath, equipped with an electric stirrer and a tap water condenser / reflux apparatus, and nitrogen gas was introduced to check the airtightness of the apparatus and whether the stirrer was functioning correctly. The heating temperature of the oil bath was set to 65°C. After the solvent temperature in the three-necked flask reached 56°C and reflux of the solvent occurred in the condenser, timing was started and reflux was carried out for 2 hours, after which heating was stopped and stirring was continued until the solvent in the three-necked flask naturally cooled to 30°C. Solid-liquid separation was performed using vacuum suction filtration, and the filtrate was washed with acetone until the filtrate was shallow. The above extraction steps were repeated three times, the filtrates were combined and rotated-dried to obtain Spirulina extract chlorophyll a.

[0042] Chlorophyll a, an extraction product of spirulina powder, was dissolved in 300 mL of ethyl ether and transferred to a 1 L three-necked flask. A mechanical stirrer and a dropping funnel were attached, nitrogen gas was introduced, and stirring was started. The mixture was cooled to -10°C in an ice salt bath environment. 150 mL of 12 M concentrated hydrochloric acid (pre-cooled to -20°C) was slowly added dropwise, and the dropping rate of the concentrated hydrochloric acid was controlled to maintain the reaction solution temperature below 0°C. After the addition of concentrated hydrochloric acid using the dropping funnel was complete, the ice salt bath cooling device at the bottom was removed, and the reaction was allowed to proceed at room temperature. The progress of the reaction was detected by thin-layer chromatography. The reaction status of the starting material chlorophyll a was detected in a petroleum ether:ethyl acetate = 3:1 developing system, and the formation status of the product pheophorbide a (CHP) was detected in a chloroform:methanol = 20:1 developing system. After the reaction was complete, the reaction solution was transferred to a 5L separatory funnel and extracted three times with 350ml of petroleum ether, and the lower acidic aqueous layer was collected.

[0043] The acidic aqueous layer solution was collected, saturated sodium carbonate solution was added dropwise to adjust the pH to approximately 4, and a turbid green solid precipitated. This was filtered by vacuum suction using a Buchner funnel, and the solid was washed off with a 1% volume fraction aqueous solution of propionic acid. The obtained solid was placed in a vacuum drying oven, and phosphorus pentoxide solid was added and dried (25°C). After the solid dried, it was dissolved in a mixed solvent of dichloromethane:methanol = 10:1, transferred to a round-bottom flask, and the solvent was rotated to obtain 1.08 g of pheophorbide a (CHP) in a total yield of 1.1%.

[0044] Synthesis of compound 11 500 mg of pheophorbide a(CHP) was methyl esterified in 40 ml of 5% sulfate-methanol solution for 4 hours, protected with N2, and after the reaction was complete, methanol was removed by rotation. The mixture was extracted three times with saturated sodium bicarbonate aqueous solution and dichloromethane, and the organic phase was extracted three times with saturated brine. The mixture was then rotated dry to obtain the methyl esterification intermediate. The obtained methyl esterification product was then dissolved in 15 ml of methanol, 1.5 ml of sodium methoxide solution was added, and the mixture was protected with N2 and reacted for 12 hours. After the reaction was complete, the pH of the solution was adjusted to neutral with 1 M HCl, methanol was rotated dry, and the mixture was separated by column chromatography under elution conditions (dichloromethane:ethyl acetate = 100:1 to 80:1). Compound 11 was obtained in 115.1 mg in yield of 23%. 1 H NMR(600MHz,Chloroform-d)δ 9.66(s,1H),9.50(s,1H),8.73(s,1H),7.98(dd,J=17.8,11.4Hz,1H),6.28(d,J=17.8Hz,1H ),6.07(d,J=11.5Hz,1H),5.29(d,J=47.0Hz,2H),4.47-4.37(m,2H),4.25(s,3H),3.76(s,3 H),3.72(q,J=7.8Hz,2H),3.62(s,3H),3.56(s,3H),3.42(s,3H),3.23(s,3H),2.60-2.51(m ,1H),2.24-2.13(m,2H),1.74(d,J=7.3Hz,4H),1.68(t,J=7.7Hz,3H),-1.47(s,1H).ESI-MS for C 37 H42 N4O6:(calculated)638.3104;(found)639.3138[M+H] +

[0045] Synthesis of Compound 1 First, THF and 1 mol / L KOH solution were sonicated for 10 minutes each. 45.5 mg of compound 11 was accurately weighed into a 25 mL round-bottom flask, 2 mL of THF was weighed out and compound 11 was dissolved, then 2 mL of 1 mol / L KOH solution was added, and the reaction was carried out at 40°C, protected with nitrogen gas, and refluxed through a condenser. The reaction was monitored by TLC, and after the reaction was complete, the reaction mixture was concentrated under reduced pressure into the remaining KOH aqueous solution. 1 mol / L HCl was added to adjust the pH to 3-4, and after a large amount of small, cloudy green solid particles precipitated, the mixture was filtered by suction using a Buchner funnel and washed with 1% propionic acid aqueous solution. The filter cake was filtered by suction until it dried and cracked, dissolved in a mixed solution of dichloromethane and methanol, transferred, concentrated under reduced pressure, and vacuum dried to obtain 34.4 mg of the crude product of compound 1, a cloudy green solid, in 81% yield. ESI-MS for C 34 H 36 N4O6:(calculated)596.2635;(found)597.21[M+H] +

[0046] Synthesis of Compound 4 45.5 mg of compound 1 was dissolved in 2 mL of anhydrous DMF, and 16.3 mg of EDCI, 12.7 mg of HOBT, and 50 μl of triethylamine were added. The mixture was stirred at 25°C, protected with a nitrogen balloon, and monitored by TLC. After 2 hours of reaction, it was found that the starting material had been converted to an intermediate. 14.8 mg of N-(2-amino)maleimide was added to the reaction mixture, stirred at 25°C, protected with a nitrogen balloon, and the reaction was detected by TLC (eluent: dichloromethane:methanol = 10:1). The reaction was stopped after 12 hours. The pH of the reaction mixture was adjusted to 3-4 with 1 M HCl, and the solid was obtained by suction filtration through a sand plate filter. The solid was then vacuum dried. Subsequently, this solid was dissolved in a dichloromethane-methanol mixed solvent (dichloromethane:methanol = 10:1), the solvent was rotary dried, and the sample was dry-packed and passed through a silica gel column. Gradient elution was performed using a dichloromethane / methanol mixed solvent containing 0.33% formic acid. The initial eluent was dichloromethane:methanol = 100:1 (v / v), and the proportion of methanol was gradually increased, sequentially to dichloromethane:methanol = 50:1 (v / v), and finally to dichloromethane:methanol = 30:1 (v / v). 19.1 mg of a turbid green solid was obtained in 35% yield. ESI-MS for C 40 H 42 N6O7:(calculated)718.3115;(found)719.43[M+H] + .

[0047] Synthesis of compound 4a 100 mg of compound 1 was dissolved in 4 mL of anhydrous DMF, and 35.4 mg of EDCI, 25.1 mg of HOBT, and 100 μl of triethylamine were added. The mixture was stirred at 25°C, protected with a nitrogen balloon, and monitored by TLC. After 2 hours of reaction, it was discovered that the starting material had been converted to an intermediate. 184.8 mg of aminopolyethylene glycol thiol (molecular weight 1000) was added to the reaction mixture, the mixture was stirred at 25°C, protected with a nitrogen balloon, and the reaction was detected by TLC (developing solvent: dichloromethane:methanol = 10:1). The reaction was stopped after 12 hours. The pH of the reaction mixture was adjusted to 3-4 with 1 M HCl, and the solid was obtained by suction filtration through a sand plate filter. The solid was then vacuum dried. Subsequently, this solid was dissolved in a dichloromethane-methanol mixed solvent (dichloromethane:methanol = 10:1), and the sample was dry-packed and passed through a silica gel column. Gradient elution was performed using a dichloromethane / methanol mixed solvent containing 0.33% formic acid. The initial eluent was dichloromethane:methanol = 100:1 (v / v), and the proportion of methanol was gradually increased, progressing through a gradient to dichloromethane:methanol = 50:1 (v / v), and finally to dichloromethane:methanol = 30:1 (v / v). 72.4 mg of a turbid green solid was obtained in a yield of 27%.

[0048] Synthesis of Compound 7 19.1 mg of compound 4 was dissolved in 2 ml of a mixed solution of dichloromethane:methanol = 10:1. After complete dissolution, 50 μl of triethanolamine was added, followed by the rapid weighing and addition of 7 mg of mercaptododecaborane disodium salt. The reaction was stirred at 25°C, protected with a nitrogen balloon, and the progress of the reaction was monitored by TLC. The reaction was allowed to proceed for 4 hours until the reaction of the reactants was complete. The reaction was stopped, a small amount of formic acid was added to neutralize the triethanolamine, the solvent was then rotated dry, and the sample was dry-packed and passed through a reversed-phase silica gel column (gradient from water:methanol = 10:1 to water:methanol = 1:3) to obtain 20.5 mg of compound 4 in 81% yield. 11H NMR (600 MHz, DMSO-d6): δ 9.77 (s, 1H, 10-H), 9.73 (s, 1H, 5-H), 9.14 (s, 1H, 20-H), 8.35 (dd, J = 17.8, 11.6 Hz, 1H, 3 1 -H), 6.46 (d, J = 17.7 Hz, 1H, 3 2a -H), 6.17 (d, J = 11.7 Hz, 1H, 3 2b -H), 5.76 (s, 1H, -CONH-), 5.68 (s, 1H, 15 1a -H), 4.88 (m, 1H, 15 1b -H), 4.59 (d, J = 7.4 Hz, 1H, 18-H), 3.83 (q, J = 7.8 Hz, 2H, 8 2 -H), 3.59 (d, J = 6.5 Hz, 1H, 17-H), 3.55 (d, J = 1.9 Hz, 6H, 12 1 -H, 2 1 -H), 3.34 (s, 3H, 7-H), 3.12 - 2.95 (m, 4H, 15 3 -H, 15 4 -H), 2.80 - 2.68 (m, 1H, 15 6 -H), 2.41 (s, 1H, 17 2a -H), 2.19 (t, J = 22.0 Hz, 3H, 17 1 -H, 17 2b -H), 1.69 (t, J = 7.3 Hz, 6H, 18 1 -H, 8 2 -H), 1.60 (s, 2H, 15 7 -2H), 1.05 (s, 11H, BSH-H), -1.92 (s, 1H, N-H), -2.44 (s, 1H, N-H). ESI-MS for C 40 H 54 B 12 N6O7S: (calculated) 940.4760; (found) 446.2500 [(M - 2Na) / 2] 2- .

[0049] Synthesis of Compound 7a 50 mg of compound 4a was dissolved in 5 ml of a mixed solution of dichloromethane:methanol = 10:1. After complete dissolution, 100 μl of triethanolamine was added, followed by the rapid weighing and addition of 8.2 mg of mercaptododecaborane disodium salt. The mixture was stirred at 25°C and the reaction was monitored by TLC under the protection of a nitrogen balloon. The reaction was allowed to proceed for 4 hours until the reaction was complete. The reaction was then stopped, a small amount of formic acid was added to neutralize the triethanolamine, the solvent was rotated dry, and the sample was dry-packed and passed through a reversed-phase silica gel column (gradient from water:methanol = 10:1 to water:methanol = 1:3) to obtain 42.7 mg of compound 4a in 75% yield.

[0050] Example 2 Synthesis of Compound 2 500 mg of pheophorbide a(CHP) was dissolved in 30 ml of pyridine in a 250 ml round-bottom flask and completely dissolved by sonication. The flask was protected with a nitrogen balloon and stirred at 115°C using a reflux condenser. The reaction was monitored by TLC and allowed to proceed for 12 hours. After the reaction was complete, the pyridine in the reaction flask was rotated dry, the sample was dry-packed and passed through a silica gel column (gradient from dichloromethane:methanol = 100:1 to dichloromethane:methanol = 30:1) to obtain 302 mg of compound 2 in 71% yield. 1 H NMR(600MHz,Chloroform-d)δ 9.36(s,1H),9.25(s,1H),8.51(s,1H),7.90(dd,J=17.9,11.5Hz,1H),6.21(d,J=17.8Hz,1H),6.11(d, J=11.5Hz,1H),5.25(d,J=44.8Hz,3H),5.08(d,J=19.1Hz,1H),4.44(q,J=7.5Hz,1H),4.27(d,J=9.4Hz ,1H),3.58(d,J=10.7Hz,5H),3.35(s,3H),3.14(s,3H),2.73-2.54(m,2H),2.41-2.29(m,1H),2.22(dt ,J=15.5,7.2Hz,1H),1.80(s,3H),1.63(d,J=15.5Hz,4H),1.26(d,J=24.3Hz,1H),-1.76(s,1H).ESI-MS for C33 H 34 N4O3:(calculated)534.2631;(found)535.2693[M+H] + .

[0051] Synthesis of Compound 5 53.6 mg of compound 2 was dissolved in 2 mL of anhydrous DMF, and 21.1 mg of EDCI, 16.7 mg of HOBT, and 50 μl of triethylamine were added. The mixture was stirred at 25°C, protected with a nitrogen balloon, and monitored by TLC. After 2 hours of reaction, it was found that the starting material had been converted to an intermediate. 20 mg of N-(2-amino)maleimide was added to the reaction mixture, stirred at 25°C, protected with a nitrogen balloon, and the reaction was detected by TLC (developing solvent: dichloromethane:methanol = 20:1). The mixture was stopped after 12 hours of reaction. The reaction mixture was transferred to a 25 ml separatory funnel using 5 ml of dichloromethane, extracted three times with 5 ml of deionized water, and then extracted twice with 5 ml of saturated saline to obtain a dichloromethane layer. The layer was dried over anhydrous sodium sulfate, filtered, and the solvent was rotary-dried. The sample was dry-packed and passed through a silica gel column (gradient from dichloromethane:methanol = 100:1 to dichloromethane:methanol = 10:1). A cloudy green solid of 42.5 mg was obtained in a yield of 65%. 1 H NMR(600MHz,Chloroform-d)δ 9.35(s,1H,10-H),9.09(s,1H,5-H),8.56(s,1H,20-H),7.98(dd,J=17.8,11.5Hz,1H,3 1 -H), 6.51(s,2H,17 7,8 -H),6.28(d,J=17.8Hz,1H,3 2a -H),6.17(d,J=11.5Hz,1H,3 2b -H),5.77(s,1H,-CONH-),5.25(d,J=19.1Hz,1H,13 2a -H), 5.03(d, J=19.1Hz, 1H, 13 2b -H),4.51(s,1H,18-H),4.31(s,1H,17-H),3.56(dd,J=14.2,7.0Hz,6H,8 1 -H,17 4 -H,17 5-H),3.40(s,3H,12-H),3.21(s,6H,2-H,7-H),2.67(s,1H,17 2a -H),2.46(s,1H,17 2b -H),2.21(s,1H,17 1a -H),1.91(s,1H,17 1b -H),1.80(d,J=6.7Hz,3H,18-H),1.57(t,J=7.7Hz,3H,8 2 -H),-1.73(s,1H,NH).ESI-MS for C 39 H 40 N6O4:(calculated)656.3111;(found)657.23[M+H] + .

[0052] Synthesis of compound 5a 100 mg of compound 5a was dissolved in 4 mL of anhydrous DMF, and 71.8 mg of EDCI, 50.6 mg of HOBT, and 100 μl of triethylamine were added. The mixture was stirred at 25°C, protected with a nitrogen balloon, and monitored by TLC. After 2 hours of reaction, it was found that the starting material had been converted to an intermediate. 206 mg of aminopolyethylene glycol thiol (molecular weight 1000) was added to the reaction mixture, the mixture was stirred at 25°C, protected with a nitrogen balloon, and the reaction was detected by TLC (eluent: dichloromethane:methanol = 20:1). The reaction was stopped after 12 hours. The reaction mixture was transferred to a 100 ml separatory funnel using 10 ml of dichloromethane, extracted three times with 20 ml of deionized water, and then extracted twice with 20 ml of saturated saline to obtain the dichloromethane layer. The layer was dried over anhydrous sodium sulfate, filtered, and the solvent was rotate-dried. The sample was dry-packed and passed through a silica gel column (gradient from dichloromethane:methanol = 100:1 to dichloromethane:methanol = 10:1). 146.5 mg of a turbid green solid was obtained in 51% yield.

[0053] Synthesis of compound 8 42.5 mg of compound 5 was dissolved in 2 ml of a mixed solution of dichloromethane:methanol = 10:1. After complete dissolution, 50 μl of triethanolamine was added, and 18.7 mg of mercaptododecaborane disodium salt was rapidly weighed and added. The mixture was stirred at 25°C and the reaction was controlled with a nitrogen balloon, and the progress of the reaction was monitored by TLC. The mixture was allowed to react for 2 hours until the reaction was complete, then the reaction was stopped, a small amount of formic acid was added to neutralize the triethanolamine, the solvent was then rotated dry, the sample was dry-packed and passed through a reversed-phase silica gel column (gradient from water:methanol = 10:1 to water:methanol = 1:3) to obtain 47.4 mg of compound 8 in 84% yield. 1 H NMR(600MHz,DMSO-d6)δ 9.65(s,1H,10-H),9.38(s,1H,5-H),8.93(s,1H,20-H),8.19(dd,J=17.8,11.6Hz,1H,3 1 -H),7.84(dt,J=22.8,6.2Hz,1H,-CONH-),6.38(d,J=17.8Hz,1H,3 2a -H),6.20(d,J=11.5Hz,1H,3 2b -H),5.26(dd,J=19.6,4.5Hz,1H,13 2a -H), 5.14(d, J=19.6Hz, 1H 13 2b -H),4.62-4.55(m,1H,18-H),4.31(t,J=8.4Hz,1H,17-H),3.67-3.65(m,6H,8 1 -H,17 4 -H,17 5 -H),3.61(s,3H,12-H),3.44(s,3H,2-H),3.18(s,3H,7-H),3.01(t,J=5.5Hz,2H,17 8 -H),2.76(ddd,J=18.4,7.3,2.8Hz,1H,17 2a -H),2.61(d,J=10.8Hz,1H,17 7 -H),2.32(d,J=10.7Hz,1H,17 2b -H),2.15-2.00(m,2H,17 1-H), 1.80 (d, J = 7.3 Hz, 3H, 18-H), 1.61 (t, J = 7.6 Hz, 3H, 8 2 -H), 1.00 (s, 11H, BSH-H), -2.01 (s, 1H, N-H). ESI-MS for C 39 H 52 B 12 N6Na2O4S: (calculated) 876.4883; (found) 877.70 [M + H] + .

[0054] Synthesis of Compound 8a 40 mg of Compound 5a was dissolved in 2 ml of a mixed solution of dichloromethane:methanol = 10:1. After complete dissolution, 50 μl of triethanolamine was added, 8 mg of sodium mercaptododecaborane was quickly weighed and added, and the reaction was stirred at 25 °C under protection with a nitrogen balloon. The progress of the reaction was monitored by TLC. The reaction was allowed to proceed for 2 hours until the reaction of the reactants was complete, the reaction was stopped, a small amount of formic acid was added to neutralize the triethanolamine, and then the solvent was rotary dried. The sample was dry-packed and passed through a reverse-phase silica gel column (gradient from water:methanol = 10:1 to water:methanol = 1:3), and 34.3 mg of Compound 8a was obtained in a yield of 76%.

[0055] Synthesis of Compound 10 4 mg of Compound 8 was dissolved in 2 ml of a solvent of dichloromethane:methanol = 1:1, 2.1 mg of copper acetate was added, and the reaction was carried out at room temperature for 2 hours under protection with nitrogen gas. The reaction was detected by TLC. After the reaction was completed, the solvent was rotary dried and passed through a silica gel column, and 2.5 mg of Compound 10 was obtained in a yield of 57%. ESI-MS for C 39 H 50 B 12 N6Na2O4SCu: (calculated) 938.4683; (found) 445.76 [M - 2Na] / 2 - .

[0056] The introduction of metal ions does not affect the targeting of porphyrin compounds to tumor cells, and Compound 10 similarly significantly increases the 10 content of B atoms in tumor cells. At the same time,64 Cu and other radioactive metal nuclides 68 The introduction of Ga etc. can be used in PET imaging technology.

[0057] Example 3 The compounds 7 and 8 prepared in Examples 1 and 2 were characterized.

[0058] (1) Performance test experiment 1: The purity of the synthesized complex was characterized by high performance liquid chromatography. As shown in Figure 1, the results showed that the purity of the obtained compound was higher than 95%.

[0059] (2) Performance test experiment 2: Measurement of the ultraviolet and fluorescence spectra of the porphyrin-BSH complex A sample solution of compounds 7 and 8 dispersed in methanol was added to a 3 ml quartz cuvette, and the absorption curve of the sample was measured with an ultraviolet-visible spectrophotometer (PerkinElmer, Lambda 750S). As shown in Figure 2, 1 mL of the sample solution of compounds 7 and 8 was added to a quartz cuvette, and the emission curve (Ex: 345 nm) of the sample was measured with a fluorescence spectrometer (Hitachi, F7000). Compared with the porphyrin parent nucleus structure, the fluorescence properties were not affected after modification.

[0060] Example 4 In vitro cell uptake experiments of the porphyrin-BSH complex prepared in Example 1 and Example 2

[0061] (1) Uptake of the porphyrin-BSH preparation by cells monitored with a fluorescence microscope GL261 cells and bEND.3 cells in the logarithmic growth phase with good growth status were selected, digested and collected, the cell density was adjusted, and a cell suspension (4×10 40.5 mL of (cells / mL) was uniformly and slowly seeded into a 24-well plate and incubated under normal conditions for 12 hours until the cells adhered and fully developed. The well plate was removed, the supernatant was discarded, and the plate was washed with PBS. Compounds 7 and 8 were then added to a final concentration of 25 μM, and the cells were incubated in the dark for different durations. Cells were removed at 2, 4, and 8 hours after complex addition, and the cell nuclei were labeled with DAPI. As shown in Figures 3 and 4, the intracellular accumulation of the porfin nuclei, which exhibit red fluorescence, was observed using a fluorescence microscope, and images were collected. The results of the fluorescence imaging analysis showed that the uptake of compounds 7 and 8 by GL261 cells and bEND.3 cells was time-dependent.

[0062] (2) Uptake of porfin-BSH by cells analyzed by flow cytometry Select GL261 cells in the logarithmic growth phase with good proliferation status, digest and collect them, adjust the cell density, and prepare a cell suspension (1 × 10⁶). 5 0.5 mL of the drug (cells / mL) was uniformly and slowly seeded into a 24-well plate and incubated under normal conditions for 12 hours until the cells adhered and fully developed. The drug was then added and incubated at 2, 4, and 8 hours, respectively. After incubation, the drug-containing medium was removed, washed three times with PBS, digested with 300 μl of trypsin, centrifuged for 3 minutes, the medium was discarded, and the cells were resuspended in PBS. As shown in Figure 5, the PBS was discarded, and the cells were fixed with 20 μl of 4% paraformaldehyde, resulting in a cell count of 2000. The red fluorescence properties of the drug were used to detect the red fluorescent APC channel (red fluorescence, excitation wavelength λex=633 nm, fluorescence intensity signal acquisition λem=660 nm). The results showed an increase in fluorescence intensity over time, indicating that the cell uptake of the complex was time-dependent.

[0063] (3) Evaluation of porfin-BSH in vitro cytotoxicity using the MTT method Mouse glioma cells (GL261) in the logarithmic growth phase were placed in 3 × 10⁶ well plates. 3Cells were seeded at a density of cells / well, 100 μL of cell suspension was added to each well, and the cells were incubated for 12 hours. Then, 100 μL of drug solution was added, resulting in final concentrations of the target compounds of 200 μmol / L, 100 μmol / L, 50 μmol / L, 25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L, respectively. A blank group (containing culture medium but no cells) and a control group (cultured cells without drug) were established. Cells were incubated in an incubator (standard environment) for 12 hours, then 20 μL of 5 mg / ml MTT solution was added to each well, and the cells were incubated for 4 hours. The supernatant was aspirated, and 100 μL of DMSO was added. The absorbance (OD value) of each well at a wavelength of 570 nm was measured using a microplate reader. The relative viability of the cells was measured (OD value of the experimental group). 570 / OD of the control group 570 The calculation was performed using ) × 100%, and the experiment was repeated three times. The toxicity of porfin-BSH to mouse brain microvascular endothelial cells (bEND.3) was measured using the same method. As shown in Figure 6, in the absence of neutron irradiation, the drug showed no apparent toxicity to bEND.3 cells within the 100 μM concentration range. In the absence of neutron irradiation, the drug showed no apparent toxicity to GL261 cells within the 100 μM concentration range.

[0064] (4) Uptake of the complex by cells detected by ICP-OES Select GL261 cells in the logarithmic growth phase with good proliferation status, digest and collect them, adjust the cell density, and prepare a cell suspension (1 × 10⁶). 61 mL of (cells / mL) was uniformly and slowly seeded into a 6-well plate and incubated under normal conditions for 12 hours until the cells adhered and fully developed. The well plate was removed, the supernatant was discarded, and the plate was washed three times with PBS. Then, 1 mL of cell medium containing compound 7 and compound 8 was added to each well plate. After 12 hours, the supernatant was removed, the plates were washed three times with PBS, and the cells were collected. The cells were digested with 1 mL of a mixed solution containing 66% HNO3 and 34% 30% H2O2 (VHNO3:V30%H2O2=3:2), and the intracellular boron content was measured by inductively coupled plasma atomic emission spectrometry (ICP-OES). As shown in Figure 7, the boron content of cells in groups treated with compound 7 and compound 8 increased over time after incubation for different durations. In the group treated with compound 8, the boron content reached 117 ppm at 2 hours, exceeding the treatment standard of 20 ppm, and reached 223 ppm after 12 hours of incubation. In the group treated with compound 7, the boron content remained above the treatment standard of 27 ppm even after 4 hours of incubation, reaching 56 ppm after 12 hours of incubation.

[0065] Example 5 Construction of an in vitro blood-brain barrier model and evaluation of the blood-brain barrier crossing ability of porfin-BSH preparations.

[0066] (1) Construction of an in vitro blood-brain barrier model and leakage experiment The Transwell chamber was removed, DMEM medium was added, and it was placed in a 24-well plate. It was then incubated in a cell culture incubator for 20 minutes to activate it. 2 × 10⁶ bEND.3 cells in the logarithmic growth phase were placed in the upper chamber of the Transwell. 4Cells were seeded at a density of cells / well, and 0.6 mL of culture medium was added to a 24-well plate in the lower chamber of the Transwell. The cells were cultured in an incubator for 5 days until they reached confluence (during this period, the cell medium in the upper chamber of the Transwell was periodically changed to ensure that the bEND.3 cells adhered closely and proliferated). 400 μL and 200 μL of culture medium were added to the upper and lower chambers of the bEND.3, respectively, with a liquid level difference of 1 cm in the Transwell chamber. The cells were then placed in the incubator and cultured. The liquid level was recorded after 1 hour, 4 hours, and 9 hours to observe whether the liquid level difference between the two chambers remained at the original height. As shown in Figure 8, there was no significant change in the liquid level difference on day 5, so the BBB in vitro modeling was tentatively judged to be successful.

[0067] (2) Fluorescein sodium permeability experiment in an in vitro blood-brain barrier model In vitro cultured BBB models were detected using fluorescein sodium (FLU). Three models each were selected for the bEND.3 monoculture model group and the blank group. Before measurement, the entire solution was replaced with serum-free DMEM medium, 50 μg / mL of fluorescein sodium was added to the donor cells, and the cells were incubated in a 37°C, 0.5% CO2 incubator for 5 minutes, 10 minutes, 20 minutes, 40 minutes, and 80 minutes. After incubation, 50 μL of culture medium was taken from the receiver cells, and the amount of FLU that passed through each model was measured using a fluorescence microplate reader. As shown in Figure 9, the bEND.3 monoculture model group was able to effectively inhibit the permeation of fluorescein sodium.

[0068] (3) Evaluation of the in vitro blood-brain barrier crossing ability of the porfin-BSH complex Wells that passed the leakage and fluorescein sodium permeability tests were selected as in vitro blood-brain barrier models, and the blood-brain barrier crossability of compounds 7 and 8 was evaluated. Briefly, 300 μL of solution containing compound 7 and compound 8, respectively, was added to donor cells, GL261 cells at 70% density were seeded in the lower chamber, incubated for 4 hours, and the uptake of the complex by GL261 cells was observed using a fluorescence microscope. As shown in Figure 10, compound 8 was able to cross the in vitro blood-brain barrier successfully.

[0069] Application Example 1 BNCT in vitro activity evaluation In vitro cell activity evaluation experiments of porfin-BSH complexes prepared in Example 1 and Example 2

[0070] (1) Evaluation of the effects of different neutron counts and irradiation times on the activity of GL261 cells. Select GL261 cells in the logarithmic growth phase with good proliferation status, digest and collect them, adjust the cell density, and prepare a cell suspension (1 × 10⁶). 6 1 mL of (cells / mL) was uniformly and slowly seeded into a 6-well plate and incubated under normal conditions for 12 hours until the cells adhered and fully developed. The well plate was removed, the supernatant was discarded, and it was washed three times with PBS. Then, 1 mL of cell medium containing 100 μM of compound 7 and compound 8 was added to each well. After 2 hours, the supernatant was removed, and it was washed three times with PBS. Then, pancreatin was added to digest the cells from the 6-well plate, the medium was added and the cells were resuspended, and finally the medium was added and counted to adjust the cell density to 166,000 / mL / 0.6 mL. A total of 100,000 cells were transferred to a 1.5 mL centrifuge tube. Subsequently, the neutron fluence was 3.46 × 10⁶. 5 n / cm 2The irradiation intensities were selected, and BNCT irradiation was performed for 16 minutes and 24 minutes, respectively, before terminating the irradiation. After irradiation was completed, the cells were incubated for 24 hours, then 50 μl of a solution containing CCK8 was added and incubated for 4 hours. Finally, the absorbance at 450 nm was measured using a microplate reader. As shown in Figures 11(a) and (b), the cell-killing effect of irradiation for different durations at the same neutron fluence was found to be 90% for compound 8 against GL261 cells, and 75% for compound 7 against GL261 cells. In in vitro activity evaluation, the two complexes exhibited good cell-killing activity after BNCT irradiation.

Claims

1. The porfin-BSH complex used in boron neutron capture therapy has the following structure: Here, R is independent of each other. or n １ n is an integer between 1 and 10. ２ is an integer between 1 and 10, The aforementioned X ＋ It is a monovalent cation, A porfin-BSH complex in boron neutron capture therapy, characterized in that M is a metal or metallic nuclide.

2. n １ n is an integer between 1 and 5. ２ The composite according to claim 1, characterized in that is an integer from 1 to 5.

3. Each R is independent of the others. n １ The composite according to claim 1, characterized in that is an integer from 1 to 10.

4. X ＋ The composite according to claim 1, characterized in that is a sodium ion, a quaternary ammonium cation, or a potassium ion.

5. M is, independently of each other, Cu, ６４ Cu, ６８ The composite according to claim 1, characterized in that it is Ga, Fe, Zn, Mg, Ni, Co, Pt, Pd, Sn or Ti.

6. The composite according to claim 1, characterized by having the following structure.

7. n １ n is an integer between 1 and 5. ２ The composite according to claim 6, characterized in that is an integer from 1 to 5.

8. Each R is independent of the others. n １ The composite according to claim 6, characterized in that is an integer from 1 to 10.

9. n １ The composite according to claim 8, characterized in that is an integer from 1 to 5.

10. X ＋ The composite according to claim 6, characterized in that is a sodium ion, a quaternary ammonium cation, or a potassium ion.

11. M is independent of Cu, ６４ Cd, ６８ The composite according to claim 6, characterized in that it is Ga, Fe, Zn, Mg, Ni, Co, Pt, Pd, Sn, or Ti.

12. A method for preparing a porfin-BSH complex in boron neutron capture therapy according to claim 1, the steps being as follows: Compound of formula I: Compound pheophorbide a is esterified and subjected to hydrolysis to obtain compound 1. Compound 1 is then subjected to amidation and thiol coupling reactions with compound A and compound BSH in that order to obtain the target compound. Compound II: Compound pheophorbide a is subjected to a degreasing reaction to obtain compound 2, and compound 2 is subjected to an amidation reaction and a thiol coupling reaction with compound A and compound BSH in that order to obtain the target compound. Compound I and compound II are reacted with salts containing M in a solvent to obtain compounds III and IV, respectively. Here, R, X ＋ A preparation method characterized in that the definition of M is the same as in claim 1.

13. The use of a porfin-BSH complex in boron neutron capture therapy according to claim 1 or 6, characterized in that the porfin-BSH complex in boron neutron capture therapy is used to prepare an antitumor drug.

14. The use according to claim 13, characterized in that the tumor includes head and neck tumors, intracranial tumors, breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, stomach cancer, and rectal cancer.

15. The aforementioned head and neck tumors include laryngeal cancer, thyroid cancer, rhinitis cancer, tonsil cancer, lymphoma, and sarcoma. The use according to claim 14, characterized in that the intracranial tumor includes glioma, medulloblastoma, ependymoma, and metastatic brain tumor.

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