Use of boron-containing amino acid analogs in the preparation of medicaments for treating tumors

Abstract

The application belongs to the technical field of boron-containing functional molecules, and relates to application of a boron-containing amino acid analogue in preparation of a medicine for treating tumors. A series of boron-containing compounds are found, as shown in formula I. In the application, in-vitro cell boron uptake of the compounds is detected by inductively coupled plasma emission spectrometry, and compounds C1 and F4, which have good solubility and have cell boron uptake better than that of BPA, are screened out, and in-vivo boron uptake of the compounds is detected in B16F10 tumor-bearing mice, and the result shows that both the two compounds have higher boron accumulation at tumor sites in vivo, which proves that the two compounds have potential for application in boron neutron capture therapy.

Description

Technical Field

[0001] This invention belongs to the field of boron-containing functional molecule technology, and relates to the application of boron-containing amino acid analogs in the preparation of drugs for treating tumors. Background Technology

[0002] Trimethylamine borane is a representative class of boron-containing compounds. Its molecules contain both a boron center and a carboxyl functional group, exhibiting both structural stability and the potential for further derivatization. Using this class of compounds as a parent compound, a series of structurally different derivatives can be constructed by modifying the carboxyl site or by changing the amine ligand coordinated to the boron center.

[0003] While the currently approved 4-boron-L-phenylalanine (BPA) has certain tumor targeting properties, its water solubility is poor, requiring fructose to aid dissolution during use, which limits its clinical application. Therefore, there is an urgent need to find boron-containing amino acid analogs that can improve water solubility while further enhancing its in vivo and in vitro boron uptake. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a boron-containing amino acid analog, its preparation method, and its application in boron neutron capture therapy.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] This invention discloses the use of boron-containing amino acid analogs in the preparation of drugs for treating tumors, wherein the boron-containing amino acid analogs have the structure shown in Formula I:

[0007] ;

[0008] in,

[0009] Y is selected from -OH, -OR1, or -NHR2;

[0010] R1 and R2 are independently selected from C1-C3 alkyl groups;

[0011] L is selected from , , or ;

[0012] R3, R4, and R5 are independently selected from hydrogen, C1-C3 alkyl, hydroxyethyl, or cyclohexyl;

[0013] m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from C1-C3 alkyl groups;

[0014] R7 is selected from hydrogen or C1-C3 alkyl;

[0015] The tumors mentioned are melanoma, human brain astrocytoma, human glioblastoma, or human pharyngeal squamous cell carcinoma.

[0016] In some embodiments, the boron-containing amino acid analog has the structure shown in Formula I:

[0017] ;

[0018] in,

[0019] Y is selected from -OH, -OR1, or -NHR2;

[0020] R1 and R2 are independently selected from methyl or ethyl;

[0021] L is selected from , , or ;

[0022] R3, R4, and R5 are independently selected from hydrogen, methyl, ethyl, hydroxyethyl, or cyclohexyl;

[0023] m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from methyl or ethyl;

[0024] R7 is selected from hydrogen, methyl, or ethyl.

[0025] In some embodiments, the boron-containing amino acid analog has the structure shown in Formula I:

[0026] ;

[0027] in,

[0028] Y is selected from -OH, -OR1, or -NHR2;

[0029] R1 and R2 are independently selected from methyl or ethyl;

[0030] L is selected from , , or ;

[0031] R3, R4, and R5 are independently selected from hydrogen, methyl, hydroxyethyl, or cyclohexyl;

[0032] m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from methyl groups;

[0033] R7 is selected from hydrogen or methyl.

[0034] In some embodiments, the boron-containing amino acid analogue is selected from compounds with any of the following structures:

[0035] .

[0036] In some embodiments, the boron-containing amino acid analogue is selected from compounds with any of the following structures:

[0037] .

[0038] In some embodiments, the boron-containing amino acid analogue is selected from compounds with the following structures:

[0039] .

[0040] In some embodiments, the melanoma is a human malignant melanoma or a mouse melanoma.

[0041] In some embodiments, the human malignant melanoma is human malignant melanoma cell A375.

[0042] In some embodiments, the mouse melanoma is mouse melanoma cell B16F10.

[0043] In some embodiments, the human astrocytoma is human astrocytoma cell U87.

[0044] In some embodiments, the human glioblastoma is a glioblastoma cell line LN229.

[0045] In some embodiments, the human laryngeal squamous cell carcinoma is a human laryngeal squamous cell carcinoma cell line FaDu.

[0046] Beneficial effects:

[0047] This invention discovered a series of boron-containing compounds. First, their in vitro cellular boron uptake was detected by inductively coupled plasma atomic emission spectrometry (ICP-AES). Compounds C1 and F4, which have good solubility and superior cellular boron uptake compared to BPA, were screened out. In vivo boron uptake was then detected in B16F10 tumor-bearing mice. The results showed that both compounds had higher boron accumulation at the tumor site in vivo, demonstrating their potential for application in boron neutron capture therapy. Attached Figure Description

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0049] Figure 1 The boron uptake of BPA and boron-containing amino acid analogs in human malignant melanoma cells A375 is given.

[0050] Figure 2The boron uptake of BPA and boron-containing amino acid analogs in mouse melanoma cells B16F10 is given.

[0051] Figure 3 The boron uptake of BPA and boron-containing amino acid analogs in human glioblastoma cells U87 is given.

[0052] Figure 4 The boron uptake of BPA and boron-containing amino acid analogs in human glioblastoma cells LN229 is represented.

[0053] Figure 5 The boron uptake of BPA and boron-containing amino acid analogs in human laryngeal squamous cell carcinoma cells (FaDu) is represented.

[0054] Figure 6 The graph shows the hemolytic activity data of BPA and boron-containing amino acid analogs at a final concentration of 25 mg / mL.

[0055] Figure 7 The values ​​represent the in vivo boron uptake of BPA and its boron-containing amino acid analogs C1, F4, and F5 in a mouse subcutaneous melanoma model.

[0056] Figure 8 The diagram shows the structure of organic amine raw materials and their corresponding target boron-containing amino acid analogs. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. All equivalent substitutions, simple modifications, condition adjustments, or conventional improvements made by those skilled in the art under the guidance of the present invention should fall within the protection scope of the present invention.

[0058] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified. The products obtained after the reaction can be purified according to their properties using conventional methods such as vacuum concentration, extraction, washing, drying, recrystallization, and column chromatography.

[0059] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0060] 1. The compounds described in the examples can be prepared using naturally abundant boron raw materials or boron-10 enriched raw materials as needed.

[0061] 2. For ease of description, some intermediates and target compounds are numbered as follows:

[0062] Intermediate A: Trimethylamine cyanoborane;

[0063] Intermediate B: A nitrile onium salt intermediate obtained by activation of trimethylamine cyanoborane;

[0064] Compound C1: Trimethylamine carboxylate borane;

[0065] Compound D1: (methoxycarbonyl)trimethylboramine;

[0066] Compound E1: ((ethylamino)carbonyl)trimethylboramine;

[0067] Compound F: A class of amine ligand-modified carboxylate boronane derivatives prepared by ligand exchange method.

[0068] 3. The target compounds described in the examples can be separated and purified using appropriate methods based on their polarity and stability. For compounds with good crystallinity, recrystallization can be performed using solvent systems such as dichloromethane / hexane, dichloromethane / diethyl ether, or ethyl acetate / petroleum ether; for products with poor crystallinity, silica gel column chromatography can be used for purification.

[0069] 4. The structures of the target compounds obtained in the examples can be confirmed by nuclear magnetic resonance, mass spectrometry, or other conventional analytical methods. For samples subsequently used in boron neutron capture therapy (BNCT) research, quality evaluation can also be performed as needed using methods such as boron content determination.

[0070] 5. The 4-boron-L-phenylalanine (BPA) used in the examples was manufactured by Anaiji Chemicals, with a purity of 98% and batch number HMDYRRU1. Its structural formula is as follows: .

[0071] Example 1: Preparation of Trimethylamine Cyanoborane (Intermediate A)

[0072]

[0073] The trimethylamine cyanoborane used in the examples is commercially available or can be prepared by the following method:

[0074] Sodium cyanoborohydride (75.40 g, 1.20 mol) and 800 mL of anhydrous tetrahydrofuran were added to a reaction flask equipped with a stirrer and stirred until uniformly dispersed. Trimethylamine hydrochloride (128.06 g, 1.34 mol) was then slowly added to the system. After the gas release from the reaction system significantly slowed down, another 200 mL of anhydrous tetrahydrofuran was added, and the reaction was continued with stirring at room temperature for 12 hours. After the reaction was complete, the inorganic salt solid was removed by filtration. The filter cake was washed with tetrahydrofuran, and the filtrates were combined and the solvent was removed under reduced pressure to obtain trimethylamine cyanoborane, denoted as intermediate A.

[0075] Example 2: Preparation of trimethylamine carboxylate borane (compound C1)

[0076]

[0077] The trimethylamine borane used in the examples is commercially available or can be prepared by the following method:

[0078] Intermediate A (32.53 g, 0.33 mol) and 2M triethyloxonium tetrafluoroborate diethyl ether solution (240 mL, containing 0.48 mol of triethyloxonium tetrafluoroborate) were added to a three-necked flask equipped with a reflux condenser and a stirrer. The mixture was heated under reflux for 12 hours to obtain intermediate B.

[0079] After the reaction was complete, the reaction system was cooled, and the solvent was removed under reduced pressure. The resulting intermediate B solid was added to 200 mL of water and stirred at room temperature for 24 hours to hydrolyze it. After the hydrolysis reaction was complete, the reaction solvent was removed under reduced pressure, and then 50 mL of dichloromethane was added to dissolve the remaining solid. The insoluble inorganic salt solid was removed by filtration, and the filter cake was washed with dichloromethane. The filtrates were combined and the solvent was removed under reduced pressure to obtain trimethylamine carboxylate borane, denoted as compound C1, which is a white solid.

[0080] The NMR and mass spectrometry data for compound C1 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ10.20 (s, 1H), 2.75 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 52.14 . 11 B NMR(128 MHz, Chloroform-d) δ -9.86 (t, J = 100.2 Hz). HRMS (ESI) m / z: [M+Na]+calcd for [C4H 12 BNO2Na]: 140.0853; Found: 140.0851.

[0081] Example 3: Preparation of (methoxycarbonyl)trimethylborane (compound D1)

[0082]

[0083] Compound C1 (1.156 g, 0.01 mol) was dissolved in 100 mL of dichloromethane and cooled to 0 °C in an ice bath. 4-Dimethylaminopyridine (DMAP, 0.122 g, 0.001 mol) and a suitable amount of the organic base triethylamine (2.024 g, 0.02 mol) were added, followed by the dropwise addition of methyl chloroformate (0.945 g, 0.01 mol). The reaction was continued at 0 °C to room temperature. After the reaction was complete, water was added to quench the reaction mixture. The liquid was separated, and the organic phase was washed, dried, and concentrated to obtain the crude product. Further purification by recrystallization yielded (methoxycarbonyl)trimethylboronamine, denoted as compound D1.

[0084] The NMR and mass spectrometry data for compound D1 are as follows: 1 H NMR (400 MHz, Chloroform-d) δ3.55 (s, 3H), 2.76 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 52.17, 47.94. 11 B NMR (128 MHz, Chloroform-d) δ -9.49 (t, J = 99.8 Hz). HRMS (ESI) m / z: [M+Na]+ calcd for [C5H 14 BNO2Na]: 154.1010; Found: 154.1006.

[0085] Example 4: Preparation of ((ethylamino)carbonyl)trimethylboramine (compound E1)

[0086]

[0087] Intermediate B was prepared according to the experimental method of Example 2.

[0088] Intermediate B was dissolved in dichloromethane, and a 1 mol / L sodium hydroxide aqueous solution was added dropwise to the reaction solution. After the pH of the reaction solution was monitored using pH paper until it became alkaline, the reaction was carried out in an ice bath for 1 hour. Under alkaline conditions, intermediate B was converted to the corresponding ((ethylamino)carbonyl)trimethylboramine. After the reaction was complete, the organic phase was separated, dried, and concentrated to obtain the crude product. If necessary, further purification was performed using vacuum distillation, recrystallization, or column chromatography to prepare ((ethylamino)carbonyl)trimethylboramine, denoted as compound E1.

[0089] The NMR and mass spectrometry data for compound E1 are as follows: 1H NMR (400 MHz, Chloroform-d) δ5.53 (s, 1H), 3.26 (qd, J = 7.3, 5.6 Hz, 2H), 2.76 (s, 9H), 1.09 (t, J = 7.3Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 52.20, 31.85, 15.16. 11 B NMR (128MHz, Chloroform-d) δ -8.24 (t, J = 97.1 Hz). HRMS (ESI) m / z: [M+H]+ calcd for[C6H 18 BN2O]: 145.1507; Found: 145.1503.

[0090] Example 5: Preparation of the target compound by ligand exchange method

[0091]

[0092] The structures of organic amine raw materials and corresponding target boron-containing amino acid analogs are as follows: Figure 8 As shown.

[0093] Compound C1 (1.0 mmol, 1.0 equivalent) and the corresponding organic amine (2.2 equivalent) were dissolved in 4.0 mL of N,N-dimethylformamide solution and reacted at 110°C for 24 hours under argon protection. After the reaction was completed, the solvent was removed, and the product was purified by recrystallization or silica gel column chromatography according to its properties to obtain the corresponding ligand exchange product, a boron-containing amino acid analog, denoted as compound F.

[0094] The NMR and mass spectrometry characterization data of the target compound prepared by the above ligand exchange method are as follows:

[0095] Compound F1: 1 H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.92 – 7.81 (m,2H), 6.81 – 6.69 (m, 2H), 3.07 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 155.41, 146.99, 107.06, 39.54. 11 B NMR (128 MHz, DMSO-d6) δ -12.57 . HRMS (ESI) m / z:[M+Na]+ calcd for [C8H13 BN2O2Na]: 203.0962; Found: 203.0965.

[0096] Compound F2: 1 H NMR (400 MHz, Methanol-d4) 3.91 (ddd, J = 12.9, 6.5, 3.3Hz, 2H), 3.80 (ddd, J = 12.9, 7.0, 3.1 Hz, 2H), 3.37 – 3.28 (m, 2H), 2.92(ddd, J = 13.1, 6.6, 3.1 Hz, 2H), 2.84 (s, 3H). 13 C NMR (101 MHz, Methanol-d4)δ 60.84, 57.30, 46.71. 11 B NMR (128 MHz, Methanol-d4) δ -11.78 (t, J =101.0 Hz). HRMS (ESI) m / z: [M+Na]+ calcd for [C6H 14 BNO3Na]: 182.0961; Found:182.0959.

[0097] Compound F3: 1 H NMR (400 MHz, Methanol-d4) δ 8.59 – 8.55 (m, 2H), 8.25 –8.19 (m, 1H), 7.79 – 7.73 (m, 2H). 13 C NMR (101 MHz, Methanol-d4) δ 148.04, 141.07, 125.81. 11 B NMR (128 MHz, Methanol-d4) δ -10.91 (t, J = 100.6 Hz).HRMS (ESI) m / z: [M+Na]+ calcd for [C6H8BNO2Na]: 160.0542; Found: 160.0540.

[0098] Compound F4: 1 H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 4.69 (s, 6H), 4.52 (d, J = 12.5 Hz, 3H), 4.42 (d, J = 11.5 Hz, 3H).13 ¹³C NMR (101 MHz, DMSO-d₆) δ 76.28, 71.53. 11 ¹¹B NMR (128 MHz, DMSO-d₆) δ -15.38. HRMS (ESI) m / z: [M+Na]⁺ calcd for [C₇H 15 BN₄O₂Na]: 221.1180; Found: 221.1184.

[0099] Compound F6: 1 ¹H NMR (400 MHz, Methanol-d₄) δ 3.95 – 3.79 (m, 2H), 3.67(td, J = 12.2, 2.3 Hz, 2H), 3.13 (dd, J = 13.2, 1.9 Hz, 2H), 2.82 – 2.67 (m, 2H). 13 ¹³C NMR (101 MHz, Methanol-d₄) δ 64.91, 50.21. 11 ¹¹B NMR (128 MHz, Methanol-d₄) δ -14.16. HRMS (ESI) m / z: [M+Na]⁺ calcd for [C₅H 12 BNO₃Na]: 168.0802; Found: 168.0804.

[0100] Compound F12: ¹H NMR (400 MHz, Methanol-d₄) δ 5.04 (s, 2H), 2.60 (dqd, J= 10.4, 6.5, 2.5 Hz, 1H), 2.14 – 2.04 (m, 2H), 1.77 (dt, J = 5.9, 3.3 Hz, 2H), 1.68 – 1.60 (m, 1H), 1.36 – 1.11 (m, 5H). ¹³C NMR (101 MHz, Methanol-d₄) δ 54.84, 31.31, 25.20, 24.54. ¹¹B NMR (128 MHz, Methanol-d₄) δ -19.32. HRMS (ESI) m / z: [M+Na]⁺ calcd for [C₇H 16 BNO₂Na]: 180.1166; Found: 180.1163.

[0101] Compound F13: 1 H NMR (400 MHz, Methanol-d4) δ 3.67 (td, J = 7.1, 5.0 Hz, 2H), 2.55 (t, J = 7.1 Hz, 2H), 2.27 (s, 6H). 13 C NMR (101 MHz, Methanol-d4) δ65.46, 58.16, 47.54. 11 B NMR (128 MHz, Methanol-d4) δ -17.36 . HRMS (ESI)m / z: [M+Na]+ calcd for [C5H 14 BNO3Na]: 170.0959; Found: :170.0965.

[0102] Example 6: Boron uptake in cells by BPA and boron-containing amino acid analogs

[0103] Boron uptake by BPA and boron-containing amino acid analogs in human malignant melanoma cells A375, mouse melanoma cells B16F10, human astroblastoma cells U87, human glioblastoma cells LN229, and human pharyngeal squamous cell carcinoma cells FaDu was determined using the ICP-OES method.

[0104] The above-mentioned boron-containing amino acid analogues are compounds C1, D1, E1, F1, F2, F3, F4, F6, F12, and F13, which were prepared according to Examples 2 to 5.

[0105] Human malignant melanoma cells A375, mouse melanoma cells B16F10, human brain astrocytoma cells U87, human glioblastoma cells LN229, and human pharyngeal squamous cell carcinoma cells FaDu were all purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0106] (1) Experimental methods

[0107] Tumor cells in the logarithmic growth phase were added to DMEM complete medium (containing 10% fetal bovine serum) at a concentration of 4 × 10⁶ cells per well. 5Cells were seeded at concentrations of 0.5 mg / mL and 1 mg / mL in 6-well plates. After culturing at 37°C for 24 h, boron-containing amino acid analogs or BPA prepared in DMEM complete medium were added to achieve final concentrations of 0.5 mg / mL and 1 mg / mL, respectively. After incubation at 37°C for 2 h, the cells were washed three times with PBS buffer. 1 mL of trypsin (Soluble, Beijing) was added to the culture dish for digestion. After complete digestion, all cells were collected. A portion of the cell suspension (20 μL) was used to detect protein content. The remaining cells were centrifuged again, the supernatant was discarded, and 3 mL of DMEM complete medium was added for digestion. 5 mL of concentrated nitric acid was added to each well for digestion. After digestion, the sample volume was adjusted to 10 mL. The boron content in the cells was measured using ICP-OES, and boron uptake was calculated. Data are expressed as ng Boron / mg Protein.

[0108] (2) Experimental results

[0109] Figure 1 The figure shows the boron uptake of BPA and boron-containing amino acid analogs in human malignant melanoma cells A375. As can be seen from the figure, in A375 cells, at 0.5 mg / mL, the boron uptake of all compounds except compound F12 was higher than that of BPA; at a concentration of 1 mg / mL, the boron uptake of all compounds was higher than that of BPA.

[0110] Figure 2 The figure shows the boron uptake of BPA and boron-containing amino acid analogs in mouse melanoma cells B16F10. As can be seen from the figure, in B16F10 cells, at concentrations of 0.5 mg / mL and 1 mg / mL, the boron uptake of all compounds was higher than that of BPA.

[0111] Figure 3 The figure shows the boron uptake of BPA and boron-containing amino acid analogs in human glioblastoma cells U87. As can be seen from the figure, in U87 cells, at concentrations of 0.5 mg / mL and 1 mg / mL, the boron uptake of all compounds was higher than that of BPA.

[0112] Figure 4 The figure shows the boron uptake of BPA and boron-containing amino acid analogs in human glioblastoma cells LN229. As can be seen from the figure, in LN229 cells, at concentrations of 0.5 mg / mL and 1 mg / mL, the boron uptake of all compounds was higher than that of BPA.

[0113] Figure 5The figure shows the boron uptake of BPA and boron-containing amino acid analogs in human laryngeal squamous cell carcinoma FaDu. As can be seen from the figure, in FaDu cells, at a concentration of 0.5 mg / mL, the boron uptake of all compounds was higher than that of BPA; at a concentration of 1 mg / mL, except for compound F13, the boron uptake of all other compounds was higher than that of BPA.

[0114] Example 7: Boron uptake in cells by BPA and boron-containing amino acid analogs

[0115] The boron-containing amino acid analogs used in this embodiment are compounds C1, D1, E1, F1, F2, F3, F4, F6, F12, and F13 prepared according to Examples 2 to 5 above.

[0116] The hemolytic activity of BPA and boron-containing amino acid analogues was determined using erythrocytes from Kunming mice (female, 6-8 weeks old, 18-22 g, purchased from the Experimental Animal Center of Lanzhou University).

[0117] Specific procedure: 1 mL of blood was collected via orbital sampling into a centrifuge tube containing heparin sodium. The tube was gently inverted to prevent blood clotting. The tube was centrifuged at 800×g, 4℃ for 10 min. The supernatant was discarded, and the red blood cells were resuspended in 1 mL of PBS buffer. The resuspending process was repeated three times until the resulting red blood cells were considered 100% pure. The red blood cells were diluted to 8% of their total volume with PBS buffer, and 100 μL was added to a 96-well plate. BPA and boron-containing amino acid analogs were prepared to a concentration of 50 mg / mL using PBS buffer, and 100 μL of this solution was added to the 96-well plate containing the red blood cells, bringing the final concentration to 25 mg / mL. 100 μL of PBS buffer was added to the negative control group. 100 μL of 4% Triton X-100 solution (Sigma-Aldrich, T8787) was added to the positive control group. Three replicates were set for each concentration. The mixture was then incubated at 37°C for 1 h, followed by centrifugation at 4°C and 1000×g for 15 min. After centrifugation, 150 μL of supernatant from each well was transferred to a new 96-well plate, and the absorbance of the supernatant at 490 nm was measured using a Thermo microplate reader. The hemolytic activity of each compound was calculated according to the formula for calculating hemolytic activity.

[0118] The formula for calculating hemolytic activity is as follows:

[0119] Hemolytic activity = (absorbance of sample group - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) × 100%.

[0120] Figure 6The graph shows the hemolytic activity data of BPA and boron-containing amino acid analogs at a final concentration of 25 mg / mL. The graph shows that at a concentration of 25 mg / mL, all compounds except compound D1 exhibited hemolytic activity below 5%. Among them, compounds C1, E1, and F4 showed lower hemolytic activity against mouse erythrocytes than BPA, indicating better biocompatibility.

[0121] Example 8: In vivo boron uptake experiment of BPA and boron-containing amino acid analogs in a mouse subcutaneous melanoma model

[0122] The boron-containing amino acid analogs used in this embodiment are compound C1 and compound F4 prepared according to Examples 2 and 5 above.

[0123] Mouse melanoma cells B16F10 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0124] B16F10 cells in the logarithmic growth phase were digested with trypsin (Solepro, Beijing), transferred to centrifuge tubes, centrifuged at 800 rpm for 5 minutes, and the supernatant was discarded. The cells were then resuspended in 1 mL of PBS buffer, counted under a microscope, and centrifuged again. Finally, the cells were centrifuged at 1×10⁻⁶. 7 Cells were resuspended in DMEM complete medium containing 10% fetal bovine serum at a density of [number] cells / mL. Before inoculation, the hair on the lower right back of mice (C57 male mice, 6-8 weeks old, 18-22 g) was first removed using a mouse shaver, and the skin on the back was disinfected with 75% alcohol. The cell suspension was then re-mixed by pipetting, and 100 μL of cells was injected into each mouse using a 1 mL syringe, resulting in an inoculation density of 1 × 102 cells per mouse. 6 After inoculating the cells, disinfect the injection site with an alcohol swab. Closely monitor tumor growth after injection, and continue inoculation until the tumor volume reaches 100 mm. 3 The experiment was then conducted.

[0125] BPA and two analogues, C1 and F4 (both prepared with physiological saline to a concentration of 33 mg / mL), with low hemolytic activity and high cellular boron uptake, were injected into tumor-bearing mice via the tail vein at a dose of 250 mg / kg, with only one injection. One hour after administration, the heart, liver, spleen, lung, kidney, stomach, blood, brain, and tumor tissue of the mice were collected, weighed, and digested. Finally, the boron content in each organ was detected by ICP-OES, and the final result was expressed as μg Boron / g tissue or mL Blood.

[0126] Figure 7The figure shows the in vivo boron uptake of BPA and its boron-containing amino acid analogs C1 and F4 in a mouse subcutaneous melanoma model. As can be seen from the figure, 1 h after tail vein injection, the boron accumulation in tumor tissues of compounds C1 and F4 was higher than that of BPA, with compound F4 exhibiting the highest tumor boron accumulation.

[0127] Example 9: Determination of the solubility of BPA, BPA-fructose, compound C1 and compound F4 in water

[0128] The boron-containing amino acid analogs used in this embodiment are compound C1 and compound F4 prepared according to Examples 2 and 5 above.

[0129] Weigh out excess BPA, BPA-fructose (BPA:fructose molar ratio 1:1.5), compound C1, and compound F4 solids (200 mg each), add to 500 μL of ultrapure water, seal, and incubate in a constant temperature water bath at 25.0 ± 0.1 ℃ with magnetic stirring or vortex shaking for 6 h, followed by standing at 25 ℃ for 2 h. Undissolved solids are visible in the system at this point, ensuring solid-liquid equilibrium. Filter the supernatant through a 0.45 μm aqueous nylon membrane, discard the initial filtrate (200 μL), and collect the intermediate filtrate to obtain saturated aqueous solutions of different compounds. Add the saturated solutions to the digestion tube, along with 5 mL of nitric acid and 0.5 mL of phosphoric acid. Cap and place in a digester for digestion. Finally, add water to a final volume of 10 mL, shake well, and analyze using ICP-OES. The concentration and solubility of the corresponding compounds in the saturated solution are determined based on the number of boron atoms detected, as shown in Table 1 below.

[0130] Table 1. Solubility data of different substances in water

[0131]

[0132] This invention provides a concept and method for the application of boron-containing amino acid analogs in the preparation of drugs for treating tumors. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of boron-containing amino acid analogs in the preparation of drugs for treating tumors, characterized in that, The boron-containing amino acid analog has the structure shown in Formula I: ； in, Y is selected from -OH, -OR1, or -NHR2; R1 and R2 are independently selected from C1-C3 alkyl groups; L is selected from , , or ; R3, R4, and R5 are independently selected from hydrogen, C1-C3 alkyl, hydroxyethyl, or cyclohexyl; m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from C1-C3 alkyl groups; R7 is selected from hydrogen or C1-C3 alkyl; The tumors mentioned are melanoma, human brain astrocytoma, human glioblastoma, or human pharyngeal squamous cell carcinoma.

2. The application according to claim 1, characterized in that, The boron-containing amino acid analog has the structure shown in Formula I: ； in, Y is selected from -OH, -OR1, or -NHR2; R1 and R2 are independently selected from methyl or ethyl; L is selected from , , or ; R3, R4, and R5 are independently selected from hydrogen, methyl, ethyl, hydroxyethyl, or cyclohexyl; m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from methyl or ethyl; R7 is selected from hydrogen, methyl, or ethyl.

3. The application according to claim 1, characterized in that, The boron-containing amino acid analog has the structure shown in Formula I: ； in, Y is selected from -OH, -OR1, or -NHR2; R1 and R2 are independently selected from methyl or ethyl; L is selected from , , or ; R3, R4, and R5 are independently selected from hydrogen, methyl, hydroxyethyl, or cyclohexyl; m is selected from 0 or 1; R6 is selected from -NR8R9; R8 and R9 are independently selected from methyl groups; R7 is selected from hydrogen or methyl.

4. The application according to claim 1, characterized in that, The boron-containing amino acid analogue is selected from compounds with any of the following structures: 。 5. The application according to claim 1, characterized in that, The boron-containing amino acid analogue is selected from compounds with any of the following structures: 。 6. The application according to claim 1, characterized in that, The boron-containing amino acid analogues are selected from compounds with the following structures: 。 7. The application according to claim 1, characterized in that, The melanoma is either a human malignant melanoma or a mouse melanoma; optionally, the human malignant melanoma is a human malignant melanoma cell A375; the mouse melanoma is a mouse melanoma cell B16F10.

8. The application according to claim 1, characterized in that, The human brain astrocytoma is human brain astrocytoma cell U87.

9. The application according to claim 1, characterized in that, The human glioblastoma is glioblastoma cell line LN229.

10. The application according to claim 1, characterized in that, The human laryngeal squamous cell carcinoma described is a human laryngeal squamous cell carcinoma cell line, FaDu.

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