A BPA oligomer, its targeting compound, preparation method and application

Abstract

The present invention discloses a BPA oligomer and its targeting compound, a preparation method and an application, belonging to the fields of medicinal chemistry and radiation medicine. The BPA oligomer and its targeting compound of the present invention can be used to prepare a boron delivery agent in tumor boron neutron capture therapy (BNCT). The boron uptake amount and T / N ratio of the BPA oligomer and its targeting compound of the present invention in tumor cells are superior to those of the positive control drug BPA; its permeability to cells is superior to that of BPA. The BPA oligomer targeting compound can be cleaved by FAPα enzyme to release the corresponding BPA oligomer; the preparation method of the BPA oligomer and its targeting compound of the present invention has the characteristics of mild reaction conditions, simple experimental steps, high yield, high product purity, economy and practicability, etc.

Description

Technical Field

[0001] The present invention relates to the fields of medicinal chemistry and radiation medicine, and particularly relates to a BPA oligomer for cancer boron neutron capture therapy, its targeting compound, and their preparation. Background Art

[0002] Boron neutron capture therapy (BNCT) is a fission reaction based on boron neutron capture. The non-radioactive isotope 10 B atom splits into two heavy particles ( 4 He and 7 Li) by absorbing low-energy (<0.5 eV) neutrons (thermal neutrons), and these particles release energy within a short distance (<10 μm). This distance is exactly equivalent to the diameter of a single cell. Based on this, if 10 B can be selectively delivered and enriched in malignant tumor cells, it is possible to selectively kill tumor cells while protecting normal tissues from damage. It can be seen that BNCT is a new type of radiotherapy technology, which is a binary therapy based on nuclear capture and fission reactions. Compared with traditional radiotherapy and chemotherapy, it has obvious advantages of low side effects, high selectivity, and high efficiency. However, this technology requires both high-quality neutron beams and highly targeted boron-containing drugs, and the two must cooperate closely and are indispensable. Only when both conditions are met can precise cell-level targeted killing of tumors be achieved. Currently, great progress has been made in the development of BNCT neutron beam equipment [Chinese Science Bulletin 2022, 67(14), 1471-1478]; in terms of boron drugs, only sodium mercaptododecaborate (BSH) and L-p-boronophenylalanine (BPA) have been approved by the FDA for clinical use, and both have problems of insufficient tumor targeting. For BPA, it also has problems such as low boron loading, narrow clinical indications (only suitable for a few tumor types such as glioma and melanoma), and large clinical application doses. It can be seen that clinically practical highly targeted boron drugs still cannot meet the requirements of BNCT treatment.

[0003] Structurally, BPA belongs to a non-natural amino acid, and its uptake in the body may be transported through L-type amino acid transporter 1 (LAT1). Studies have found that the absorption process of amino acids consumes a lot of energy, the carrier is easily saturated, the absorption is slow, and there is also absorption competition between amino acids; while the absorption of oligopeptides, especially dipeptides and tripeptides, consumes less energy, has a faster transport speed, the carrier is not easily saturated, and oligopeptides also have a lower osmotic pressure. Therefore, converting BPA into an oligomer may increase the accumulation amount and selectivity of boron-containing compounds in tumor cells, and at the same time improve the ability to penetrate the blood-brain barrier (BBB).

[0004] In recent years, a large number of studies have shown that fibroblast activation protein α (FAPα) is a tumor stromal antigen molecule specifically highly expressed by tumor-associated fibroblasts, which plays an important promoting role in the occurrence and development of tumors. FAPα has specific peptidase activity and can selectively hydrolyze N-terminally blocked glycylproline dipeptide sequences, such as substrates carrying the Z-Gly-Pro (Z-GP) dipeptide. Therefore, selecting fibroblast activation protein α (FAPα) as a target may also be an effective strategy to increase the accumulation and selectivity of boron in tumor cells. Summary of the Invention

[0005] In order to improve the tumor targeting, the ability to penetrate the blood-brain barrier (BBB), and the ability of active absorption of L-p-boronophenylalanine (BPA), the object of the present invention is to provide a BPA oligomer, its targeting compound, and a preparation method thereof.

[0006] Another object of the present invention is to provide the application of the above BPA oligomer and its targeting compound in the treatment of cancer by boron neutron capture therapy.

[0007] In order to achieve the above object, the specific solution adopted by the present invention is as follows:

[0008] A BPA oligomer and its targeting compound, the structure of which is shown in Formulas I and II,

[0009]

[0010] wherein, BPA is (S)-2-amino-3-[4-( 10 B)dihydroxyboranylphenyl]propionic acid; n = 0, 1, 2; Z-GP is benzyloxycarbonylglycylprolyl, and its structure is as follows:

[0011]

[0012] Preferably, the BPA oligomer and its targeting compound include the following specific compounds:

[0013]

[0014] The preparation method of the BPA oligomer compound includes the following steps:

[0015] (1-1) Preparation of N-Boc-BPA-OH: Weigh N-tert-butoxycarbonyl-4-iodo-L-phenylalanine into a reaction flask, and sequentially add tributyl borate at room temperature 10 B(O nBu)3], NaH, bis(2-dimethylaminoethyl) ether, and the reaction flask was placed in an ice bath. Under nitrogen protection, the organometallic reagent was slowly added dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 16 h. Under ice bath conditions, ice water was added and stirred for 10 min to quench the reaction. Methyl tert-butyl ether was added, and the pH value was adjusted to 3 with concentrated hydrochloric acid. The mixture was extracted twice with ethyl acetate, and the organic solvent was removed by concentration under reduced pressure. Water was added to the concentrate, and the pH value was adjusted to 12 with 1 mol / L aqueous NaOH solution. The aqueous layer was washed twice with n-butanol, and then the pH value was adjusted to 3 with concentrated hydrochloric acid. A white precipitate was formed by stirring, and it was washed by slurrying with dichloromethane, filtered, and dried to obtain the product.

[0016] The synthetic route is as follows:

[0017]

[0018] In the above steps, the molar ratios of N-tert-butoxycarbonyl-4-iodo-L-phenylalanine to tributyl borate 10 B(O n Bu)3], NaH, bis(2-dimethylaminoethyl) ether, and the organometallic reagent are 1:1.0 - 5.0, 1:1.0 - 3.0, 1:1.0 - 10.0, 1:1.0 - 10.0, respectively;

[0019] The organometallic reagent is any one of isopropylmagnesium chloride and isopropylmagnesium chloride-lithium chloride.

[0020] (1 - 2) Preparation of N-Boc-BPA-OMe: Weigh N-Boc-4-dihydroxyboronyl-L-phenylalanine (N-Boc-BPA-OH) and dissolve it in DMF. At room temperature, an inorganic base was added, and after stirring for 30 min, methyl iodide was added dropwise. After the addition was complete, the reaction was continued by stirring for 24 hours. After the reaction ended, water was added, and the mixture was extracted with ethyl acetate. The organic layers were combined and washed with saturated sodium bicarbonate and saturated brine respectively, dried over anhydrous Na2SO4, and the organic solvent was removed by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the target product.

[0021] The synthetic route is as follows:

[0022]

[0023] In the above steps, the molar ratios of N-Boc-4-dihydroxyboronyl-L-phenylalanine to the inorganic base and methyl iodide are 1:1.0 - 5.0, 1:1.0 - 5.0, respectively; the inorganic base is any one of potassium bicarbonate, sodium bicarbonate, and lithium bicarbonate.

[0024] (1-3) Preparation of H-BPA-OMe: Weigh N-Boc-BPA-OMe, add an acidic reagent, and stir the reaction at room temperature for 3 to 5 hours. After the reaction is completed, remove the solvent by rotary evaporation, add petroleum ether for pulping, and obtain the product after vacuum drying.

[0025] The synthesis steps are as follows:

[0026]

[0027] In the above steps, the molar ratio of N-Boc-BPA-OMe to the acidic reagent is 1:4.0 to 10.0; the acidic reagent is any one of trifluoroacetic acid and 4 mol / L HCl methanol solution.

[0028] (1-4) Preparation of N-Boc-(BPA) n -OMe (n = 2, 3, 4): Weigh N-Boc-BPA-OH, a condensing agent, and an organic base, dissolve them in an appropriate amount of organic solvent, stir for 30 min, and then add H-(BPA) m -OMe (m = 1, 2, 3) portionwise. After the addition is complete, stir the reaction at room temperature for 12 hours. After the reaction is completed, add an organic solvent. The organic phase is washed with saturated sodium bicarbonate, water, saturated brine, dried over anhydrous Na2SO4, the organic solvent is removed by rotary evaporation, and the residue is purified by silica gel column chromatography to obtain the target product.

[0029] The synthesis route is as follows:

[0030]

[0031] In the above steps, the molar ratios of N-Boc-BPA-OH to the condensing agent, organic base, and H-(BPA) m -OMe (m = 1, 2, 3) are 1:1.1 to 1.5, 1:2.0 to 5.0, and 1.1 to 1.5:1.0 respectively; the condensing agent is any one of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), ethyl chloroformate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N,N'-diisopropylcarbodiimide (DIC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), and tetramethylchlorouronium hexafluorophosphate (TCFH); the organic base is any one of N,N-diisopropylethylamine (DIPEA) and N-methylimidazole (NMI).

[0032] (1-5) Preparation of H-(BPA) n -OMe (n = 2, 3, 4): Weigh the compound N-Boc-(BPA) n-OMe (n = 2, 3, 4), an acidic reagent was added, and the reaction was stirred at room temperature for 3 - 5 hours. After the reaction was completed, the solvent was removed by rotary evaporation, petroleum ether was added for pulping, and the product was obtained after vacuum drying.

[0033] The synthetic route is as follows:

[0034]

[0035] In the above steps, the compound N-Boc-(BPA) n -OMe (n = 2, 3, 4) and the acidic reagent were fed in a molar ratio of 1.0:5.0 - 10.0; the acid was any one of trifluoroacetic acid (TFA) and 4 mol / L HCl methanol solution.

[0036] (1 - 6) Preparation of BPA oligomers [(BPA)2 - (BPA)4]: Weigh the compound H-(BPA) n -OMe (n = 2, 3, 4), add a basic reagent, stir at room temperature for 1 - 4 hours, monitor by TLC until the hydrolysis is complete, add 1M HCl solution to adjust the pH value to 6, precipitate a white solid, filter, wash, and vacuum dry to obtain the target product.

[0037] The synthetic route is as follows:

[0038]

[0039] In the above steps, the compound H-(BPA) n -OMe (n = 2, 3, 4) and the basic reagent were fed in a molar ratio of 1.0:5.0 - 15.0; the basic reagent could be any one of the aqueous solutions of NaOH, KOH, and LiOH.

[0040] The preparation method of the BPA oligomer targeting compound includes the following steps:

[0041] (2 - 1) Preparation of N-carbobenzoxy-glycyl-proline methyl ester (Z-GP-OMe): Replace N-Boc-L-BPA-OH with N-Z-Gly-OH, and H-(BPA) m -OMe with H-Pro-OMe, and prepare according to the method described in step (1 - 4).

[0042] The synthetic route is as follows:

[0043]

[0044] (2 - 2) Preparation of N-carbobenzoxy-glycyl-proline (Z-GP-OH): Replace the hydrolysis substrate with N-carbobenzoxy-glycyl-proline methyl ester (Z-GP-OMe), and prepare according to the method described in step (1 - 6).

[0045] The synthetic route is as follows:

[0046]

[0047] (2-3) Preparation of Z-GP-BPA oligomer methyl ester [Z-GP-(BPA) n -OMe, n = 2, 3, 4]: Replace N-Boc-L-BPA-OH with Z-GP-OH and prepare according to the method described in steps (1-4). The synthetic route is as follows:

[0048]

[0049] (2-4) Preparation of targeting compounds of BPA oligomers [Z-GP-(BPA) n -OH, n = 2, 3, 4]: Replace the hydrolysis substrate with Z-GP-BPA oligomer methyl ester [Z-GP-(BPA) n -OMe, n = 2, 3, 4] and prepare according to the method described in steps (1-6). The synthetic route is as follows:

[0050]

[0051] The present invention detected the cytotoxicity of a series of BPA oligomers and their targeting compounds against human umbilical vein endothelial cells (HUVEC), human normal liver cells (LO2), and human hepatocellular carcinoma cells (HepG2) by the MTT method, and found that these compounds had no toxicity or very low toxicity within the concentration range of 500 μM.

[0052] The present invention detected the uptake and T / N ratio (boron uptake in tumor cells / boron uptake in normal cells) of a series of BPA oligomers and their targeting compounds in human umbilical vein endothelial cells (HUVEC), human hepatocellular carcinoma cells (HepG2), human hepatocellular carcinoma cells transfected with FAPα (HepG2 / FAPα), human breast cancer cells (MDA-MB-231), and human glioma cells (U87) by ICP-MS method. The results showed that the boron uptake of a series of BPA oligomers and their targeting compounds in the cells investigated was greater than 10 9 B / cell, T / N > 3; compared with BPA, for different tumor cells, BPA oligomers and their targeting compounds showed superior boron uptake characteristics to BPA.

[0053] The present invention detected the hydrophilic-lipophilic balance coefficients of a series of BPA oligomers and their targeting compounds by the shake-flask method combined with HPLC technology. The results showed that all BPA oligomers and their targeting compounds exhibited superior hydrophilic-lipophilic characteristics to BPA.

[0054] As a preferred embodiment of the application of the present invention, the targeting compound of the BPA oligomer is a specific hydrolysis substrate of fibroblast activation protease α (FAPα) in the tumor stroma.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The compounds of general formulas I and II and their synthesis methods described in the present invention are reported for the first time in the present invention. The synthesis method of the present invention has the characteristics of mild reaction conditions, simple experimental steps, high yield, high product purity, economy and practicability;

[0057] (2) Compared with BPA, the BPA oligomer and its targeting compound of the present invention have a higher T / N ratio and boron loading in tumor cells;

[0058] (3) Compared with BPA, the BPA oligomer and its targeting compound of the present invention have more excellent lipophilic and hydrophilic characteristics. Description of the Drawings

[0059] Figure 1 shows the boron loading of the BPA oligomer and its targeting compound in human hepatoma cells HepG-2 and HepG-2 transfected with FAPα.

[0060] Figure 2 shows the T / N ratio of the BPA oligomer and its targeting compound in human hepatoma cells HepG-2 and HepG-2 transfected with FAPα and human umbilical vein endothelial cells HUVEC.

[0061] Figure 3 is the enzymatic hydrolysis rate curve of FΑPα on the compound Z-GP-(BPA)2.

[0062] Figure 4 is the enzymatic hydrolysis rate curve of FΑPα on the compound Z-GP-(BPA)3.

[0063] Figure 5 is the enzymatic hydrolysis rate curve of FΑPα on the compound Z-GP-(BPA)4. Detailed Embodiments

[0064] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0065] Example 1. Preparation of N-Boc-BPA-OH

[0066] Weigh 1.17 g (3.0 mmol) of N-tert-butoxycarbonyl-4-iodo-L-phenylalanine into a 100 ml three-necked flask. At room temperature, successively add tributyl borate 10 B(O nBu)3]2.14 g (9.3 mmol), NaH 0.24 g (60%) (6.0 mmol), bis(2-dimethylaminoethyl) ether 3.85 g (24.0 mmol). Under nitrogen protection, isopropylmagnesium chloride (1.7 M in THF) 14 mL (24.0 mmol) was slowly added under ice bath conditions. The addition was completed in 30 min. After removing the ice bath, the mixture was stirred at room temperature for 16 h. Under ice bath conditions, ice water was added and stirred for 10 min to quench the reaction. Methyl tert-butyl ether was added, and the pH value was adjusted to 3 with concentrated hydrochloric acid. Extraction was carried out twice. The organic layer was separated and an aqueous NaOH solution was added to the organic layer to adjust the pH value to 12. The aqueous layer was extracted and washed twice with n-butanol, and then the pH value was adjusted to 3 with concentrated hydrochloric acid. A white precipitate was precipitated by stirring, filtered, and dried in vacuo to obtain 653.0 mg of a white solid with a yield of 70.4%. 1 HNMR (400 MHz, DMSO-d6) δ 7.96 (s, 2H), 7.71 (d, J = 6.6 Hz, 2H), 7.21 (d, J = 6.5 Hz, 2H), 7.05 (d, J = 7.7 Hz, 1H), 4.12 (s, 1H), 3.03 (d, J = 12.4 Hz, 1H), 2.94 - 2.73 (m, 1H), 1.32 (s, 9H); 13 CNMR (101 MHz, DMSO-d6) δ 174.09, 155.91, 140.39, 134.51, 132.34, 128.62, 78.56, 55.50, 36.96, 28.61.

[0067] Example 2. Preparation of H-BPA-OMe hydrochloride

[0068] Synthesized in two steps.

[0069] 2.1. Preparation of N-Boc-BPA-OMe: Weigh 2 g (6.5 mmol) of N-Boc-BPA-OH into a 25 mL round-bottom flask, add 10 mL of DMF to dissolve it. At room temperature, add 1.3 g of KHCO3 and stir for half an hour. Then add 0.8 mL of methyl iodide and stir at room temperature for 24 h. After the reaction is completed, add 20 mL of ethyl acetate. The organic layer was washed successively with saturated brine (3 × 10 mL), 1 M hydrochloric acid (3 × 10 mL), and saturated sodium bicarbonate (3 × 10 mL). The organic phases were combined, concentrated, and purified by column chromatography (DCM / MeOH = 30 / 1, R f = 0.36), and dried in vacuo to obtain 1.786 g of a white solid with a yield of 85.4%. 11H NMR (400 MHz, CDCl3) δ 7.93 (s, 2H), 7.52 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 5.04 (d, J = 7.4 Hz, 1H), 4.64 (d, J = 7.0 Hz, 1H), 3.75 (s, 3H), 3.14 (ddd, J = 31.9, 13.8, 5.9 Hz, 2H), 1.44 (s, 9H); 13 13C NMR (101 MHz, CDCl3) δ 169.45, 161.86, 136.90, 136.44, 135.51, 128.58, 84.08, 54.27, 53.27, 36.33, 24.71.

[0070] 2.2. Preparation of H-BPA-OMe hydrochloride: Weigh 0.97 g (3.0 mmol) of N-Boc-BPA-OMe into a 25 mL round-bottom flask, add 4 mL of 4 M hydrochloric acid methanol solution, stir at room temperature for 4 h, concentrate under reduced pressure, and dry in vacuo to obtain 0.762 g of a white solid with a yield of 97.9%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.84 (br, 2H), 8.04 (s, 2H), 7.77 (d, J = 7.5 Hz, 2H), 7.20 (d, J = 7.6 Hz, 2H), 4.34 - 4.07 (s, 1H), 3.59 (s, 3H), 3.32 - 2.97 (m, 2H); 13 13C NMR (101 MHz, DMSO-d6) δ 169.64, 137.00, 134.96, 133.25, 128.92, 105.12, 53.77, 53.03, 36.27.

[0071] Example 3. Preparation of Z-GP-OH

[0072] Synthesized in two steps.

[0073] 3.1 Preparation of Z-GP-OMe: Weigh 0.497 g (3.0 mmol) of L-proline methyl ester hydrochloride and 0.628 g (3.0 mmol) of N-carbobenzoxy glycine into a 50 ml reaction flask, add 20 mL of acetonitrile and stir to dissolve. At room temperature, add 0.739 g (9.0 mmol) of NMI and 287.6 mg (3.0 mmol) of TCFH, and stir the reaction at room temperature for 4 h. After monitoring the reaction by TLC until it is completed, remove the solvent by rotary evaporation. After adding 20 mL of DCM, wash the organic layer successively with distilled water (3×10 mL), 1 M hydrochloric acid (3×10 mL), and saturated sodium bicarbonate (3×10 mL). Combine the organic phases, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify the residue by column chromatography (DCM / MeOH = 20 / 1, R f = 0.28) to obtain 902.0 mg of a colorless oily liquid with a yield of 93.6%. 1 1H NMR (400 MHz, CDCl3) δ 7.27 (m, 5H), 5.87 (s, 1H), 5.03 (s, 2H), 4.42 (d, J = 7.6 Hz, 1H), 3.92 (dd, J = 10.1, 3.8 Hz, 2H), 3.62 (s, 1H), 3.55 - 3.27 (m, 2H), 2.07 (m, 1H), 1.91 (m, 3H); 13 13C NMR (101 MHz, CDCl3) δ 172.36, 167.19, 156.35, 136.54, 128.41, 127.95, 127.87, 66.67, 58.84, 52.24, 45.83, 43.26, 28.91, 24.57.

[0074] 3.2 Preparation of Z-GP-OH: Dissolve 0.96 g (3.0 mmol) of Z-GP-OMe in 10 ml of THF, add 10 ml of 1 M NaOH solution, and stir at room temperature for 12 hours. Monitor the reaction by TLC until the substrate reacts completely. Add 1 M HCl solution to adjust the pH value to 2, concentrate under reduced pressure to remove THF, precipitate a white solid, filter, and purify by column chromatography (DCM / MeOH = 10 / 1, R f = 0.31). Dry in vacuo to obtain 0.823 g of a white solid with a yield of 95.4%. 1 1H NMR (300 MHz, DMSO-d6) δ 12.64 (s, 1H), 7.37 (m, 5H), 5.05 (s, 2H), 4.25 (dd, J = 8.7, 3.2 Hz, 1H), 3.84 (ddd, J = 33.3, 17.2, 6.0 Hz, 2H), 3.60 - 3.28 (m, 2H), 2.13 (m, 1H), 1.91 (m, 3H); 1313C NMR (75 MHz, DMSO-d6) δ 173.82, 167.61, 156.94, 137.59, 128.87, 128.24, 128.14, 65.97, 59.06, 45.90, 43.08, 29.07, 24.83.

[0075] Example 4. Preparation of BPA oligomers [(BPA)2 to (BPA)4]

[0076] 4.1. Preparation of BPA dimer [(BPA)2]

[0077] Synthesized in 3 steps.

[0078] 4.1.1. Preparation of N-Boc-(BPA)2-OMe: Weigh 0.831 g (3.0 mmol) of H-BPA-OMe hydrochloride and 0.99 g (3.0 mmol) of N-Boc-BPA-OH into a 50 ml reaction flask, add 15 mL of dry redistilled DCM and 1.5 mL of DMF to dissolve, place it in an ice bath, slowly add 1.255 g (3.3 mmol) of HATU, add 4.5 ml of DIPEA (9.0 mmol) after 5 min, stir for 30 mim and then transfer to room temperature and stir for 12 h. After monitoring the reaction by TLC until it is completed, add 10 mL of DCM to the reaction solution, wash the organic layer successively with distilled water (3 × 10 mL), 1 M hydrochloric acid (3 × 10 mL), and saturated sodium bicarbonate (3 × 10 mL), combine the organic phases, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography (PE / EA = 2 / 1, V / V, R f = 0.23) with gradient elution to obtain 1.279 g of white solid, with a yield of 82.9%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.31 (d, J = 7.2 Hz, 1H), 7.96 (s, 2H), 7.93 (s, 2H), 7.70 (dd, J = 13.5, 7.4 Hz, 4H), 7.20 (d, J = 6.9 Hz, 4H), 6.84 (d, J = 8.5 Hz, 1H), 4.52 (m, 1H), 4.20 (m, 1H), 3.59 (s, 3H), 3.13 - 2.79 (m, 3H), 2.69 (m, 1H), 1.29 (s, 9H); 1313C NMR (101 MHz, DMSO-d6) δ 172.34, 172.25, 155.57, 140.45, 139.26, 134.66 (2), 134.38 (2), 128.69 (2), 128.64 (2), 103.48, 103.23, 78.53, 60.23, 53.92, 52.32, 37.88, 37.27, 28.59 (3).

[0079] 4.1.2. Preparation of H-(BPA)2-OMe hydrochloride: Weigh 1.028 g (2.0 mmol) of N-Boc-(BPA)2-OMe into a 25 mL round-bottom flask, add 3 mL of 4 M hydrochloric acid methanol solution, stir at room temperature for 4 h, concentrate under reduced pressure, and purify by column chromatography (DCM / MeOH = 5 / 1, V / V, R f = 0.11) and dry under vacuum to obtain 0.893 g of a white solid with a yield of 99.1%. 1 1H NMR (400 MHz, DMSO-d6) δ 9.22 (br, 2H), 8.33 (m, 1H), 8.14 (s, 4H), 7.72 (m, 4H), 7.23 (dd, J = 11.8, 7.0 Hz, 4H), 4.54 (m, 1H), 4.06 (m, 1H), 3.59 (s, 3H), 3.15 (m, 1H), 3.00 (m, 3H); 13 13C NMR (101 MHz, DMSO-d6) δ 171.62, 168.63, 139.10, 137.06, 134.79 (2), 134.73 (2), 129.14 (2), 128.66 (2), 103.96, 103.82, 54.32, 53.51, 52.54, 37.26, 37.17.

[0080] 4.1.3. Preparation of BPA dimer [(BPA)2]: Weigh 0.45 g (1.0 mmol) of H-(BPA)2-OMe hydrochloride, add 6 ml of 1 M NaOH solution, stir at room temperature for 1 hour, monitor the hydrolysis of the substrate by TLC, add 1 M HCl solution to adjust the pH to 6, precipitate a white solid, filter, wash, and dry under vacuum to obtain 0.342 g of a white solid with a yield of 85.5%. 1 1H NMR (400 MHz, CD3OD) δ 8.92 (br, 2H), 8.31 (m, 1H), 7.76 (m, 4H), 7.69 (dd, J = 20.6, 7.3 Hz, 4H), 7.22 (t, J = 7.8 Hz, 4H), 4.50 (m, 1H), 3.71 (m, 1H), 3.22 (m, 1H), 3.08 (m, 2H), 2.74 (m, 1H);13 CNMR(101MHz,CD3OD)δ178.48,176.53,140.47,139.03,134.14(2),133.70(2),128.65(2),128.50(2),111.12,107.52,55.84,55.69,48.64,39.77; TOF-MS ES(m / z): calcd. for C 18 H 21 10 B2N2O7([M-H] - )397.1613, found: 397.1615. All data confirmed that the compound was BPA dimer [(BPA)2], and its structure was as follows:

[0081]

[0082] 4.2. Preparation of BPA trimer [(BPA)3]

[0083] Synthesized in three steps.

[0084] 4.2.1. Preparation of N-Boc-(BPA)3-OMe: Replace 0.831 g (3.0 mmol) of H-BPA-OMe hydrochloride with 1.344 g (3.0 mmol) of H-(BPA)2-OMe hydrochloride, and prepare according to the method in step 4.1.1. 1.753 g of white solid was obtained, and the yield was 83.2%. 1 H NMR(400MHz,DMSO-d6)δ8.33(m,2H),7.98(s,2H),7.94(m,4H),7.73(dd,J = 13.5,7.4Hz,6H),7.23(d,J = 6.9Hz,6H),6.85(m,1H),4.55(m,2H),4.23(m,1H),3.61(s,3H),3.15 - 2.81(m,4H),2.72(m,2H),1.31(s,9H); 13 C NMR(101MHz,DMSO-d6)δ172.35,172.27,172.23,155.57,140.45,140.31,139.27,134.71(3),134.42(3),128.65(3),128.62(3),103.51,103.35,103.27,78.56,60.21,59.16,56.75,52.18,37.79,37.56,36.92,28.57(3).

[0085] Preparation of 4.2.2.H-(BPA)3-OMe hydrochloride: Weigh 1.405 g (2.0 mmol) of N-Boc-(BPA)3-OMe and prepare it according to the method in step 4.1.2. 1.20 g of white solid was obtained with a yield of 99.3%. 1 H NMR (400 MHz, DMSO-d6) δ 9.22 (br, 2H), 8.31 (m, 2H), 8.11 (m, 6H), 7.73 (m, 6H), 7.25 (m, 6H), 4.85 (m, 1H), 4.57 (m, 1H), 4.06 (m, 1H), 3.63 (s, 3H), 3.19 (m, 2H), 3.03 (m, 4H); 13 C NMR (101 MHz, DMSO-d6) δ 171.62, 170.93, 169.12, 139.11, 138.63, 137.11, 134.76(3), 134.75(3), 129.15(3), 128.67(3), 103.96, 103.82, 103.53, 55.23, 54.35, 53.56, 52.52, 38.65, 37.25, 37.18.

[0086] Preparation of 4.2.3. BPA trimer [(BPA)3]: Weigh 0.64 g (1.0 mmol) of H-(BPA)3-OMe hydrochloride and prepare it according to the method in step 4.1.3. 0.50 g of white solid was obtained with a yield of 84.6%. 1 H NMR (400 MHz, CD3OD) δ 8.93 (br, 2H), 8.34 (m, 2H), 7.75 (m, 6H), 7.69 (m, 6H), 7.22 (m, 6H), 4.91 (m, 1H), 4.72 (m, 1H), 3.87 (m, 1H), 3.23 (m, 2H), 3.11 (m, 2H), 2.76 (m, 2H); 13 C NMR (101 MHz, CD3OD) δ 178.45, 176.51, 176.37, 140.52, 140.34, 139.15, 134.15(3), 133.72(3), 128.64(3), 128.51(3), 111.15, 107.53, 107.45, 57.32, 55.84, 55.72, 48.65, 39.82, 39.36; TOF-MS ES (m / z): calcd. for C 27 H 31 10 B3N3O 10 ([M - H] -)587.2403, found: 587.2405. All data confirmed that this compound is BPA trimer [(BPA)3], and its structure is as follows:

[0087]

[0088] 4.3. Preparation of BPA tetramer [(BPA)4]

[0089] Synthesized in 3 steps.

[0090] 4.3.1. Preparation of N-Boc-(BPA)4-OMe: Replace 0.831 g (3.0 mmol) of H-BPA-OMe hydrochloride with 1.873 g (3.0 mmol) of H-(BPA)3-OMe hydrochloride, and prepare according to the method in step 4.1.1. Obtained 2.184 g of white solid, with a yield of 81.6%. 1 H NMR (400 MHz, DMSO-d6) δ 8.31 (m, 3H), 7.95 (m, 4H), 7.93 (m, 4H), 7.76 (m, 8H), 7.25 (m, 8H), 6.93 (m, 1H), 4.63 (m, 3H), 4.27 (m, 1H), 3.63 (s, 3H), 3.17 - 2.83 (m, 5H), 2.73 (m, 3H), 1.31 (s, 9H); 13 C NMR (101 MHz, DMSO-d6) δ 172.46, 172.31, 172.27, 171.93, 155.59, 140.47, 140.33, 139.44, 139.27, 134.68(4), 134.43(4), 128.67(4), 128.65(4), 103.52, 103.36(2), 103.25, 78.58, 60.13, 59.17, 58.67, 56.74, 52.16, 37.83, 37.56, 37.33, 36.94, 28.55(3).

[0091] 4.3.2. Preparation of H-(BPA)4-OMe hydrochloride: Weigh 1.785 g (2.0 mmol) of N-Boc-(BPA)4-OMe and prepare according to the method in step 4.1.2. Obtained 1.562 g of white solid, with a yield of 98.6%. 11H NMR (400 MHz, DMSO-d6) δ 9.18 (br, 2H), 8.34 (m, 3H), 8.05 (m, 8H), 7.75 (m, 8H), 7.21 (m, 8H), 4.87 (m, 1H), 4.83 (m, 1H), 4.56 (m, 1H), 4.02 (m, 1H), 3.65 (s, 3H), 3.23 (m, 3H), 3.05 (m, 5H); 13 13C NMR (101 MHz, DMSO-d6) δ 171.73, 171.61, 170.95, 169.21, 139.13, 138.65, 137.11, 136.58, 134.69 (4), 134.73 (4), 129.12 (4), 128.65 (4), 103.94, 103.78, 103.68, 103.46, 55.67, 55.34, 54.32, 53.54, 52.31, 38.72, 37.37, 37.25, 36.83.

[0092] 4.3.3. Preparation of BPA tetramer [(BPA)4]: Weigh 0.83 g (1.0 mmol) of H-(BPA)4-OMe hydrochloride and prepare it according to the method in step 4.1.3. 663.9 mg of white solid was obtained with a yield of 85.3%. 1 1H NMR (400 MHz, CD3OD) δ 8.89 (br, 2H), 8.36 (m, 3H), 7.79 (m, 8H), 7.65 (m, 8H), 7.19 (m, 8H), 4.94 (m, 1H), 4.92 (m, 1H), 4.74 (m, 1H), 3.93 (m, 1H), 3.25 (m, 3H), 3.13 (m, 2H), 2.75 (m, 3H); 13 13C NMR (101 MHz, CD3OD) δ 178.52, 176.51, 176.37, 176.15, 140.61, 140.26, 139.54, 139.21, 134.17 (4), 133.75 (4), 128.62 (4), 128.53 (4), 110.85, 109.32, 106.54, 106.18, 58.21, 57.36, 55.78, 55.66, 48.33, 39.73, 39.28, 36.76; TOF-MS ES (m / z): calcd. for C 36 H 41 10 B4N4O 13 ([M - H] -)777.3193, found: 777.3187. All data confirmed that the compound is BPA tetramer [(BPA)4], and its structure is as follows:

[0093]

[0094] Example 5. Preparation of BPA oligomer targeting compounds [Z-GP-(BPA)2~Z-GP-(BPA)4]

[0095] 5.1. Preparation of BPA dimer targeting compound [Z-GP-(BPA)2]

[0096] Synthesized in 2 steps.

[0097] 5.1.1. Preparation of Z-GP-(BPA)2-OMe: Weigh 0.613 g (2.0 mmol) of compound Z-GP-OH into a 50 mL reaction flask, add 10 mL of redistilled DCM and 1 mL of DMF to dissolve it, place it in an ice bath, slowly add 0.76 g (2.2 mmol) of HATU, add 3 ml of DIPEA (6.0 mmol) after 5 min, add 0.9 g (2.0 mmol) of H-(BPA)2-OMe hydrochloride after 5 min, stir for 30 mim and then transfer it to room temperature and stir for 12 h. After monitoring the reaction by TLC until it is completed, add 10 mL of DCM to the reaction solution, wash the organic layer successively with distilled water (3×10 mL), 1 M hydrochloric acid (3×10 mL), and saturated sodium bicarbonate (3×10 mL), combine the organic phases, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography (DCM / MeOH = 20 / 1, V / V, R f = 0.23) with gradient elution to obtain 1.163 g of white solid, and the yield is 83.0%. 1 H NMR (400 MHz, DMSO-d6) δ 8.22 (m, 2H), 7.95 (m, 1H), 7.92 (s, 4H), 7.77 - 7.63 (m, 4H), 7.33 (m, 5H), 7.18 (m, 4H), 5.03 (s, 2H), 4.90 (m, 1H), 4.51 (m, 1H), 4.43 (m, 1H), 3.85 (m, 1H), 3.53 (s, 3H), 3.45 (m, 2H), 3.18 (m, 2H), 3.00 (m, 3H), 2.78 (m, 1H), 1.62 (m, 2H); 1313C NMR (101 MHz, DMSO-d6) δ 172.11, 171.54, 171.43, 168.43, 156.95, 139.32, 137.57, 137.42, 134.62 (2), 134.38 (2), 128.78, 128.68, 128.59 (2), 128.50 (2), 128.24, 128.15, 128.11, 105.24, 104.92, 65.96, 60.22, 56.81, 56.73, 54.29, 53.85, 52.19, 43.29, 37.24, 36.85, 29.17, 24.46。

[0098] 5.1.2. Preparation of Z-GP-(BPA)2: Weigh 0.70 g (1.0 mmol) of Z-GP-(BPA)2-OMe, add 6 ml of 1 mol / L NaOH solution, stir at room temperature for 1 hour. Monitor the hydrolysis of the substrate by TLC. After the substrate is completely hydrolyzed, add 1 mol / L HCl solution to adjust the pH value to 2. A precipitate will form. Filter it and purify it by column chromatography (DCM / MeOH = 9 / 1, V / V, R f = 0.21), and then dry it under vacuum to obtain 0.523 g of white solid with a yield of 75.9%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.36 (m, 2H), 7.96 (m, 1H), 7.93 (s, 4H), 7.67 (d, J = 7.4 Hz, 4H), 7.33 (m, 5H), 7.17 (d, J = 7.5 Hz, 4H), 5.02 (s, 2H), 4.93 (m, 1H), 4.74 (m, 1H), 4.45 (m, 1H), 3.84 (s, 2H), 3.47 (m, 2H), 3.19 (m, 2H), 2.96 (m, 3H), 2.78 (m, 1H), 1.62 (m, 2H); 13 13C NMR (101 MHz, DMSO-d6) δ 172.17, 171.48, 171.32, 168.57, 156.93, 139.35, 137.54, 137.52, 134.71 (2), 134.42 (2), 128.82, 128.73, 128.61 (2), 128.53 (2), 128.22, 128.16, 128.13, 105.25, 104.93, 65.97, 60.23, 56.84, 56.75, 54.32, 53.86, 43.33, 37.37, 36.83, 29.22, 24.51; TOF-MS ES-(m / z): calcd. for C 33 H 37 10 B2N4O11 ([M-H] - )685.2723, found: 685.2726. All data confirm that this compound is a BPA dimer targeting compound [Z-GP-(BPA)2], and its structure is as follows:

[0099]

[0100] 5.2. Preparation of BPA trimer targeting compound [Z-GP-(BPA)3]

[0101] Synthesized in 2 steps.

[0102] 5.2.1. Preparation of Z-GP-(BPA)3-OMe: Replace 0.9 g (2.0 mmol) of H-(BPA)2-OMe hydrochloride with 1.276 g (2.0 mmol) of H-(BPA)3-OMe hydrochloride, and prepare according to the method in step 5.1.1. Obtain 1.467 g of white solid, and the yield is 82.4%. 1 H NMR (400 MHz, DMSO-d6) δ 8.24 (m, 3H), 7.95 (m, 1H), 7.91 (s, 6H), 7.78 - 7.65 (m, 6H), 7.33 (m, 5H), 7.19 (m, 6H), 5.05 (s, 2H), 4.91 (m, 2H), 4.53 (m, 1H), 4.41 (m, 1H), 3.87 (m, 1H), 3.61 (s, 3H), 3.47 (m, 2H), 3.19 (m, 3H), 3.05 (m, 3H), 2.83 (m, 2H), 1.62 (m, 2H); 13 C NMR (101 MHz, DMSO-d6) δ 172.13, 171.57, 171.46, 171.34, 168.62, 156.68, 139.37, 138.74, 137.62, 137.53, 134.62(3), 134.38(3), 128.92, 128.75, 128.59(3), 128.50(3), 127.67, 127.26, 127.18, 105.25, 104.93, 104.68, 66.87, 59.36, 58.41, 56.83, 56.72, 54.83, 52.16, 42.41, 37.44(2), 36.87, 29.52, 24.36.

[0103] 5.2.2. Preparation of Z-GP-(BPA)3: Weigh 0.89 g (1.0 mmol) of Z-GP-(BPA)3-OMe, and prepare according to the method in experimental step 5.1.2. Obtain 0.671 g of white solid, and the yield is 76.6%.1 1H NMR (400 MHz, DMSO-d6) δ 8.32 (m, 3H), 7.94 (m, 1H), 7.91 (s, 6H), 7.73 (d, J = 7.3 Hz, 6H), 7.33 (m, 5H), 7.19 (d, J = 7.4 Hz, 6H), 5.04 (s, 2H), 4.92 (m, 2H), 4.73 (m, 1H), 4.45 (m, 1H), 3.84 (s, 2H), 3.49 (m, 2H), 3.21 (m, 3H), 2.95 (m, 3H), 2.81 (m, 2H), 1.63 (m, 2H); 13 13C NMR (101 MHz, DMSO-d6) δ 172.19, 171.52, 171.37, 171.25, 168.64, 156.89, 139.28, 137.43 (2), 137.31, 134.68 (3), 134.35 (3), 128.79, 128.68, 128.57 (3), 128.45 (3), 128.31, 128.22, 128.15, 105.26, 104.92 (2), 66.72, 60.12, 58.76, 58.32, 56.83, 54.84, 43.35, 37.41 (2), 36.58, 29.54, 24.33; TOF-MS ES- (m / z): calcd. for C 42 H 47 10 B3N5O 14 ([M-H] - ) 875.3513, found: 875.3517. All data confirmed that the compound was the BPA trimer targeting compound [Z-GP-(BPA)3], and its structure was as follows:

[0104]

[0105] 5.3. Preparation of BPA tetramer targeting compound [Z-GP-(BPA)3]

[0106] Synthesized in 2 steps.

[0107] 5.3.1. Preparation of Z-GP-(BPA)4-OMe: Replace 0.9 g (2.0 mmol) of H-(BPA)2-OMe hydrochloride with 1.657 g (2.0 mmol) of H-(BPA)4-OMe hydrochloride, and prepare according to the method in step 5.1.1. 1.467 g of white solid was obtained, and the yield was 81.8%. 11H NMR (400 MHz, DMSO-d6) δ 8.32 (m, 4H), 7.97 (m, 1H), 7.93 (s, 8H), 7.79 - 7.67 (m, 8H), 7.32 (m, 5H), 7.21 (m, 8H), 5.06 (s, 2H), 4.92 (m, 3H), 4.56 (m, 1H), 4.42 (m, 1H), 3.84 (m, 2H), 3.65 (s, 3H), 3.43 (m, 2H), 3.16 (m, 4H), 3.03 (m, 3H), 2.85 (m, 3H), 1.64 (m, 2H); 13 13C NMR (101 MHz, DMSO-d6) δ 172.15, 171.61 (2), 171.43, 171.32, 168.91, 156.34, 139.41, 138.72 (2), 137.58, 137.47, 134.61 (4), 134.36 (4), 128.89, 128.72, 128.58 (4), 128.46 (4), 127.65, 127.23, 127.14, 105.24, 104.92, 104.65 (2), 66.86, 59.34, 58.46 (2), 56.81, 56.69, 54.85, 52.18, 42.36, 37.43 (2), 36.85 (2), 29.48, 24.33。

[0108] 5.3.2. Preparation of Z-GP-(BPA)4: Weigh 1.08 g (1.0 mmol) of Z-GP-(BPA)4-OMe and prepare it according to the method in Experimental Procedure 5.1.2. 0.834 g of white solid was obtained, with a yield of 78.2%. 1 1H NMR (400 MHz, DMSO-d6) δ 8.34 (m, 4H), 7.98 (m, 1H), 7.93 (s, 8H), 7.74 (d, J = 7.4 Hz, 8H), 7.33 (m, 5H), 7.21 (d, J = 7.3 Hz, 8H), 5.05 (s, 2H), 4.93 (m, 3H), 4.71 (m, 1H), 4.43 (m, 1H), 3.86 (s, 2H), 3.48 (m, 2H), 3.26 (m, 4H), 2.96 (m, 4H), 2.83 (m, 2H), 1.65 (m, 2H); 1313C NMR(101MHz,DMSO-d6)δ172.21,171.46,171.35(2),171.23,168.55,156.82,139.23,137.47(2),137.36(2),134.64(4),134.32(4),128.81,128.73,128.54(4),128.42(4),128.35,128.23,128.17,105.24(2),104.89(2),66.83,60.21,58.82,58.31(2),56.85,54.86,42.33,37.45(3),36.54,29.52,24.28; TOF-MS ES-(m / z): calcd. for C 51 H 57 10 B4N6O 17 ([M-H] - )1065.4303, found: 1065.4301. All data confirmed that the compound was a BPA tetramer targeting compound [Z-GP-(BPA)4], and its structure was as follows:

[0109]

[0110] Example 6. Cytotoxicity of BPA oligomers and their targeting compounds

[0111] The cell culture medium containing peptide bovine serum was incubated at 37 °C for 24 h. The human normal liver cells LO2 cells in subculture were made into a cell suspension of 1×10 5 cells / mL with tissue culture medium. The cell suspension was inoculated into a 96-well cell culture plate (100 μl / well) and incubated in a 37 °C carbon dioxide incubator for 24 h. After the cells adhered and grew, the supernatant was removed, and the tissue culture medium of the control group (without drug) and the test group (with drug) was added for incubation. The concentration of the drug in the cell culture medium was 500 μmol / L, and the incubation was continued in a 37 °C carbon dioxide incubator. After 2 days of incubation, it was taken out. MTT solution was added and incubation continued for 4 h. The cell incubation solution was removed, and then DMSO was added and shaken for 10 min. Finally, the absorbance value was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm, and the relative growth rate (RGR) of the cells was calculated according to the absorbance using the formula. The formula for calculating the relative growth rate of the cells is:

[0112]

[0113] The results are shown in Table 1. It can be seen that after incubating the series of BPA oligomers and their targeted compounds with human normal liver cells LO2 for 2 days, their RGRs are all close to 100%, indicating that these compounds have no toxicity or very low toxicity to human normal liver cells at a concentration of 500 μmol / L.

[0114] Table 1. Relative growth rate (RGR) of cells measured by MTT colorimetric method

[0115]

[0116] Example 7. Boron uptake characteristics of the series of BPA oligomers and their targeted compounds in cells

[0117] The ICP-MS method was used to determine the boron uptake characteristics of the series of BPA oligomers and their targeted compounds in human umbilical vein endothelial cells (HUVEC), human hepatoma cells HepG2, human hepatoma cells HepG2 transfected with FAPα (HepG2 / FAPα), human breast cancer cells MDA-MB-231, and human glioma cells U87.

[0118] Specific process: Add an appropriate amount of RPMI 1640 culture medium (10% fetal bovine serum, 100 U / ml penicillin) to tumor cells in the logarithmic growth phase, adjust the cell concentration to 1×10 5 cells / ml, inoculate into a 6-well culture plate, and inoculate 100 μl of tumor cell suspension into each well. After culturing for 24 h, add the prepared test drug (no drug is added to the control group), make the final concentration of the drug 100 μM, place it in a 5% CO2 incubator, and culture at 37 °C for 24 h. Then, use a pipette gun to remove the culture medium in the culture dish, wash it with PBS (3×1 mL), add 0.5 mL of trypsin to the culture dish, and then place it in a 37 °C constant temperature CO2 incubator for 3 min. During this period, observe under a microscope whether the cells shrink and become round. After the cells shrink and become round, quickly add 0.6 ml of culture medium to terminate the digestion, and gently pipette the adherent cells to make all the cells fall off the bottom of the culture dish. Then, transfer the cell suspension to a centrifuge tube, dilute it, and count the number of cells in each well using a hemocytometer. Centrifuge the above cell suspension, remove the supernatant, and wash it with PBS (3×1 mL). Add 500 μL of concentrated nitric acid to the washed centrifuge tube containing cells for digestion; use the ICP-MS method to measure the boron content in the cells and calculate the T / N ratio. The definition of the T / N ratio is: the amount of boron uptake in tumor cells / the amount of boron carried in HUVEC cells.

[0119] The results are shown in Tables 2 to 5. It can be seen that among the four strains of tumor cells investigated, the boron-carrying amounts of all BPA oligomers and their targeted compounds are greater than 10 9Individual B / cells with a T / N ratio greater than 3.0 all meet the prerequisite conditions as boron delivery agents. All BPA oligomers and their targeting compounds exhibited boron uptake characteristics superior to those of the positive control drug BPA; among tumor cells without FAPα transfection, BAP oligomers all showed boron uptake characteristics superior to those of the corresponding targeting compounds; however, in FAPα-transfected HepG-2 cells, BPA oligomer targeting compounds all showed higher boron loading and T / N ratios than the corresponding BPA oligomers.

[0120] Table 2. Boron uptake characteristics of a series of BPA oligomers and their targeting compounds in human hepatocellular carcinoma HepG2 cells

[0121]

[0122] a The T / N ratio is defined as: boron uptake amount of human hepatocellular carcinoma cells HepG2 / boron uptake amount of human umbilical vein endothelial cells HUVEC

[0123] Table 3. Boron uptake characteristics of a series of BPA oligomers and their targeting compounds in FAPα-transfected human hepatocellular carcinoma HepG2 cells

[0124]

[0125] a The T / N ratio is defined as: boron uptake amount of FAPα-transfected human hepatocellular carcinoma cells HepG2 / boron uptake amount of human umbilical vein endothelial cells HUVEC

[0126] Table 4. Boron uptake characteristics of a series of BPA oligomers and their targeting compounds in human breast cancer cells MDA-MB-231

[0127]

[0128] a The T / N ratio is defined as: boron uptake amount of human breast cancer cells MDA-MB-231 / boron uptake amount of human umbilical vein endothelial cells HUVEC

[0129] Table 5. Boron uptake characteristics of a series of BPA oligomers and their targeting compounds in human glioma cells U87

[0130]

[0131] a The T / N ratio is defined as: boron uptake amount of human glioma cells U87 / boron uptake amount of human umbilical vein endothelial cells HUVEC

[0132] Example 8. Lipophilic and hydrophilic characteristics of a series of BPA oligomers and their targeting compounds

[0133] Experimental method: a) Establish the detection standard curve for each sample to be tested. Dissolve the series of BPA oligomers and their target compounds in the enzymatic hydrolysis buffer (50 mM Tris-HCl, 1.0 M NaCl, pH 7.4), and set 5 concentration gradients of 0.012725, 0.006363, 0.003181, 0.001591, and 0.000795 μg / ml respectively. Plot the standard curve with the peak area as the ordinate and the drug concentration as the abscissa. This experiment is repeated 3 times; b) Prepare n-octanol saturated with aqueous solution and aqueous solution saturated with n-octanol. After mixing n-octanol and water in equal proportions, stir for 24 h to make the two phases saturate each other. After standing and separating the layers, separate the two phases and store them for later use; c) Weigh 10 mg of the sample to be tested precisely, place it in a 10 mL volumetric flask, add n-octanol saturated with aqueous solution to dissolve the sample and dilute it to the scale. Precisely pipette 8 mL from it into a 25 mL volumetric flask, and add 8 mL of aqueous solution saturated with n-octanol. After shaking the 25 mL volumetric flask for 3 h, stand and separate the aqueous phase and the oil phase. Filter through a 0.22 μm filter membrane, inject 10 μl each time, and use HPLC to measure the peak area. Substitute it into the standard curve equation to calculate the content of the sample to be tested in each phase. The calculation formula for the oil-water distribution coefficient:

[0134]

[0135] Experimental results: The results are shown in Table 6. It can be seen that the LogP value of the positive control drug BPA is -1.72 ± 0.12, which is less than 1, indicating that BPA has high solubility in water, low passive diffusion and penetration, poor absorption and brain permeability, and high renal clearance. The LogP values of the compounds of the present invention all fall between 1 and 3, indicating that such compounds have good lipophilic and hydrophilic characteristics, better permeability than BPA, increased binding to metabolic enzymes, and higher metabolic degree.

[0136] Table 6. Lipophilic and hydrophilic coefficients (LogP) of the series of BPA oligomers and their target compounds

[0137]

[0138] Example 9. Experiment on the targeted release characteristics of the BPA oligomer target compound

[0139] Experimental method: The HPLC chromatographic conditions are as follows: Agilent 1200 high-performance liquid chromatograph; Cosmosil C18 reverse-phase chromatographic column (4.6×250 mm, 5 μm); mobile phase (at 0 min, 55% methanol and 45% water (containing 2 mM ammonium formate); at 10 min, 65% methanol and 35% water (containing 2 mM ammonium formate); at 15 min, 75% methanol and 25% water (containing 2 mM ammonium formate); at 30 min, 85% methanol and 15% water (containing 2 mM ammonium formate); at 40 min, 85% methanol and 15% water (containing 2 mM ammonium formate); flow rate 1 mL / min; detection wavelength: 254 nm; injection volume 2 μL.

[0140] Establish the Z-GP-(BPA) n (n = 2, 3, 4) The method for detecting the standard curve is as follows: Dissolve Z-GP-(BPA)2, Z-GP-(BPA)3 and Z-GP-(BPA)4 in the enzymatic hydrolysis buffer (50 mM Tris-HCl, 1.0 M NaCl, pH 7.4) respectively. Set 5 concentration gradients, which are 0.012725, 0.006363, 0.003181, 0.001591, 0.000795 μg / ml respectively. Plot the standard curve with the peak area as the ordinate and the drug concentration as the abscissa. This experiment is repeated 3 times. Incubate the Z-GP-(BPA) n (n = 2, 3, 4) in the buffer containing 5 μg / ml and 10 μg / ml of rhFΑPα respectively. The reaction temperature is 37°C. At the time points of 0, 4, 8, 12, 16, 24 h of incubation, take the supernatant and detect the free diarylhydrazine by HPLC method.

[0141] Calculate the enzymatic hydrolysis rate according to the following formula:

[0142]

[0143] Among them, [S]0 is the initial concentration of the substrate, and [S] t represents the concentration of the substrate at time t.

[0144] Experimental results: The results of the enzymatic hydrolysis experiment show that FΑPα can catalyze the hydrolysis of Z-GP-(BPA) n (n = 2, 3, 4) to release (BPA) n (n = 2, 3, 4), and its enzymatic hydrolysis rate is positively correlated with the enzyme concentration ( Figures 3 to 5 ). FΑPα is an activating protease secreted by tumor-associated fibroblasts and plays an important role in the occurrence and development of tumors. FΑPα is highly specifically expressed on the surface of tumor-associated fibroblasts in more than 90% of cancer types in the human body. Z-GP-(BPA) n(n = 2, 3, 4) can be hydrolyzed by FAPα, indicating that it can be enzymatically released in tumor tissues to produce (BPA). n (n = 2, 3, 4).

[0145] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are taken as the protection scope.

Claims

1. A BPA oligomer or its targeting compound, characterized in that: The structure of the BPA oligomer is shown in Formula I, and the structure of the targeting compound is shown in Formula II; the structures of Formula I and Formula II are as follows: Among them, BPA is (S)-2-amino-3-[4-( 10 B)dihydroxyboranylphenyl]propionic acid; n = 1, 2; Z-GP is benzyloxycarbonylglycylprolyl group, and its structure is as follows:

2. The preparation method of the BPA oligomer according to claim 1, characterized in that, It includes the following steps: (1) Preparation of N-Boc-BPA-OH: Weigh N-tert-butoxycarbonyl-4-iodo-L-phenylalanine into a reaction flask. At room temperature, successively add tributyl borate, i.e., 10 B(O n (Bu)3, NaH, and bis(2-dimethylaminoethyl) ether. Place the reaction flask in an ice bath; Under nitrogen protection, a metal-organic reagent was slowly added. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 16 h. Under ice bath conditions, ice water was added and stirred for 10 min to quench the reaction. Methyl tert-butyl ether was added, and the pH value was adjusted to 3 with concentrated hydrochloric acid. Extraction was performed twice with ethyl acetate. The organic solvent was removed by concentration under reduced pressure. Water was added to the concentrate, and the pH value was adjusted to 12 with 1 mol / L aqueous NaOH solution. The aqueous layer was washed twice with n-butanol, and then the pH value was adjusted to 3 with concentrated hydrochloric acid. A white precipitate was precipitated by stirring, washed by pulping with dichloromethane, filtered and dried to obtain the product; The synthesis route is as follows: (2) Preparation of N-Boc-BPA-OMe: Weigh N-Boc-BPA-OH and dissolve it in DMF. An inorganic base was added at room temperature, and methyl iodide was added dropwise after stirring for 30 min. After the addition was complete, the reaction was continued by stirring for 24 hours. After the reaction ended, water was added, and extraction was performed with ethyl acetate. The organic layers were combined and washed with saturated sodium bicarbonate and saturated brine respectively, dried over anhydrous Na2SO4, and the organic solvent was removed by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the target product; The synthesis route is as follows: (3) Preparation of H-BPA-OMe: Weigh N-Boc-BPA-OMe, add an acidic reagent, and stir the reaction at room temperature for 3 - 5 hours. After the reaction ended, the solvent was removed by rotary evaporation, slurried with petroleum ether, and dried under vacuum to obtain the product; The synthesis steps are as follows: (4)N-Boc-(BPA) n Preparation of -OMe: Weigh N-Boc-BPA-OH, condensing agent and organic base, dissolve them in an appropriate amount of organic solvent. After stirring for 30 min, add H-(BPA) m -OMe in batches. After the feeding is completed, stir and react at room temperature for 12 hours. After the reaction is completed, add an organic solvent. The organic phase is washed with saturated sodium bicarbonate, water, saturated brine, dried over anhydrous Na2SO4, and the organic solvent is removed by rotary evaporation. The residue is purified by silica gel column chromatography to obtain the target product; The synthesis route is as follows: (5)H-(BPA) n Preparation of -OMe: Weigh the compound N-Boc-(BPA) n -OMe, add an acidic reagent, stir and react at room temperature for 3 - 5 hours. After the reaction is completed, remove the solvent by rotary evaporation, add petroleum ether for pulping, and obtain the product after vacuum drying; The synthesis route is as follows: In steps (4) - (5), n = 2, 3, 4; m = 1, 2, 3; (6) Preparation of BPA oligomers, namely (BPA)3 to (BPA)4: Weigh the compound H-(BPA) n -OMe, add a basic reagent, stir at room temperature for 1 to 4 hours, monitor by TLC until the hydrolysis is complete, add 1M HCl solution to adjust the pH value to 6, precipitate a white solid, filter, wash, and dry under vacuum to obtain the target product; The synthesis route is as follows: In step (6), n = 3, 4; m = 2, 3.

3. The preparation method of the BPA oligomer according to claim 2, wherein: In step (1), the molar ratio of N-Boc-4-iodo-L-phenylalanine, tributyl borate, i.e., 10 B(O n Bu)3, NaH, bis(2-dimethylaminoethyl) ether, and the organometallic reagent is 1:1.0 to 5.0, 1:1.0 to 3.0, 1:1.0 to 10.0, 1:1.0 to 10.0, respectively; the organometallic reagent is any one of isopropylmagnesium chloride and isopropylmagnesium chloride lithium chloride; In step (2), the molar ratios of N-Boc-BPA-OH to the inorganic base and methyl iodide in terms of feeding amount are 1:1.0 - 5.0 and 1:1.0 - 5.0 respectively; the inorganic base is any one of potassium bicarbonate, sodium bicarbonate, and lithium bicarbonate; In step (3), the molar ratio of N-Boc-BPA-OMe to the acidic reagent in terms of feeding amount is 1:4.0 - 10.0; the acidic reagent is any one of trifluoroacetic acid and 4 mol / L HCl methanol solution; In step (4), the molar ratios of N-Boc-BPA-OH to the condensing agent, organic base, and H-(BPA) m -OMe are 1:1.1 to 1.5, 1:2.0 to 5.0, and 1.1 to 1.5:1.0 respectively, where m = 1, 2, 3 in H-(BPA) m -OMe; the condensing agent is any one of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, ethyl chloroformate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, and tetramethylchlorouronium hexafluorophosphate; the organic base is any one of N,N-diisopropylethylamine and N-methylimidazole; In step (5), the molar ratio of the compound N-Boc-(BPA) n -OMe to the acidic reagent is 1.0:5.0 to 10.0, and in N-Boc-(BPA) n -OMe, n = 2, 3, or 4; the acidic reagent is any one of trifluoroacetic acid and 4 mol / L HCl methanol solution; In step (6), the molar ratio of the feed of compound H-(BPA) n -OMe to the basic reagent is 1.0:5.0 to 15.0, and in H-(BPA) n -OMe, n = 3, 4; the basic reagent is any one of aqueous solutions of NaOH, KOH, and LiOH.

4. The preparation method of the targeting compound of the BPA oligomer according to claim 1, characterized in that, It includes the following steps: (1) Preparation of N-benzyloxycarbonylglycylproline methyl ester, i.e., Z-GP-OMe: Replace N-Boc-BPA-OH with N-Z-Gly-OH, and H-(BPA) m -OMe with H-Pro-OMe, and prepare according to the method described in step (4) of claim 2; The synthesis route is as follows: (2) Preparation of N-carbobenzoxy-glycyl-proline, i.e., Z-GP-OH: The hydrolysis substrate was replaced with N-carbobenzoxy-glycyl-proline methyl ester, i.e., Z-GP-OMe, and it was prepared according to the method described in step (6) of claim 2; the synthesis route is as follows: (3) Z-GP-BPA oligomer methyl ester, namely Z-GP-(BPA) n -OMe preparation: Replace N-Boc-BPA-OH with Z-GP-OH and prepare according to the method described in step (4) of claim 2; The synthetic route is as follows: (4)Targeting compound of BPA oligomer, namely Z-GP-(BPA) n Preparation of -OH: Replace the hydrolysis substrate with Z-GP-BPA oligomer methyl ester, namely Z-GP-(BPA) n -OMe, prepared according to the method described in step (6) of claim 2; The synthetic route is as follows: In steps (3) - (4), n = 3, 4.

5. Use of the BPA oligomer or its targeting compound according to claim 1 in the preparation of a boron delivery agent for use in boron neutron capture therapy for treating tumors.

6. A boron delivery agent applied in boron neutron capture therapy for treating tumors, characterized in that, It includes the BPA oligomer or its targeting compound according to claim 1.

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