A cisplatin-boron compound, its synthesis method and application

Abstract

The application relates to the technical field of tumor treatment drugs, and particularly discloses a cisplatin-boron compound, a synthesis method and application thereof. The cisplatin-boron compound has a structural formula as shown in (a) in the specification. The cisplatin-boron compound has a certain targeted killing effect on tumor cells, can present a higher boron concentration in cancer cells, is easy to be enriched in the cancer cells, and has an ideal value for BNCT treatment.

Description

Technical Field

[0001] This application relates to the field of tumor therapeutic drug technology, specifically to a cisplatin-boron compound, its synthesis method, and its application. Background Technology

[0002] With the development of medical technology, the technique of radiation-based tumor killing has evolved from non-selective X-ray and gamma radiation to proton and heavy ion radiation therapy with a certain degree of longitudinal energy selectivity (Borager peak). Especially utilizing... 10 Boron neutron capture therapy (BNCT) uses boron molecules to capture ultrathermal low-energy neutrons (1eV~10keV) and generate nuclear reactions (~3 MeV) in tumor cells, resulting in a higher order of selective killing of tumors.

[0003] Although platinum-based drugs and boron drugs used in BNCT (Boron-Neutralizing Injection) have vastly different mechanisms of action, they are both key areas of focus in precision oncology. However, clinically available boron drugs are still limited to 4-boronic acid-L-phenylalanine (BPA) and disodium mercaptododecanoate (BSH), which have low enrichment and short retention rates, making it difficult to meet the requirement of ≥20 μg per gram of tumor tissue. 10 The theoretical threshold for B atoms, and a flux ≥10 is required for deep tumors. 9 n / (cm) 2 The accelerator for the superheated neutrons (S) is very expensive.

[0004] Research suggests that sequential or simultaneous administration of cisplatin, carboplatin, or oxaliplatin with BNCT, utilizing the synergistic effect of DNA cross-linking and alpha particle double-strand breaks, can increase the objective response rate for recurrent head and neck cancer by 20%–20% in phase I / II trials. However, the overlapping effects of peripheral neurotoxicity, nephrotoxicity, and radiation-induced mucositis create dose limitations. Therefore, no platinum-boron combination drug has yet entered clinical trials. Developing third-generation boron drugs with high-load targeted antibody-polymer, boron nanoclusters, and oral Pt(IV)-boron prodrugs is therefore essential. Summary of the Invention

[0005] The purpose of this application is to provide a cisplatin-boron compound, its synthesis method, and its application, in order to solve the problem that there is currently no high-load targeted antibody-polymer, boron nanocluster, or oral Pt(IV)-boron prodrug.

[0006] In order to achieve the above-mentioned objectives, in a first aspect, this application provides a cisplatin-boron compound.

[0007] The cisplatin-boron compound of this application has the following structural formula (a): ; HSA stands for human serum albumin.

[0008] In a second aspect of this application, the method for synthesizing the cisplatin-boron compound in the embodiments is as follows, comprising the following steps: A carborane solution was placed at -85℃ to -70℃, a lithium reagent was added, and the mixture was stirred to obtain a nucleophilic carborane alkyl lithium intermediate. 4-(bromomethyl)pyridine hydrobromide was added, the temperature was raised to room temperature, and the reaction was carried out for 12 h. The solvent was then removed to obtain the first crude product. The first crude product was purified to obtain the first intermediate product. Cisplatin and silver nitrate were added to N,N-dimethylformamide and stirred for 24 hours under light-protected conditions and at a temperature of 40℃~50℃. The mixture was then centrifuged to obtain a mixed solution. Under conditions of light protection and a temperature of 40℃~50℃, the first intermediate product was added to the mixed solution, stirred for 24 hours, the solvent was removed, and then methanol was added to precipitate and crystallize to obtain PtCB. PtCB and human serum albumin were dissolved in pH buffer and mixed at 35℃~40℃ for 24 h. The mixture was then purified by dialysis to obtain a compound with the structure shown in (a).

[0009] In some embodiments, according to stoichiometric ratios, the reaction process of the structure shown in (a) is as follows: The ratio of carborane to 4-(bromomethyl)pyridine hydrobromide is 1:1.1~1.5; The ratio of cisplatin to silver nitrate is 1:1 to 1.5; The ratio of PtCB to human serum albumin is 2:1~1.5.

[0010] In some embodiments, a cutoff membrane with a molecular weight of 3.0 kDa to 4 kDa is used in the dialysis purification.

[0011] In some embodiments, the lithium reagent is selected from any one of n-butyllithium, sec-butyllithium, and tert-butyllithium.

[0012] A third aspect of this application provides the use of a cisplatin-boron compound as described above, or a cisplatin-boron compound synthesized by the method described above, as a component of a boron neutron capture therapeutic agent for tumors.

[0013] Beneficial technical effects: Compared with the prior art, the cisplatin-boron compound of this application has a good killing effect on cancer cells and is almost non-toxic to normal fibroblasts, indicating that it has a certain targeted killing effect on tumor cells. At the same time, it can show a higher concentration of boron in cancer cells and is easily enriched in cancer cells, with a better ideal value for BNCT treatment. Therefore, it can be used as a component of high-load targeted antibody-polymer, nano boron cluster, and oral Pt(IV)-boron prodrug. Attached Figure Description

[0014] To make the objectives, technical solutions, and advantages of this application clearer, a preferred description of this application will be provided below with reference to the accompanying drawings, wherein: Figure 1 The image shows the 1H NMR spectrum of the cisplatin-boron compound in methanol-d4 in Example 1 of this application. Figure 2 This is the mass spectrum of the cisplatin-boron compound in Example 1 of this application in a mobile phase of methanol / water; Figure 3 This is a high-performance liquid chromatography (HPLC) chromatogram of the cisplatin-boron compound in Example 1 of this application in methanol / water as the mobile phase; Figure 4 This is a graph showing the ratio of platinum to boron in cisplatin-boron compounds quantified by ICP-MS in Example 1 of this application. Figure 5 This is a diagram showing the toxicity of the cisplatin-boron compound to HSC-3 oral tumor cells and normal fibroblast L929 cells in Example 1 of this application; Figure 6 The graph shows the uptake of the cisplatin-boron compound in HSC-3 tumor cells and normal fibroblast L929 cells by ICP-MS. Figure 7 This is a diagram showing the in vivo distribution of the cisplatin-boron compound in mouse HSC-3 oral cancer xenografts, determined by ICP-MS. Detailed Implementation

[0015] To make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the following detailed description of this application is provided in conjunction with embodiments.

[0016] In the embodiments of this application, DMF represents N,N-dimethylformamide; HSA represents human serum albumin; BPA represents 4-boron-L-phenylalanine; DMSO represents dimethyl sulfoxide; and MTT represents 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide.

[0017] This application provides a cisplatin-boron compound having the structural formula shown in (a) below: ; HSA stands for human serum albumin.

[0018] Some methods for preparing the above-mentioned cisplatin-boron compounds are as follows: The synthesis method of the above-mentioned cisplatin-boron compound includes the following steps: A carborane solution was placed at -85℃ to -70℃, a lithium reagent was added, and the mixture was stirred to obtain a nucleophilic carborane alkyl lithium intermediate. 4-(bromomethyl)pyridine hydrobromide was added, the temperature was raised to room temperature, and the reaction was carried out for 12 h. The solvent was then removed to obtain the first crude product. The first crude product was purified to obtain the first intermediate product. Cisplatin and silver nitrate were added to N,N-dimethylformamide and stirred for 24 hours under light-protected conditions and at a temperature of 40℃~50℃. The mixture was then centrifuged to obtain a mixed solution. Under conditions of light protection and a temperature of 40℃~50℃, the first intermediate product was added to the mixed solution, stirred for 24 hours, the solvent was removed, and then methanol was added to precipitate and crystallize to obtain PtCB. PtCB and human serum albumin were dissolved in pH buffer and mixed at 35℃~40℃ for 24 h. The mixture was then purified by dialysis to obtain a compound with the structure shown in (a).

[0019] In some embodiments, the lithium reagent is selected from any one of n-butyllithium, sec-butyllithium, and tert-butyllithium.

[0020] In some embodiments, when the reaction produces a compound having structural formula (a) by stoichiometric ratio, the ratio of the carborane to the 4-(bromomethyl)pyridine hydrobromide during the reaction is 1:1.1 to 1.5. For example, 1:1.1, or 1:1.2, or 1:1.25, or 1:1.3, or 1:1.35, or 1:1.4, or 1:1.45, or 1:1.5, and any ratio between any two of these ratios.

[0021] In some embodiments, when the reaction produces a compound having structural formula (a) by stoichiometric ratio, the ratio of cisplatin to silver nitrate is 1:1 to 1.5 during the reaction. For example, 1:1, or 1:1.1, or 1:1.2, or 1:1.25, or 1:1.3, or 1:1.35, or 1:1.4, or 1:1.45, or 1:1.5, and any ratio between any two of these ratios.

[0022] In some embodiments, when the reaction produces a compound having structural formula (a) by means of a stoichiometric ratio, the ratio of PtCB to human serum albumin during the reaction is 2:1 to 1.5. For example, 2:1, or 2:1.1, or 2:1.2, or 2:1.25, or 2:1.3, or 2:1.35, or 2:1.4, or 2:1.45, or 2:1.5, and any ratio between any two of these ratios.

[0023] In some embodiments, the cisplatin-boron compound shown in structural formula (a) is prepared using a cutoff membrane with a molecular weight of 3.0 kDa to 4 kDa during the dialysis purification process. For example, the cutoff membrane has a molecular weight of 3.5 kDa.

[0024] This application further provides the use of the cisplatin-boron compound as described above, or the cisplatin-boron compound synthesized by the method described above, as a component of a boron neutron capture therapeutic agent for tumors.

[0025] The following specific examples illustrate the synthesis method of cisplatin-boron compounds according to this application.

[0026] Example 1 The method for synthesizing cisplatin-boron compounds with the structural formula shown in (a) is as follows: Step S11: Dissolve 1 equiv of carborane in 4 mL of ultra-dry tetrahydrofuran, stir at -78°C for 20 min, add n-butyllithium and stir for 2 h, then add 1.2 equiv of 4-(bromomethyl)pyridine hydrobromide, gradually raise the temperature to room temperature, and continue stirring for 24 h.

[0027] Step S12: The solvent was then removed to obtain the crude product, which was purified by silica gel column chromatography. The yellow precipitate was collected to obtain PYCB. The PYCB was then analyzed, and the results were as follows: 1 H NMR (500 MHz, methanol-d4, C8H 17 B 10 N) δ8.54 (d, J = 6.3 Hz, 2H), 7.33 (d, J = 6.1 Hz, 2H), 4.58 (s, 1H), 3.66 (s,2H), 1.50-2.65 (m, BH, 10H).

[0028] Step S13: Mix 1 equiv of cisplatin and 1 equiv of silver nitrate in 4 mL of ultra-dry N,N-dimethylformamide (DMF), stir at 45°C for 24 h in the dark, and centrifuge to obtain a mixed solution (pale yellow).

[0029] Step S14: Under light-protected conditions at 45°C, PYCB and the mixed solution are mixed and stirred for 24 hours to remove the solvent. Then, methanol is added to precipitate the mixture, which is then removed. Recrystallization is performed using methanol and acetone to obtain the precipitate. The precipitate is white and was detected by 1H NMR spectroscopy. The results are as follows: Figure 1 As shown, 1 H NMR (500 MHz, methanol-d4, [Pt (NH3)2(C8H17 B 10 N)Cl] + ) δ8.75 (d, J = 6.0 Hz, 2H), 7.42 (d, J = 6.0 Hz, 2H), 4.63 (s, 1H), 3.75(s, 2H), 1.50-2.65 (m, BH, 10H). ESI-MS: calcd. for [Pt (NH3)2(C8H 17 B 10 [N)Cl]NO3[M-NO3] + The values ​​were 500.04 and 500.10, respectively, and HPLC and mass spectrometry tests were performed. The results are as follows: Figure 2 and Figure 3 As shown, from Figures 1 to 3 This indicates that the obtained compound is PtCB.

[0030] Step S15: Dissolve 2 equiv of PtCB and 1 equiv of human serum albumin (HSA) in pH buffer (HEPES), stir at 37°C for 24 h, then dialysis with a 3.5 kDa cutoff membrane for 48 h to purify the product. Collect the purified product, freeze-dry it, and then analyze it by inductively coupled plasma mass spectrometry (ICP-MS). The results are as follows. Figure 4 As shown, from Figure 4 It can be identified as a compound having the structural formula shown in (a), abbreviated as PtCBHSA.

[0031] The specific synthetic route for the cisplatin-boron compound with the structural formula (a) is shown below: .

[0032] Performance verification 1. Cytotoxicity of the cisplatin-boron compound PtCBHSA to HSC-3 and L929 cells.

[0033] The cytotoxicity of this compound against HSC-3 and L929 cells was determined using the MTT assay. Approximately 5,000 cells per well were seeded into 96-well plates and cultured for 24 h to promote cell adhesion. Subsequently, cells were exposed to different concentrations of the compound (Cisplatin / BPA / PtCB / PtCBHSA = 0–100 μM, PtCBHSA was quantified by PtCB). The compound was diluted with serum-free medium and co-incubated with cells for 2 h and 24 h (IC50 was measured at the end of 2 h and 24 h). 50After removing the drug, each well was washed with PBS and incubated for 24 hours with fresh bovine serum-containing medium. Then, 25 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for 4 hours. Subsequently, the medium was removed, and 125 μL of DMSO was added. The absorbance of the solution at 490 nm was monitored using a microplate reader, and the results are shown in Table 1. Figure 5 As shown.

[0034] From Table 1 and Figure 5 It is known that PtCBHSA has a certain degree of toxicity to HSC-3, with a 2hIC value. 50 (Hardest inhibitory concentration) = 63.2 ±0.7 μM, 24hIC 50 = 12.5 ±2.9 μM; low toxicity to L929, IC50 50 >100 μM.

[0035] Table 1. IC50 values ​​for various cancer cell lines 50 Value (μM) Where nd indicates that it was not detected.

[0036] 2. The uptake of cisplatin-boron compounds in HSC-3 tumor cells and normal fibroblast L929 cells was determined by ICP-MS.

[0037] Cell uptake was determined by ICP-MS. Cells were loaded at a concentration of 1 × 10⁶ cells / mL. 6 Cells were seeded in culture dishes at a density of 1 / mL. The drug was added to the culture medium and incubated for 2 h, after which the cells were collected. Samples were digested with aqua regia at room temperature for 24 h. Each sample was then diluted with Milli-Q water to prepare a 3% (w / w) HNO3 sample solution. The boron content was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7850), and the results are as follows: Figure 6 As shown.

[0038] from Figure 6 It can be seen that, at the same drug concentration, the boron content of cisplatin-boron compounds in HSC-3 cells is significantly higher than that of BPA, the clinical drug of BNCT, and is even higher than that in L929 cells, indicating that cisplatin-boron compounds are more easily enriched in HSC-3 cells.

[0039] 3. The distribution of cisplatin-boron compounds in HSC-3 oral cancer xenograft mice was determined by ICP-MS.

[0040] HSC-3 cells were subcutaneously injected into the legs of mice. Mice were then administered 40 μg B / kg (BPA / PtCB / PtCBHSA) via tail vein injection. Mice were then euthanized at 2 h, and tumors, blood, heart, liver, spleen, lungs, and kidneys were collected and digested with aqua regia at room temperature for 72 h. Each sample was subsequently diluted with ultrapure water, and the B content was measured by ICP-MS. The ratio of tumor to normal tissue (T / N ratio) and the ratio of tumor to blood (T / B ratio) were calculated, and the results are shown below. Figure 7 As shown.

[0041] from Figure 7 It can be seen that the boron content in PtCBHSA tumors is much higher than that in blood and various organs, and the T / B and T / N values ​​are both greater than 3, reaching the ideal values ​​for BNCT treatment.

[0042] The above embodiments merely illustrate several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A cisplatin-boron compound, characterized in that, The cisplatin-boron compound has the following structural formula (a): ， HSA stands for human serum albumin.

2. A method for synthesizing the cisplatin-boron compound as described in claim 1, characterized in that, Includes the following steps: A carborane solution was placed at -85℃ to -70℃, a lithium reagent was added, and the mixture was stirred to obtain a nucleophilic carborane alkyl lithium intermediate. 4-(bromomethyl)pyridine hydrobromide was added, the temperature was raised to room temperature, and the reaction was carried out for 12 h. The solvent was then removed to obtain the first crude product. The first crude product was purified to obtain the first intermediate product. Cisplatin and silver nitrate were added to N,N-dimethylformamide and stirred for 24 hours under light-protected conditions and at a temperature of 40℃~50℃. The mixture was then centrifuged to obtain a mixed solution. Under conditions of light protection and a temperature of 40℃~50℃, the first intermediate product was added to the mixed solution, stirred for 24 hours, the solvent was removed, and then methanol was added to precipitate and crystallize to obtain PtCB. PtCB and human serum albumin were dissolved in pH buffer and mixed at 35℃~40℃ for 24 h. The mixture was then purified by dialysis to obtain a compound with the structural formula shown in (a). The structural formula of the first intermediate product is shown below: ； The structural formula of PtCB is shown below: 。 3. The method for synthesizing the cisplatin-boron compound as described in claim 2, characterized in that, Based on stoichiometric ratios, the reaction process of the structure shown in (a) is as follows: The ratio of carborane to 4-(bromomethyl)pyridine hydrobromide is 1:1.1~1.5; The ratio of cisplatin to silver nitrate is 1:1 to 1.5; The ratio of PtCB to human serum albumin is 2:1~1.

5.

4. The method for synthesizing the cisplatin-boron compound as described in claim 2, characterized in that, In the dialysis purification, a cutoff membrane with a molecular weight of 3.0 kDa to 4 kDa is used.

5. The method for synthesizing the cisplatin-boron compound as described in claim 2, characterized in that, The lithium reagent is selected from any one of n-butyllithium, sec-butyllithium, and tert-butyllithium.

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