Porphyrin-BSH Conjugates in Boron Neutron Capture Therapy and Their Applications

Abstract

Porphyrin-BSH conjugate and its application in boron neutron capture therapy, belonging to the field of medical technology. The porphyrin-BSH conjugate of the present invention is synthesized by coupling with an amino-substituted maleimide compound, and the method is simple; the porphyrin compound has the characteristics of tumor cell targeting, good tumor cell uptake, safety and low toxicity, and the fluorescence phenomenon of the photosensitizer itself, and can realize the targeted delivery of the second-generation boron carrier sodium mercaptododecaborate (BSH) to tumor cells. Compared with BSH, it can increase the content of <supgt;10< / supgt;B atoms in cells to varying degrees; at the same time, the fluorescence characteristics of porphyrin itself are used to monitor the in vivo distribution of the conjugate in real time, so as to formulate the best neutron irradiation conditions; the conjugate is not only safe and low-toxic, but also greatly increases the content of <supgt;10< / supgt;B in cells; at the same time, this type of conjugate has obtained good results in the in vitro activity test of BNCT, so it can be studied as a potential boron carrier for BNCT in the future.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a class of porphyrin-BSH conjugates in boron neutron capture therapy and their applications. Background Art

[0002] Glioblastoma is the most common primary intracranial tumor caused by the carcinogenesis of glial cells in the brain and spinal cord. Its strong metastatic ability, high invasiveness, strong drug resistance and other characteristics lead to relatively high recurrence and fatality rates. It is reported that the annual incidence of glioblastoma in China is 5-8 per 100,000 people, and the 5-year fatality rate ranks the third among all tumors in the body, second only to pancreatic cancer and lung cancer. Traditional treatment methods mainly include surgical resection, radiotherapy and chemotherapy. However, due to problems such as the diffusion and infiltration of tumor cells and unclear infiltration boundaries during the development of glioblastoma, it is difficult to completely remove it by surgery. Radiotherapy has poor treatment effects due to the limitation of the radiation dose in the brain and the easy generation of tolerance. In addition, the blood-brain barrier (BBB) present in the brain restricts the enrichment of more than 95% of small molecule drugs and biological macromolecules to the brain tissue, making it difficult for drug treatment to play its role. Therefore, it is of great significance to develop an efficient and low-toxic glioblastoma treatment strategy.

[0003] A two-dimensional tumor radiotherapy boron neutron capture therapy (BNCT) developed based on the concept of precision medicine is a non-invasive tumor treatment method at the cellular scale, with higher tumor targeting. At the same time, the stronger neutron penetration ability provides the possibility for the treatment of deep tumors, and its treatment time and cycle are short, and the side effects are lower. Its mechanism of action is that the isotope boron-10 ( 10 B) undergoes a neutron capture fission reaction after being irradiated by low-energy (0.025 eV) thermal neutrons or epithermal neutrons (10,000 eV), and two high-linear energy transfer rays, alpha particles (4He 2+ ) and 7Li 3+ particles, are generated through the nuclear fission reaction, thereby playing a role in killing tumor cells. The killing range of these two killing rays is only between 5 and 8 micrometers, limited to the diameter of a single cell. Therefore, while causing damage to tumor cells, it will not have a great impact on surrounding normal cells.

[0004] The advantages of BNCT treatment also include: ① The α rays produced can destroy both dividing and dormant tumor cells at the same time (traditional radiotherapy and chemotherapy mainly act on actively dividing cells and are insensitive to dormant tumor cells); ② No additional oxygen is required, and hypoxic tumor cells can be killed at the same time (traditional radiotherapy is not very sensitive to hypoxic cell treatment); ③ The lethal and potentially lethal damage produced cannot be repaired by DNA, and it has a significant therapeutic effect on refractory tumors that can repair DNA damage after chemotherapy and radiotherapy, and can effectively inhibit tumor recurrence.

[0005] At present, BNCT has not been widely used in tumor treatment. The reasons include that it takes a certain amount of time to build BNCT treatment equipment and that there are not many boron carriers that can be used in clinical treatment. 10 The targeted delivery of B atoms to tumor cells and their large accumulation in tumor cells are currently the great challenges faced by boron carriers. Porphine has many advantages as a carrier for targeted delivery of boron agents: on the one hand, porphine, as a safe and low-toxic molecule, can target tumor cells and accumulate in tumor cells in large quantities; on the other hand, this type of molecule is a photosensitizer with good fluorescence properties, which can be used to perform fluorescent tracing of compounds in the body and observe the distribution of drugs in the body.

[0006] At present, there are no reports at home or abroad on the use of porphine as a carrier to carry the second-generation boron carrier BSH to penetrate the blood-brain barrier and deliver it to glioma cells in a targeted manner. Summary of the invention

[0007] The purpose of the present invention is to provide a class of tumor-targeting amphiphilic porphin-BSH conjugates to achieve 10 The efficient delivery of B atoms and the tracing function of compounds in vivo are applied to BNCT treatment. Porphine core compounds are connected to linkers and then BSH is coupled to water-soluble target molecules. This type of conjugate can not only efficiently and safely transport a large number of 10 B to tumor cells, and the drug distribution is observed by fluorescence tracing.

[0008] The technical solution of the present invention:

[0009] A type of porphine-BSH conjugate in boron neutron capture therapy has the following structure:

[0010]

[0011] Among them, R is independent of each other. or n1 is an integer of 1-10, and n2 is an integer of 1-10.

[0012] The X +X is a monovalent cation; M is a metal or a metal nuclide.

[0013] Specifically, each R is independently or n1 is an integer from 1 to 5, and n2 is an integer from 1 to 5.

[0014] Specifically, X + is sodium ion, quaternary ammonium salt cation or potassium ion.

[0015] Specifically, each M is independently Cu, 64 Cu, 68 Ga, Fe, zn, Mg, Ni, Co, Pt, Pd, Sn or Ti.

[0016] Specifically, the quaternary ammonium salt cation is selected from tetramethyl quaternary ammonium salt cation, tetraethyl quaternary ammonium salt cation, tetrapropyl quaternary ammonium salt cation, tetrabutyl quaternary ammonium salt cation.

[0017] The porphine nuclei are respectively

[0018]

[0019] The linking group is:

[0020]

[0021] The boron agent is the second-generation boron carrier mercaptododecaborane disodium salt, the second-generation boron carrier mercaptododecaborane diquaternary ammonium salt or the second-generation boron carrier mercaptododecaborane dipotassium salt:

[0022]

[0023] After the porphine nucleus of the conjugate reacts with a metal salt (such as a metal chloride or a metal acetate complex), a conjugate chelated with a divalent metal or a tetravalent metal is obtained.

[0024] A preparation method of a porphine-BSH conjugate in boron neutron capture therapy, characterized in that the steps are as follows:

[0025]

[0026] Compound pheophytin a is subjected to esterification and hydrolysis reactions to obtain compound 1, and compound 1 is successively subjected to amidation reaction and thiol coupling reaction with compound A and compound BSH to obtain the target compound I; or, compound pheophytin a is subjected to a deacylation reaction to obtain compound 2, and compound 2 is successively subjected to amidation reaction and thiol coupling reaction with compound A and compound BSH to obtain the target compound II.

[0027] Among them, R and X+ It has the same definition as that in the structural formula.

[0028]

[0029] Compound I and Compound II react with the salt containing metal M in a solvent respectively to obtain Compound III and Compound IV.

[0030] Among them, R and X + and the definition of M are the same as those in the structural formula.

[0031] Specific preparation methods of two boron neutron capture therapy chlorin-BSH conjugates include the following steps:

[0032] ① Using compound pheophytin a as a raw material, dissolve it in a 5% sulfuric acid methanol solution with a concentration of 0.01 M, react at 25 °C for 4 h to obtain a methyl esterification product. Subsequently, dissolve the obtained methyl esterification product in methanol, add 2 equivalents of sodium methoxide and react at 25 °C for 12 h to obtain Compound 1; dissolve Compound 11 in a mixed solution of 5% KOH∶THF = 1∶1, ultrasonicate for 5 min, and react at 40 °C for 12 h to obtain Compound 2; dissolve Compound 2 in DMF and successively add EDCI, N-(2-amino)maleimide and triethylamine. Among them, the molar ratio of Compound 2∶EDCI∶N-(2-amino)maleimide is 1∶1.1∶1.1, and react at room temperature for 12 - 24 h to obtain Compound 12; dissolve Compound b in a mixed solution of DCM∶EtOH = 1∶1 and successively add disodium mercaptododecaborane and triethanolamine. Among them, the molar ratio of Compound b∶BSH is 1∶1.2, and react at room temperature for 4 - 8 h; the reaction formula is as follows:

[0033]

[0034] ② Using compound pheophytin a as a raw material, dissolve it in pyridine with a concentration of 0.01 M, and react at 115 °C for 12 - 18 h to obtain Compound 1; dissolve Compound 1 in a DMF solution, and successively add EDCI, N-(2-amino)maleimide and triethylamine. Among them, the molar ratio of Compound 2∶EDCI∶N-(2-amino)maleimide is 1∶2∶2, and react at room temperature for 12 - 24 h to obtain Compound 5; dissolve Compound 5 in a mixed solution of DCM∶EtOH = 1∶1 and successively add disodium mercaptododecaborane and triethanolamine. Among them, the molar ratio of Compound 5∶BSH is 1∶1.2, and react at room temperature for 4 - 8 h. The reaction formula is as follows:

[0035]

[0036] The reaction formulas of the other two porphyrin-BSH conjugates in boron neutron capture therapy are as follows:

[0037]

[0038] The specific reaction steps are the same as those in the preparation processes of Compound 4 and Compound 7.

[0039] Specifically, the reaction formula for chelating metal ions onto the porphyrin mother nucleus is as follows:

[0040]

[0041] Compound 8 was added to a mixed solvent of dichloromethane:methanol = 1:1, copper acetate solid was added, and the reaction was carried out at room temperature for 1.5 h. Compound 10 was obtained by passing through a silica gel column.

[0042] The above-mentioned porphyrin-BSH conjugate is used as an active part of a targeted boron agent for BNCT to prepare a targeted anti-tumor drug. The tumors include head and neck tumors (including laryngeal cancer, thyroid cancer, nasopharyngeal cancer, tonsillar cancer, lymphoma, sarcoma), intracranial tumors (including glioma, medulloblastoma, ependymoma, brain metastases), breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, gastric cancer, rectal cancer.

[0043] Advantages of the present invention: The porphyrin-BSH conjugate molecule of the present invention is in the form of a salt, effectively improving the water solubility of the overall molecule. Using the porphyrin mother nucleus as a carrier, the porphyrin compound has the characteristics of tumor cell targeting, good tumor cell uptake, low safety and toxicity, and the fluorescence phenomenon of the photosensitizer itself, and can achieve the targeted delivery of the second-generation boron carrier sodium mercaptododecaborane disodium salt (BSH) to tumor cells, effectively solving the problems of insufficient tumor targeting and poor accumulation ability in tumor cells during the clinical use of BSH. The introduction of the porphyrin mother nucleus significantly increases the 10 content of B atoms in tumor cells. At the same time, the fluorescence characteristics of porphyrin itself are used to monitor the in vivo distribution of the conjugate in real time, so as to formulate the best neutron irradiation conditions. Combining the results of dark toxicity and BNCT in vitro activity tests, the porphyrin-BSH conjugate has a strong killing effect on tumor cells while having little toxicity to normal cells. Such molecules can be used for the preparation of the third-generation boron carrier in BNCT treatment. Description of the Drawings

[0044] Figure 1 It is a characterization diagram of the compound.

[0045] Among them, (a) is the high-performance liquid chromatography diagram of Compound 8;

[0046] (b) is the high-performance liquid chromatography diagram of Compound 7.

[0047] Figure 2 Spectral data graph of porphyrin-BSH conjugate.

[0048] Among them, (a) is the ultraviolet-visible absorption spectrum of porphyrin-BSH;

[0049] (b) is the fluorescence spectrum of porphyrin-BSH.

[0050] Figure 3 Fluorescence microscopy monitoring of the uptake of porphyrin-BSH conjugate by GL261 cells.

[0051] Figure 4 Fluorescence microscopy monitoring of the uptake of porphyrin-BSH conjugate by bEND.3 cells.

[0052] Figure 5 Flow cytometry analysis of the uptake of porphyrin-BSH by GL261 cells.

[0053] Figure 6 MTT assay for evaluating the toxicity of porphyrin-BSH conjugate preparations to bEND.3 cells and GL261 cells.

[0054] Figure 7 ICP OES detection of the 10 accumulation of B in GL261 cells after incubation with the conjugate for different times.

[0055] Figure 8 Photograph of the liquid surface of the Transwell chamber on the fifth day and graph of the change in the liquid surface height of the chamber at 1, 3, and 5 days at 1, 4, and 9 h.

[0056] Figure 9 In vitro blood-brain barrier model fluorescein sodium permeability experiment graph.

[0057] Figure 10 Graph for evaluating the ability of porphyrin-BSH conjugate preparations to cross the in vitro blood-brain barrier.

[0058] Figure 11 Graph of the activity data of porphyrin-BSH conjugate on GL261 cells after BNCT irradiation.

[0059] Among them, (a) is the total neutron fluence of 5.54×10 6 n / cm 2 after 16 min of BNCT irradiation;

[0060] (b) is the total neutron fluence of 8.31×10 6 n / cm 2 after 24 min of BNCT irradiation. Detailed implementation method

[0061] Example 1

[0062] Extraction and Synthesis of Compound Pheophytin a

[0063] Accurately weigh 100 g of Spirulina platensis powder from Chenghai Lake, soak the powder with 500 mL of acetone and transfer it to a 1-L three-necked flask. Place the three-necked flask in an oil bath, equipped with an electric stirring device and a tap water reflux condenser, and introduce nitrogen gas to check the airtightness of the device and the normal operation of the stirring device. Set the oil bath to heat at 65 °C. Start timing after the solvent temperature in the three-necked flask reaches 56 °C and solvent reflux occurs in the condenser. Reflux for 2 h, then stop heating and continue stirring until the solvent in the three-necked flask cools naturally to 30 °C. Use vacuum filtration to separate the solid from the liquid, and wash the residue with acetone until the filtrate becomes lighter. Repeat the above extraction steps three times, combine the filtrates and rotary evaporate to obtain Spirulina extract chlorophyll a.

[0064] Dissolve the Spirulina powder extract chlorophyll a with 300 mL of ether and transfer it to a 1-L three-necked flask. Install a mechanical stirring device and a dropping funnel device, introduce nitrogen gas, start stirring and cool down to -10 °C in an ice-salt bath environment. Slowly add 150 ml of 12 M concentrated hydrochloric acid (pre-cooled to -20 °C) dropwise, and control the dropping rate of the concentrated hydrochloric acid to keep the temperature of the reaction solution below 0 °C. After the concentrated hydrochloric acid is added dropwise through the dropping funnel, remove the ice-salt bath cooling device below and react at room temperature. Use thin-layer chromatography to detect the reaction process. Use a developing system of petroleum ether∶ethyl acetate = 3∶1 to detect the reaction of the raw material chlorophyll a, and use a developing system of chloroform∶methanol = 20∶1 to detect the formation of the product CHP. After the reaction is completed, transfer the reaction solution to a 5-L separatory funnel, extract it three times with 350 ml of petroleum ether, and collect the lower acid aqueous layer.

[0065] Collect the acid aqueous layer solution, gradually add saturated sodium carbonate solution to adjust the pH to about 4, precipitate a dark green solid, perform vacuum filtration with a Buchner funnel, wash the solid with a 1% (v / v) propionic acid aqueous solution, and place the obtained solid in a vacuum drying oven and add solid phosphorus pentoxide for drying (25 °C). After the solid is dried, dissolve it with a mixed solvent of dichloromethane∶methanol = 10∶1, transfer it to a round-bottomed flask and rotary evaporate the solvent to obtain 1.08 g of pheophorbide a (CHP), with a total yield of 1.1%.

[0066] Synthesis of Compound 11

[0067] CHP (500 mg) was methyl esterified with 40 ml of 5% sulfuric acid methanol solution for 4 h under N₂ protection. After the reaction, methanol was removed by rotary evaporation. The mixture was extracted three times with saturated sodium bicarbonate aqueous solution and dichloromethane, and then the organic phase was extracted three times with saturated brine. Then it was dried by rotary evaporation to obtain the methyl esterified intermediate. Subsequently, the obtained methyl esterified product was dissolved in 15 ml of methanol, 1.5 ml of sodium methoxide solution was added, and the reaction was carried out under N₂ protection for 12 h. After the reaction, the pH of the solution was adjusted to neutral with 1 M HCl, methanol was removed by rotary evaporation, and column chromatography separation was carried out. The elution conditions were (dichloromethane∶ethyl acetate = 100∶1 - 80∶1) to obtain 11115.1 mg of compound 1 with a yield of 23%. 1 H NMR (600 MHz, Chloroform-d) δ 9.66 (s, 1H), 9.50 (s, 1H), 8.73 (s, 1H), 7.98 (dd, J = 17.8, 11.4 Hz, 1H), 6.28 (d, J = 17.8 Hz, 1H), 6.07 (d, J = 11.5 Hz, 1H), 5.29 (d, J = 47.0 Hz, 2H), 4.47 - 4.37 (m, 2H), 4.25 (s, 3H), 3.76 (s, 3H), 3.72 (q, J = 7.8 Hz, 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 = 7.3 Hz, 4H), 1.68 (t, J = 7.7 Hz, 3H), -1.47 (s, 1H). ESI-MS for C 37 H 42 N4O6: (calculated) 638.3104; (found) 639.3138 [M + H] + .

[0068] Synthesis of Compound 1

[0069] First, THF and 1 mol / L KOH solution were ultrasonically degassed for 10 minutes respectively. 45.5 mg of compound 11 was accurately weighed in a 25 mL eggplant-shaped flask. 2 mL of THF was measured to dissolve compound 11, and then 2 mL of 1 mol / L KOH solution was added. The reaction was carried out at 40 °C under nitrogen protection with a reflux condenser. The reaction was monitored by TLC. After the reaction was completed, when the reaction solution was concentrated under reduced pressure to the remaining KOH aqueous solution, 1 mol / L HCl was added to adjust the pH value to 3 - 4. A large amount of dark green solid small particles precipitated, and then suction filtration was carried out with a Buchner funnel and washed with 1% propionic acid aqueous solution. Suction filtration was continued until the filter cake cracked. The mixture was dissolved and transferred with a mixed solution of dichloromethane and methanol, concentrated under reduced pressure and dried in vacuo to obtain 34.4 mg of the crude product of dark green solid compound 1 with a yield of 81%. ESI-MS for C 34 H36 N4O6: (calculated) 596.2635; (found) 597.21 [M+H] +

[0070] Synthesis of Compound 4

[0071] Dissolve 45.5 mg of Compound 1 in 2 mL of anhydrous DMF, add 16.3 mg of EDCI, 12.7 mg of HOBT and 50 μl of triethylamine, stir at 25 °C under a nitrogen balloon protection, monitor by TLC. After the reaction proceeds for 2 h, it is found that the raw material is converted into an intermediate. Add 14.8 mg of N-(2-amino) maleimide to the reaction solution and stir at 25 °C under a nitrogen balloon protection. Detect the reaction by TLC (developing agent: dichloromethane: methanol = 10:1). Stop the reaction after it proceeds for 12 h. Adjust the pH of the reaction solution to 3-4 with 1 M HCl, filter with a sand plate filter to obtain a solid, and dry it under vacuum to obtain a solid. Dissolve it with a solvent of dichloromethane: methanol = 10:1, rotary evaporate the solvent, load the sample dry onto a silica gel column (gradient from dichloromethane containing 0.33% formic acid to dichloromethane: methanol = 100:1 containing 0.33% formic acid, then gradient to 50:1 containing 0.33% formic acid, and then gradient to 30:1 containing 0.33% formic acid). Obtain 19.1 mg of dark green solid with a yield of 35%. ESI-MS for C 40 H 42 N6O7: (calculated) 718.3115; (found) 719.43 [M+H] + .

[0072] Synthesis of Compound 4a

[0073] Dissolve 100 mg of Compound 1 in 4 mL of anhydrous DMF, add 35.4 mg of EDCI, 25.1 mg of HOBT and 100 μl of triethylamine, stir at 25 °C under a nitrogen balloon protection, monitor by TLC. After the reaction proceeds for 2 h, it is found that the raw material is converted into an intermediate. Add 184.8 mg of amino polyethylene glycol thiol (molecular weight 1000) to the reaction solution and stir at 25 °C under a nitrogen balloon protection. Detect the reaction by TLC (developing agent: dichloromethane: methanol = 10:1). Stop the reaction after it proceeds for 12 h. Adjust the pH of the reaction solution to 3-4 with 1 M HCl, filter with a sand plate filter to obtain a solid, and dry it under vacuum to obtain a solid. Dissolve it with a solvent of dichloromethane: methanol = 10:1, rotary evaporate the solvent, load the sample dry onto a silica gel column (gradient from dichloromethane containing 0.33% formic acid to dichloromethane: methanol = 100:1 containing 0.33% formic acid, then gradient to 50:1 containing 0.33% formic acid, and then gradient to 30:1 containing 0.33% formic acid). Obtain 72.4 mg of dark green solid with a yield of 27%.

[0074] Synthesis of Compound 7

[0075] Dissolve 19.1 mg of Compound 4 in 2 ml of a mixed solution of dichloromethane∶methanol = 10∶1. After complete dissolution, add 50 μl of triethanolamine, then quickly weigh and add 7 mg of sodium dodecaboranethiolate. Stir and react at 25 °C under the protection of a nitrogen balloon, and monitor the reaction progress by TLC. After the reaction proceeds for 4 h, the reactants are completely reacted. Stop the reaction, add a small amount of formic acid to neutralize triethanolamine, then rotary evaporate the solvent, and load the sample by dry method onto a reverse-phase silica gel column (gradient from water∶methanol = 10∶1 to water∶methanol = 1∶3) to obtain 20.5 mg of Compound 4 with a yield of 81%. 1 H NMR(600 MHz, DMSO-d6) δ 9.76 (d, J = 29.5 Hz, 2H), 9.14 (s, 1H), 8.35 (dd, J = 17.8, 11.6 Hz, 1H), 6.46 (d, J = 17.9 Hz, 1H), 6.17 (d, J = 11.8 Hz, 1H), 5.76 (s, 1H), 5.02 (s, 1H), 4.59 (d, J = 7.2 Hz, 1H), 3.83 (d, J = 7.7 Hz, 2H), 3.83 (d, J = 7.7 Hz, 2H), 3.55 (s, 5H), 3.55 (s, 6H), 3.40 (s, 2H), 1.70 (t, J = 7.3 Hz, 6H), 1.70 (t, J = 7.3 Hz, 8H), 0.85 (s, 11H). ESI-MS for C 40 H 54 B 12 N6O7S: (calculated) 940.4687; (found) 446.83 [(M - 2Na - H) / 2] 3- .

[0076] Synthesis of Compound 7a

[0077] Dissolve 50 mg of Compound 4a in 5 ml of a mixed solution of dichloromethane∶methanol = 10∶1. After complete dissolution, add 100 μl of triethanolamine, then quickly weigh and add 8.2 mg of sodium dodecaboranethiolate. Stir and react at 25 °C under the protection of a nitrogen balloon, and monitor the reaction progress by TLC. After the reaction proceeds for 4 h, the reactants are completely reacted. Stop the reaction, add a small amount of formic acid to neutralize triethanolamine, then rotary evaporate the solvent, and load the sample by dry method onto a reverse-phase silica gel column (gradient from water∶methanol = 10∶1 to water∶methanol = 1∶3) to obtain 42.7 mg of Compound 4a with a yield of 75%.

[0078] Example 2

[0079] Synthesis of Compound 2

[0080] Dissolve 500 mg of CHP in 30 ml of pyridine in a 250 ml round-bottom flask and ultrasonically dissolve it thoroughly. Protect with a nitrogen balloon and stir the reaction using a condenser heat reflux device at 115 °C. Monitor the reaction progress by TLC and react for 12 h. After the reaction is completed, rotary evaporate the pyridine in the reaction flask and load the sample onto a silica gel column by dry method (gradient from dichloromethane∶methanol = 100∶1 to dichloromethane∶methanol = 30∶1) to obtain 302 mg of compound 2 with a yield of 71%. 1H NMR (600 MHz, Chloroform-d) δ 9.36 (s, 1H), 9.25 (s, 1H), 8.51 (s, 1H), 7.90 (dd, J = 17.9, 11.5 Hz, 1H), 6.21 (d, J = 17.8 Hz, 1H), 6.11 (d, J = 11.5 Hz, 1H), 5.25 (d, J = 44.8 Hz, 3H), 5.08 (d, J = 19.1 Hz, 1H), 4.44 (q, J = 7.5 Hz, 1H), 4.27 (d, J = 9.4 Hz, 1H), 3.58 (d, J = 10.7 Hz, 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, 7.2 Hz, 1H), 1.80 (s, 3H), 1.63 (d, J = 15.5 Hz, 4H), 1.26 (d, J = 24.3 Hz, 1H), -1.76 (s, 1H). ESI-MS for C 33 H 34 N4O3: (calculated) 534.2631; (found) 535.2693 [M + H] + .

[0081] Synthesis of Compound 5

[0082] Dissolve 53.6 mg of compound 2 in 2 mL of anhydrous DMF, add 21.1 mg of EDCI, 16.7 mg of HOBT and 50 μl of triethylamine, stir at 25 °C, protect with a nitrogen balloon, monitor by TLC. After reacting for 2 h, it is found that the raw material is converted into an intermediate. Add 20 mg of N-(2-amino) maleimide to the reaction solution and stir at 25 °C, protect with a nitrogen balloon, detect the reaction by TLC (developer: dichloromethane∶methanol = 20∶1), and stop the reaction after reacting for 12 h. Transfer the reaction solution to a 25 ml separatory funnel with 5 ml of dichloromethane, extract three times with 5 ml of deionized water, and then extract twice with 5 ml of saturated brine to obtain the dichloromethane layer. Dry it with anhydrous sodium sulfate, filter and rotary evaporate the solvent, and load the sample onto a silica gel column by dry method (gradient from dichloromethane∶methanol = 100∶1 to dichloromethane∶methanol = 10∶1). Obtain 42.5 mg of dark green solid with a yield of 65%.1 1H NMR (600 MHz, Chloroform-d) δ 9.36 (s, 1H), 9.10 (s, 1H), 8.57 (s, 1H), 7.98 (dd, J = 17.8, 11.5 Hz, 2H), 6.51 (s, 3H), 6.28 (d, J = 17.8 Hz, 2H), 6.17 (d, J = 11.4 Hz, 2H), 5.78 (s, 1H), 5.27 (s, 1H), 5.24 (s, 1H), 5.04 (s, 1H), 5.01 (s, 1H), 4.52 (s, 1H), 4.33 - 4.30 (m, 2H), 3.57 (dd, J = 14.2, 7.3 Hz, 2H), 3.50 (d, J = 8.5 Hz, 2H), 3.41 (s, 8H), 3.21 (s, 11H), 2.67 (s, 1H), 2.46 (s, 1H), 2.21 (s, 1H), 1.92 (s, 1H), 1.81 (d, J = 6.7 Hz, 6H), 1.58 (t, J = 7.7 Hz, 8H), 1.26 (s, 4H), 0.91 - 0.83 (m, 2H), -1.73 (s, 1H). ESI-MS for C 39 H 40 N6O4: (calculated) 656.3111; (found) 657.23 [M + H] + .

[0083] Synthesis of Compound 5a

[0084] Dissolve 100 mg of Compound 5a in 4 mL of anhydrous DMF, add 71.8 mg of EDCI, 50.6 mg of HOBT and 100 μl of triethylamine, stir at 25 °C under nitrogen balloon protection, monitor by TLC. After 2 h of reaction, it was found that the raw material was converted into an intermediate. Add 206 mg of amino polyethylene glycol thiol (molecular weight 1000) to the reaction solution, stir at 25 °C under nitrogen balloon protection, and detect the reaction by TLC (developing solvent: dichloromethane:methanol = 20:1). Stop the reaction after 12 h. Transfer the reaction solution to a 100 ml separatory funnel with 10 ml of dichloromethane, extract three times with 20 ml of deionized water, and then extract twice with 20 ml of saturated brine to obtain the dichloromethane layer. Dry it with anhydrous sodium sulfate, filter and rotary evaporate the solvent, and load the sample onto a silica gel column by dry method (gradient from dichloromethane:methanol = 100:1 to dichloromethane:methanol = 10:1). Obtain 146.5 mg of dark green solid, with a yield of 51%.

[0085] Synthesis of Compound 8

[0086] Dissolve 42.5 mg of Compound 5 in 2 ml of a mixed solution of dichloromethane∶methanol = 10∶1. After complete dissolution, add 50 μl of triethanolamine, then quickly weigh and add 18.7 mg of sodium dodecaborane mercaptide. Stir and react at 25 °C under nitrogen balloon protection, and monitor the reaction progress by TLC. After 2 h, the reactants are completely reacted. Stop the reaction, add a small amount of formic acid to neutralize triethanolamine, then evaporate the solvent to dryness. Load the sample by dry method and pass through a reverse-phase silica gel column (gradient from water∶methanol = 10∶1 to water∶methanol = 1∶3) to obtain 47.4 mg of Compound 8, with a yield of 84%. 1 H NMR(600MHz, DMSO-d6)δ9.65(s, 1H), 9.38(s, 1H), 8.93(s, 1H), 8.19(dd, J = 17.8, 11.6Hz, 1H), 7.84(dt, J = 22.8, 6.2Hz, 1H), 6.38(d, J = 17.8Hz, 1H), 6.20(d, J = 11.5Hz, 1H), 5.26(dd, J = 19.6, 4.5Hz, 1H), 5.21(t, J = 5.0Hz, 1H), 5.16(s, 1H), 4.61 - 4.54(m, 1H), 4.31(t, J = 8.4Hz, 1H), 3.69 - 3.63(m, 6H), 3.63 - 3.56(m, 5H), 3.51(q, J = 4.5, 3.9Hz, 2H), 3.44(s, 4H), 3.18(s, 4H), 3.12(dq, J = 20.2, 6.2Hz, 3H), 3.02(q, J = 6.0Hz, 3H), 2.97(dd, J = 16.3, 5.8Hz, 1H), 2.76(ddd, J = 18.4, 7.3, 2.8Hz, 1H), 2.61(d, J = 10.8Hz, 1H), 2.55(s, 10H), 2.33(s, 1H), 2.13(ddd, J = 13.9, 8.9, 4.8Hz, 1H), 1.80(d, J = 7.3Hz, 3H), 1.61(t, J = 7.6Hz, 3H), 1.00(s, 11H), 0.20(s, 1H), -2.01(s, 1H).ESI-MS for C 39 H 52 B 12 N6Na2O4S:(calculated)878.4683;(found)877.70[M - H] - .

[0087] Synthesis of Compound 8a

[0088] Dissolve 40 mg of compound 5a in 2 ml of a mixed solution of dichloromethane:methanol = 10:1. After complete dissolution, add 50 μl of triethanolamine, then quickly weigh and add 8 mg of disodium mercaptododecaborane. Stir and react at 25 °C under a nitrogen balloon protection, and monitor the reaction progress by TLC. After 2 h of reaction, the reactants are completely reacted, stop the reaction, add a small amount of formic acid to neutralize triethanolamine, then rotary evaporate the solvent, and load the sample onto a reversed-phase silica gel column (gradient from water:methanol = 10:1 to water:methanol = 1:3) to obtain 34.3 mg of compound 8a with a yield of 76%.

[0089] Synthesis of Compound 10

[0090] Dissolve 4 mg of compound 8 in 12 ml of a solvent of dichloromethane:methanol = 1:1, add 2.1 mg of copper acetate, react at room temperature for 2 h under nitrogen protection, and detect the reaction by TLC. After the reaction is completed, rotary evaporate the solvent and pass through a silica gel column to obtain 2.5 mg of compound 10 with a yield of 57%. ESI-MS for C 39 H 50 B 12 N6Na2O4SCu: (calculated) 938.4683; (found) 445.76 [M - 2Na] / 2 - .

[0091] The introduction of metal ions does not affect the tumor cell targeting of porphyrin compounds. Compound 10 also significantly increases the content of 10 B atoms in tumor cells. At the same time, 64 the introduction of Cu and other radioactive metal nuclides 68 such as Ga can be used in PET imaging technology.

[0092] Example 3

[0093] Characterize the compounds 7 and 8 prepared in Examples 1 and 2:

[0094] (1) Performance test experiment 1: Characterize the purity of the synthesized conjugate by high performance liquid chromatography, as Figure 1 , and the results show that the purity of the obtained compounds is greater than 95%.

[0095] (2) Performance test experiment 2: UV and fluorescence spectroscopy determination of porphyrin - BSH conjugate

[0096] In a 3-ml quartz cuvette, add the sample solutions of Compound 7 and Compound 8 dispersed in methanol, and use a UV-visible spectrophotometer (PerkinElmer, Lambda 750S) to measure the absorption curves of the samples. Add 1 mL of the sample solutions of Compound 7 and Compound 8 to the quartz cuvette, and use a fluorescence spectrometer (Hitachi, F7000) to measure the emission curves of the samples (Ex: 345 nm), as Figure 2 shown. Comparing with the porphin mother nucleus structure, its fluorescence properties are not affected after modification.

[0097] Example 4

[0098] Perform in vitro cell uptake experiments on the porphin-BSH conjugates prepared in Example 1 and Example 2

[0099] (1) Monitor the uptake of porphin-BSH preparations by cells using a fluorescence microscope

[0100] Select GL261 cells and bEND.3 cells in the logarithmic growth phase with good growth status, digest and collect them, and adjust the cell density. Add 0.5 mL of the cell suspension (4×10 4 cell / mL) evenly and slowly into a 24-well plate, and culture it routinely for 12 h until the cells adhere to the wall and fully spread. Take out the plate, discard the supernatant, wash it with PBS, and then add Compound 7 and Compound 8 respectively to make the final concentration 25 μM, and incubate in the dark for different times. At 2 h, 4 h, and 8 h after adding the conjugate, take out the cells and use DAPI to label the cell nuclei. Observe the enrichment of the porphin mother nucleus showing red fluorescence in each group of cells under a fluorescence microscope, and collect images, as Figure 3 and Figure 4 shown. The analysis results of fluorescence imaging show that the uptake of Compound 7 and Compound 8 by GL261 cells and bEND.3 cells is time-dependent.

[0101] (2) Analyze the uptake of porphin-BSH by cells using a flow cytometer

[0102] Select GL261 cells in the logarithmic growth phase with good growth status, digest and collect them, and adjust the cell density. Add 0.5 mL of the cell suspension (1×10 5 cell / mL) evenly and slowly into a 24-well plate, and culture it routinely for 12 h until the cells adhere to the wall and fully spread. Then select the time points 2 h, 4 h, and 8 h to add drugs for incubation respectively. After the incubation is over, remove the drug-containing medium, wash it three times with PBS, add 300 μl of trypsin for digestion, centrifuge for 3 min, discard the medium, and resuspend it with PBS. Discard the PBS, fix it with 20 μl of 4% paraformaldehyde, and the number of cells is 2000, as Figure 5As shown, the red fluorescence property of the drug was used to detect the red fluorescence APC channel (red fluorescence, excitation wavelength λex = 633 nm, fluorescence intensity signal collection λem = 660 nm). The results showed that with the passage of time, the fluorescence intensity continuously increased, indicating that the uptake of the conjugate by cells was time-dependent.

[0103] (3) Evaluation of the in vitro cytotoxicity of porphine-BSH by MTT method

[0104] Mouse glioma cells (GL261) in the logarithmic growth phase were seeded in 96-well plates at a density of 3×10 3 cells / well, and 100 μL of cell suspension was added to each well. After culturing for 12 h, 100 μL of the drug solution was added, and the final concentrations of the test compounds were 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 without cells) and a control group (cultured cells without adding drugs) were set up. After the cells were incubated in the incubator (standard environment) for 12 h, 20 μL of MTT solution with a concentration of 5 mg / ml was added to each well, and the cells were cultured for another 4 h. Then 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 by an enzyme-linked immunosorbent assay (ELISA) reader. The relative survival rate of the cells was calculated according to the following formula: (OD of the experimental group 570 / OD of the control group 570 ) × 100%, and the experiment was repeated 3 times. The toxicity of porphine-BSH to mouse brain microvascular endothelial cells (bEND.3) was measured by the same method. As Figure 6 shown, it can be seen that the drug had no obvious toxicity to bEND.3 cells within the concentration range of 100 μM without neutron irradiation. The drug had no obvious toxicity to GL261 cells within the concentration range of 100 μM without neutron irradiation.

[0105] (4) Detection of the uptake of the conjugate by cells by ICP-OES

[0106] GL261 cells in the logarithmic growth phase with good growth status were digested, collected, and the cell density was adjusted. 1 mL of cell suspension (1×10 6 cell / mL) was evenly and slowly inoculated in six-well plates and cultured routinely for 12 h until the cells adhered to the wall and fully spread. The plates were taken out, the supernatant was discarded, and the cells were washed 3 times with PBS and then 1 mL of cell culture medium containing compound 7 and compound 8 was added respectively. After 12 h of addition, the supernatant was removed, the cells 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% of 30% H2O2 (VHNO3∶V30% H2O2 = 3∶2), and the boron content in the cells was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). AsFigure 7 As shown, the boron content in the cells in the compound 7 and compound 8 groups continued to accumulate with time after incubation for different times. Among them, the boron content in the compound 8 group reached 117 ppm at the 2nd hour, which was greater than the treatment standard of 20 ppm, and reached 223 ppm after incubation for 12 hours. The boron content in the compound 7 group was also higher than the amount of one treatment standard, 27 ppm, at the 4th hour of incubation and reached 56 ppm at the 12th hour of incubation.

[0107] Example 5

[0108] Construction of an in vitro blood-brain barrier model and evaluation of the ability of porphine-BSH preparation to cross the blood-brain barrier

[0109] (1) Construction of an in vitro blood-brain barrier model and leak test

[0110] Take out the Transwell chamber, add DMEM medium and place it in a 24-well plate, and let it stand and activate in the cell culture incubator for 20 min. Seed bEND.3 cells in the logarithmic growth phase at a density of 2×10 4 cell / well on the upper chamber of the Transwell, and add 0.6 mL of medium to the 24-well plate in the lower chamber of the Transwell, and place it in the incubator for 5 days until the cells are in a confluent state (during this period, regularly change the cell medium in the upper chamber of the Transwell to make the bEND.3 cells grow tightly attached to the wall). Add 400 μL and 200 μL of medium to the upper and lower chambers of bEND.3 respectively. At this time, the liquid level difference in the Transwell chamber is 1 cm. Place it in the incubator and continue to culture. Record the liquid level height at 1, 4, and 9 h, and observe whether the liquid level difference between the two chambers remains the original height. As Figure 8 shown, the liquid level difference did not change significantly on the 5th day, that is, it was initially judged that the in vitro BBB modeling was successful.

[0111] (2) Fluorescein sodium permeability experiment of the in vitro blood-brain barrier model

[0112] Use fluorescein sodium (FLU) to detect the in vitro cultured BBB model. Select the bEND.3 single-culture model group and the blank group, with 3 models in each group. Replace the whole liquid with serum-free DMEM culture medium before measurement. Add 50 μg / mL fluorescein sodium to the donor pool, and place it in a 37 °C, 0.5% CO2 incubator for 5 min, 10 min, 20 min, 40 min, and 80 min. Then take 50 μL of the culture medium from the receiver pool and measure the amount of FLU passing through different models with a fluorescence microplate reader. As Figure 9 shown, the bEND.3 single-culture model group can well block the permeation of fluorescein sodium.

[0113] (3) Evaluate the ability of porphine-BSH conjugate to cross the in vitro blood-brain barrier

[0114] The wells that passed the leak detection experiment and the fluorescein sodium penetration experiment were selected as the in vitro blood-brain barrier model to evaluate the ability of Compound 7 and Compound 8 to cross the blood-brain barrier. Briefly, 300 μL of the solutions containing Compound 7 and Compound 8 were added to the donor chambers respectively. The lower chamber was paved with GL261 cells at 70% density and incubated for 4 h. The uptake of the conjugate by GL261 cells was observed under a fluorescence microscope. As Figure 10 shown, Compound 8 can cross the in vitro blood-brain barrier better.

[0115] Application Example 1

[0116] In vitro Activity Evaluation of BNCT

[0117] An in vitro cell activity evaluation experiment was conducted on the porphine-BSH conjugates prepared in Example 1 and Example 2.

[0118] (1) Evaluate the effects of different neutron numbers and different irradiation durations on the activity of GL261 cells.

[0119] GL261 cells in the logarithmic growth phase with good growth status were digested, collected and adjusted to the cell density. 1 ml of cell suspension (1×10 6 cell / ml) was evenly and slowly inoculated into a six-well plate and cultured routinely for 12 h until the cells adhered and fully spread. The plate was taken out, the supernatant was discarded, and after washing 3 times with PBS, 1 ml of cell culture medium containing 100 μM of Compound 7 and Compound 8 was added respectively. After 2 h of addition, the supernatant was removed, washed three times with PBS, and then trypsin was added to digest the cells from the six-well plate, resuspended with the culture medium, and finally the culture medium was added for counting and adjusted to a cell density of 166,000 / ml. 0.6 ml was transferred to a 1.5 ml centrifuge tube, totaling 100,000 cells. Subsequently, the irradiation intensities with neutron fluences of 3.46×10 5 n / cm 2 were used to perform BNCT irradiation for 16 min and 24 min respectively, and then the irradiation was terminated. After the irradiation was completed, the cells were incubated for another 24 h, then 50 μl of the solution containing CCK8 was added and incubated for 4 h. Finally, the absorbance at 450 nm was measured using an enzyme-labeled instrument. As Figure 11 (a)(b) shown, for the cell killing effects caused by irradiating for different times under the same neutron fluence, we can see from the figure that the killing effect of Compound 8 on GL261 reached 90%, and the killing effect of Compound 7 on GL261 cells also reached 75%. In the in vitro activity evaluation, both conjugates had good cell killing effects after BNCT irradiation.

Claims

1. A class of porphine-BSH conjugates in boron neutron capture therapy, characterized in that, Its structure is as follows: wherein each R independently is n1 is an integer from 1 to 5; X + is sodium ion, quaternary ammonium cation or potassium ion; M is independently Cu, 64 Cu, Fe or Zn.

2. The preparation method of a porphine-BSH conjugate in boron neutron capture therapy according to claim 1, characterized in that, The steps are as follows: Compound of formula I: Compound Pheophorbide a is subjected to esterification and hydrolysis reactions to obtain Compound 1, and Compound 1 is successively subjected to amidation reaction and thiol coupling reaction with Compound A and Compound BSH to obtain the target compound; Compound of formula II: Compound Pheophorbide a is subjected to a deacylation reaction to obtain Compound 2, and Compound 2 is successively subjected to amidation reaction and thiol coupling reaction with Compound A and Compound BSH to obtain the target compound; The compound of formula I and the compound of formula II are respectively reacted with a salt containing M in a solvent to obtain the compound of formula III and the compound of formula IV; Among them, the definitions of R and X + are the same as those in claim 1 for M.

3. Use of the porphine-BSH conjugate in boron neutron capture therapy according to claim 1, characterized in that, The boron neutron capture therapy porphyrin-BSH conjugate is used for preparing an anti-tumor drug; the tumor is a head and neck tumor, an intracranial tumor, breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, gastric cancer, rectal cancer.

4. The application according to claim 3, wherein The head and neck tumor is laryngeal cancer, thyroid cancer, tonsillar cancer, lymphoma; The intracranial tumor is glioma, medulloblastoma, ependymoma, brain metastasis tumor.

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