Albumin-binding bodipy derivatives, preparation and use

Abstract

This invention relates to albumin-bound BODIPY derivatives, their preparation, and applications, belonging to the field of pharmaceutical technology. The albumin-bound BODIPY derivative of this invention couples BODIPY with the albumin-free non-covalent binder 4-(p-iodophenyl)butyric acid (IP), and can be further coupled with a boron thiol compound (BSH). 4-(p-iodophenyl)butyric acid has excellent albumin-binding ability; by utilizing the EPR effect of albumin, tumor-targeted delivery of boron-containing modules (BODIPY or BSH) can be achieved, overcoming the dependence of traditional boron drugs 4-dihydroxyboron-L-phenylalanine (BPA) on the LAT1 transporter protein, and exhibiting a certain degree of broad-spectrum targeting. Simultaneously, the fluorine-18 (…) introduced into the BODIPY structure… 18 The F) atom allows for real-time monitoring of the distribution of boron-containing drugs in vivo via PET-CT, enabling integrated diagnosis and treatment of tumors. This conjugate exhibits high safety and low toxicity, high in vitro cellular uptake, and in vivo animal distribution experiments confirming its excellent tumor targeting properties. This invention can serve as a broad-spectrum targeted boron-containing drug in integrated BNCT diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to an albumin-bound BODIPY derivative for boron neutron capture therapy, its preparation method, and its application. Background Technology

[0002] Boron neutron capture therapy (BNCT) is a binary targeted radiotherapy technique based on the principle of nuclear reaction. Its basic principle is to utilize non-radioactive boron-10 (… 10 B) The high capture cross-section of isotopes for thermal neutrons, when 10 After B accumulates in tumor cells, it is then exposed to a superheated neutron beam. 10 B. Neutron capture generation 11 B, followed by nuclear fission, produces alpha rays with high energy transfer density. 4 He) and recoil lithium core ( 7 Li). The range of these two types of rays is only 5-9 μm, which is smaller than the diameter of a single tumor cell, so they can precisely kill tumor cells while minimizing damage to surrounding normal tissues.

[0003] The clinical efficacy of BNCT is highly dependent on the performance of the boron carrier. An ideal boron carrier for BNCT should meet the following requirements: (1) low non-neutron irradiation toxicity; (2) high tumor selectivity: the ratio of intratumoral boron concentration to normal tissue boron concentration (T / N) and the ratio of intratumoral boron concentration to blood boron concentration (T / B) should both be greater than 3:1; (3) ideal metabolic kinetics: slow metabolism within the tumor and rapid metabolism in normal tissue and blood; (4) good water solubility, facilitating large-volume administration via intravenous injection. Currently, the only boron carrier used in clinical research is disodium mercaptododecanoate (Na2B). 12 H 11While both hydroxylamine (BSH) and 4-dihydroxyboron-L-phenylalanine (BPA) are effective treatments, they also have limitations. BSH lacks active targeting, has a low tumor uptake rate, and cannot achieve T / N and T / B values ​​greater than 3 simultaneously. Although BPA can be taken up by tumor cells via the L-amino acid transporter (LAT1), its over-reliance on LAT1 is also a drawback. In tumors with low LAT1 expression levels (such as pancreatic cancer and ovarian cancer), the efficacy of BPA becomes very limited. In addition, there are significant individual differences in LAT1 expression levels, which means that even in tumor types traditionally considered to have high LAT1 expression (such as head and neck cancer and glioma), some patients cannot achieve ideal treatment results due to insufficient LAT1 expression. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a class of albumin-bound BODIPY derivatives and their isotope products. BODIPY is coupled with the albumin non-covalent binder 4-(p-iodophenyl)butyric acid (IP), and can be further coupled with BSH. Utilizing the EPR effect of albumin, tumor-targeted delivery of boron-containing modules (BODIPY or BSH) can be achieved. The albumin delivery system does not rely on specific amino acid transport proteins, but rather utilizes the biological characteristics prevalent in solid tumors, making it a broad-spectrum boron carrier platform. Furthermore, the imaging monitoring of boron distribution using BODIPY can be applied to integrated diagnosis and treatment of BNCT (Boron-Neuro-Traumatic Brain Tissue Catalysis).

[0005] The technical solution of this invention: a class of albumin-bound BODIPY derivatives, wherein the albumin-bound BODIPY derivatives have the structure and isotopic structure shown in Formula I or Formula II:

[0006] ;

[0007] Among them, R 0 for or m is an integer from 1 to 10, and n is an integer from 1 to 10;

[0008] X is NH, O, or S;

[0009] Y is F or 18 F;

[0010] R 1 R 2 R 3 R 4 R 5 R 6Each of these elements is independent of the others: hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. or Z represents hydrogen, sodium, potassium, lithium, cesium, rubidium, or... ;

[0011] R 7 for or q is an integer from 0 to 5, and t is an integer from 1 to 10;

[0012] A and G can be independently represented as NH, O, or S;

[0013] k is an integer from 1 to 10;

[0014] R 8 It is a C1-C6 alkyl, C1-C6 alkoxy or ;

[0015] p is an integer between 0 and 5;

[0016] E + Sodium ions, potassium ions, lithium ions, cesium ions, rubidium ions, or .

[0017] Furthermore, in Equation I, R 0 for m is an integer between 2 and 6;

[0018] X is NH;

[0019] Y is F;

[0020] R 1 R 3 R 4 R 6 Each is independently selected from methyl and ethyl;

[0021] R 2 R 5 Each independently selected from hydrogen or Z represents sodium or potassium.

[0022] Furthermore, in Equation II, R 0 for m is an integer between 2 and 6;

[0023] X is NH;

[0024] Y is F;

[0025] R 1 R 3 R6 Each is independently selected from methyl and ethyl;

[0026] R 2 R 5 Each independently selected from hydrogen or Z represents sodium or potassium;

[0027] R 7 for q is an integer between 0 and 2;

[0028] A is NH;

[0029] k is an integer between 2 and 4;

[0030] R 8 for ;

[0031] G is S;

[0032] p is 0;

[0033] E + It consists of sodium ions, potassium ions, or lithium ions.

[0034] In the preferred formulas I and II of this invention, R 0 for m is 4; X is NH; Y is F; R1, R3, R4, and R6 are each independently selected from methyl and ethyl; methyl is preferred.

[0035] R 2 For hydrogen or Z represents sodium; R 5 For hydrogen or Z represents sodium.

[0036] R 7 for q is 0; A is NH; k is 2; R 8 for G is S; p is 0; E + It is a sodium ion.

[0037] The albumin-bound BODIPY derivative has the following structure:

[0038] .

[0039] A specific preparation method for a class of albumin-bound BODIPY derivatives includes the following steps:

[0040] Starting with 2,4-dimethylpyrrole, dissolved in anhydrous dichloromethane, 5-bromopentanoyl chloride was added, and the reaction was carried out at 60°C for 1 h to give 2. BF3·OEt2 was added, and the reaction was carried out at room temperature for 12 h to give 3. 3 was synthesized via the Gabriel reaction (nucleophilic substitution of phthalimide, dephthalimide removal by hydrazine hydrate). 5 was dissolved in DMF and reacted with 4-(p-iodophenyl)butyric acid at room temperature to synthesize IP-BDP. The reaction formula is as follows:

[0041]

[0042] A specific preparation method for a class of water-soluble albumin-bound BODIPY derivatives includes the following steps:

[0043] IP-BDP was dissolved in anhydrous dichloromethane, and chlorosulfonic acid was added at -15°C. After reacting for 1 h, NaHCO3 was added to convert the sulfonic acid group into a sodium salt, thus obtaining IP-sulfo-BDP and IP-disulfo-BDP.

[0044]

[0045] A specific preparation method for a class of albumin-bound BODIPY derivatives with high boron loading includes the following steps:

[0046] Using compound 5 as a starting material, the amino group was protected with benzyloxycarbonyl, and the methyl group outside the BODIPY ring was oxidized to an aldehyde group with pyridinium chlorochromate. A benzyl-protected carboxyl group was introduced through a Witting reaction. The benzyloxycarbonyl group and benzyl protection were removed simultaneously by hydrogenation of palladium on carbon. 4-(p-iodophenyl)butyric acid and N-(2-aminoethyl)maleimide were introduced through a two-step amide condensation reaction. BSH was introduced through Michael addition of the mercapto group to maleimide, thus synthesizing IP-BDP-BSH.

[0047]

[0048] A specific preparation method for a class of water-soluble albumin-bound BODIPY derivatives with high boron loading includes the following steps:

[0049] Using compound 11 as a raw material, it was dissolved in anhydrous dichloromethane, and chlorosulfonic acid was added at -15℃. After reacting for 1 h, NaHCO3 was added to convert the sulfonic acid group into a sodium salt, yielding compounds 12 and 13. BSH was introduced by Michael addition of the mercapto group with maleimide to synthesize IP-sulfo-BDP-BSH and IP-disulfo-BDP-BSH.

[0050]

[0051] The aforementioned albumin-bound BODIPYs: IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP, IP-BDP-BSH, IP-sulfo-BDP-BSH, and IP-disulfo-BDP-BSH are used as the active component in the preparation of targeted antitumor drugs for BNCT. The tumors include head and neck tumors (including laryngeal cancer, thyroid cancer, nasal cancer, tonsil cancer, lymphoma, and sarcoma), intracranial tumors (including glioma, medulloblastoma, ependymoma, and brain metastases), breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, gastric cancer, and rectal cancer.

[0052] The beneficial effects of this invention are as follows: This invention provides a class of albumin-bound BODIPY derivatives, which couple IP with albumin non-covalent binding to BODIPY, and can be further coupled to BSH. Utilizing the albumin EPR effect, tumor targeting of boron-containing modules (BODIPY or BSH) can be achieved. This strategy does not rely on specific amino acid transporters and has a certain broad-spectrum targeting ability. Simultaneously, BODIPY, as a fluorescence / PET imaging group, can monitor the distribution of boron carriers in vivo. These molecules can serve as novel molecular tools in the integrated diagnosis and treatment of BNCT (Boron-Neuro-Cellular Transplantation). Attached Figure Description

[0053] Figure 1 shows the proton NMR spectrum of the compound IP-BDP.

[0054] Figure 2 shows the proton NMR spectrum of the compound IP-sulfo-BDP.

[0055] Figure 3 shows the 1H NMR spectrum of the compound IP-disulfo-BDP.

[0056] Figure 4 shows the proton NMR spectrum of the compound IP-BDP-BSH.

[0057] Figure 5 shows the UV-Vis spectra (A) and fluorescence spectra (B) of compounds IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP and IP-BDP-BSH.

[0058] Figure 6 shows the high-performance liquid chromatogram of compound IP-BDP.

[0059] Figure 7 shows the high-performance liquid chromatogram of compound IP-sulfo-BDP.

[0060] Figure 8 shows the high-performance liquid chromatogram of compound IP-disulfo-BDP.

[0061] Figure 9 shows the high-performance liquid chromatogram of compound IP-BDP-BSH.

[0062] Figure 10 shows the non-neutron irradiation toxicity of IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP, and IP-BDP-BSH to MOC-1 cells as evaluated by the MTT assay.

[0063] Figure 11 shows the non-neutron irradiation toxicity of IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP and IP-BDP-BSH to SAS cells as evaluated by the MTT assay.

[0064] Figure 12 shows the uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells as captured by fluorescence microscopy.

[0065] Figure 13 shows the uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells as captured by fluorescence microscopy.

[0066] Figure 14 shows the boron uptake of IP-disulfo-BDP, IP-BDP-BSH and BSH by MOC-1 cells as detected by ICP-OES.

[0067] Figure 15 shows the boron uptake of IP-disulfo-BDP, IP-BDP-BSH and BSH by SAS cells as detected by ICP-OES.

[0068] Figure 16 shows the boron distribution of IP-disulfo-BDP (A) and BPA-F (B) in various organs and tissues of tumor-bearing mice as detected by ICP-OES.

[0069] Figure 17 shows the comparison of boron concentration in tumors of tumor-bearing mice by IP-disulfo-BDP and BPA-F (A) and T / N (B) and T / B (C) graphs.

[0070] Figure 18 shows the boron distribution of IP-BDP-BSH (A) and BSH (B) in various organs and tissues of tumor-bearing mice as detected by ICP-OES.

[0071] Figure 19 shows the comparison of boron concentration in tumors of tumor-bearing mice (A) and T / N (B) and T / B (C).

[0072] Figure 20 shows the serum levels of ALT (A), AST (B), BUN (C), and Cr (D) in mice 14 days after tail vein injection of IP-disulfo-BDP and IP-BDP-BSH. Detailed Implementation

[0073] Example 1

[0074]

[0075] Synthesis of Compound 2

[0076] Take a 1000 mL round-bottom flask, add 200 mL of anhydrous dichloromethane, then add 10 mL of 2,4-dimethylpyrrole (compound 1, 97.12 mmol, 2 eq.) and 6.5 mL of 5-bromopentanoyl chloride (48.56 mmol, 1 eq.). Add a magnetic stir bar, purge with nitrogen three times, reflux at 60 °C for 1 h, and analyze by TLC (petroleum ether:dichloromethane = 2:1). Compound 1 has reacted completely. Stop heating and cool to room temperature to obtain intermediate compound 2. Without purification, proceed directly to the next reaction.

[0077] Synthesis of Compound 3

[0078] The flask containing compound 1 was placed in an ice-water bath. 27.0 mL of Et3N (194.24 mmol, 4 eq.) was added, and the reaction was continued in the ice-water bath for 30 min. Then, 24.5 mL of BF3·Et2O (194.24 mmol, 4 eq.) was added, and the reaction was continued in the ice-water bath for 10 min. The ice-water bath was removed, and the reaction was carried out at 25°C for 12 h. TLC analysis (petroleum ether:dichloromethane = 2:1) showed the formation of a large amount of fluorescent material, at which point the reaction was stopped. The excess BF3·Et2O was neutralized by adding saturated NaHCO3 solution while stirring until no more bubbles were produced. The organic phase was washed with water (200 mL × 3) and saturated NaCl aqueous solution (200 mL × 3), dried with anhydrous Na2SO4, filtered, and the filtrate was collected, concentrated under reduced pressure, and separated by column chromatography (SiO2, petroleum ether: dichloromethane = 1:1~1:5) to give 34.5958 g of orange-yellow solid compound, with a yield of 25%. 1H NMR (400 MHz, Chloroform-d) δ 6.06 (s, 2H), 3.45 (t, J = 6.5 Hz, 2H), 2.97 (m, 2H), 2.52(s, 6H), 2.42 (s, 6H), 2.05 (m, 2H), 1.81 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 154.23, 145.43, 140.39, 131.51, 121.90, 33.23, 32.88, 30.29,27.63, 16.54, 14.59. HRMS (ESI, m / z): C 17 H 22 BBrF2N2Na + for [M+Na] + , calcd405.0925, found 405.0930.

[0079] Synthesis of Compound 4

[0080] Weigh 4.5958 g (12.03 mmol, 1 eq.) of compound 3, add 4.4562 g (24.06 mmol, 2 eq.) of potassium phthalimide, dissolve in 80 mL of DMF, add a magnetic buoy, purge with nitrogen three times, and react at 60 °C for 12 h. TLC analysis (petroleum ether: dichloromethane = 1:5) showed that compound 3 had reacted completely, and the reaction was stopped. The reaction solution was divided into four portions, and each portion was extracted to remove DMF as follows: 600 mL of ethyl acetate was added for dilution, followed by extraction with water (200 mL × 3), washing the organic phase with saturated NaCl aqueous solution (200 mL × 3), drying the organic phase with anhydrous Na2SO4, filtering, collecting the filtrates, combining all organic phases, concentrating under reduced pressure, and separating by column chromatography (SiO2, petroleum ether: dichloromethane = 1:1~1:30) to obtain an orange-yellow solid compound 4 3.7935 g, yield 70%. 1H NMR (400 MHz, Chloroform-d) δ 7.84(dd, J = 5.4, 3.1 Hz, 2H), 7.72 (dd, J = 5.5, 3.0 Hz, 2H), 6.03 (s, 2H), 3.73(t, J = 7.3 Hz, 2H), 3.07 - 2.89 (m, 2H), 2.50 (s, 6H), 2.38 (s, 6H), 1.89(p, J = 7.8 Hz, 2H), 1.74 - 1.62 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ168.41, 154.13, 145.52, 140.37, 134.16, 132.09, 131.49, 123.41, 121.84,37.77, 29.11, 29.04, 27.98, 16.50, 14.55. HRMS (ESI, m / z): C 25 H 26 BF2N3NaO2 + for[M+Na] + , calcd 472.1984, found 472.1981.

[0081] Synthesis of Compound 5

[0082] 3.7935 g (8.44 mmol, 1 eq.) of compound 4 was weighed and dissolved in 100 mL of DCM and 100 mL of LEtOH. 6.6 mL (135.11 mmol, 16 eq.) of NH₂NH₂·H₂O was added, and the mixture was heated under reflux at 80 °C for 4 h, producing a large precipitate. TLC analysis (DCM) showed that compound 4 had reacted completely, and the reaction was stopped. The precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure and separated by column chromatography (SiO₂, dichloromethane:methanol = 100:1~50:1, 50:1 + 1% NH₃·H₂O) to obtain 2.1363 g of orange-yellow solid compound 5, with a yield of 79%. 1 H NMR (400 MHz, Chloroform-d) δ 6.01 (s, 2H), 2.85 (dt, J = 8.0, 3.9 Hz, 2H), 2.78 - 2.66 (m, 2H), 2.49 (s, 6H), 2.34 (s,6H), 1.71 - 1.51 (m, 4H), 1.35 (s, 2H).13 C NMR (101 MHz, Chloroform-d) δ153.60, 146.25, 140.32, 131.32, 121.51, 41.74, 33.99, 29.06, 27.97, 16.15,14.31. HRMS (ESI, m / z): C 17 H 24 BFN3 + for [MF] + , calcd 300.2047, found 300.2052;C 17 H 25 BF2N3 for [M+H] + , calcd 320.2110, found 320.2111.

[0083] Synthesis of compound IP-BDP

[0084] Weigh out 543.3 mg (1.88 mmol, 1.5 eq.) of 4-(p-iodophenyl)butyric acid, 362.3 mg (1.88 mmol, 1.5 eq.) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 255.2 mg (1.88 mmol, 1.5 eq.) of 1-hydroxybenzotriazole (HOBt). Dissolve in 5 mL of DMF, add 4 drops of Et3N, add a magnetic stir bar, purge with nitrogen three times, and react at 25 °C for 1 h. TLC analysis (dichloromethane:methanol = 50:1) showed that a large amount of intermediate was generated. Add 403.6 mg (1.25 mmol, 1 eq.) of compound 5, and continue the reaction at 25 °C for 12 h. TLC analysis (D:M = 50:1) showed that compound 5 had reacted completely, and the reaction was stopped. Dilute with 150 mL of EA, extract with water (50 mL × 3), wash the organic phase with saturated NaCl aqueous solution (50 mL × 3), dry the organic phase with anhydrous Na2SO4, filter, collect the filtrate, and concentrate under reduced pressure. Separate by column chromatography (SiO2, dichloromethane:methanol = 500:1~100:1) to obtain 556.2 mg of orange-yellow solid compound IP-BDP, yield 74%. The 1H NMR spectrum of IP-BDP is shown below. Figure 1 As shown, 1H NMR (400 MHz, Chloroform-d) δ 7.55 (d, J = 8.2 Hz, 2H), 6.87 (d, J = 8.0 Hz, 2H), 6.01 (s,2H), 5.81 (t, J = 5.8 Hz, 1H), 3.19 (q, J = 6.4 Hz, 2H), 2.93 - 2.81 (m, 2H), 2.52 (t, J = 7.5 Hz, 2H), 2.48 (s, 6H), 2.32 (s, 6H), 2.07 (t, J = 7.4 Hz, 2H), 1.86 (p, J = 7.5 Hz, 2H), 1.67 - 1.49 (m, 4H). 13 C NMR (101 MHz, Chloroform-d) δ 172.66, 153.91, 145.84, 141.14, 140.31, 137.41, 131.37,130.59, 121.77, 91.04, 39.15, 35.59, 34.66, 30.03, 29.15, 27.82, 26.90,16.35, 14.45. HRMS (ESI, m / z): C 27 H 33 BF2IN3NaO + for [M+Na] + ,calcd 614.1627,found 614.1624.

[0085] Example 2

[0086]

[0087] Synthesis of compound IP-sulfo-BDP

[0088] Weigh 50.2 mg (0.08 mmol, 1 eq.) of IP-BDP and dissolve it in 3 mL of anhydrous DCM. Place the flask in an ice-salt bath (-15℃) for 10 min, then add 6 μL (0.09 mmol, 1.1 eq.) of ClSO3H2O and continue reacting in an ice-water bath for 10 min. Remove the ice-salt bath and react at 25℃ for 1 h. An orange-red precipitate forms, and the DCM layer shows no fluorescence. Perform TLC analysis on the DCM layer (dichloromethane:methanol = 50:1). No IP-BDP remains. Stop the reaction and concentrate under reduced pressure to remove DCM. Dissolve the precipitate again in 3 mL of MeOH and 1 mL of H2O, then add 7.6 mg (0.09 mmol, 1.1 eq.) of NaHCO3 and react at 25℃ for 1 h. Concentrate under reduced pressure to remove the solvent, and separate by reversed-phase column chromatography (C10). 18 (SiO2, H2O:MeOH = 20:1~1:1) yielded 44.5 mg of an orange-yellow solid compound IP-sulfo-BDP, with a yield of 82%. The 1H NMR spectrum of IP-sulfo-BDP is shown below. Figure 2 As shown, 1 H NMR (400 MHz, Methanol-d4) δ 7.54 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.9 Hz, 2H), 6.13 (s, 1H), 3.17 (t, J = 6.6 Hz, 2H), 2.88(t, J = 8.4 Hz, 2H), 2.73 (s, 3H), 2.65 (s, 3H), 2.50 (t, J = 7.7 Hz, 2H), 2.44 (s, 3H), 2.31 (s, 3H), 2.16 (t, J = 7.5 Hz, 2H), 1.82 (p, J = 7.6 Hz,2H), 1.63 (p, J = 6.9, 6.5 Hz, 2H), 1.58 - 1.45 (m, 2H). 13C NMR (101 MHz, Methanol-d4) δ 175.69, 158.33, 151.70, 149.33, 144.78, 142.74, 138.48,134.12, 133.90, 131.72, 130.45, 124.41, 91.52, 39.93, 36.47, 35.66, 30.67,30.04, 28.93, 28.49, 16.93, 14.77, 14.37, 14.19. HRMS (ESI, m / z):C 27 H 32 BF2IN3O4S - for [M-Na] - , calcd 670.1219, found 670.1231.

[0089] Synthesis of compound IP-disulfo-BDP

[0090] The synthesis of IP-disulfo-BDP is the same as that of IP-sulfo-BDP, except that disulfonic acid substitution can be achieved by increasing the sulfonating agent ClSO3H to 2 equivalents. 400.1 mg (0.68 mmol, 1 eq.) of IP-BDP was weighed, 90 μL (1.35 mmol, 2 eq.) of ClSO3H was added, and 114.7 mg (1.35 mmol, 2 eq.) of NaHCO3 was added to obtain 403.1 mg of the orange-yellow solid compound IP-disulfo-BDP, with a yield of 75%. The 1H NMR spectrum of IP-disulfo-BDP is shown below. Figure 3 As shown, 1 H NMR (400 MHz, Methanol-d4) δ 7.58 (d, J = 8.3 Hz, 2H), 6.98 (d, J= 8.3 Hz, 2H), 3.23 (t, J = 5.4 Hz, 4H), 2.79 (s, 6H), 2.75 (s, 6H), 2.59 -2.54 (m, 2H), 2.18 (t, J = 7.5 Hz, 2H), 1.87 (p, J = 7.6, 7.2 Hz, 2H), 1.75 -1.67 (m, 4H). 13C NMR (101 MHz, Methanol-d4) δ 175.75, 154.99, 151.68, 142.70,141.00, 138.45, 135.81, 131.89, 131.72, 91.45, 39.96, 36.31, 35.60, 30.64,30.18, 29.08, 28.36, 14.77, 14.40. HRMS (ESI, m / z): C 27 H 31 BF2IN3O7S2 2- for [M-2Na] 2- , calcd 374.5355, found 375.0387.

[0091] Example 3

[0092]

[0093] Synthesis of Compound 6

[0094] 1007.1 mg (3.13 mmol, 1 eq.) of compound 5 and 789.9 mg (9.40 mmol, 3 eq.) of NaHCO3 were weighed and dissolved in 10 mL of tetrahydrofuran (THF) and 10 mL of H2O. The flask was placed in an ice-water bath, and after 10 min, 835 μL (9.40 mmol, 3 eq.) of benzyl chloroformate (CbzCl) was added. After 10 min, the flask was removed, and the reaction was carried out at 25 °C for 12 h. TLC analysis (dichloromethane:methanol = 10:1) showed that compound 5 had completely reacted, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the mixture was separated by column chromatography (SiO2, dichloromethane:methanol = 500:1) to give 802.8 mg of orange-yellow oily liquid compound 6, with a yield of 56%. 1 H NMR (400 MHz, Chloroform-d) δ 7.38- 7.28 (m, 5H), 6.02 (s, 2H), 5.08 (s, 2H), 4.86 (s, 1H), 3.21 (q, J = 6.4Hz, 2H), 2.90 (t, J = 8.1 Hz, 2H), 2.50 (s, 6H), 2.35 (s, 6H), 1.73 - 1.54(m, 4H). 13C NMR (101 MHz, Chloroform-d) δ 156.36, 153.64, 145.80, 140.30,136.53, 131.21, 128.34, 127.93, 127.86, 121.54, 66.35, 40.42, 30.10, 28.46,27.61, 15.96, 14.23. HRMS (APCI, m / z): C 25 H 29 BF2N3O2 - for [MH] - , calcd452.2321, found 452.2335.

[0095] Synthesis of Compound 7

[0096] 802.8 mg (1.77 mmol, 1 eq.) of compound 6 and 3435.7 mg (15.94 mmol, 9 eq.) of pyridinium chlorochromate (PCC) were weighed and dissolved in 25 mL of THF. A magnetic stir bar was added, and the mixture was purged three times with nitrogen. The reaction was carried out at 25 °C for 12 h. TLC analysis (dichloromethane:methanol = 100:1) showed that compound 6 had completely reacted, and the reaction was stopped. Excess PCC was removed by filtration, and the filtrate was concentrated under reduced pressure and separated by column chromatography (SiO2, dichloromethane:methanol = 500:1) to give 7486.1 mg of orange-yellow solid compound, with a yield of 59%. 1 H NMR (400 MHz, Chloroform-d) δ 10.22 (s, 1H), 7.38 - 7.31 (m, 5H), 6.85 (s, 1H), 6.29 (s,1H), 5.08 (s, 2H), 4.99 (s, 1H), 3.24 (t, J = 6.4 Hz, 2H), 3.03 (t, J = 8.3Hz, 2H), 2.61 (s, 3H), 2.45 (s, 3H), 2.39 (s, 3H), 1.81 - 1.60 (m, 4H). 13CNMR (101 MHz, Chloroform-d) δ 184.43, 164.25, 156.47, 148.18, 142.65, 136.50,135.63, 135.28, 134.65, 132.79, 128.46, 128.12, 128.00, 125.80, 120.41,67.84, 46.36, 40.54, 32.66, 30.31, 28.62, 28.33, 16.85, 15.94, 15.26. HRMS(ESI, m / z): C 25 H 28 BF2N3O3Na + for [M+Na] + , calcd 490.2089, found 490.2080.

[0097] Synthesis of Compound 8

[0098] 486.1 mg (1.04 mmol, 1 eq.) of compound 7 and 853.9 mg (2.08 mmol, 2 eq.) of (benzyloxycarbonylmethylene)triphenylphosphine were weighed and dissolved in 5 mL of DCM. A magnetic stir bar was added, and the mixture was purged three times with nitrogen. The reaction was carried out at 25 °C for 12 h. TLC analysis (dichloromethane:methanol = 100:1) showed that compound 7 had completely reacted, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the mixture was separated by column chromatography (SiO2, dichloromethane:methanol = 500:1~200:1) to give 8429.8 mg of a purple-red solid compound, with a yield of 69%. 1 H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 16.3 Hz, 1H), 7.43 - 7.32 (m, 10H), 6.59 (s, 1H), 6.44 (d, J = 16.0 Hz, 1H), 6.32 (p, J =1.8 Hz, 2H), 6.16 (s, 1H), 5.26 (s, 2H), 5.09 (s, 2H), 4.84 (s, 1H), 3.25 (d,J = 6.9 Hz, 2H), 2.99 (t, 2H), 2.57 (s, 3H), 2.40 (s, 6H), 1.74 - 1.65 (m,4H). 13C NMR (101 MHz, Chloroform-d) δ 166.30, 165.64, 159.19, 156.48, 145.15,144.01, 141.06, 136.52, 135.84, 134.43, 132.91, 128.56, 128.50, 128.45,128.31, 128.24, 128.18, 128.13, 128.06, 124.48, 123.83, 121.36, 119.95,66.31, 65.07, 40.59, 30.27, 28.73, 28.02, 16.54, 16.16, 14.86. HRMS (ESI, m / z): C 34 H 35 BF2N3O4 - for [MH] - , calcd 598.2689, found 598.2683.

[0099] Synthesis of Compound 9

[0100] 429.8 mg (0.72 mmol, 1 eq.) of compound 8 and 43.7 mg of palladium / carbon (palladium loading 10%, water content approximately 55%) were weighed and dissolved in 9 mL of MeOH. Three drops of AcOH were added, a magnetic stir bar was added, and the mixture was purged three times with hydrogen gas. The reaction was carried out at 25 °C for 4 h under a hydrogen atmosphere. The fluorescence of the solution changed from orange-red to yellow-green. TLC analysis (dichloromethane:methanol = 100:1) showed that compound 8 had completely reacted, and the reaction was stopped. The palladium / carbon was removed by diatomaceous earth filtration, and the filtrate was concentrated under reduced pressure. The concentrated solid was washed three times with DCM to obtain an orange-yellow solid, compound 9. Without further column chromatography purification, this solid was directly used for the next reaction, yielding 149.4 mg (55% yield). HRMS (ESI, m / z): C 19 H 25 BF2N3O2 - for [MH] - ,calcd 376.2008,found 376.2011.

[0101] Synthesis of Compound 10

[0102] Weigh out 173.4 mg (0.59 mmol, 1.5 eq.) of 4-(p-iodophenyl)butyric acid, 116.3 mg (0.59 mmol, 1.5 eq.) of EDCI, and 81.4 mg (0.59 mmol, 1.5 eq.) of HOBt. Dissolve in 5 mL of DMF, add 2 drops of Et3N, add a magnetic stir bar, purge with nitrogen three times, and react at 25 °C for 1 h. TLC analysis (D:M = 50:1) showed that a large amount of intermediate was generated. Add 149.4 mg (0.40 mmol, 1 eq.) of compound 9, and continue the reaction at 25 °C for 12 h. TLC analysis (dichloromethane:methanol = 50:1) showed that compound 9 had completely reacted, and the reaction was stopped. Dilute with 150 mL of EA, extract with water (50 mL × 3), wash the organic phase with saturated NaCl aqueous solution (50 mL × 3), dry the organic phase with anhydrous Na2SO4, filter, collect the filtrate, concentrate under reduced pressure, and separate by column chromatography (SiO2, dichloromethane:methanol = 100:1~50:1, 50:1 + 1% formic acid) to obtain 10,213.4 mg of orange-yellow solid compound, yield 83%. 1 H NMR (400 MHz, Chloroform-d) δ 7.57 (d, J = 7.7 Hz, 2H), 6.90 (d, J = 7.8 Hz, 2H), 6.10 (s, 1H), 6.04(s, 1H), 5.66 (s, 1H), 3.23 (m, 4H), 2.90 (t, J = 7.5 Hz, 2H), 2.76 (t, J =7.4 Hz, 2H), 2.56 (t, J = 6.9 Hz, 2H), 2.49 (s, 3H), 2.35 (s, 6H), 2.13 (t,2H), 1.91 (m, 2H), 1.64 (m, 4H). HRMS (ESI, m / z): C 29 H 34 BF2IN3O3 - for [MH] - ,calcd 648.1706, found 648.1717.

[0103] Synthesis of Compound 11

[0104] Weigh out 213.4 mg (0.33 mmol, 1 eq.) of compound 10, 77.0 mg (0.39 mmol, 1.2 eq.) of EDCI, and 55.5 mg (0.39 mmol, 1.2 eq.) of HOBt. Dissolve in 5 mL of DMF, add 2 drops of Et3N, add a magnetic stir bar, purge with nitrogen three times, and react at 25 °C for 1 h. TLC analysis (dichloromethane:methanol = 10:1) showed that a large amount of intermediate was generated. Add 69.1 mg (0.39 mmol, 1.2 eq.) of N-(2-aminoethyl)maleimide hydrochloride, and continue the reaction at 25 °C for 12 h. TLC analysis (D:M = 10:1) showed that compound 10 had completely reacted, and the reaction was stopped. Dilute with 150 mL of EA, extract with water (50 mL × 3), wash the organic phase with saturated NaCl aqueous solution (50 mL × 3), dry the organic phase with anhydrous Na2SO4, filter, collect the filtrate, concentrate under reduced pressure, and separate by column chromatography (SiO2, D:M = 300:1~50:1) to obtain 11,196.2 mg of orange-yellow solid compound, yield 77%. 1 H NMR (400 MHz, Chloroform-d) δ 7.59 (d,J = 8.2 Hz, 2H), 6.91 (d, J = 8.1 Hz, 2H), 6.66 (s, 2H), 6.10 (s, 1H), 6.06(s, 1H), 3.64 (t, J = 5.6 Hz, 2H), 3.44 (t, J = 5.7 Hz, 2H), 3.28 (d, J = 6.0Hz, 2H), 3.16 (t, J = 7.5 Hz, 2H), 2.97 (d, J = 8.7 Hz, 2H), 2.58 (td, J =7.5, 4.2 Hz, 4H), 2.49 (s, 3H), 2.39 (s, 3H), 2.38 (s, 3H), 2.15 (t, J = 7.4Hz, 2H), 1.92 (p, 2H), 1.73 - 1.60 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ171.66, 171.16, 171.09, 156.24, 153.57, 146.96, 141.59, 141.15, 140.59,137.00, 134.53, 130.84, 130.43, 121.93, 119.85, 91.32, 54.95, 38.13, 37.21,36.95, 34.73, 34.11, 33.53, 29.80, 29.03, 27.59, 26.89, 23.46, 15.96, 15.85,14.17. HRMS (ESI, m / z): C 35 H 41 BF2IN5NaO4 + for [M+Na] + , calcd 794.2162, found794.2160.

[0105] Synthesis of compound IP-BDP-BSH

[0106] Weigh 50.6 mg (0.06 mmol, 1 eq.) of compound 11 and 22.5 mg (0.10 mmol, 1.5 eq.) of BSH, dissolve them in 3 mL of DCM and 0.3 mL of MeOH, add 13.5 μL (0.10 mmol, 1.5 eq.) of Et3N, add a magnetic stir bar, purge three times with nitrogen, and react at 25 °C for 36 h. TLC analysis (dichloromethane:methanol = 10:1) showed that compound 11 had completely reacted, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the mixture was separated by reversed-phase column chromatography (C10). 18 (SiO2, H2O:MeOH = 10:1~1:1) yielded 49.8 mg of an orange-yellow solid compound IP-BDP-BSH, with a yield of 77%. The 1H NMR spectrum of IP-BDP-BSH is shown below. Figure 4 As shown, 1H NMR (400 MHz, Methanol-d4) δ 7.58 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.29 (s, 1H), 6.07 (s, 1H), 3.75 (dd, J = 8.7, 4.0Hz, 1H), 3.60 (m, 2H), 3.43 (dd, J = 12.6, 5.7 Hz, 2H), 3.24 (m, 3H), 3.16 (t, J = 7.8 Hz, 2H), 3.04 (dd, J = 18.7, 8.8 Hz, 1H), 2.95 (d, J = 9.1 Hz,2H), 2.60 (m,2H), 2.54 (t, J = 7.7 Hz, 2H), 2.43 (s, 3H), 2.41 (s, 3H), 2.38(s, 3H), 2.20 (t, J = 7.4 Hz, 2H), 1.86 (q, J = 7.6 Hz, 2H), 1.66 (m, 4H),1.61 - 0.64 (m, 11H). HRMS (ESI, m / z): C 35 H 53 B 13 F2IN5O4S1 2- for [M-2Na] 2- , calcd472.6657, found 472.7067.

[0107] Example 4

[0108]

[0109] Synthesis of Compound 12

[0110] Weigh 70.3 mg (0.09 mmol, 1 eq.) of compound 11, dissolve it in 3 mL of anhydrous DCM, place the flask in an ice-salt bath (-15℃), add 7 μL (0.10 mmol, 1.1 eq.) of ClSO3H after 10 min, and continue to react in an ice-water bath for 10 min. Remove the ice-salt bath and react at 25℃ for 1 h. An orange-red precipitate is formed, and the DCM layer shows no fluorescence. Perform TLC analysis on the DCM layer (dichloromethane:methanol = 10:1). No compound 11 remains. Stop the reaction, concentrate under reduced pressure to remove DCM. Dissolve it again in 3 mL of MeOH and 1 mL of H2O, add 9.1 mg (0.10 mmol, 1.1 eq.) of NaHCO3, react at 25℃ for 1 h, concentrate under reduced pressure to remove the solvent, and separate by reversed-phase column chromatography (C10). 18 The reaction mixture of SiO2, H2O:MeOH (10:1~1:2) yielded 1245.6 mg of an orange-yellow solid compound, with a yield of 57%. 1 H NMR (400 MHz, Methanol-d4) δ7.60 - 7.55 (m, 2H), 6.96 (d, J = 8.5 Hz, 2H), 6.79 (s, 2H), 6.24 (s, 1H),3.88 (s, 1H), 3.61 (dd, J = 6.6, 4.9 Hz, 2H), 3.41 - 3.34 (m, 2H), 3.22 (s,2H), 3.13 (t, J = 7.6 Hz, 4H), 2.76 - 2.69 (m, 6H), 2.55 (t, J = 7.7 Hz, 2H), 2.49 (t, J = 7.6 Hz, 2H), 2.45 (d, J = 5.2 Hz, 3H), 2.17 (t, J = 7.5 Hz, 2H),1.90 - 1.82 (m, 2H), 1.69 (s, 4H).HRMS (ESI, m / z): C 35 H 40 BF2IN5O7S - for [M-Na] - ,calcd 850.1754, found 850.1764.

[0111] Synthesis of Compound 13

[0112] The synthesis of compound 13 was performed in the same manner as compound 12, with disulfonation substitution achieved by increasing the sulfonating agent ClSO3H to 2 equivalents. 70.0 mg (0.09 mmol, 1 eq.) of compound 11 was weighed, 12 μL (0.18 mmol, 2 eq.) of ClSO3H was added, and 16.4 mg (0.18 mmol, 2 eq.) of NaHCO3 was added, yielding 27.4 mg of orange-yellow solid compound 13, with a yield of 31%. HRMS (ESI, m / z): C 35 H 39 BF2IN5O 10 S2 2- for [M-2Na] 2- , calcd464.5622, found 464.5627.

[0113] Synthesis of compound IP-sulfo-BDP-BSH

[0114] Weigh 45.6 mg (0.05 mmol, 1 eq.) of compound 12 and 17.2 mg (0.08 mmol, 1.5 eq.) of BSH, dissolve them in 3 mL of MeOH, add 11 μL (0.08 mmol, 1.5 eq.) of Et3N, add a magnetic stir bar, purge with nitrogen three times, and react at 25 °C for 30 h. TLC analysis (dichloromethane:methanol = 5:1) showed that compound 12 had completely reacted, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the mixture was separated by reversed-phase column chromatography (C10). 18 (SiO2, H2O:MeOH = 5:1~1:5) yielded 40.1 mg of orange-yellow solid IP-sulfo-BDP-BSH, with a yield of 70%. HRMS (ESI, m / z): C 35 H 52 B 13 F2IN5O7S2 3- for [M-3Na] 3- , calcd 349.1233, found 349.1234.

[0115] Synthesis of compound IP-disulfo-BDP-BSH

[0116] Weigh 27.4 mg (0.03 mmol, 1 eq.) of compound 13 and 10.6 mg (0.04 mmol, 1.5 eq.) of BSH, dissolve in 3 mL of MeOH, add 6 μL (0.04 mmol, 1.5 eq.) of Et3N, add a magnetic stir bar, purge with nitrogen three times, and react at 25 °C for 36 h. TLC analysis (ethyl acetate:methanol = 3:1) showed that compound 13 had completely reacted, and the reaction was stopped. The solvent was removed by concentration under reduced pressure, and the mixture was separated by reversed-phase column chromatography (C10). 18 (SiO2, H2O:MeOH = 5:1~1:8) yielded 16.3 mg of orange-yellow solid IP-disulfo-BDP-BSH, with a yield of 48%. HRMS (ESI, m / z): C 35 H 51 B 13 F2IN5O 10 S3 4- for [M-4Na] 4- , calcd 275.8573, found 275.8571.

[0117] Example 5

[0118] The compounds IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP, and IP-BDP-BSH synthesized in Examples 1, 2, and 3 were characterized as follows:

[0119] Spectroscopic testing experiments: The ultraviolet-visible spectra of the compounds were measured using a UV-Vis spectrophotometer, and the fluorescence spectra of the compounds were measured using a fluorescence spectrometer. The results are shown in Figure 5. The results show that the maximum absorption wavelengths of IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP, and IP-BDP-BSH are all around 500 nm, and the maximum emission wavelengths are 519, 512, 504, and 513 nm, respectively, and they exhibit yellow fluorescence.

[0120] Purity analysis experiment: The purity of the synthesized compounds was characterized by high performance liquid chromatography, as shown in Figure 6-9. The results showed that the purity of the obtained compounds was greater than 95%.

[0121] Example 6

[0122] Non-neutron irradiation toxicity evaluation was performed on the compounds IP-BDP, IP-sulfo-BDP, IP-disulfo-BDP, and IP-BDP-BSH synthesized in Examples 1, 2, and 3:

[0123] Weigh 11.8 mg of IP-BDP and dissolve it in 1 mL of DMSO solution to prepare a 20 mM stock solution. Weigh 13.4 mg of IP-sulfo-BDP, 15.0 mg of IP-disulfo-BDP, and 19.8 mg of IP-BDP-BSH and dissolve them separately in 1 mL of PBS solution to prepare 20 mM stock solutions. Take different volumes of the stock solutions of the four drugs and prepare drug solutions with concentrations of 12.5, 25, 50, 100, 200, and 400 μM using culture medium.

[0124] (1) Evaluation of non-neutron irradiation toxicity of the test drug to mouse oral squamous cell carcinoma cells (MOC-1) using the MTT assay

[0125] MOC-1 cells in logarithmic growth phase were digested with 0.25% trypsin, the trypsin was neutralized with culture medium, centrifuged, the supernatant was discarded, and the cells were resuspended in culture medium to a density of 1×10⁶ cells / year. 5 A cell suspension of 1 × 10⁶ cells / mL was seeded into 96-well plates, with 1 × 10⁶ cells / mL per well. 4 Cells were dispersed evenly by shaking and incubated in a constant temperature incubator for 12 h. Cells were observed to adhere well and grow well. The original culture medium was removed from the 96-well plate, and 100 μL of the prepared drug solution was added to each well. After shaking, the plate was incubated with the drug-containing medium for 12 h. After incubation, the drug-containing medium was removed, and cell growth was observed. Each well was rinsed with 100 μL of PBS three times. The PBS was removed, and 200 μL of MTT solution (0.5 mg / mL) was added to each well under dark conditions. After shaking, the plate was incubated for 4 h, resulting in the formation of blue-purple formazan crystals. The solution in the wells was carefully removed under dark conditions, and 200 μL of chromatographic grade DMSO was added to each well to dissolve the formazan crystals. The plate was then placed in a multi-plate reader, shaken for 5 min, and the absorbance (OD) of each well was measured at 570 nm. Four parallel replicates were set up for each drug delivery well, and the average value was taken. Cell viability was calculated according to the following formula: Cell viability (%) = (OD of test drug group / OD of blank group) × 100%. The results are shown in Figure 10. The results show that under non-neutron irradiation conditions, IP-BDP had no significant toxicity to MOC-1 cells at concentrations below 25 μM, IP-sulfo-BDP had no significant toxicity to MOC-1 cells at concentrations below 50 μM, and IP-disulfo-BDP and IP-BDP-BSH had no significant toxicity to MOC-1 cells at concentrations below 100 μM.

[0126] (2) Evaluation of non-neutron irradiation toxicity of the test drug to human tongue squamous cell carcinoma (SAS) using the MTT assay

[0127] The non-neutron toxicity of the compounds to SAS cells was determined using the same method. The results are shown in Figure 11. The results indicate that, under non-neutron irradiation conditions, IP-BDP showed no significant toxicity to SAS cells at concentrations below 25 μM, IP-sulfo-BDP showed no significant toxicity to SAS cells at concentrations below 50 μM, and IP-disulfo-BDP and IP-BDP-BSH showed no significant toxicity to SAS cells at concentrations below 200 μM.

[0128] Example 7

[0129] Qualitative monitoring of in vitro cellular uptake of IP-disulfo-BDP and IP-BDP-BSH synthesized in Examples 2 and 3 was performed.

[0130] (1) Qualitative monitoring of the uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells using fluorescence microscopy

[0131] MOC-1 cells in logarithmic growth phase were digested with 0.25% trypsin, the trypsin was neutralized with culture medium, centrifuged, the supernatant was discarded, and the cells were resuspended in culture medium to a density of 1×10⁶ cells / year. 6 A cell suspension of 5 × 10⁶ cells / mL was seeded into 12-well plates, with 5 × 10⁶ cells / mL per well. 5Cells were dispersed evenly by shaking and incubated in a constant temperature incubator for 12 h. Cells were observed to adhere well and grow well. IP-disulfo-BDP and IP-BDP-BSH solutions were prepared at 100 μM concentrations using culture medium. The original culture medium was removed from 12-well plates, and 1 mL of the prepared drug solution was added to each well. After shaking, the plates were incubated in a constant temperature incubator for 0.5, 1, 2, 4, 8, and 12 h, with four parallel wells at each time point. After incubation, the drug-containing culture medium was removed, and cell growth was observed in the wells. Each well was rinsed with 500 μL of PBS three times, and the PBS was removed. Each well was then fixed with 500 μL of 4% paraformaldehyde for 20 min at room temperature. The paraformaldehyde was removed, and each well was rinsed with 500 μL of PBS three times, and the PBS was removed. Perform the following procedures under light-protected conditions: Add 500 μL of DAPI solution (0.5 μg / mL) to each well, stain cell nuclei at room temperature for 20 min, aspirate the DAPI solution, add 500 μL of PBS to each well for rinsing three times, aspirate the PBS, and finally add 500 μL of PBS to each well to fix the cells. Use a fluorescence microscope to capture intracellular fluorescence images, where IP-disulfo-BDP exhibits yellow-green fluorescence (excitation wavelength λ). ex = 496 nm, emission wavelength λ em = 504 nm), IP-BDP-BSH is a yellow-green fluorescence (λ). ex = 495 nm, λ em = 513 nm), DAPI is a blue fluorescent (λ) ex = 340 nm, λ em = 488 nm). The results are shown in Figure 12. The results show that the yellow fluorescence in MOC-1 cells became increasingly stronger during the co-incubation period of 0.5 h to 12 h after drug administration, with the strongest fluorescence intensity at 12 h. This indicates that the uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells increased in a time-dependent manner.

[0132] (2) Qualitative monitoring of the uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells using fluorescence microscopy

[0133] The uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells was monitored using the same method, and the results were as follows: Figure 13As shown, the results indicated that the yellow fluorescence in SAS cells became increasingly intense during the co-incubation period of 0.5 h to 12 h after drug administration, with the strongest fluorescence intensity at 12 h, indicating that the uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells increased in a time-dependent manner.

[0134] Example 8

[0135] In vitro cellular uptake quantitative analysis was performed on the IP-disulfo-BDP and IP-BDP-BSH synthesized in Examples 2 and 3:

[0136] In this embodiment, BSH (disodium mercaptododecylene) was purchased from Katchem, Czech Republic.

[0137] (1) Quantitative analysis of the uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells using inductively coupled plasma optical emission spectrometry (ICP-OES).

[0138] MOC-1 cells in logarithmic growth phase were digested with 0.25% trypsin, the trypsin was neutralized with culture medium, centrifuged, the supernatant was discarded, and the cells were resuspended in culture medium to a density of 1×10⁶ cells / year. 6 A cell suspension of 5 × 10⁶ cells / mL was seeded into 12-well plates, with 5 × 10⁶ cells / mL per well. 5Cells were dispersed evenly by shaking and incubated in a constant temperature incubator for 12 h. Cells were observed to adhere well and grow well. IP-disulfo-BDP, IP-BDP-BSH, and BSH were prepared into 100 μM drug solutions using culture medium. The original culture medium was removed from the 12-well plates, and 1 mL of the prepared drug solution was added to each well. After shaking, the plates were placed in a constant temperature incubator, and MOC-1 cells were co-incubated with the drug-containing medium for 0.5, 1, 2, 4, 8, and 12 h, with 5 parallel wells at each time point. After incubation, the drug-containing medium was removed, and cell growth was observed in the wells. Each well was rinsed with 500 μL PBS three times, and the PBS was removed. 1 mL of digestion buffer (concentrated nitric acid:concentrated sulfuric acid = 1:1, v:v) was added to each well, the plates were capped, and digestion was carried out at room temperature for 72 h. After digestion, deionized water was added to each well to a final volume of 3 mL. The solution was then filtered into EP tubes using a 5 mL syringe and a 0.22 μm pore size polyethersulfone membrane. The boron concentration in the solution of the EP tubes was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The boron uptake of cells after co-incubation with the drug-containing culture medium for different time periods was calculated using the following formula: Cellular boron uptake (ng B / 10) 6 (cells) = (boron content in the treatment group - boron content in the control group) / cell number. The results are shown in Figure 14. The results indicate that the boron uptake of IP-disulfo-BDP by MOC-1 cells showed a slow increasing trend within the test range, reaching exactly 20 ng B / 10 at 2 h. 6 Cells, reaching peak at 12h (23 ng B / 10) 6 The boron uptake of IP-BDP-BSH by MOC-1 cells also showed a slow upward trend, exceeding 20 ng B / 10 at 0.5 h. 6 Cells, reaching peak at 12 h (43 ng B / 10) 6 The uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells showed a time-dependent increase, consistent with the uptake observed under fluorescence microscopy. Furthermore, the uptake of IP-disulfo-BDP and IP-BDP-BSH by MOC-1 cells was significantly superior to that of BSH at all time points.

[0139] (2) ICP-OES quantitative analysis of the uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells

[0140] The uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells was quantitatively determined using the same method. The results are shown in Figure 15. The results showed that the uptake of IP-disulfo-BDP by SAS cells reached 50 ngB / 10 at 0.5 h. 6 The number of cells exceeded 20 ng B / 10. 6 Cells showed a slow upward trend in uptake from 0.5 to 4 hours with prolonged co-incubation time, and the upward trend slowed further from 4 to 12 hours, reaching the maximum uptake value (73 ng B / 10) at 12 hours. 6 The uptake of IP-BDP-BSH by SAS cells increased slowly over 0.5–4 h, surged sharply between 4–8 h, and then slowly increased again to a maximum of 144 ng B / 10⁻¹⁰ between 8–12 h. 6 Cells. The uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells showed a time-dependent increase, consistent with the uptake observed under fluorescence microscopy. Furthermore, the uptake of IP-disulfo-BDP and IP-BDP-BSH by SAS cells was significantly superior to that of BSH at all time points.

[0141] Example 9

[0142] Evaluation of boron distribution in tumor-bearing mice using IP-disulfo-BDP synthesized in Example 2:

[0143] In this embodiment, the BPA-fructose complex (BPA-F, with a molar ratio of BPA to fructose of 1:1.125) was purchased from Zhongbo (Xiamen) Medical.

[0144] Fifty-two 6-7 week old male C57BL / 6 black mice, weighing 20-22 g, were selected and acclimatized for one week. MOC-1 subcutaneous tumors were implanted in the nape of the neck. Seven days after implantation, a spherical protrusion approximately 0.3 cm in diameter appeared on the skin at the implantation site, indicating successful establishment of the MOC-1 subcutaneous tumor model in mice. The tumor-bearing mice were randomly divided into 13 groups (n = 4). One group received a tail vein injection of PBS as a blank control group, six groups received a tail vein injection of IP-disulfo-BDP (600 mg / kg, 0.75 mmol / kg), and the remaining six groups received a tail vein injection of BPA-F (309 mg / kg, containing 0.75 mmol / kg BPA). Mice in each group were euthanized by cervical dislocation at the corresponding time after drug administration. Blood was collected by removing the eyeballs. The heart, liver, spleen, lungs, kidneys, brain, tumors, blood, adjacent muscle and skin tissues were dissected and removed. A total of 10 organs or tissues were collected from each mouse. The liver was placed in a 50 mL centrifuge tube, and the other organs or tissues were placed in 15 mL centrifuge tubes. All organs or tissues were weighed. 5 mL of concentrated nitric acid was added to each centrifuge tube containing liver and blood, and 2 mL of concentrated nitric acid was added to each centrifuge tube containing heart, spleen, lungs, kidneys, brain, tumors, adjacent muscle and skin. After digestion at 90°C for 2.5 h, the liquid in the centrifuge tube containing liver and blood was diluted to 10 mL with purified water, and the liquid in the centrifuge tube containing other organs or tissues was diluted to 5 mL with purified water. All test sample liquids in the centrifuge tubes were filtered through a 0.22 μm pore size polyethersulfone filter membrane into EP tubes. The boron content of each organ was determined using ICP-OES. The boron content of each organ of the mouse after drug administration was calculated according to the following formula: Organ boron content (μg B / g) = (Bron content of organs in the drug administration group - Boron content of organs in the blank group) / Organ mass.

[0145] As shown in Figure 16, within 0.5–2 h, IP-disulfo-BDP was mainly distributed in the liver, lungs, kidneys, tumors, and blood. The liver and kidneys, as important metabolic organs, distributed large quantities of the compound through their metabolism. Boron concentrations in the lungs and blood continuously decreased during this period, while the boron concentration in the tumor remained between 6 and 8 ppm, reaching a maximum of 7.2 ± 0.5 ppm at 2 h, demonstrating a certain accumulation and retention capacity in the tumor. After 2 h, IP-disulfo-BDP was continuously metabolized, and the boron concentrations in various organs and tissues continued to decrease. After binding to albumin, IP-disulfo-BDP can endow it with a certain tumor accumulation and retention capacity, exhibiting slow metabolism within tumors and rapid metabolism in normal organs and tissues. At the same dosage, BPA-F was mainly distributed in the kidneys and tumors. The boron concentration in the tumor reached a maximum of 9.0 ± 2.4 ppm at 2 h, while the boron concentration in other organs or tissues hardly exceeded 5 ppm. After 2 h, the boron in the tumor and other organs and tissues was rapidly cleared.

[0146] A significant difference analysis was performed on the boron concentrations of IP-disulfo-BDP and BPA-F in tumors, and the results are shown in Figure 17. The results showed no significant difference between IP-disulfo-BDP and BPA-F within the tumor over a period of 0.5–12 h. The T / N and T / B values ​​were obtained by comparing the boron concentrations in the tumor with those in muscle and blood. The positive control drug BPA-F showed T / N and T / B values ​​exceeding 3 at 2 h, as did IP-disulfo-BDP, meeting the requirements for boron carriers in BNCT. The fact that both drugs achieved similar tumor boron concentrations and T / N and T / B values ​​at the same dosage indicates that IP-disulfo-BDP has similar effects to BPA-F, but with a broader spectrum.

[0147] Example 10

[0148] Evaluation of boron distribution in tumor-bearing mice using IP-BDP-BSH synthesized in Example 3:

[0149] In this embodiment, BSH (disodium mercaptododecylene) was purchased from Katchem, Czech Republic.

[0150] Fifty-two 6-7 week old male C57BL / 6 black mice, weighing 20-22 g, were selected and acclimatized for one week. A mouse MOC-1 subcutaneous tumor model was constructed using the same method as in Example 9. The tumor-bearing mice were randomly divided into 13 groups (n = 4). One group received a tail vein injection of PBS as a blank control group, six groups received a tail vein injection of IP-BDP-BSH (141 mg / kg, with a boron dose of 20 mg B / kg), and the remaining six groups received a tail vein injection of BSH (34 mg / kg, with a boron dose of 20 mg B / kg). Mouse organs and tissues were treated using the same method as in Example 9, and the boron content of each organ and tissue was determined using ICP-OES.

[0151] As shown in Figure 18, IP-BDP-BSH was widely distributed in the liver, lungs, kidneys, and blood at 0.5 h. The boron concentration in these organs showed a strict decreasing trend over time, especially in the liver and blood, indicating that IP-BDP-BSH was rapidly cleared from these organs. The boron concentration within the tumor remained relatively stable at around 10 ppm from 0.5 to 2 h, reaching a maximum of 13.1 ± 1.6 ppm at 1 h, indicating that IP-BDP-BSH could stably persist within the tumor for approximately 2 h, demonstrating a certain ability to accumulate and remain in the tumor. At the same dosage, the boron distribution trend of BSH in various organs and tissues was similar to that of IP-BDP-BSH. At 0.5 h, it was mainly distributed in the liver, lungs, kidneys, and blood, with less distribution in other organs and tissues. The boron concentration in each organ and tissue showed a strict decreasing trend over time. When IP-BDP-BSH binds to albumin, it can prolong the accumulation of albumin in tumors and slow down intratumoral metabolism, while metabolism in normal organs and tissues remains rapid. Conjugating IP-BDP to BSH can improve the tumor targeting, accumulation, and retention capabilities of BSH to some extent.

[0152] A significant difference analysis of boron concentrations in tumors using IP-BDP-BSH and BSH was performed, and the results are shown in Figure 19. The results showed significant differences in boron concentrations between IP-BDP-BSH and BSH in tumors from 0.5 to 4 hours. The T / N and T / B values ​​were obtained by comparing the boron concentration in the tumor with those in muscle and blood. The T / N value of the positive control drug BSH remained above 3 from 0.5 to 1 hour, then continuously decreased, while the T / B value showed a continuous upward trend but never exceeded 3. The T / N and T / B values ​​of IP-BDP-BSH both exceeded 3 at 2 hours, meeting the requirements for boron carriers in BNCT, while the T / N and T / B values ​​of BSH did not simultaneously exceed 3 at any time point. This indicates that conjugating IP-BDP with BSH can endow BSH with certain tumor targeting and accumulation, thus meeting the requirements for BNCT irradiation.

[0153] Example 11

[0154] The in vivo safety of IP-disulfo-BDP and IP-BDP-BSH synthesized in Examples 2 and 3 was evaluated in mice:

[0155] The reagent kit in this embodiment was purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd.

[0156] Fifteen male C57BL / 6 mice, aged 6–7 weeks and weighing 20–22 g, were selected and randomly divided into three groups (n = 5) after one week of acclimatization. The first group received a tail vein injection of 200 μL PBS as a blank control; the second group received a tail vein injection of 600 mg / kg IP-disulfo-BDP; and the third group received a tail vein injection of 141 mg / kg IP-BDP-BSH. Fourteen days after administration, the mice were anesthetized with ether, and blood was collected by enucleation. The blood was incubated at 4°C for 12 h, then centrifuged at 3000 rpm for 15 min at 4°C, and the supernatant serum was separated. Serum samples were collected, and serum ALT and AST levels were measured according to the instructions for the Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) test kits to assess liver function after drug administration. Serum samples were also collected, and serum BUN and Cr levels were measured according to the instructions for the Blood Urea Nitrogen (BUN) and Creatinine (Cr) test kits to assess kidney function after drug administration. As shown in Figure 20, there were no significant differences in ALT, AST, BUN, and Cr levels in the serum of mice in the IP-disulfo-BDP group, IP-BDP-BSH group, and control group. The dosage administered in this experiment did not cause an increase in serum transaminase, urea nitrogen, creatinine, or other indicators in mice, indicating that IP-disulfo-BDP and IP-BDP-BSH had no significant toxic effects on the liver and kidneys of mice within 14 days after administration, and had certain tissue compatibility and safety.

[0157] This invention designs and synthesizes a class of albumin-bound BODIPY derivatives. These compounds use BODIPY as the core, introducing the albumin-free non-covalent binder 4-(p-iodophenyl)butyric acid (IP) into the molecule. BSH can be further introduced to provide additional boron sources. Utilizing the EPR effect of albumin, tumor-targeted delivery to boron-containing modules (BODIPY or BSH) can be achieved. This strategy does not rely on specific amino acid transporters and exhibits broad-spectrum targeting. Simultaneously, the imaging capabilities of BODIPY enable both therapeutic and diagnostic functions for BNCT (Brain-Neuro-Traumatic Cognitive Therapy), providing a new molecular tool for the integrated diagnosis and treatment of BNCT.

[0158] Theranostics is a crucial direction in precision medicine, its core concept being the integration of diagnostic and therapeutic functions onto a single molecular platform to achieve "what is seen is what is treated." In the field of boron non-invasive tumor therapy (BNCT), the development of theranostics-based boron carriers has significant clinical implications: firstly, real-time monitoring of boron drug distribution in vivo via fluorescence or radionuclide imaging allows for optimization of neutron irradiation timing, improving treatment efficacy; secondly, it enables the assessment of tumor heterogeneity in boron drug uptake, screening for suitable BNCT patients and achieving personalized treatment.

[0159] Fluoroboropyrrole (BODIPY) fluorescent dyes possess excellent photophysical properties, such as high molar extinction coefficient, high fluorescence quantum yield, good photostability, and easily modifiable chemical structures. The application of BODIPY in BNCT will... 10 B, introduced into the BODIPY structure, not only achieves... 10 The delivery of B can also monitor the distribution of boron in tumor tissue in real time, providing a visualization tool for the precise implementation of BNCT; and realize the targeted delivery of BODIPY-type boron carriers using albumin.

[0160] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of albumin-bound BODIPY derivatives, characterized in that, The albumin-bound BODIPY derivative has the structure and isotopic structure shown in Formula I or Formula II: ； Among them, R 0 for or m is an integer from 1 to 10, and n is an integer from 1 to 10; X is NH, O, or S; Y is F or 18 F; R 1 R 2 R 3 R 4 R 5 R 6 Each of these elements is independent of the others: hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy. or Z represents hydrogen, sodium, potassium, lithium, cesium, rubidium, or... ; R 7 for or q is an integer from 0 to 5, and t is an integer from 1 to 10; A and G can be independently represented as NH, O, or S; k is an integer from 1 to 10; R 8 It is a C1-C6 alkyl, C1-C6 alkoxy or ; p is an integer between 0 and 5; E + Sodium ions, potassium ions, lithium ions, cesium ions, rubidium ions, or .

2. The albumin-bound BODIPY derivative according to claim 1, characterized in that, In formula I, R 0 for m is an integer between 2 and 6; X is NH; Y is F; R 1 R 3 R 4 R 6 Each is independently selected from methyl and ethyl; R 2 R 5 Each independently selected from hydrogen or Z represents sodium or potassium.

3. The albumin-bound BODIPY derivative according to claim 1, characterized in that, In Formula II, R 0 for m is an integer between 2 and 6; X is NH; Y is F; R 1 R 3 R 6 Each is independently selected from methyl and ethyl; R 2 R 5 Each independently selected from hydrogen or Z represents sodium or potassium; R 7 for q is an integer between 0 and 2; A is NH; k is an integer between 2 and 4; R 8 for ; G is S; p is 0; E + It consists of sodium ions, potassium ions, or lithium ions.

4. The albumin-bound BODIPY derivative according to claim 1, characterized in that, The BODIPY derivative has the following structure: 。 5. The method for preparing a class of albumin-bound BODIPY derivatives according to claim 1, characterized in that: Preparation of compound I, R 2 and R 5 When all hydrogen atoms are present, the synthetic route is as follows: ， Among them, R 0 R 1 R 3 R 4 R 6 The definition of X is the same as in claim 1; Preparation of compound of formula I, R 2 R 5 When the hydrogen atoms are not both present, the synthetic route is as follows: ， Among them, R 0 R 1 R 3 R 4 R 6 The definition of X is the same as in claim 1; Preparation of compound of formula II, R 2 and R 5 When both are hydrogen, the synthetic route is as follows: ； Among them, R 0 R 1 R 3 R 6 R 7 R 8 X, A, G, k, p, E + The definition is the same as that in claim 1; Preparation of compound of formula II, R 2 R 5 If the hydrogen atoms are not both present, the synthetic route is as follows: ， Among them, R 0 R 1 R 3 R 6 R 7 R 8 X, A, G, k, p, E + The definition is the same as in claim 1.

6. The application of the albumin-bound BODIPY derivative according to any one of claims 1-5, characterized in that, The application of the derivative in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that: The derivative is used in the preparation of antitumor drugs in which the targeted boron agent of BNCT is the active component.

8. The application according to claim 7, characterized in that, The tumors include head and neck tumors, intracranial tumors, breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, prostate cancer, pancreatic cancer, uterine cancer, stomach cancer, and rectal cancer.

9. The application according to claim 8, characterized in that, The head and neck tumors mentioned include laryngeal cancer, thyroid cancer, nasal cancer, tonsil cancer, lymphoma, and sarcoma.

10. The application according to claim 8, characterized in that, The intracranial tumors mentioned include gliomas, medulloblastomas, ependymomas, and brain metastases.

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