Boron phenylalanine dipeptide composition based on LAT1 targeting as well as preparation method and application of boron phenylalanine dipeptide composition

Abstract

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a boron phenylalanine dipeptide composition based on LAT1 targeting as well as a preparation method and application of the boron phenylalanine dipeptide composition. The boron phenylalanine dipeptide composition based on LAT1 targeting is prepared from the following components: boron phenylalanine dipeptide marked by 131I and a stabilizing agent, the mass ratio of the 131I labeled boron phenylalanine dipeptide to the stabilizer is 1: (0.4-0.6); the structural formulas of the 131I-labeled boron phenylalanine dipeptide and the stabilizer are respectively as follows: # imgabs0 #. After the composition is injected into a body, the lifetime can be prolonged, the stable boron concentration of a patient can be kept for a long time, and the treatment effect can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a boron-phenylalanine dipeptide composition targeting LAT1, and a preparation method and application thereof. Background Art

[0002] Cancer is a serious health problem globally. Thyroid cancer is one of the most common malignant tumors in the endocrine system and head and neck, accounting for 1% of all systemic malignant tumors, and also faces challenges in diagnosis and treatment. Molecular probes and radionuclide labeling techniques play a key role in cancer diagnosis and treatment. New molecular probes and labeling techniques are constantly emerging, which can provide highly specific information to help doctors better understand the characteristics and metabolism of tumors. Therefore, the application of molecular probes and radionuclide labeling techniques can effectively track tumor cells, facilitate clinical treatment, and promote the improvement of the prognosis of patients with iodine-refractory thyroid cancer.

[0003] The existing molecular probes and labeling techniques are widely and deeply applied in tumor imaging. Molecular probes are compounds designed to specifically bind to tumor cells or their related biomarkers. These probes usually carry radioactive, fluorescent or other detectable labels, enabling tumors to be clearly identified and located in imaging examinations. Labeling techniques involve connecting these probes to specific detection signal sources, such as radioactive isotopes, fluorescent molecules or MRI contrast agents, to ensure high enough signal intensity and specificity during in vivo detection. In clinical applications, molecular probes and labeling techniques are widely used for the early diagnosis, condition monitoring and efficacy evaluation of tumors. For example, PET and SPECT use radioactively labeled probes to achieve highly sensitive tumor imaging, helping doctors detect tumors and their metastases at an early stage. However, some molecular probes and labeling techniques involve cumbersome preparation reactions, which not only increase the difficulty of laboratory operations, but also raise production costs and time consumption, posing certain challenges to their popularization in practical applications. Therefore, it is very important to break through the limitations of some molecular probes and labeling techniques and obtain a simple preparation process.

[0004] Boron neutron capture therapy (BNCT) is a promising treatment method that uses the boron-10 isotope to capture neutron beams, releasing α particles and Li-7 nuclear reaction products, thereby achieving selective destruction of tumors. However, to achieve effective BNCT, a specific carrier is needed to deliver the boron-10 isotope to tumor tissues. 4-Boron-L-phenylalanine (BPA) is a promising molecule for cancer treatment and imaging, but its limitations include low water solubility, easy excretion by tumor cells, and short residence time in tumor cells, etc.

[0005] Therefore, finding a method to reduce the limitations of BPA treatment and using its specific binding to LAT1 for thyroid cancer imaging and BNCT has become an important technical challenge. Summary of the Invention

[0006] One object of the present invention is to provide a LAT1-targeted boron phenylalanine dipeptide composition.

[0007] Another object of the present invention is to provide a preparation method of the LAT1-targeted boron phenylalanine dipeptide composition.

[0008] A third object of the present invention is to provide the application of the above-mentioned LAT1-targeted boron phenylalanine dipeptide composition.

[0009] To achieve the above objects, the present invention adopts the following technical solutions:

[0010] The LAT1-targeted boron phenylalanine dipeptide composition includes the following components: 131 I-labeled boron phenylalanine dipeptide ( 131 I-labeled BPA-BPA), a stabilizer; the 131 mass ratio of the I-labeled boron phenylalanine dipeptide to the stabilizer is 1:0.4 - 0.6; the 131 structural formulas of the I-labeled boron phenylalanine dipeptide and the stabilizer are shown as follows:

[0011]

[0012] Further, the preparation process of the stabilizer includes the following steps:

[0013]

[0014] (1) Add 4-boron-L-phenylalanine to the quercetin solution, heat for reaction, and after the reaction is completed, purify the reaction solution to obtain intermediate 1;

[0015] (2) Add intermediate 1 to a solvent, add N,N'-dicyclohexylcarbodiimide and L-glutamine for reaction, and after the reaction is completed, filter the reaction solution, concentrate and purify to obtain.

[0016] Further, in step (1), the molar ratio of quercetin to 4-boron-L-phenylalanine is 1:1 - 2, and the concentration of the quercetin solution is 0.4 mol / L.

[0017] Even further, the solvent of the quercetin solution in step (1) is tetrahydrofuran.

[0018] Further, the heating temperature in step (1) is 70 - 75 °C, and the time is 6 - 8 h.

[0019] Further, the molar ratio of intermediate 1, N,N'-dicyclohexylcarbodiimide, and L-glutamine in step (2) is 1:1.2 - 2:1 - 1.5; the reaction time is 16 - 18 h.

[0020] More specifically, the solvent in step (2) is dichloromethane.

[0021] Further, the 131 Preparation of I-labeled BPA - BPA includes the following steps:

[0022]

[0023] S1. Add 4-boron-L-phenylalanine and thionyl chloride to methanol for reaction, and concentrate the reaction solution to obtain the 4-boron-L-phenylalanine carboxyl esterification product.

[0024] S2. Add the 4-boron-L-phenylalanine carboxyl esterification product, triethylamine, (S)-BOC-4-(dihydroxyboronyl)phenylalanine, hydroxybenzotriazole, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N,N-dimethylformamide for reaction, and obtain the peptide bond coupling product after post-treatment of the reaction solution.

[0025] S3. Add the peptide bond coupling product and lithium hydroxide to an aqueous solution of methanol, heat for reaction, concentrate, extract, wash, dry, filter, and concentrate the reaction solution to obtain the demethyl-protected peptide bond coupling product; add water and a 1,4-dioxane solution of hydrochloric acid to the demethyl-protected peptide bond coupling product, stir for reaction, and obtain the boronophenylalanine dipeptide after purification of the reaction solution.

[0026] S4. Dissolve the boronophenylalanine dipeptide in PBS solution, add Na 131 I, use Iodogen as a mediator for catalytic reaction to obtain the reactant, and treat the above reactant with an activated C18 column; evaporate ethanol to dryness, dissolve with normal saline, and then elute with ethanol to obtain the sample to be separated.

[0027] S5. Separate the sample to be separated by thin layer chromatography to obtain 131 I-labeled boronophenylalanine dipeptide.

[0028] Further, in step S2, the molar ratio of the 4-boron-L-phenylalanine carboxyl esterification product, triethylamine, (S)-BOC-4-(dihydroxyboronyl)phenylalanine, hydroxybenzotriazole, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:3 - 4:1 - 1.6:1.1 - 1.6:0.9 - 1.5; the reaction time is 11 - 13 h.

[0029] Further, in step S3, the molar ratio of the peptide bond coupling product to lithium hydroxide is 1:1 - 2; the dosage ratio of the demethylated protected peptide bond coupling product, water, and the 1,4 - dioxane solution of hydrochloric acid is 2.14 mmol: 2.2 - 3 mL: 10 - 11 mL; the temperature of the heating reaction is 60 - 70 °C, and the time is 3 - 5 h.

[0030] For the preparation method of the above LAT1 - targeted boron phenylalanine dipeptide composition, weigh the above - mentioned 131 I - labeled boron phenylalanine dipeptide and stabilizer according to the above mass ratio, and mix them evenly to obtain the product.

[0031] The application of the above LAT1 - targeted boron phenylalanine dipeptide composition includes the application of the composition in the preparation of drugs for specific detection and treatment of thyroid cancer.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. The BPA dipeptide composition of the present invention is prepared from a stabilizer and 131 I - labeled BPA - BPA. After being injected into the body, this composition can prolong the survival period, is beneficial to maintaining a stable boron concentration in the body for a long time, and improves the treatment effect.

[0034] 2. The present invention first synthesizes a relatively large molecule from BPA small molecules to improve its stability and bioavailability in tumor cells, and combines iodine radioisotope labeling, providing a highly efficient, stable and low - cost labeling method. 131 I - labeled BPA - BPA can be used as a new molecular composition for nuclear medicine imaging by binding to LAT1, with high specificity and targeting ability. It can accurately observe and monitor cancer cells in the patient's body, which is beneficial for clinicians' diagnostic decisions and the subsequent treatment of patients; the iodine isotope 131 I - labeled BPA - BPA is achieved by reacting iodine isotope with a polymer to form a BPA - iodine isotope complex. The labeling process needs to be carried out under specific experimental conditions to ensure the stability and high efficiency of labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the ITLC - SG thin - layer chromatogram analysis result diagram of 131 I - labeled boron phenylalanine dipeptide before purification;

[0036] Figure 2 It is the ITLC - SG thin - layer chromatogram analysis result diagram of 131 I - boron phenylalanine dipeptide after purification;

[0037] Figure 3It is a physical diagram of the detection result of macroscopic thin-layer chromatography analysis;

[0038] Figure 4 It is a diagram of the detection result of the expression of LAT1 in tumor-bearing nude mice by immunohistochemical staining method. Specific implementation manners

[0039] The technical solutions of the present invention will be further explained below in conjunction with specific examples, comparative examples, test examples and the accompanying drawings.

[0040] In the following examples, comparative examples and test examples, unless otherwise specified, the raw materials and preparation methods used are all conventional materials and techniques in the art.

[0041] Preparation Example 1

[0042] The preparation process of borophenylalanine dipeptide (BPA-BPA) includes the following steps:

[0043]

[0044] S1. At 0 °C, 4-boron-L-phenylalanine (4.78 mmol) was added to 11 mL of methanol, and then thionyl chloride (SOCl 2 , 8.61 mol) was added dropwise, and the reaction was carried out at 0 °C for 2 h; after concentrating the reaction solution, the 4-boron-L-phenylalanine carboxyl esterification product was obtained;

[0045] S2. At room temperature, triethylamine (TEA, 18.01 mmol), (S)-BOC-4-(dihydroxyboron)phenylalanine (6.12 mmol), BPA carboxyl esterification product (5.56 mmol), hydroxybenzotriazole (HOBt, 7.45 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC hydrochloride, 6.62 mmol) were added to 15 mL of dimethylformamide (DMF), and the reaction was carried out at room temperature for 12 h; the reaction solution was poured into 50 mL of 1M HCl, and the mixed system was washed twice with ethyl acetate. The combined organic layers were washed with saturated brine, dried over magnesium sulfate after layering, and the organic layer was filtered and concentrated to obtain the peptide bond coupling product;

[0046] S3. At room temperature, add the peptide bond coupling product (4.28 mmol) and lithium hydroxide (6.28 mmol) to a solution of 36 mL of methanol and 4 mL of water, and react at 65 °C for 4 hours; concentrate the reaction solution to remove the methanol therein, and acidify the remaining aqueous layer with 20 mL of 1 M HCl solution. Extract the aqueous phase three times with ethyl acetate. Wash the combined organic layers with saturated brine, dry over magnesium sulfate, filter, and concentrate to obtain the peptide bond coupling product with the methyl protection removed; at room temperature, add 2.6 mL of water to the above-mentioned peptide bond coupling product with the methyl protection removed (2.14 mmol), and slowly add a 1,4-dioxane solution of 4 M hydrochloric acid (10.6 mL), and stir at room temperature for 2 h; purify the reaction solution by reverse-phase high-performance liquid chromatography to obtain BPA-BPA. 1 H NMR(C 18 H 22 B 2 N 2 O 7 ,400MHz,d 6 -DMSO)δ12.90(s,1H),8.86(s,2H),8.32(s,1H),7.75(d,4H),7.20(d,4H),4.72(t,1H),4.21(s,4H),3.95(t,1H),3.44(q,1H),3.19 - 3.12(m,2H),2.87(q,1H).

[0047] Preparation Example 2

[0048] The preparation process of BPA-BPA includes the following steps:

[0049] S1. The preparation process of the 4-boron-L-phenylalanine (BPA) carboxyl esterification product is the same as that in Preparation Example 1;

[0050] S2. At room temperature, add triethylamine (TEA, 16.68 mmol), (S)-BOC-4-(dihydroxyboron)phenylalanine (5.56 mmol), BPA carboxyl esterification product (5.56 mmol), hydroxybenzotriazole (HOBt 6.12 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC hydrochloride, 5.01 mmol) to 15 mL of dimethylformamide (DMF), and react at room temperature for 11 h; pour the reaction solution into 50 mL of 1 M HCl, and wash the mixed system twice with ethyl acetate. Wash the combined organic layers with saturated brine, dry over magnesium sulfate after layering, filter the organic layer and concentrate to obtain the peptide bond coupling product;

[0051] S3. At room temperature, add the peptide bond coupling product (4.28 mmol) and lithium hydroxide (4.28 mmol) to a solution of 36 mL of methanol and 4 mL of water, and react at 60 °C for 5 h; concentrate the reaction solution to remove the methanol therein, and acidify the remaining aqueous layer with 20 mL of 1 M HCl solution. Extract the aqueous phase three times with ethyl acetate, wash the combined organic layers with saturated brine, dry over magnesium sulfate, filter, and concentrate to obtain the peptide bond coupling product with the methyl protection removed; at room temperature, add 2.2 mL of water to the above peptide bond coupling product with the methyl protection removed (2.14 mmol), and slowly add a 1,4-dioxane solution of 4 M hydrochloric acid (10 mL), and stir at room temperature for 2 h; purify the reaction solution by reverse-phase high performance liquid chromatography to obtain BPA-BPA; 1 The HNMR results are the same as those in Preparation Example 1.

[0052] Preparation Example 3

[0053] The preparation process of BPA-BPA includes the following steps:

[0054] S1. The preparation process of the 4-boron-L-phenylalanine (BPA) carboxyl esterification product is the same as that in Preparation Example 1;

[0055] S2. At room temperature, add triethylamine (TEA, 22.24 mmol), (S)-BOC-4-(dihydroxyboryl)phenylalanine (5.56 mmol), BPA carboxyl esterification product (5.56 mmol), hydroxybenzotriazole (HOBt 8.89 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC hydrochloride, 8.34 mmol) to 15 mL of dimethylformamide (DMF), and react at room temperature for 13 h; pour the reaction solution into 50 mL of 1 M HCl, and wash the mixed system twice with ethyl acetate. Wash the combined organic layers with saturated brine, dry over magnesium sulfate after separation, filter the organic layer and concentrate to obtain the peptide bond coupling product;

[0056] S3. At room temperature, add the peptide bond coupling product (4.28 mmol) and lithium hydroxide (8.56 mmol) to a solution of 36 mL of methanol and 4 mL of water, and react at 70 °C for 3 h; concentrate the reaction solution to remove the methanol therein, and acidify the remaining aqueous layer with 20 mL of 1 M HCl solution. Extract the aqueous phase three times with ethyl acetate, wash the combined organic layers with saturated brine, dry over magnesium sulfate, filter, and concentrate to obtain the peptide bond coupling product with the methyl protection removed; at room temperature, add 3 mL of water to the above peptide bond coupling product with the methyl protection removed (2.14 mmol), and slowly add a 1,4-dioxane solution of 4 M hydrochloric acid (11 mL), and stir at room temperature for 2 h; purify the reaction solution by reverse-phase high performance liquid chromatography to obtain BPA-BPA;1 The 1H NMR result was the same as that in Preparation Example 1.

[0057] Preparation Example 4

[0058] Prepared by the Iodogen iodine labeling method 131 125I-labeled boron phenylalanine dipeptide ( 131 125I-labeled BPA-BPA); its structure is

[0059]

[0060] The 131 Preparation process of 125I-labeled BPA-BPA includes the following steps:

[0061] 1) Preparation of Iodogen-coated tube: Weigh 2 mg of Iodogen powder, dissolve it in 2 mL of dichloromethane solution according to a ratio of 1:1. After complete dissolution, aliquot 50 μL into different EP tubes; blow the bottom of each EP tube with nitrogen to make it volatilize as soon as possible, and a uniform thin film is formed at the bottom of the tube; seal the mouth of each EP tube with a sealing film. Store it in a -20 °C refrigerator for later use;

[0062] 2) Take Na 131 125I solution, and measure its radioactivity and volume using a radioactivity meter;

[0063] 3) Dissolve 1 mg of BPA-BPA (abbreviated as BPA dipeptide) in 1 mL of phosphate buffer (PBS, concentration 1 mol / L, pH 7.4), take 20 μL and put it into a clean EP tube, named EP-1;

[0064] 4) Add 185 MBq (<1 mL) of Na 131 125I to EP-1, use Iodogen as a mediator to catalyze the reaction, mix the reaction vigorously for 10 min (constant temperature reaction instrument, vortex mixer (automatic mode): temperature 20 - 35 °C, RPM 600), and obtain 125I-labeled BPA-BPA before purification; 131 125I-labeled BPA-BPA;

[0065] 5) Activate the C18 column with 5 mL of ethanol, and treat the 125I-labeled BPA-BPA before purification through the activated C18 column. Evaporate the ethanol to dryness at 90 °C, then redissolve it with physiological saline, and then take 2 mL of ethanol for elution to obtain 125I-labeled BPA-BPA after column purification; 131 125I-labeled BPA-BPA; 131 125I-labeled BPA-BPA;

[0066] 6) Use ITLC-SG thin layer chromatography for the 125I-labeled BPA-BPA after column purification 131Separation and purification of I-labeled BPA-BPA was carried out with ethyl acetate as the developing agent. A 3200 radiation probe was used (the labeled BPA dipeptide was absorbed with a capillary with a diameter of 0.5 mm, spotted at the zero point of the pre-prepared chromatography paper. After drying, the chromatography paper below the zero point was placed into a developing tank filled with ethyl acetate to allow the liquid to move up along the chromatography paper); when the developing agent moved up to about 10 cm on the chromatography paper, the chromatography paper was taken out, dried, and I-labeled BPA-BPA was separated and obtained. 131 I-labeled BPA-BPA.

[0067] Test Example 1

[0068] 1. Analysis of I-labeled BPA-BPA before purification was carried out by ITLC-SG thin-layer chromatography technology, and the results are shown in 131 . Figure 1 .

[0069] Figure 1 Figure for the radioactive TLC analysis results of I-labeled BPA-BPA before purification. In 131 , two radioactive peaks were observed: the first free I peak appeared at a retention time of 0.34 min, with a peak width of 11.0 s and an area of 127690.1 mAU·s, accounting for 59.51% of the total area. The second peak was the radioactive nuclide-labeled compound peak, which appeared at a retention time of 0.89 min, with an area of 29345.4 mAU·s, accounting for 13.68% of the total area. Figure 1 131

[0070] 2. Detection by ITLC-SG thin-layer chromatography analysis

[0071] (1) Radiochemical purity detection of I-BPA-BPA obtained in step 5) was carried out by ITLC-SG thin-layer chromatography. A gamma-ray counter (3200 radiation probe) was used to measure its radioactivity, monitor the formed peaks, and calculate the percentage of each peak to evaluate the purity of BPA-BPA. 131 Figure 2 Figure for the radioactive TLC analysis results of I-labeled BPA-BPA after column purification in step 5). Moreover, the radiochemical purity of this compound was accurately calculated to be (75.54 ± 3.9)%. 131

[0072] (2) The visualization results obtained by thin-layer chromatography in step 6) are as shown in Figure 3 .

[0073] As can be seen from Figure 3 , two obvious bands were seen in the thin-layer chromatography (TLC) analysis. The running distance of the sample in TLC is mainly affected by its polarity. The iodination product ( 131 ​​​​I-labeled BPA-BPA exhibits higher non-polar characteristics due to the addition of iodine atoms, resulting in its moving further in a non-polar solvent system relative to BPA-BPA. Therefore, based on the migration distance on thin-layer chromatography, the shorter band was identified as unreacted BPA-BPA, while the longer band was considered the iodination product( 131 I-labeled BPA-BPA).

[0074] Example 1

[0075] A LAT1-targeted boron phenylalanine dipeptide composition, comprising the following components: 131 I-labeled BPA-BPA, a stabilizer; the 131 mass ratio of I-labeled BPA-BPA to the stabilizer is 1:0.5; the structural formula of the stabilizer is as follows:

[0076]

[0077] The preparation process of the stabilizer includes the following steps:

[0078]

[0079] (1) Dissolve quercetin (10 mmol) in 25 mL of tetrahydrofuran (THF), stir at 72 °C for 10 min to obtain a mixture, then add 4-boron-L-phenylalanine (BPA, 15 mmol) to the mixture, and react at 72 °C for 7 h; after the reaction is completed, concentrate the reaction solution to remove tetrahydrofuran and then purify by distillation to obtain intermediate 1; 1H NMR (C 24 H 18 BNO 9 , 400 MHz, d6-DMSO) δ 16.45 (s, 1H), 12.88 (s, 1H), 10.68 (s, 1H), 10.20 (s, 1H), 8.72 (s, 1H), 7.75 (d, 2H), 7.20 (d, 2H), 7.04 (t, 1H), 6.60 (d, 1H), 6.52 (s, 1H), 6.03 (s, 1H), 5.94 (s, 1H), 4.18 (t, 1H), 3.42 (q, 1H), 3.17 (q, 1H); MS (ESI) m / z = 476.11 [M + H] + . The above results confirm that the obtained product is the target product.

[0080] (2) Add intermediate 1 (10 mmol) to 25 mL of dichloromethane (DCM), then add N,N'-dicyclohexylcarbodiimide (DCC, 15 mmol) and L-glutamine (12 mmol), and react at 28 °C for 17 h; filter the reaction solution, concentrate the filtrate to remove dichloromethane, and purify the crude product by reverse-phase high-performance liquid chromatography to obtain the stabilizer. 1H NMR (C 29 H 26 BN 3 O 11 , 400 MHz, d6-DMSO) δ 16.45 (s, 1H), 12.66 (s, 1H), 10.68 (s, 1H), 10.20 (s, 1H), 8.86 (s, 2H), 8.32 (s, 1H), 7.75 (d, 2H), 7.20 (d, 2H), 7.04 - 7.02 (m, 3H), 6.60 (d, 1H), 6.52 (s, 1H), 6.03 (s, 1H), 5.94 (s, 1H), 4.56 - 4.54 (m, 1H), 3.95 (t, 1H), 3.42 (q, 1H), 3.17 (q, 1H), 2.07 - 2.05 (m, 4H); MS (ESI) m / z = 604.17 [M + H] + . The above results confirm that the obtained product is the target product.

[0081] The preparation method of the LAT1-targeted boron phenylalanine dipeptide composition specifically includes the following steps:

[0082] Weigh the 131 I-labeled BPA-BPA and the stabilizer obtained in Preparation Example 4 according to the above mass ratio, and mix them evenly to obtain the BPA dipeptide composition, denoted as the BPA dipeptide composition.

[0083] Example 2

[0084] The LAT1-targeted boron phenylalanine dipeptide composition includes the following components: 131 I-labeled BPA-BPA, stabilizer; the 131 mass ratio of I-labeled BPA-BPA to the stabilizer is 1:0.4; the structural formula of the stabilizer is the same as that in Example 1.

[0085] The preparation process of the stabilizer includes the following steps:

[0086] (1) Dissolve quercetin (10 mmol) in 25 mL of tetrahydrofuran (THF), stir at 70 °C for 10 min to obtain a quercetin solution, then add 4-boron-L-phenylalanine (BPA, 10 mmol) to the quercetin solution, and continue to react at 70 °C for 8 h; after the reaction is completed, concentrate the reaction solution to remove tetrahydrofuran and then purify it by distillation to obtain intermediate 1; the 1H NMR of intermediate 1 is the same as that in Example 1.

[0087] (2) Add intermediate 1 (10 mmol) to 25 mL of dichloromethane (DCM), then add N,N'-dicyclohexylcarbodiimide (DCC, 12 mmol) and L-glutamine (10 mmol), and react at 25 °C for 18 h; filter the reaction solution, concentrate the filtrate to remove dichloromethane, and purify the crude product by reverse-phase high-performance liquid chromatography technology to obtain the stabilizer. The 1H NMR of the stabilizer is the same as that in Example 1.

[0088] The preparation method of the boron phenylalanine dipeptide composition targeting LAT1 is the same as that in Example 1.

[0089] Example 3

[0090] The boron phenylalanine dipeptide composition targeting LAT1 includes the following components: 131 I-labeled BPA-BPA, stabilizer; the 131 mass ratio of I-labeled BPA-BPA to the stabilizer is 1:0.6; the structural formula of the stabilizer is the same as that in Example 1.

[0091] The preparation process of the stabilizer includes the following steps:

[0092] (1) Dissolve quercetin (10 mmol) in 25 mL of tetrahydrofuran (THF), stir at 75 °C for 10 min to obtain a mixture, then add 4-boron-L-phenylalanine (BPA, 20 mmol) to the mixture, and continue to react at 75 °C for 6 h; after the reaction is completed, concentrate the reaction solution to remove tetrahydrofuran and then purify it by distillation to obtain intermediate 1; the 1H NMR of intermediate 1 is the same as that in Example 1.

[0093] (2) Add intermediate 1 (10 mmol) to 25 mL of dichloromethane (DCM), then add N,N'-dicyclohexylcarbodiimide (DCC, 20 mmol) and L-glutamine (15 mmol), and react at 30 °C for 16 h; filter the reaction solution, concentrate the filtrate to remove dichloromethane, and purify the crude product by reverse-phase high-performance liquid chromatography technology to obtain the stabilizer. The 1H NMR of the stabilizer is the same as that in Example 1.

[0094] The preparation method of the boron phenylalanine dipeptide composition targeting LAT1 is the same as that in Example 1.

[0095] Comparative Example 1

[0096] The boron phenylalanine dipeptide targeting LAT1 was basically the same as Example 1, except that the stabilizer was omitted.

[0097] Test Example

[0098] Test procedure: (1) Establish a tumor-bearing nude mouse model of human medullary thyroid carcinoma TT cell line: Select 51 BALB / c mice, randomly divide them into Example 1 group, Example 2 group, Example 3 group, and Comparative Example 1 group. Each of the above groups was further divided into four batches (for example, the Example 1 group was divided into four batches), with 3 mice in one batch and 12 mice in each group. In addition, a model group with 3 mice was set up. The human medullary thyroid tumor TT cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (placed in a cell culture flask) until 90% confluence. Add 2 mL of trypsin to the cell culture flask and gently shake, then add 6 mL of DMEM / F12 medium to the cell culture flask and pipette the flask wall to collect the cell suspension. Centrifuge at 1000 rpm for 5 min at 4 °C, discard the medium, resuspend the precipitate with DMEM / F12 medium, then continue to centrifuge at 1000 rpm for 5 min at 4 °C, discard the medium, and then add DMEM / F12 medium to resuspend to obtain a cell suspension with a concentration of 5×10 7 cells / mL. Aspirate 0.2 mL of the cell suspension with a syringe and inject it into the left axilla of the mouse. When the tumor volume reaches 1.2 - 1.5 cm 3 , it is used for subsequent experiments.

[0099] (2) To detect the expression of LAT1 in the tumor-bearing nude mouse model, take the tumor sections of the tumor-bearing nude mice of the established human medullary thyroid carcinoma TT cell line in the model group for immunohistochemical staining, and the staining results are shown in Figure 4 .

[0100] Result analysis: L-type amino acid transporters (LATs) are members of the solute carrier family 7 (SLC7), and among them, LAT1 (also known as SLC7A5) is a heteromeric amino acid transporter (HAT), and its function and expression are of great significance under various physiological and pathological conditions. Especially in the tumor microenvironment, the expression of LAT1 is closely related to the processes of tumor growth, invasion, and metastasis.

[0101] Figure 4The immunohistochemical staining results of LAT1 in tumor-bearing nude mice are shown. The immunohistochemical staining results show that SLC7A5 is mainly localized in the cytoplasm and appears as distinct yellow to brown color. This color indicates positive immunoreactivity in the cytoplasm and cell membrane, suggesting the presence of a large amount of SLC7A5 protein in these regions and its possible involvement in the active transport process of amino acids. LAT1 has a specific distribution pattern in the tumor microenvironment, with differences in staining intensity among different cells. Some cells show strong signals while some show weak signals. The staining pattern of the sections is consistent, indicating the stability and reliability of the experimental operation and also providing the distribution of SLC7A5 protein in the tumor microenvironment.

[0102] (3) Experimental procedure: Select the tumor-bearing nude mice established with the above-mentioned human medullary thyroid carcinoma TT cell line and fix them in the prone position. Expose the tails of the nude mice, wipe and disinfect them with alcohol cotton balls. Then, the compositions prepared in Examples 1-3 and Comparative Example 1 are respectively compounded with normal saline to obtain the injection solutions to be used. Each group of nude mice is injected with the composition injection solution with a radioactivity of 20 μCi / 200 μL through the tail vein using a 1 mL disposable syringe (for example, the group of Example 1 is injected with the injection solution to be used obtained by compounding the composition prepared in Example 1 and normal saline). After injection, press the puncture point with an alcohol cotton ball.

[0103] The nude mice are sacrificed at 1 h, 3 h, 5 h, and 24 h after injection respectively. Remove the tumors and other important organs and measure their weights; use a gamma counter to measure the radioactive counts of each group of nude mice at different time points. After radioactive decay correction, calculate the percentage of the injection dose per gram of tissue in each organ (%ID / g). The results are shown in Tables 1 to 4, and the results are expressed in the form of mean ± standard deviation (means ± SD).

[0104] %ID / g = Radioactive count of each tissue organ group / (Total injected radioactive count × Tissue weight) × 100%.

[0105] Table 1 In vivo biodistribution of the composition obtained in Example 1 in BALB / c nude mice bearing medullary thyroid carcinoma %ID / g (x ± s, n = 3)

[0106]

[0107]

[0108] Table 2 In vivo biodistribution of the composition obtained in Example 2 in BALB / c nude mice bearing medullary thyroid carcinoma %ID / g (x ± s, n = 3)

[0109]

[0110] Table 3 In vivo biodistribution of the composition obtained in Example 3 in BALB / c nude mice bearing medullary thyroid carcinoma %ID / g (x±s, n = 3)

[0111]

[0112] Table 4 In vivo biodistribution of the boron phenylalanine dipeptide obtained in Comparative Example 1 in BALB / c nude mice bearing medullary thyroid carcinoma %ID / g (x±s, n = 3)

[0113]

[0114] Result analysis:

[0115] The longer the accumulation time of the radiopharmaceutical or labeled compound in the tumor, the longer the residence time of the drug in the target tissue, which helps the drug to exert a more lasting therapeutic effect at the target site. On the contrary, if the accumulation time of the radiopharmaceutical in the target tissue is too short, it may lead to insufficient drug efficacy and failure to achieve the expected therapeutic effect.

[0116] One hour after intravenous injection of the BPA dipeptide compositions of Examples 1-3, the radioactivity mainly accumulated in the stomach, kidneys and tumor sites, and the radioactivity gradually decreased with the extension of time. Among them, in Example 1 at 5 h, the radioactivity in the stomach decreased to 19.34% ID / g, which was significantly lower than that at 1 h; the radioactivity in the kidney decreased to 1.24% ID / g. The radioactivity uptake in the tumor site at the 1 h time point was 6.76% ID / g, and the tumor radioactivity uptake was only 1.85% ID / g at 24 h.

[0117] In Comparative Example 1, after injection of 131 1h after I-BPA-BPA, the radioactivity mainly accumulated in the stomach and kidney sites, which were 65.68% ID / g and 9.19% ID / g respectively. After that, at 5h, the radioactivity in the stomach decreased to 11.30% ID / g, and the kidney site decreased to 0.98% ID / g. The radioactivity uptake in the tumor site at the 1 h time point was 6.14% ID / g, and the tumor radioactivity uptake was only 0.44% ID / g at 24 h. Comparative analysis found that at 24 h after injection, the radioactivity in the tumor site of Example 1 was still much higher than that of Comparative Example 1. This is because a stabilizer was added to the BPA dipeptide composition of Example 1. The above results show that the BPA dipeptide composition of the present invention can extend the survival period after being injected into the body, is conducive to maintaining a stable boron concentration for a long time, and improves the therapeutic effect.

[0118] The above are only the preferred embodiments of the present invention, not limited to the above examples. For those skilled in the art, various changes and modifications can be made under the principle of the present invention. Any modifications, improvements, etc. should be regarded as within the protection scope of the present invention.

Claims

1. A borophenylalanine dipeptide composition based on LAT1 targeting, characterized in that: The components include: 131 I-labeled borophenylalanine dipeptide, stabilizer; 131 The mass ratio of I-labeled borophenylalanine dipeptide to the stabilizer is 1:0.4-0.6; 131 The structural formulas of the I-labeled borophenylalanine dipeptide and the stabilizer are shown below:

2. The LAT1-targeted borophenylalanine dipeptide composition according to claim 1, characterized in that: The preparation process of the stabilizer comprises the following steps: (1) adding 4-boron-L-phenylalanine to a quercetin solution, heating for reaction, and after the reaction is completed, purifying the reaction solution to obtain intermediate 1; (2) The intermediate 1 is added to a solvent, and N,N'-dicyclohexylcarbodiimide and L-glutamine are added to react. After the reaction is completed, the reaction solution is filtered, concentrated, and purified to obtain the product.

3. The LAT1-targeted borophenylalanine dipeptide composition according to claim 2, characterized in that: In step (1), the molar ratio of quercetin to 4-boron-L-phenylalanine is 1:1-2, and the concentration of the quercetin solution is 0.4 mol / L.

4. The LAT1-targeted borophenylalanine dipeptide composition according to claim 2, characterized in that: The heating temperature in step (1) is 70-75° C. and the heating time is 6-8 hours.

5. The LAT1-targeted borophenylalanine dipeptide composition according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate 1, N,N'-dicyclohexylcarbodiimide and L-glutamine is 1:1.2-2:1-1.5; and the reaction time is 16-18 hours.

6. The LAT1-targeted borophenylalanine dipeptide composition according to claim 1, characterized in that: Said 131 The preparation process of I-labeled borophenylalanine dipeptide comprises the following steps: S1. Adding 4-boron-L-phenylalanine and thionyl chloride to methanol to react, and concentrating the reaction solution to obtain a carboxyl esterification product of 4-boron-L-phenylalanine; S2. adding the carboxyl esterification product of 4-boryl-L-phenylalanine, triethylamine, (S)-BOC-4-(dihydroxyboryl)phenylalanine, hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N,N-dimethylformamide for reaction, and post-treating the reaction solution to obtain a peptide bond coupling product; S3. adding the peptide bond coupling product and lithium hydroxide to an aqueous solution of methanol, heating the reaction, concentrating the reaction solution, extracting, washing, drying, filtering, and concentrating to obtain a demethylated peptide bond coupling product; Adding water and a 1,4-dioxane solution of hydrochloric acid to the demethylated peptide bond coupling product, stirring for reaction, and purifying the reaction solution to obtain borophenylalanine dipeptide; S4. Dissolve the borophenylalanine dipeptide in PBS solution and add Na 131 I, using Iodogen as a medium to carry out a catalytic reaction to obtain a reactant, and treating the reactant with an activated C18 column; evaporating ethanol to dryness, dissolving with physiological saline, and then eluting with ethanol to obtain a sample to be separated; S5. Use thin layer chromatography to separate the sample to be separated, and obtain 131 I-labeled borophenylalanine dipeptide.

7. The LAT1-targeted borophenylalanine dipeptide composition according to claim 6, characterized in that: In step S2, the molar ratio of the 4-boron-L-phenylalanine carboxyl esterification product, triethylamine, (S)-BOC-4-(dihydroxyboronyl)phenylalanine, hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:3-4:1-1.6:1.1-1.6:0.9-1.5; and the reaction time is 11-13h.

8. The LAT1-targeted borophenylalanine dipeptide composition according to claim 6, characterized in that: In step S3, the molar ratio of the peptide bond coupling product and lithium hydroxide is 1:1-2; the dosage ratio of the demethylated peptide bond coupling product, water, and 1,4-dioxane solution of hydrochloric acid is 2.14mmol:2.2-3mL:10-11mL; the temperature of the heating reaction is 60-70°C, and the time is 3-5h.

9. The method for preparing the LAT1-targeted borophenylalanine dipeptide composition according to any one of claims 1 to 8, characterized in that: According to the above mass ratio, weigh the 131 The I-labeled borophenylalanine dipeptide and the stabilizer are mixed evenly to obtain the product.

10. The use of the LAT1-targeted borophenylalanine dipeptide composition according to any one of claims 1 to 8, characterized in that: The composition is used in preparing drugs for specific detection and treatment of thyroid cancer.

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