PEGylated boron tryptophan derivative, preparation method thereof, pharmaceutical composition containing PEGylated boron tryptophan derivative and application of pharmaceutical composition

Abstract

The invention provides a pegylated boron tryptophan derivative and a preparation method thereof. Pharmaceutical compositions comprising pegylated boron tryptophan derivatives and their use for the treatment of tumors and for positron tomography scanning to assess the pharmacokinetics of boron neutron capture therapy of tumors and to quantify to calculate the drug concentration of the pharmacokinetics. The pegylated boron tryptophan derivative and the pharmaceutical composition containing the pegylated boron tryptophan derivative disclosed by the invention pass various efficacy experiments; the effects of treating tumors and evaluating pharmacokinetics of boron neutron capture therapy of the tumors through positron tomography scanning and quantitatively calculating the drug concentration of the pharmacokinetics are achieved.

Description

Polyethylene glycol boron tryptophan derivative and preparation method thereof, pharmaceutical composition containing the polyethylene glycol boron tryptophan derivative and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on U.S. patent application No. 63 / 619,755, filed on January 11, 2024, and the entire contents disclosed in the corresponding U.S. patent application file are incorporated as part of this specification. Technical Field

[0003] The present invention relates to a pegylated boron tryptophan derivative, a pharmaceutical composition comprising the pegylated boron tryptophan derivative, and its use in treating tumors and in positron emission tomography scanning to evaluate the pharmacokinetics of boron neutron capture therapy of tumors and to quantify the drug concentration for calculating the pharmacokinetics. Background Art

[0004] As the most malignant primary central nervous system tumor, glioblastoma (GBM) often carries a poor prognosis. In the United States, it is projected that by 2022, 40,594 children and adolescents aged 0-19 will be diagnosed with primary brain and other central nervous system tumors, nearly equivalent to the 40,738 cases of leukemia diagnosed in children and adolescents aged 0-19. Furthermore, between 2014 and 2018, 2,693 children and adolescents aged 0-19 died from malignant brain and other central nervous system tumors, demonstrating the incurability of these diseases. This equates to an average annual mortality rate of 0.66 per 100,000 population, or an average of 539 deaths annually from malignant brain and other central nervous system tumors.

[0005] GBM is currently treated with surgery, radiation therapy, and temozolomide (TMZ) chemotherapy as standard treatment. However, the median overall survival is only 14.6 months. Overall, the 5-year survival rate is only 6.8%. This extremely low survival rate may be due to the inability to completely resect the invasive growth. Other factors include drug resistance or radiation insensitivity.

[0006] In contrast to drugs targeting sites outside the brain, central nervous system drugs must cross the blood-brain barrier (BBB). The BBB is responsible for nutrient transport, homeostasis, and communication between the body and the brain, and it also prevents foreign substances from reaching the brain. Two types of cell junctions, such as intercellular adherens junctions and paracellular tight junctions, block passive diffusion and prevent leakage between endothelial cells.

[0007] Nutrients and small molecules can be transported into and out of the brain through a variety of methods, including passive diffusion, carrier-mediated transport, endocytosis, and active transport. Carrier-mediated transport is driven by two major protein families: the solute carrier (SLC) superfamily and the ATP-binding cassette (ABC) transporters. The primary function of these transporters is to transport essential amino acids and glucose from the blood to the brain. SLC7A5 is a transporter specific for essential amino acids. In the pre-genomic era, it was known as the large protein transporter 1 (LAT1). LAT-1 mediates reverse transport of amino acids. Furthermore, LAT-1 has been found to be overexpressed in brain tumors. Phenylalanine and tryptophan, both essential amino acid nutrients for brain metabolism, have been proposed as carriers for the delivery of boron-10 atoms for targeted boron neutron capture therapy (BNCT). Boron phenylalanine (BPA) is currently under intensive preclinical investigation (see Figure 1).

[0008] Therefore, tryptophan, as an essential amino acid, may be able to cross the BBB via the LAT-1 transporter, thereby achieving nutritional interactions. Increased lipophilicity through protective groups can improve permeability across the blood-brain barrier, but it can also show unwanted nonspecific accumulation in other organs (such as the liver). On the other hand, poor hydrophilicity makes it unsuitable for formulations injected through the tail vein. Tryptophan is used as a platform to deliver boron-10 to the tumor site for BNCT. BNCT has been intensively studied for the treatment of recurrent brain tumors. Enrichment of covalently bound molecules with biological functions 10 B atoms can be accumulated at the tumor site and then bombarded with epithermal neutrons with a penetration range of 10 cm. 7 Li and α ions, two high-energy particles, split and can damage surrounding tumor cells within the range of cell diameter.

[0009] To improve the limited hydrophilicity of these tri-Boc 5- and 6-boronopinacol tryptophan compounds, prodrug concepts have been reported through conjugation with polyethylene glycol (PEG). PEG is an FDA-approved polymer that enhances surface hydrophilicity and reduces phagocyte binding and uptake. While PEG's primary role in nanomedicine is through coagulation with bioactive agents or drugs, covalently bonded PEG-drug molecules have also been reported.

[0010] Although there are some methods to date that can indirectly quantify the uptake of PEGylated nanoparticles by cells, such as hybridization of metal nanoparticles and fluorescent dye labels, the quantitative range of PEGylated small molecules in animals is relatively limited. PEGylated nanoparticles can be labeled or labelled with dyes, isotopes or metal nanoparticles, which usually leads to significant changes in physicochemical properties such as size, stiffness and surface groups. Due to the disadvantages brought by the lack of these qualitative tools, radioactive transition metals (e.g.67 Ga) is therefore suitable for evaluating high molecular weight biomolecules, such as proteins or peptides, through complex chelation. However, the geometric requirements of chelation limit the biorecognition of small molecules.

[0011] To address the above-mentioned issues, those skilled in the art are in urgent need of developing novel pegylated boron tryptophan derivatives, pharmaceutical compositions comprising pegylated boron tryptophan derivatives, and methods for evaluating the pharmacokinetics of boron neutron capture therapy for tumors (e.g., brain tumors), and quantifying drug concentrations that can be used to calculate pharmacokinetic data, to benefit the vast population in need.

[0012] Summary of the Invention

[0013] In view of this, the object of the present invention is to provide a pegylated boron tryptophan derivative having the following chemical formula (I):

[0014] Wherein, R is polyethylene glycol (PEG).

[0015] In an embodiment of the present invention, the PEG is PEG 200 , and when the PEG is PEG 200 When the PEGylated boron tryptophan derivative has the following chemical formula (Ia):

[0016] In an embodiment of the present invention, the PEG is PEG 600 , and when the PEG is PEG 600 When the PEGylated boron tryptophan derivative has the following chemical formula (Ib):

[0017] In an embodiment of the present invention, the PEG is PEG 1000 , and when the PEG is PEG 1000 When the PEGylated boron tryptophan derivative has the following chemical formula (Ic):

[0018] Another object of the present invention is to provide a pegylated boron tryptophan derivative having the following chemical formula (II):

[0019] Wherein, R is polyethylene glycol (PEG).

[0020] In an embodiment of the present invention, the PEG is PEG 200 , and when the PEG is PEG 200When the PEGylated boron tryptophan derivative has the following chemical formula (IIa):

[0021] In an embodiment of the present invention, the PEG is PEG 600 , and when the PEG is PEG 600 When the PEGylated boron tryptophan derivative has the following chemical formula (IIb):

[0022] Another object of the present invention is to provide a pegylated boron tryptophan derivative having the following chemical formula (III):

[0023] Wherein, R is OTs or F, and the PEGylated boron tryptophan derivative comprises PEG 200 .

[0024] In an embodiment of the present invention, when R is OTs, the pegylated boron tryptophan derivative has the following chemical formula (IIIa):

[0025] In an embodiment of the present invention, when R is F, the pegylated boron tryptophan derivative has the following chemical formula (IIIb):

[0026] In an embodiment of the present invention, the F is fluorine-18 ( 18 F) Radioactive isotopes.

[0027] Another object of the present invention is to provide a method for preparing the pegylated boron tryptophan derivative as described above, comprising adding HBTU, DMAP and NEt3 to a compound of formula (I), wherein R of the compound of formula (I) is H.

[0028] Another object of the present invention is to provide a method for preparing the pegylated boron tryptophan derivative as described above, comprising adding HBTU, DMAP and NEt3 to a compound of formula (II), wherein R of the compound of formula (II) is H.

[0029] Another object of the present invention is to provide a pharmaceutical composition comprising the aforementioned pegylated borotryptophan derivative and a pharmaceutically acceptable carrier.

[0030] In an embodiment of the present invention, the pharmaceutical composition is in a dosage form for parenteral administration.

[0031] Another object of the present invention is to provide a use of the aforementioned pegylated borotryptophan derivative for preparing a pharmaceutical for treating tumors.

[0032] Another object of the present invention is to provide a use of the aforementioned pegylated boron tryptophan derivative for preparing a formulation for positron emission tomography (PET) scanning to evaluate the pharmacokinetics of boron neutron capture therapy (BNCT) of tumors and quantify the drug concentration for calculating the pharmacokinetics.

[0033] In an embodiment of the invention, the tumor is a brain tumor.

[0034] In an embodiment of the invention, the brain tumor is a glioma.

[0035] Another object of the present invention is to provide a method for evaluating the pharmacokinetics of boron neutron capture therapy for tumors, comprising using the aforementioned pegylated boron tryptophan derivative for positron emission tomography (PET) scanning.

[0036] In summary, the pegylated borotryptophan derivatives and pharmaceutical compositions comprising the pegylated borotryptophan derivatives of the present invention are effective in the development of highly accumulated drug molecules suitable for both diagnostic and therapeutic purposes in live animal tumor models, as demonstrated in the following examples. The high tumor accumulation dose, i.e., 18% of the drug accumulates in the tumor, with a high tumor-to-normal tissue ratio of 100:1, is also demonstrated. Furthermore, the pegylated borotryptophan derivatives exhibit extremely low toxicity, with cell viability exceeding 90% at 1000 μM. These compounds are effective in treating tumors (e.g., brain tumors) and in assessing the pharmacokinetics of boron neutron capture therapy (BNCT) for tumors using positron emission tomography (PET) scanning, allowing for quantitative calculation of drug concentrations for pharmacokinetic calculations.

[0037] The following will further illustrate the embodiments of the present invention. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Any person skilled in the art may make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the content defined in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 shows the higher expression of large protein transporter 1 (LAT1) in two glioma cell lines, U87 and LN229, based on genomic databases and the structure of boronic acid phenylalanine (BPA), which is used for boron neutron capture therapy (BNCT).

[0039] FIG2 shows the preparation process of N2O4 saturated nitric acid (red smoke HNO3) required for the nitration step.

[0040] Figure 3 shows the synthetic route 1 of boron tryptophan compound 1 (TB-6-BTme1), wherein (a) LiOH, THF / H2O2:1, 0°C, room temperature for 4 to 16 hours, yield 73 to 93% (TB-6-BT 3 and TB-5-BT 4), (b) HBTU (4 equivalents), DMAP (4 equivalents), NEt3 (100 equivalents), PEG200-1000 (2 equivalents), 20 hours, yield 30 to 60% (TB-6-BT-PEG 200 5-200, TB-6-BT-PEG 600 5-600, TB-6-BT-PEG 1000 5-1000, TB-5-BT-PEG 200 6-200 and TB-5-BT-PEG 600 6-600).

[0041] Figure 4 shows the preparation of tosylated polyethylene glycol (PEG) TB-6-BT 7, followed by fluorination and radiofluorination. Pathway 2, wherein (a) TsCl, toluene, room temperature for 3 hours, yield 61% (TB-6-BT-PEG 200 OTs 7), (b) KF, DMF, 100 °C for 20 min, (c) H[ 18 F]F, K2CO3, Kryptofix [2,2,2], CH3CN, 1.7% corrected radiochemical yield (EOB) (radioactive compound [ 18 F]F-8).

[0042] Figure 5 shows a cell survival assay using various boranotryptophan analogs and a control group. Due to different water solubilities, three different concentrations (one for each compound, determined by its water solubility) were used, designated (a), (b), and (c). (a) 500 μM + 0.8% DMSO, (b) 1000 μM + 0.8% DMSO, and (c) 2000 μM + 0.8% DMSO.

[0043] Figures 6A and 6B show the intracellular accumulation of boron-containing compounds at three different concentrations (one concentration for each compound, determined by the compound's water solubility) designated as (a) 100 μM + 0.8% DMSO, (b) 250 μM + 0.8% DMSO, and (c) 482 μM + 0% DMSO. Two human glioblastoma cell lines, U87 (Figure 5A) and LN229 (Figure 5B), were incubated for 1 hour, 2 hours, and 4 hours, respectively.

[0044] Figure 7 shows the effect of TB-6-BTPEG on the growth of U87 tumor cell line xenograft model mice 200[ 18 F]F([ 18 F]F-8) of a series of PET images.

[0045] FIG8A shows TB-6-BTPEG 200 [ 18 F]F([ 18 Figure 8B shows the radioactivity uptake data from the tumor and muscle to generate a time-based uptake ratio.

[0046] Figure 9 shows the effect of TB-6-BTPEG in a xenograft model mouse model using LN229 tumor cell line. 200 [ 18 F]F([ 18 F]F-8) of a series of PET images.

[0047] FIG. 10A shows TB-6-BTPEG 200 [ 18 F]F([ 18 Figure 10B shows the radioactivity uptake data from the tumor and muscle to generate a time-based uptake ratio.

[0048] Figure 11 shows TB-6-BTPEG 200 [ 18 [F]F fusion PET imaging from 0 to 60 minutes in xenograft model mice with U87 and LN229 tumor cell lines.

[0049] FIG12 shows the particle size distribution of the borotryptophan derivative measured by a laser scattering particle size analyzer.

[0050] Figure 13 shows a comparison of the aqueous solubility of PEGylated tryptophan analogs.

[0051] Figure 14 shows the proposed coagulation of PEGylated borotryptophan analogs. DETAILED DESCRIPTION

[0052] definition

[0053] The numerical values ​​used herein are approximate, and all experimental data are expressed within a range of ±20%, preferably within a range of ±10%, and most preferably within a range of ±5%.

[0054] Unless otherwise specified herein, the terms "a", "an", "the" and similar terms used in this specification (especially in the scope of the following patent application) should be understood to include both singular and plural forms.

[0055] As used herein, "treating" or "treatment" means alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, as well as lowering, stopping, or reversing the progression of the severity of the condition or symptom being treated.

[0056] According to the present invention, the pharmaceutical composition can be manufactured into dosage forms suitable for parenteral administration using techniques well known to those skilled in the art, including, but not limited to, injections, such as sterile aqueous solutions or dispersions, sterile powders, tablets, troches, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like.

[0057] The pharmaceutical composition according to the present invention can be administered via a parenteral route selected from the group consisting of intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0058] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a chelating agent, a diluent, a gelling agent, a preservative, a lubricant, an absorption delaying agent, a liposome, and the like. The selection and amount of these agents are within the professional knowledge and routine skills of those skilled in the art.

[0059] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), a sugar solution, an aqueous solution containing alcohol, and combinations thereof.

[0060] According to the present invention, as a member of the second period of atoms, radioactive fluorine, e.g. 18 F atoms (t 1 / 2 =110 minutes) can be covalently bonded to small molecules or drugs without interfering with the molecular shape, thus still retaining biorecognition. In addition, like Ga-67, F-18 is a positron emitter that captures surrounding electrons to emit two beams of coherent gamma radiation at an angle of 180°, thus achieving internal calibration for reconstructing 3D images and drug dynamics. Therefore, the effect of small molecule boron compounds in tumor sites can be obtained. 10 B concentration, and develop a treatment plan to optimize the neutron dose before BNCT. 18 F atoms were introduced into 6-boron tryptophan. Due to the unstable boron pinacol group, [ 18 Electrophilic fluorination of [F]F2 / Ne failed to produce any trace of fluorinated products, resulting only in decomposition. Only by introducing a PEG group could a suitable nucleophilic fluorination site be achieved. This not only provided terminal hydroxyl groups for tosyl functionalization but also improved its water solubility.

[0061] The present invention is further illustrated by the following examples. These examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the present invention is as set forth in the appended claims.

[0062] [Corrected 26.04.2024 according to Rule 26] The general experimental procedures are as follows. All reagents and solvents were purchased from Acros (Geal, Belgium) and Alfa (Binfield, Berkshire, UK). PEG 200 and 600 were purchased from First Chemicals Ltd, a local company in Taiwan, China. PEG 1000 was purchased from Acros (Geal, Belgium). Unless otherwise stated, the preparation of all compounds was routinely carried out in dry glassware at room temperature under positive nitrogen pressure. CH2Cl2 and CH3CN were dried over CaH2, and CH3OH was dried over Mg and distilled before use in the reaction. THF was distilled over Na before use. Solvents such as DMF and N(i-Pr)2Et were distilled under reduced pressure. Dimethylaminopyridine (DMAP) was purified by recrystallization from EtOAc and n-hexane before use. Flash chromatography eluents such as EtOAc, acetone, and n-hexane were all industrial grade and distilled before use; other chromatography solvents such as CH3OH, CH2Cl2, and CHCl3 were all reagent grade and used without further purification. NMR spectra include 1 H-NMR (500MHz), 13 C-NMR (125 MHz, DEPT-135) and 19 F-NMR (470 MHz) was performed on a Unity Inova 500 MHz instrument (Varian, USA). The deuterated solvent CDCl3 for NMR spectroscopy was purchased from Aldrich (St. Louis, MO, USA). Low-resolution mass spectrometry (LRMS) was performed using a Varian 901-MS liquid chromatography tandem mass spectrometry Q-TOF spectrometer or a Quattro Micro LC / MS / MS on an ESI-MS spectrometer. High-resolution mass spectrometry (HRMS) analysis was performed using a Varian HPLC (prostar series ESI / APCI) system in combination with a Varian 901-MS (FT-ICR Mass) mass detector and a triple quadrupole instrument. TLC silica gel 60F was used. 254 Thin-layer chromatography (TLC) was performed on pre-coated glass plates (Machery-Nagel, Dueren, Germany) to visually monitor starting materials and products under UV light (254 nm). Further confirmation was achieved by staining the TLC plates with ninhydrin or ceric ammonium molybdate under heating. Flash chromatography was performed using Silicycle 60 silica gel (70-230 mesh, Quebec City, Quebec, Canada).

[0063] Normal-phase HPLC consisted of an Agilent isocratic 1100 pump connected to a UVIS detector (254 nm) and a ZORBAX SIL column (9.4 mm × 250 mm, 5 μm). A 1:19 mixture of CH₃OH and CH₂Cl₂ was used as the mobile phase at a flow rate of 3 mL / min. A Rheodyne syringe with a 0.5 mL loop was used.

[0064] [Corrected 26.04.2024 in accordance with Regulation 26] Boron concentration was determined by ICP-mass spectrometry using the services provided by Thermo Fisher Scientific iCAP TQ (Germany).

[0065] Radiofluorination was performed at the Longtan Nuclear Energy Research Institute in Taoyuan, Taiwan. The hot cell contained a remote control module, and the detailed synthesis process is described below. Radioactive compounds were purified at NERI using a first HPLC system for initial fractionation, consisting of an Eldex Model 2SMP pump with a 1 / 8-inch head and stainless steel damper and a ZORBAX SIL column (9.4 mm × 250 mm, 5 μm) at a flow rate of 3 mL / min. A second HPLC system, used for mass analysis or purification, consisted of a Waters Model 6CE controller equipped with a Model 60F pump, a Waters 2489 UV detector, a radioactivity detector, and the same ZORBAX SIL column (9.4 mm × 250 mm, 5 μm) at a flow rate of 1 mL / min, with a preparative flow rate of 3 mL / min.

[0066] Positron emission tomography (PET) scanning experiments were performed using a Spanish SuperArgus PET / CT scanner Inc., Taipei Medical University, China.

[0067] The general deprotection process for the carboxyl group is as follows. At 0°C, LiOH (29 mg, 1.21 mmol, 3 equivalents) was added to a mixture of TB-5-BTme 2 (dissolved in 1.5 mL THF / H2O 2:1). The mixture was stirred at room temperature for 4 hours. In the case of TB-6-BTme 1, a reaction time of 16 hours was used. TLC (CH3OH / CH2Cl2=1 / 19) showed that the starting material 2 was consumed (R f = 0.98, 0.76) and formed product 4 (R f= 0.32, 0.08). After concentration under reduced pressure, the residue was chromatographed with an eluent (CH3OH / CH2Cl2 = 1 / 19) to obtain a viscous product TB-5-BT 4 (237 mg, 0.38 mmol) with a yield of 92% and a melting point of 122 to 124°C. As for TB-6-BT 3, column chromatography extraction or cation exchange resin (H + The deprotected products were purified by treatment with 1% HCl (Dowex) and filtration. The desired products, TB-6-BT 3 and TB-5-BT 4, were obtained in 73% and 93% yields, respectively. NMR spectra of these deprotected products were measured in CD3OD. Even in the presence of silver foil, they were unstable in CDCl3.

[0068] TB-5-BT 4

[0069] 1 H-NMR(500MHz,CD3OD)δ1.29(s,18H,N-Boc),1.35(s,12H,Bpin),1.65(s,9H,N-Boc),3.44-3.46(m,2H,H-1'a,H-1'b),5.18(dd,J 2’,1’b =10.0,J 2’,1’a =6.5Hz,1H,H-2'),7.39(s,1H,H-2),7.67(d,J 6,7 =9.0Hz,1H,H-6),7.98(s,1H,H-4),8.07(d,J 7,6 =9.0 Hz, 1H, H-7); 13 C-NMR(125MHz,CD3OD)δ25.22(CH3,Bpin),26.39(CH2,C-1'),27.98(CH3,N-Boc), 28.05(CH3,N-Boc),60.06(CH,C-2'),75.84(C,C-3),84.31(C,N-Boc),85.04(C,Bp in),115.49(CH,C-7),118.39(C,C-9),125.65(CH,C-6),127.27(CH,C-4),131.66( CH,C-2),138.95(C,C-8),150.81(C,N-Boc),153.19(C,N-Boc),173.52(C,COOH);C 32 H 47 BN2O 10 Analysis, calculation results are [M+Na] +(m / z) = 653.3221 (100.0%), 654.3255 (34.6%), 652.3258 (24.8%), found ESI-Q-TOF HR-ESI-MS: [M+Na] + =653.3225 (29.8%), 654.3276 (10.0%), 652.3285 (6.8%), δ [ppm] = 0.6.

[0070] TB-6-BT 3

[0071] 1 H-NMR(500MHz,CD3OD)δ1.28(s,18H,N-Boc),1.36(s,12H,Bpin),1.65-1.67(m,9H,N-Boc),3.38-3.48(m,2H,H-1'a,H-1'b),5.15(dd,J 2’,1’a =10.5,J 2’,1’b =4.5Hz,1H,H-2'),7.44(s,1H,H-2),7.54(d,J 5,4 =8.0Hz,1H,H-5),7.60(d,J 4,5 =8.0Hz,1H,H-4),8.53(s,1H,H-7); 13 C-NMR(125MHz,CD3OD)δ25.22(CH3,Bpin),26.35(CH2,C-1'),27.99(CH3,N-Boc),28.3 9(CH3,N-Boc),59.81(CH,C-2'),75.84(C,N-Boc),84.29(C,N-Boc),85.03(C,Bpin),11 8.22(C,C-3),119.46(CH,C-7),122.76(CH,C-4),126.68(CH,C-5),129.55(CH,C-2),13 4.45(C,C-8),136.55(C,C-9),150.80(C,N-Boc),153.28(C,N-Boc),173.45(C,COOH);C 32 H 47 BN2O 10 Analysis, calculation results are [M+Na] + (m / z) = 653.3221 (100.0%), 654.3255 (34.6%), 652.3258 (24.8%), found ESI-Q-TOF HR-ESI-MS: [M+Na] +=653.3224 (100.0%), 654.3228 (38.6%), 652.3253 (22.4%), δ [ppm] = 0.5.

[0072] The PEGylation reaction protocol is as follows. A mixture of TB-6-BT 3 (120 mg, 0.189 mmol) in CH3CN (3 mL) was placed in a two-necked round-bottom flask (50 mL) under N2 protection and equipped with a relatively large stir bar. A mixture of hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) (4 equivalents, 0.756 mmol, 286 mg) and diethylaminopyridine (DMAP) (4 equivalents, 0.756 mmol, 92 mg) in CH3CN (3 mL) was then added. NEt3 (100 equivalents, 5.3 mL) was added and the mixture was stirred for 10 minutes. Two equivalents of PEG 200 were added per 2 mL of CH3CN, and the mixture was co-distilled twice using NEt3 (2 mL). The reaction was allowed to proceed for 20-24 hours. The entire mixture was then concentrated under reduced pressure. Column chromatography was then performed using CH3OH / CH2Cl2 (1:19) to obtain a product mixture with a range of MW distributions of the PEG moiety, with a 30% yield. Yields of 40-60% were achieved for PEG200 and PEG1000. Some of the chromatographic samples were further purified using normal-phase HPLC with CH3OH / CH2Cl2 (1:19) as the eluent. The PEGylated product mixtures consistently exhibited a pink or purple color and a pleasant odor.

[0073] The preparation process for the N2O4-saturated nitric acid (red-fume HNO3) required for the nitration step is as follows (see Figure 2). Add the copper wire cut from a PC cable into small pieces to an Erlenmeyer flask. Add 30 mL of thick, yellow-fume HNO3 prepared from NH4NO3 or NaNO3 and H2SO4. While stirring and optionally heating, allow the released red-fume N2O4 to bubble through the right-hand foaming bottle. When gas evolution ceases (approximately 5 minutes), water can be added and the solution heated (approximately 60°C) to drive gas evolution. After 20 minutes, foaming is complete and ceases, and the solution turns reddish-brown. Note that the N2O4-saturated HNO3 will occasionally release N2O4 gas as it returns to room temperature. The bottle should not be tightly capped for storage.

[0074] Boron tryptophan compound 1 (TB-6-BTme 1) can be prepared via a six-step synthesis involving nitration of the starting tryptophan, introduction of Boc, reduction, iodination, and borylation. Nitration is optimized using red-fuming nitric acid, prepared by bubbling N₂O₄ through a mixture of cut copper wire in concentrated nitric acid with the addition of H₂O. Brown N₂O₄-saturated nitric acid produces the desired 6-nitrotryptophan, along with a minor amount of the 4-nitro isomer, in 65% overall yield. Subsequent deprotection using LiOH(aq) is mild, while purification varies, using either direct concentration or extraction followed by chromatography (Pathway 1, see Figure 3).

[0075] Figure 3 shows the synthetic route 1 of boron tryptophan compound 1 (TB-6-BTme 1), wherein (a) LiOH, THF / H2O 2:1, 0°C, room temperature for 4 to 16 hours, yield 73 to 93% (TB-6-BT 3 and TB-5-BT 4), (b) HBTU (4 equivalents), DMAP (4 equivalents), NEt3 (100 equivalents), PEG200-1000 (2 equivalents), 20 hours, yield 30 to 60% (TB-6-BT-PEG 200 5-200, TB-6-BT-PEG 600 5-600, TB-6-BT-PEG 1000 5-1000, TB-5-BT-PEG 200 6-200 and TB-5-BT-PEG 600 6-600).

[0076] Direct fluorination of the terminal hydroxyl group of PEGylated tryptophan using diethylaminosulfur trifluoride failed, while the introduction of a tosyl group made subsequent fluorination and radiofluorination feasible (Pathway 2, see Figure 4). On the other hand, although radiofluoride chemistry uses common CH3CN, it still requires optimization of solvent conditions. In the present invention, dimethylformamide sensitized the reaction and obtained a satisfactory radiochemical yield (1.7%) (radiocompound [ 18 F]F-8).

[0077] Figure 4 shows the preparation of tosylated PEGylated TB-6-BT 7, followed by fluorination and radiofluorination, where (a) TsCl, toluene, room temperature for 3 hours, yield 61% (TB-6-BT-PEG 200 OTs 7), (b) KF, DMF, 100℃ for 20 minutes, (c) H[ 18 F]F, K2CO3, Kryptofix [2,2,2], CH3CN, 1.7% corrected radiochemical yield (EOB) (radioactive compound [ 18 F]F-8).

[0078] First, benzoic acid is used to regulate PEGylation. Due to the characteristics of the molecular weight distribution of PEG reagents, the product mixture will distribute and tail on its own when developed by TLC, increasing the difficulty of column chromatography. Although common PEGylation uses excess adducts to consume a large amount of PEG reagents, potential hydrogen bonds through nucleophilic OH groups will lead to incompleteness. Although the common preparation method uses DCC as a coupling agent and separates the PEGylation product from the remaining reagent by dialysis, the results are not satisfactory. For example, 1 H-NMR spectroscopy does not provide a satisfactory integration ratio to estimate the amount of PEG reagent that remains. The presence of a significant amount of PEG reagent in solution will cause the PEGylated product to solidify. Furthermore, due to overlap with other chromatographic reagents, such as DMAP and HBTU, the purification process is cumbersome and requires optimization through repeated chromatography. HPLC further confirms these PEGylated products and allows differentiation between a series of PEGylated product mixtures.

[0079] Figure 5 shows a cell survival assay using various boranotryptophan analogs and a control group. Due to different water solubilities, three different concentrations (one for each compound, determined by its water solubility) were used, designated (a), (b), and (c). (a) 500 μM + 0.8% DMSO, (b) 1000 μM + 0.8% DMSO, and (c) 2000 μM + 0.8% DMSO.

[0080] Subsequent bioassays were performed at low DMSO concentrations (0.8%) to reduce potential toxic side effects (Figure 5). The improved water-soluble BPA-fructose complex, complexed with fructose, allowed the PEGylated compounds to reach concentrations twice as high (the increased water solubility allowed for higher concentrations without precipitation), reaching 482 μM and 250 μM, respectively.

[0081] All of these PEGylated borotryptophan analogs showed no significant toxicity (1 mM), which means that the boron dose is sufficient for BNCT (0.5-1 g / kg).

[0082] Figures 6A and 6B show the intracellular accumulation of boron-containing compounds at three different concentrations (one concentration for each compound, determined by the compound's water solubility) designated as (a) 100 μM + 0.8% DMSO, (b) 250 μM + 0.8% DMSO, and (c) 482 μM + 0% DMSO. Two human glioblastoma cell lines, U87 (Figure 6A) and LN229 (Figure 6B), were incubated for 1 hour, 2 hours, and 4 hours, respectively.

[0083] The uptake of these PEGylated borotryptophan compounds was determined in glioma cells expressing LAT-1 (e.g., human glioblastoma cell lines U87 and LN229) (Figures 6A and 6B). The accumulation of boron atoms in the PEGylated borotryptophan series of compounds in U87 cells was higher than that in BPA-fructose. 1000 The most prominent one is close to 30ppm. However, the corresponding accumulation amount in LN229 is different. Only TB-6-BTPEG 1000 and TB-5-BTPEG 200 Better results were shown for 50ppm and 25ppm, respectively. BPA-fructose showed marginal uptake in U87 cells (<3ppm) and more uptake in LN229 cells (25ppm), which corresponds to its LAT-1 dependent characteristics. LN229 cells with higher LAT-1 expression were set as the positive control, however, U-87 cells are not considered to be LAT-1 independent. The increasing trend of accumulation in U87 cells reflects the preferential cell type for these pegylated borotryptophan compounds. In contrast, the initial compound uptake in LN229 was the highest, but the gradual pumping out characteristics may indicate that it is not suitable as a target cell. For the feasibility of the experiment, TB-6-BTPEG was chosen. 200 Radiofluorination was performed for in vivo studies.

[0084] Figure 7 shows the effect of TB-6-BTPEG on the growth of U87 tumor cell line xenograft model mice 200 [ 18 F]F([ 18 F]F-8) time distribution of a series of PET imaging, observed [ 18 F] Image of the distribution of F-8 within tissues or organs.

[0085] FIG8A shows TB-6-BTPEG 200 [ 18 F]F([ 18 Figure 8B shows the radioactivity distribution derived from PET imaging of U87 tumor cell line xenograft mice. Figure 8B shows the radioactivity uptake data from the tumor and muscle sites to generate a time-based uptake ratio. A trend of increasing tumor uptake and a maximum radioactivity dose accumulation (18%) were found in U87 mice (Figures 8A and 8B). Within 50 minutes after injection, [ 18 The highest tumor-to-normal tissue uptake ratio of F]F-8 was 170 (T / N), indicating a high tumor cumulative dose, or 18% of the drug, accumulated in the tumor, and a high tumor-to-normal tissue ratio of approximately 100-170:1.

[0086] Figure 9 shows the effect of TB-6-BTPEG in a xenograft model mouse model using LN229 tumor cell line. 200 [ 18 F]F([ 18 F]F-8) time distribution of a series of PET imaging, observed [ 18 F] Image of the distribution of F-8 within tissues or organs.

[0087] FIG. 10A shows TB-6-BTPEG 200 [ 18 F]F([ 18 Figure 10B shows the radioactivity uptake data from the tumor and muscle to generate a time-based uptake ratio.

[0088] Figure 11 shows TB-6-BTPEG 200 [ 18 F]F fusion PET imaging from 0 to 60 min in xenograft model mice with U87 and LN229 tumor cell lines, respectively.

[0089] By using TB-6-BTPEG 200 [ 18 F]F([ 18 F]F-8) coupled to PET to achieve in vivo determination of these pegylated borotryptophan compounds (Figures 7, 9, and 11). Dynamic PET imaging of xenograft mice inoculated with the two cell lines spanned from 0 to 60 minutes, and tumor sites were marked, which could be detected by 18 The characteristics of F are quantified to calculate the pharmacokinetic drug concentration.

[0090] In contrast, LN229 [ 18 The uptake of F]F-8 was low (3% radioactivity) (Figures 10A and 10B). This showed an acceptable T / N ratio of 8 at 60 minutes after injection. 18 The uptake of F]F-8 in the brain may be due to its molecular shape.

[0091] Figure 12 shows the particle size distribution of the boron tryptophan derivatives measured by laser scattering particle size analyzer. The dynamic light scattering results of these PEGylated boron tryptophan compounds show that TB-6-BTPEG 200 The average diameter of the PEG-6 nanoparticles was 173.1±25.0 nm, which was larger than that of its precursor TB-6-BTme 1 (ID=78.7 nm), but smaller than that of PEG-6 nanoparticles. 200 (ID = 297.4 nm). and PEG at 263.6 nm 600Compared with TB-6-BTPEG 600 The aggregation of PEG200 (ID=505.4 nm) was significant. 200 The main structural features are determined by the shape of the tryptophan core. 600 Shows about PEG 600 The size is twice that of TB-5-BTPEG, indicating that the uncoating effect occurs through bimolecular coagulation. 200 The satisfactory in vitro uptake results may be potential candidates for future radiofluorinated target molecules.

[0092] TB-6-BT PEG 200 5-200 has the following chemical formula (Ia):

[0093] 1 H-NMR (500 MHz, CDCl3) δ 1.31, 1.32, 1.33 (s×3, 30H, CO2C(CH3)3, pinacol), 1.62 (s, 9H, CO2C(CH3)3), 2.78 (s, 5.9H, OH), 3.33 (dd, J 1a',1b' =15.0,J 1a',2' =10.0Hz,1H,H-1a'),3.49(dd,J 1b',1a' =15.0,J 1b',2' =5.0Hz,1H,H-1b'),3.57(t,J=4.0Hz,1.69H,PEG),3.62(s,5.10H,PEG),3.70(t,J=4.5Hz,2.94H,PE G),4.23-4.27(m,0.81H,PEG),4.33-4.38(m,0.78H,PEG),4.46(t,J=5.0Hz,2.12H,PEG),5.20(dd,J 2',1a' =10.0,J 2',1b' =5.0Hz,1H,H-2'),7.41(s,1H,arom,H-2),7.50(dd,J=7.5,3.5Hz,1H,arom,H-4),7.62(d,J=8.0Hz,1H,arom,H-5)8.60(bs,1H,arom,H-7); 13C-NMR (125MHz, CDCl3) δ24.88 (CH3, pinacol), 25.36 (CH2, C-1'), 27.67 (CH3, CO2C (CH3) 3), 27.77 (CH3, CO2C (CH3) 3), 28.1 2(CH3,CO2C(CH3)3),58.16(CH,C-2'); CH2(PEG):61.74,61.77,64.61,68.85,70.27,70.34,70.41,70.55,70.65,70.6 8,70.76,72.48,72.54;82.97(C,CO2C(CH)3),83.34(C,CO2C(CH)3),83.60(C,CO2C(CH)3),CH(arom):118.21,121.75, 125.41,128.51,C(arom):116.47,132.94,135.26;149.41(C,CO2C(CH3)3),151.79(C,CO2C(CH3)3),170.28(C,CO2PEG 200 ).

[0094] TB-6-BT PEG 600 5-600 has the following chemical formula (Ib):

[0095] 1 H-NMR (500 MHz, CDCl3) δ 1.31, 1.32 (s × 2, 30H, CO2C (CH3) 3, pinacol), 1.63 (s, 9H, CO2C (CH3) 3), 3.34 (dd, J 1a',1b' =15.0,J 1a',2' =10.5Hz,1H,H-1a'),3.57(dd,J 1b',1a' =15.0,J 1b',2' =5.0Hz,1H,H-1b'),3.68-3.71(m,32.2H,PEG),3.68-3.71(m,3.4H,PEG),4.22-4.35(m,2H,PEG),5.19(dd,J 2',1a' =10.5,J 2',1b' =5.0Hz,1H,H-2'),7.40(s,1H,arom,H-2),7.49(d,J=8.0Hz,1H,arom,H-4),7.62(d,J=8.0Hz,1H,arom,H-5)8.59(s,1H,arom,H-7); 13C-NMR(125MHz, CDCl3)δ24.88(CH3, pinacol),25.35(CH2,C-1'),27.66(CH3,CO2C(CH3)3),27.76(CH3,CO2C(CH3)3),28 .11(CH3,CO2C(CH3)3),58.13(CH,C-2'); CH2(PEG):61.50,64.57,68.80,69.98,70.26,70.31,70.40,70.47,70.61 ,72.36;82.96(C,CO2C(CH)3),83.35(C,CO2C(CH)3),83.59(C,CO2C(CH)3);CH(arom):118.17,121.75,125.39,128 .50; C(arom):116.44,124.70,132.92,135.23;149.39(C,CO2C(CH3)3),151.79(C,CO2C(CH3)3),170.27(C,CO2PEG 600 ).

[0096] TB-6-BT PEG 1000 5-1000 has the following chemical formula (Ic):

[0097] 1 H-NMR (500 MHz, CDCl3) δ1.26, 1.28, 1.30 (s×2, 33H, CO2C(CH3)3, pinacol), 1.58 (s, 13H, CO2C(CH3)3), 1.75 (bs, 6H, H2O), 2.73 (s, 1.54H, OH), 3.29 (dd, J 1a',1b' =15.0,J 1a',2' =10.0Hz,1.44H,H-1a'),3.44(dd,J 1b',1a' =15.0,J 1b',2' =5.0Hz,2.09H,H-1b'),3.57(s,63.01H,PEG),3.64-3.67(m,3.54H,PEG),4.19-4.30(m,2.64H,PEG),5.14(dd,J 2',1a' =10.0,J 2',1b' =5.0Hz,1H,H-2'),7.36(s,1H,arom,H-2),7.44(d,J=7.5Hz,1H,arom,H-4),7.58(d,J=7.5Hz,1H,arom,H-5)8.55(s,1H,arom,H-7).; 13C-NMR (125MHz, CDCl3) δ24.83(CH3, pinacol),25.30(CH2,C-1'),27.71(CH3,CO2C(CH3)3),28.06(CH3,CO2C(CH3)3),28.26(CH3 ,CO2C(CH3)3),29.61(CH3,CO2C(CH3)3),58.08(CH,C-2'); CH2(PEG):61.40,64.53,68.75,69.88,70.16,70.25,70.41,70. 56,72.25;82.91(C,CO2C(CH)3),83.30(C,CO2C(CH)3),83.55(C,CO2C(CH)3),83.60(C,CO2C(CH)3); CH(arom):118.13,121 .71,125.35,128.45;C(arom):116.40,132.87,135.19;149.34(C,CO2C(CH3)3),151.74(C,CO2C(CH3)3),170.22(C,CO2PEG 1000 ).

[0098] TB-5-BT PEG 200 6-200 has the following chemical formula (IIa):

[0099] 1 H-NMR (500 MHz, CDCl3) δ 1.31, 1.33 (s × 2, 30H, CO2C (CH3) 3, pinacol), 1.62 (s, 9H, CO2C (CH3) 3), 3.36 (dd, J 1a',1b' =15.0,J 1a',2' =10.5Hz,1H,H-1a'),3.51(dd,J 1b',1a' =15.0,J 1b',2' =4.0Hz,1H,H-1b'),3.56-3.72(m,J=4.0Hz,16H,PEG),4.24-4.28(m,1H,PEG),4.34-4.40(m,1H,PEG),4.46(t,J=5.0Hz,2.12H,PEG),5.23(dd,J 2',1a' =10.5,J 2',1b' =4.0Hz,1H,H-2'),7.34(s,1H,arom,H-2),7.72(dd,J=8.5Hz,1H,arom,H-4),7.97(s,1H,arom,H-5)8.08(d,J=7.5Hz,1H,arom,H-7);13 C-NMR (125 MHz, CDCl3) δ 24.86 (CH3, pinacol), 24.90 (CH3, pinacol), 25.28 (CH2, C-1'), 27.76 (CH3, CO2C(CH3)3), 28.15 (CH3, CO2C(CH3)3), 58.10 (CH, C-2'); CH2 (PEG): 61.74, 64.64, 68.84, 70.27, 70.35, 70.41, 70.52, 70.55, 70.64, 70.69, 70.80, 72.49 ,72.54;82.92(C,CO2C(CH)3),82.98(C,CO2C(CH)3),83.51(C,CO2C(CH)3),83.64(C,CO2C(CH)3),CH(arom):114.49,124. 40,126.02,130.86,C(arom):116.68,130.02,137.62;149.46(C,CO2C(CH3)3),151.63(C,CO2C(CH3)3),170.33(C,CO2PEG 200 ).

[0100] TB-5-BT PEG 600 6-600 has the following chemical formula (IIb):

[0101] 1 H-NMR (500 MHz, CDCl3) δ 1.30, 1.33 (s × 2, 30H, CO2C (CH3) 3, pinacol), 1.62 (s, 9H, CO2C (CH3) 3), 3.34 (dd, J 1a',1b' =14.5,J 1a',2' =10.5Hz,1H,H-1a'),3.57(dd,J 1b',1a' =15.0,J 1b',2' =5.0Hz,1H,H-1b'),3.68-3.71(m,32.2H,PEG),3.68-3.71(m,3.4H,PEG),4.22-4.35(m,2H,PEG),5.19(dd,J 2',1a' =10.5,J 2',1b' =5.0Hz,1H,H-2'),7.40(s,1H,arom,H-2),7.49(d,J=8.0Hz,1H,arom,H-4),7.62(d,J=8.0Hz,1H,arom,H-5)8.59(s,1H,arom,H-7); 13C-NMR (125MHz, CDCl3) δ24.87 (CH3, pinacol), 24.90 (CH3, pinacol), 25.28 (CH2, C-1'), 27.77 (CH3, CO2C(CH3)3), 28.16 (CH3, CO2C(CH3)3), 58.09 (CH, C-2'); CH2 (PEG): 61.66, 64.61, 68.81, 70.22, 70.67, 72.55; 82.91 (C,CO2C(CH)3),83.51(C,CO2C(CH)3),83.63(C,CO2C(CH)3); CH(arom):114.49,124.39,126.01,130.8 6;C(arom):116.68,130.02,137.61;149.46(C,CO2C(CH3)3),151.63(C,CO2C(CH3)3),170.32(C,CO2PEG 600 ).

[0102] TB-6-BT-PEG 200 -OTs 7 has the following chemical formula (IIIa):

[0103] The starting materials TB-6-BT-PEG200 (160 mg, 216 mmol, 1 eq) and TsCl (60 mg, 0.324 mmol, 1.5 eq) were treated with toluene (3 mL x 3) to distil H2O. These were then transferred sequentially to a two-necked round-bottom flask under N2 with CH2Cl2 (5 mL). Et3N (0.06 mL, 0.44 mmol, 2 eq) was then added, and the mixture was monitored by TLC (CH3OH / CH2Cl2 = 1 / 19). The molecular weight distribution of the starting materials (R f =0.32~0.61) consumption and molecular weight distribution product information (R f =0.83-0.96). After 3 hours, the mixture was concentrated under reduced pressure. Flash chromatography was then performed using CH3OH / CH2Cl2 = 1:79 to obtain a light yellow oil with a sawdust odor in a 61% yield (120 mg).

[0104] 1 H-NMR(500MHz, CDCl3)δ1.31(s,27H,CO2C(CH3)3),1.33,1.62(s,12.0H,CO2C(CH3)3),2.41(s,3.0H,-CH3),3.33(dd,J 1a',1b' =15.0,J 1a',2'=4.5Hz,1H,H-1a'),3.48(dd,J 1b',1a' =15.0,J 1b',2' =10.0Hz,1H,H-1b'),3.54-3.55(m,3.7H,PEG),3.58-3.59(m,3.3H,PEG),3.64-3.66(m,4.2H, PEG),4.12-4.14(m,2.1H,PEG),4.23-4.25(m,1.3H,PEG),4.24-4.32(m,1.1H,PEG),5.20(dd,J 2',1a' =10.0,J 2',1b' =5.0Hz,1H,H-2'),7.41(s,1H,),7.48(dd,J=7.5,3.7Hz,1H),7.62(d,J=8.0Hz,1H)8.60(s,1H). 13 C-NMR(125MHz,CDCl3)δ24.79(CH3,CH3CCH3),24.80(CH3,CH3CCH3),25.28(CH2,C-1'),27.59(CH3,CO2C(CH3)3),27.68(CH3,CO 2C(CH3)3)28.03(CH3,CO2C(CH3)3),53.36(CH,C-2'),58.06(CH,C-2'); CH2(PEG):61.74,61.77,64.61,68.85,70.27,70.34,70 .41,70.55,70.65,70.68,70.76,72.48,72.54;82.97(C,CO2C(CH)3),83.34(C,CO2C(CH)3),83.60(C,CO2C(CH)3),CH(arom):11 8.21,121.75,125.41,128.51,C(arom):116.47,132.94,135.26;149.41(C,CO2C(CH3)3),151.79(C,CO2C(CH3)3),170.28(C,CO2 PEG 200 ).

[0105] TB-6-BT-PEG200-F 8 has the following chemical formula (IIIb):

[0106] KF (14.5 mg, 0.25 mmol, 5 equivalents) in a two-necked round-bottom flask was dried in an oven at 120°C for 24 hours. 200-OTs (50 mg, 0.05 mmol, 1 equivalent) was distilled from toluene (3 mL × 3). 200 -OTs was transferred to a two-necked round-bottom flask along with DMF (1.5 mL). Kryptofix [2,2,2] (26 mg, 0.07 mmol, 1.4 equiv) was then added. The mixture was stirred at 100°C for 20 minutes. Flash chromatography was then performed using a CH3OH / CH2Cl2 (1 / 19) eluent to afford a pale yellow oil in a 51% yield (48 mg). 1 H-NMR showed the presence of the hydrolyzed analog in a 50% fraction.

[0107] 1 H-NMR(500MHz, CDCl3)δ1.31(s,27H,CO2C(CH3)3),1.33,1.62(s,12.0H,CO2C(CH3)3),3.33(dd,J 1a',1b' =15.0,J 1a',2' =4.5Hz,2H,H-1a'),3.48(dd,J 1b',1a' =15.0,J 1b',2' =10.0Hz,2H,H-1b'),3.57-3.7(m,21.0H,PEG),4.23-4.34(m,3.8H,PEG),4.47-4.57(2.0H,PEG),5.20(dd,J 2',1a' =10.0,J 2',1b' =5.0Hz,1.6H,-CH),7.41(s,1H,),7.49(dd,J=7.5Hz,1.4H),7.63(d,J=8.0Hz,1.4H)8.60(s,1H)

[0108] 19 F-NMR (470 MHz, CDCl3)

[0109] δ-222.93

[0110] Preparation of PEGylated Benzoate

[0111] Take benzoyl-PEG600-OH as an example. To a mixture of PEG-600 (909 μL, 1.7 mmol, 1 eq) in a round-bottom flask (50 mL) were added Et3N (2 mL) and CH2Cl2 (2 mL) in sequence and co-evaporated. The mixture was then protected with N2, followed by the addition of DMAP (415 mg, 3.4 mmol, 2 eq) and Et3N (0.2 mL, 1.4 mmol, 0.8 eq). The mixture was homogenized with CH2Cl2 (5 mL). A solution of benzoyl chloride (454 μL, 3.91 mmol, 2.3 eq) (dissolved in CH2Cl2 (5 mL)) was added. After 90 minutes, the white turbid mixture was monitored by TLC (CH3OH / CH2Cl2=0.8:9.2). It was observed that the main product mixture had a less polar portion (R f =0.90~0.65) and the minor products have more polar parts (R f =0.56). After 2.5 hours of reaction, the mixture was concentrated under reduced pressure. The snow-white crude mixture (2.54 g) was chromatographed using a gradient of CH3OH / CH2Cl2 = 1 / 32 to 1 / 19 to obtain the major monobenzoylated product in 50% yield (1.3 g) and the minor dibenzoylated product in 2% yield (73 mg). These two products were confirmed to be monobenzoylated PEG-OH and dibenzoylated PEG. In fact, the less polar product was the monobenzoylated benzoyl-PEG600-OH. The more polar product was the dibenzoylated product.

[0112] Benzoyl PEG200 9-200 has the following chemical formula (IV):

[0113] 1 H-NMR (500MHz, CDCl3) δ1.63 (s, 1H, OH), 3.64-3.72 (m, 4.60H, PEG), 3.81-3.85 (m, 2.33H, PEG), 4.64 (t, J = 5.0 Hz,2.28H,PEG),7.43(t,J=7.5Hz,2H,arom-H),7.55(t,J=7.5Hz,1H,arom-H),8.05(d,J=7.5Hz,2H,arom-H); 13 C-NMR (125 MHz, CDCl3) δCH2(PEG):64.07,69.19,69.26,70.57,70.61,70.65,70.68,CH(arom):128.28,129.63,132.92,C(arom):130.09,CO(ester):166.49.

[0114] Benzoyl PEG 400 9-400 has the following chemical formula (V):

[0115] 1 H-NMR (500 MHz, CDCl3) δ 3.60-3.62 (m, 7.32H, PEG), 3.63-3.65 (m, 2.48H, PEG), 3.67-3.69 (m, 2.20H, PEG), 3.81 (t, J = 5.0 Hz, 2.18H, PEG), 4.45 (t, J = 5.0 Hz, 2.15H, PEG), 7.41 (t, J = 7.5 Hz, 2.13H, arom-H, one site + two sites ), 7.45 (t, J = 7.5 Hz, 2H, arom-H, two sites), 7.53 (t, J = 7.5 Hz, 1.02H, arom-H, one site + two sites), 7.58 (t, J = 7.5 Hz, 0.18H, arom-H, two sites), 8.03 (d, J = 7.5 Hz, 2.00H, arom-H, one site + two sites), 8.08 (d, J = 7.5 Hz, 0.35H, arom-H, two sites); 13 C-NMR (125 MHz, CDCl3) δCH2(PEG):64.07,69.16,70.49,70.54,70.57,70.63,CH(arom):128.26,128.33,129.61,130.00,C(arom):130.06,CO(ester):166.48.

[0116] Benzoyl PEG600 9-600 has the following chemical formula (VI):

[0117] 1 H-NMR (500MHz, CDCl3) δ2.31(s,1.2H,OH),3.61(s,18.62H,PEG),3.63-3.65(m,2.44H,PEG),3.67-3.68(m,2.18H,PEG),3.81(t,J=5.0Hz,2.16 H,PEG),4.45(t,J=5.0Hz,2.11H,PEG),7.41(t,J=7.5Hz,2.05H,arom-H),7.53(t,J=7.5Hz,1.01H,arom-H),8.03(d,J=7.5Hz,2.00H,arom-H); 13C-NMR (125 MHz, CDCl3) δCH2(PEG):64.06,69.15,70.49,70.54,70.57,70.63,CH(arom):128.25,129.60,132.89,C(arom):130.06,CO(ester):166.45.

[0118] Benzoyl PEG1000 9-1000 has the following chemical formula (VII):

[0119] 1 H-NMR(500MHz, CDCl3)δ2.21(bs,4.58H,OH),3.62(s,31.95H,PEG),3.64-3.66(m,2.66H,PEG),3. 68-3.69(m,2.30H,PEG),3.82(t,J=5.0Hz,2.16H,PEG),4.46(t,J=5.0Hz,2.12H,PEG),7.42(t,J= 7.5Hz,2.12H,arom-H),7.45(t,J=7.5Hz,0.28H,arom-H),7.54(t,J=7.5Hz,1.02H,arom-H),7.58 (t,J=7.5Hz,0.18H,arom-H),8.03(d,J=7.5Hz,2.00H,arom-H),8.06(d,J=7.5Hz,0.33H,arom-H); 13 C-NMR (125 MHz, CDCl3) δCH2(PEG):64.04,69.13,70.46,70.51,70.54,70.60,CH(arom):128.24,129.58,129.88,132.88,133.01,C(arom):130.03,CO(ester):166.44.

[0120] Size Distribution Measurement of PEGylated Boron Tryptophan Derivatives

[0121] Take samples TB-6-BTMe, TB-6-BT-PEG 200 TB-6-BT-PEG 600 PEG 200 and PEG 6001 mg of each was dissolved in CH₃CH₂OH (3 mL vial) and ultrasonically shaken for 5 minutes. Particle size was measured using a Zetasizer Ultra-Red using laser irradiation to extract diffraction pattern information. Data were numerically analyzed using the manufacturer's software. Size distribution was plotted using GraphPad Prism 5 software.

[0122] Bioassay

[0123] Due to the viscous nature of these PEGylated tryptophan compounds, all samples were initially weighed and treated with EtOH (20 μL) and ddH2O (200 μL), then freeze-dried. The freeze-dried product was dissolved in an aqueous solution containing DMSO (13.6% v / v) as a stock solution (8.2-33 mM). Serial dilutions were then performed to the desired concentrations for subsequent bioanalysis.

[0124] The protocol for in vitro cytotoxicity is as follows. The cytotoxicity of tryptophan conjugates against U87 and LN229 cells was measured in monolayer culture. Cells were seeded in 96-well plates (5000 cells / 100 μL per well) to allow attachment. Due to the advantages of these PEGylated compounds with reduced toxicity and enhanced solubility, the most concentrated formulations included TB-6-BT-PEG. 600 TB-6-BT-PEG 1000 TB-5-BT-PEG 600 and TB-5-BT-PEG 1000 (2mM). Moderately water-soluble TB-6-BT-PEG 200 and TB-5-BT-PEG 200 Prepared to 1mM, TB-6-BT, TB-5-BT and BPA, which have the lowest water solubility, were prepared to 0.5mM. The above cell culture medium (100μM) was removed and supplemented with the same volume of the corresponding medium containing the PEGylated tryptophan derivative and BPA to obtain a final mixture containing 0.8% DMSO. After 24 hours, the cell viability in all wells was determined using the MTT assay and a microplate reader (Spectramax 190, Molecular Devices Corp., Sunnyvale, CA). The medium was supplemented with 10% MTT reagent (100μL) and incubated for 4 hours. After replacing with DMSO (100μL), the cells were plated at λ abs =570 nm. The fraction of viable cells was calculated by dividing the mean optical density produced by the treated cells by the mean optical density of the untreated control cells.

[0125] The procedure for cell accumulation assay is as follows. Two cell lines, U87 and LN229, were cultured at 2.5×10 5 cells / well and 5×10 5 The cells were seeded at a concentration of 10 cells / well in a culture dish (ID: 6 cm) containing 2 mL of culture medium. After 24 hours of adhesion to the inner wall, the culture medium was replaced with a new culture medium containing a tryptophan derivative and BPA (50 μM, 10 mL). The supernatant at each time point of 0.05, 1, 2 and 4 hours of incubation was removed, and the cells were washed twice with PBS solution (5 mL). The remaining cell mixture was lysed for 5 minutes using a mixture of trypsin and EDTA (2 mL). The cell pellet mixture was washed 3 times with culture medium (5 mL) and then transferred to a centrifuge tube and centrifuged at 1200 rpm for 5 minutes. The supernatant was removed and 1 mL of culture medium was added for dissolution. A 20 μL aliquot was mixed with trypan blue (20 uL) and 10 μL was extracted for cell counting. The centrifuge tube was centrifuged again. The supernatant was removed and the precipitate was treated with 70% Wt. HNO 3(aq) (75 μL) was used for digestion for 48 hours. Additional distilled water (1.42 mL) was added to obtain a solution (1.5 mL) with a concentration of 3.5% HNO3 (aq). The concentration of the sample was analyzed using inductively coupled plasma mass spectrometry (ICP-MS). All data were obtained in triplicate. The detection limit of the instrument was 0.399 ppb.

[0126] 10 The concentration of B atoms was calculated according to the following equation:

[0127] radiochemistry

[0128] About activated K+kryptofix[2,2,2][ 18 F]F - The process is as follows. 18 Aqueous solution of [F]F (262 mCi) was captured on a QMA-Light Sep Park anion exchange column. A mixture of K2CO3 (5.8 mg) and Kryptofix [2,2,2] (10.4 mg) in a solvent combination of CH3CN (1 mL) and H2O (0.5 mL) was used to elute the captured [ 18 F]F - , yielding >90% (262 mCi) of radioactive fluoride. The mixture was heated to 95°C under a nitrogen purge for 3 minutes. After cooling to 50°C, the radiochemical activity was 246 mCi. This process was repeated, adding CH3CN (1 mL), heating to 95°C, purging with nitrogen, and cooling to 50°C, yielding 208 mCi of radioactivity.

[0129] The radiofluorination protocol is as follows: TB-6-BT-PEG tosylate was dissolved in DMF (1 mL). 200 -OTs (20 mg) was transferred to the above-described reaction vessel. After heating to 100°C for 10 minutes, the vessel pressure reached 188 kPa and the activity was 248 mCi. The reaction was then cooled to 50°C and the vessel returned to ambient pressure. The mixture was eluted through an Al2O3 column and washed with CH3CN, yielding an eluent with 8.79 mCi of activity and 197 mCi of activity retained on the solid support. The mixture was dried at 55°C and dissolved in CH3OH / CH2Cl2 (1:49). The product solution was fractionated by HPLC using CH3OH / CH2Cl2 (1:49) as the eluent at a flow rate of 3 mL / min, yielding a product solution with 3.11 mCi of activity. A corrected radiochemical yield of 1.7% was obtained after a 1-hour run (EOB). Following concentration and further drying, the sample was submitted for animal PET imaging studies. An analytical amount of the sample was further submitted to analytical HPLC. The injection, prepared with 10% DMSO and saline, had an activity of greater than 11 μCi and was used for PET scans of two tumor-bearing mice.

[0130] The procedures for the positron emission tomography (PET) study are as follows. All in vivo experiments were conducted in accordance with the NHMRC Taiwan Standard for the Care and Use of Animals for Scientific Purposes. Animal Use Protocol No. 111022 of the Institute of Nuclear Energy was approved prior to the experiment. Rats were housed under fixed environmental conditions and had free access to food and water throughout the experiment.

[0131] LN229 tumor-bearing mice (n=1, 6 weeks after administration) and U87MG tumor-bearing mice (n=1, 6 weeks after administration) were used to evaluate the efficacy of [ 18 F]F-TB-6-BT-PEG 200 [ 18 F]F-8 in vivo PET studies.

[0132] for[ 18 F]F-TB-6-BT-PEG 200 [ 18 For dynamic PET studies of F]F-8, mice were anesthetized with 1-2% isoflurane and the tumor was located in the center of the field of view. 1.0±0.6MBq / 0.1mL of [ 18 F]F-TB-6-BT-PEG 200 [ 18 After 8-hour PET scans, the mice were subjected to a 1-hour dynamic PET scan to collect 6 × 10-minute frames. The 6 × 10-minute frames were either analyzed directly or merged to obtain a scan set from 0 to 60 minutes.

[0133] Six frames are used to draw activity-time curves, where regions of interest (ROIs) cover the tumor site or adjacent normal area and the time course. The merged data set from frame 4 to frame 6 can also be used as a static image frame (0-60 minutes) for comparison with other static scan data. 18 F]F-TB-6-BT-PEG 200 In the 24-group, mice were imaged using a static imaging mode that collected imaging data from 0 to 60 minutes after injection.

[0134] In total, the PET study included two tumor-bearing mice. Small animal PET scanning studies were performed using a PET scanner (SuperArgus PET Inc., Spain). Images were acquired using 2-D ordered subset expectation maximization (OSEM 2D) reconstruction and processed using PMOD 3.2 imaging analysis software. ROIs were drawn on the tumor using a threshold of (maximum intensity minus minimum intensity) × 50%, and ROIs were drawn on the liver, kidney, and muscle regions. The mean intensity (kBq / cc) of the ROIs was measured.

[0135] [ 18 F]F-TB-6-BT-PEG 200 [ 18 Determination of the partition coefficient of F-8

[0136] Dissolve the salt Na3PO4 (409 mg, 2.5 mmol) in a volumetric flask (50 mL) to make a 50 mM solution and titrate to pH 7.4 with a 100-fold diluted H3PO4 solution. Mix 3 mL of Na3PO4 (aq) and an aliquot of n-octanol in a round-bottom flask (10 mL) and stir for 10 minutes. Add the radioactive tracer [ 18 F]F-TB-6-BT-PEG 200 Continue stirring for 20 minutes. Transfer 0.2 mL of the organic layer and the aqueous layer to the test tubes respectively, repeat four times. Then add 1 mL of CH3CH2OH. Then use Radioactivity was measured using an automated gamma counter. The logarithm of the counts in the organic layer was divided by the counts in the aqueous layer.

[0137] Physical properties and description

[0138] Figure 13 shows a comparison of the water solubility of PEGylated tryptophan analogs. Table 1 describes the water solubility of PEGylated borotryptophan compounds. The small logP value of 0.65 ± 0.04 indicates that [ 18 The lower lipophilicity of F]F-8 improves its water solubility.

[0139] Table 1

[0140] Figure 14 shows the proposed coagulation of PEGylated borotryptophan analogs.

[0141] The current study provides a new class of borotryptophan compounds through PEGylation to improve their water solubility for use in injectable formulations, while the Boc-protected core structure is still able to cross cell membranes. Due to the instability of the ester group, once the compound circulates through the bloodstream, the borotryptophan core structure can be released through esterification by lipase. This prodrug concept has been shown to be effective in the treatment of U87 xenograft tumors in mice [ 18 F] F-8 showed significant cumulative positive results. Also addressed the high T / N ratio of 170.

[0142] As one of the rare reports on imaging of PEGylated compounds to date, [ 18 F]F-PEGylated borotryptophan compounds have acceptable radiochemical activity (<100 μCi) and have been used to generate in vivo images to rapidly bridge a compound from fresh laboratory synthesis to preliminary in vivo evaluation of its efficacy.

[0143] In summary, the pegylated boron tryptophan derivatives and pharmaceutical compositions comprising the pegylated boron tryptophan derivatives of the present invention can achieve the efficacy of treating tumors (e.g., brain tumors) and can be used for positron emission tomography (PET) scanning to evaluate the pharmacokinetics of boron neutron capture therapy (BNCT) of tumors and quantify the drug concentration for calculating the pharmacokinetics, as shown in the results illustrated in the above examples.

[0144] The above description is for illustrative purposes only and is not intended to be limiting. Any equivalent modifications or variations that do not depart from the spirit and scope of the present invention should be included in the appended claims.

Claims

1. A polyethylene glycolated boron tryptophan derivative having the following chemical formula (I): Among them, R is polyethylene glycol (PEG).

2. The polyethylene glycolated boron tryptophan derivative according to claim 1, wherein, The PEG is PEG 200 , and when the PEG is PEG 200 , the polyethylene glycolated boron tryptophan derivative has the following chemical formula (Ia):

3. The polyethylene glycolated boron tryptophan derivative according to claim 1, wherein, The PEG is PEG 600 , and when the PEG is PEG 600 , the polyethylene glycolated boron tryptophan derivative has the following chemical formula (Ib):

4. The polyethylene glycolated boron tryptophan derivative according to claim 1, wherein, The PEG is PEG 1000 , and when the PEG is PEG 1000 , the polyethylene glycolated boron tryptophan derivative has the following chemical formula (Ic):

5. A polyethylene glycolated boron tryptophan derivative having the following chemical formula (II): Among them, R is polyethylene glycol (PEG).

6. The polyethylene glycolated boron tryptophan derivative according to claim 5, wherein, The PEG is PEG 200 , and when the PEG is PEG 200 , the polyethylene glycolated boron tryptophan derivative has the following chemical formula (IIa):

7. The polyethylene glycolated boron tryptophan derivative according to claim 5, wherein, The PEG is PEG 600 , and when the PEG is PEG 600 , the polyethylene glycolated boron tryptophan derivative has the following chemical formula (IIb):

8. A polyethylene glycolated boron tryptophan derivative having the following chemical formula (III): Among them, R is OTs or F, and the polyethylene glycolated boron tryptophan derivative contains PEG 200 .

9. The polyethylene glycolated boron tryptophan derivative according to claim 8, wherein, When R is OTs, the polyethylene glycolated boron tryptophan derivative has the following chemical formula (IIIa):

10. The polyethylene glycolated boron tryptophan derivative according to claim 8, wherein, When R is F, the polyethylene glycolated boron tryptophan derivative has the following chemical formula (IIIb):

11. The polyethylene glycolated boron tryptophan derivative according to claim 10, wherein, The F is a fluorine-18 ( 18 F) radioisotope.

12. A method for preparing a polyethylene glycolated boron tryptophan derivative according to any one of claims 1 to 4, comprising adding HBTU, DMAP and NEt3 to a compound having the chemical formula (I), wherein, R of the compound having the chemical formula (I) is H.

13. A method for preparing a polyethylene glycolated boron tryptophan derivative according to any one of claims 5 to 7, comprising adding HBTU, DMAP and NEt3 to a compound having the chemical formula (II), wherein, R of the compound having the chemical formula (II) is H.

14. A pharmaceutical composition comprising a polyethylene glycolated boron tryptophan derivative according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition is in a dosage form for parenteral administration.

16. Use of a polyethylene glycolated boron tryptophan derivative according to any one of claims 1 to 11 for the preparation of a medicament for treating tumors.

17. Use of a polyethylene glycolated boron tryptophan derivative according to any one of claims 1 to 11 for the preparation of a formulation for positron emission tomography (PET) scanning to evaluate the pharmacokinetics of boron neutron capture therapy (BNCT) of tumors and to quantify the drug concentration for calculating the pharmacokinetics.

18. The use according to claim 16 or 17, wherein, The tumor is a brain tumor.

19. The use according to claim 18, wherein, The brain tumor is glioma.

20. A method for evaluating the pharmacokinetics of boron neutron capture therapy of tumors, comprising using a polyethylene glycolated boron tryptophan derivative according to any one of claims 1 to 11 for positron emission tomography (PET) scanning.

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