Self-assembled drug molecule as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of biological medicine, and provides a self-assembled drug molecule and a preparation method and application thereof, and the preparation method comprises the following steps: synthesizing a compound Py-S-S-COOH; the method comprises the following steps: synthesizing a compound Py-S-S-B1RA; synthesizing a compound A; synthesizing a compound B; synthesizing a compound C; synthesizing a compound D; synthesizing a compound E; synthesizing a compound F; and the compound CBT-Gem-B1RA is synthesized through the synthesis of A chemotherapeutic drug gemcitabine and a targeted peptide bradykinin B1 receptor stimulant are coupled with a peptide fragment with self-assembly performance, so that the molecule has the capabilities of penetrating a blood brain barrier and targeting brain glioma, can smoothly enter a brain glioma part and can be self-assembled into nano particles in brain glioma cells, and the nano particles can be used for preparing a drug for treating brain glioma. The residence time of the drug in tumor cells is prolonged, the drug enrichment concentration is increased, and finally the glioma specific targeted therapy is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a self-assembled drug molecule, its preparation method, and its application. Background Technology

[0002] Gliomas are the most common malignant brain tumors and the most prevalent primary brain tumors, yet to date, there is still no treatment that significantly improves the survival of glioma patients. Malignant gliomas often grow into normal brain tissue, exhibiting diffuse and infiltrative growth, resulting in unclear boundaries between tumor and normal tissue. With current technology, surgical removal of the lesion is often difficult. Therefore, chemotherapy, as a non-invasive treatment, is a commonly used adjuvant therapy after glioma surgery, playing a crucial role in prolonging patient survival. However, traditional small-molecule chemotherapy drugs are easily metabolized and cleared during circulation; furthermore, due to the blood-brain barrier, traditional chemotherapy drug molecules often struggle to effectively cross the blood-brain barrier to reach the lesion area, resulting in low bioavailability and significant tissue toxicity. Therefore, overcoming the blood-brain barrier and improving the tumor targeting of chemotherapy drugs are crucial for enhancing the efficacy of chemotherapy for brain tumors.

[0003] The effective penetration of the blood-brain barrier by therapeutic drugs has drawn significant attention to various receptors on the barrier. The bradykinin B1 receptor (B1R), a G protein-coupled receptor, is overexpressed under pathological and inflammatory conditions. Studies have demonstrated its expression in various malignant tumors, including breast cancer, prostate cancer, and glioma. Notably, the inflammatory environment surrounding gliomas promotes B1R activation in the brain tumor microenvironment, leading to upregulated B1R expression in glioma cells and surrounding brain capillary endothelial cells. Furthermore, activation of the bradykinin receptor increases blood-brain barrier permeability, facilitating drug penetration and entry into the glioma site. Therefore, B1R is expected to become an ideal target for the treatment of glioma; however, natural bradykinin is easily metabolized during circulation in the body, which is not conducive to its application. Therefore, Guillaume Amouroux et al. modified it. The modified bradykinin B1 receptor agonist (B1RA) structure not only has a high affinity for B1R, but also has better metabolic stability.

[0004] Regarding the issue of easy metabolism of small molecule chemotherapy drugs, existing technologies have disclosed numerous targeted nanoparticle delivery systems that can effectively reduce the metabolism of chemotherapy drugs during in vivo circulation, thereby improving disease diagnosis and treatment outcomes. However, in vitro prepared nanoparticles consistently face problems such as easy liver metabolism and poor bioavailability in practical applications. Enzyme-controlled self-assembly strategies have been extensively studied in disease diagnosis and cancer treatment. Enzyme-controlled self-assembled small molecules can be prepared through chemical synthesis, exhibiting good modifiability. Different functions can be assigned to target molecules through chemical coupling, demonstrating good potential for large-scale production. Furthermore, the strategy of in-situ self-assembly of small molecules into nanoparticles through enzyme control can avoid liver metabolism of target compounds due to size issues, exhibiting higher delivery efficiency, and can prolong the residence time of compounds at the lesion site, improving diagnostic and treatment outcomes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing self-assembled drug molecules, thereby addressing the problems mentioned in the background section.

[0006] The present invention is implemented as follows: a method for preparing self-assembled drug molecules includes the following steps:

[0007] 2,2'-dithiodipyridine was dissolved in methanol, 3-mercaptopropionic acid dissolved in methanol was added dropwise, and then acetic acid was added to react. The reaction was carried out by rotary evaporation to obtain a yellow oily crude product, which was purified to obtain compound Py-SS-COOH.

[0008] The resin loaded with 2-chlorotriphenylmethyl chloride was dispersed in DMF and shaken. The DMF was then extruded, and fluorenemethoxycarbonyl-D-phenylalanine and N,N-diisopropylethylamine dissolved in DMF were added. The reaction was initiated. After washing, DMF was added to disperse the resin, followed by methanol. The reaction solution was extruded, washed, and then reacted with a DMF solution containing piperidine. This process was repeated. After deprotection, the resin was washed with DMF, and fluorenemethoxycarbonyl-L-proline, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine dissolved in DMF were added. The reaction was initiated, washed, and the Fmoc protecting group was removed with a DMF solution containing piperidine. The same procedure was repeated to sequentially attach fluorenemethoxycarbonyl-O-tert-methylalanine to the resin. Butyl-L-serine, fluorenylmethoxycarbonyl-3-cyclohexyl-L-alanine, fluorenylmethoxycarbonyl-glycine, fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, fluorenylmethoxycarbonyl-tert-butoxycarbonyl-L-lysine, and Py-SS-COOH were used. The resin was washed, and the polypeptides were cleaved from the resin with a dichloromethane solution containing trifluoroacetic acid. All reaction solutions were collected, rotary evaporated, and then lyophilized. The resulting solid was dissolved in a DCM solution containing trifluoroacetic acid, and triisopropylsilane was added. After reaction, rotary evaporation was used to obtain a yellow oily crude product, which was purified to obtain compound Py-SS-B1RA.

[0009] The resin containing 2-chlorotriphenylmethyl chloride was dispersed in DMF and shaken. After extruding the DMF, fluorenemethoxycarbonyl-lysine and N,N-diisopropylethylamine were added, reacted, washed, methanol was added, washed with DMF, piperidine-containing DMF solution was added, washed with DMF, fluorenemethoxycarbonyl-S-triphenylmethyl-L-cysteine, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were added, reacted, washed, and piperidine-containing DMF solution was added. In the same operation, fluorenemethoxycarbonyl-L-citrulline and tert-butyloxycarbonyl-L-valine were added to the resin. The resin was washed, and the polypeptide was cleaved from the resin with DCM solution containing trifluoroacetic acid. The polypeptide was precipitated with diethyl ether, centrifuged, collected, and evaporated to obtain a powdery solid, which yielded compound A.

[0010] Compound A and 6-amino-2-cyanobenzothiazole were lyophilized. Compound A was dissolved under nitrogen protection, and N-methylmorpholine was added. Isobutyl chloroformate was added under ice bath conditions. After stirring, dissolved 6-amino-2-cyanobenzothiazole was added, and isobutyl chloroformate was added to the mixture. The mixture was stirred and reacted at room temperature overnight. After the reaction was completed, a yellow solid was obtained by rotary evaporation and purified to obtain compound B.

[0011] Compound B was lyophilized, dissolved under nitrogen protection, and then phenylsilane and tetra(triphenylphosphine)palladium were added. After reaction, rotary evaporation was performed to obtain a powdered solid, which was then purified to obtain compound C.

[0012] Compound C and succinic anhydride were lyophilized, and then dissolved separately in DMF. The pH of the DMF solution containing succinic anhydride was adjusted with N,N-diisopropylethylamine, and then mixed with the DMF solution containing compound C. The mixture was stirred and purified to obtain compound D.

[0013] Compound D, gemcitabine, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were lyophilized. Compound D, gemcitabine, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were dissolved together in anhydrous DMF, stirred overnight, and purified to obtain compound E.

[0014] Compound E was lyophilized, dissolved in DCM solution containing trifluoroacetic acid, and triisopropylsilane was added. After stirring, the mixture was rotary evaporated. The product was precipitated with diethyl ether, and the precipitate was collected by low-temperature centrifugation and evaporated to dryness to obtain compound F.

[0015] Py-SS-B1RA and compound F were dissolved in DMF, respectively. Under nitrogen protection, the DMF solution containing compound F was added dropwise to the DMF solution containing Py-SS-B1RA. The reaction was allowed to proceed overnight, and the compound CBT-Gem-B1RA was obtained after purification.

[0016] Another objective of this invention is to provide a self-assembled drug molecule prepared by the above-described preparation method.

[0017] Another objective of this invention is to provide the application of a self-assembled drug molecule in the preparation of an antitumor prodrug.

[0018] This invention provides a method for preparing a self-assembled drug molecule, which couples the chemotherapy drug gemcitabine (Gem) with a peptide with self-assembly properties to design a therapeutic probe that can self-assemble into nanoparticles within glioma cells. This probe is then combined with a bradykinin B1 receptor agonist. Utilizing the high affinity of the bradykinin B1 receptor agonist for bradykinin B1 receptors in cerebral vascular endothelial cells and glioma cells, the molecule is endowed with the ability to penetrate the blood-brain barrier and target gliomas. This allows it to smoothly enter the glioma site and self-assemble into nanoparticles within the glioma cells, prolonging the drug's residence time in tumor cells, increasing drug concentration, and ultimately achieving specific targeted therapy for gliomas.

[0019] Specifically, this self-assembled drug molecule has the following advantages: Utilizing the targeting effect of bradykinin B1 receptor agonists, it helps the drug penetrate the blood-brain barrier to reach the glioma site, enhancing drug targeting; the GSH and CTSB enzyme-responsive self-assembly capability allows the compound to self-assemble in situ within glioma cells, thereby increasing the drug concentration within tumor cells, prolonging drug retention time, and enhancing therapeutic efficacy. Furthermore, compared to other nanomedicines, the self-assembled compound CBT-Gem-B1RA is not metabolized by the liver due to size issues, improving drug delivery efficiency and bioavailability.

[0020] The drug molecules prepared in this invention can be used in antitumor prodrugs. After intravenous injection, they can bind to bradykinin B1 receptors with the help of bradykinin B1 receptor agonists, thereby penetrating the blood-brain barrier and reaching the brain tumor site. After entering brain tumor cells, the self-assembled peptides in CBT-Gem-B1RA can be reduced by the highly expressed GSH in brain tumor cells and cleaved by CTSB enzyme. Subsequently, they form dimers through click reaction, and then further self-assemble into nanoparticles CBT-Gem-NPs. This increases the concentration of the chemotherapeutic drug Gem in tumor cells, prolongs the drug retention time, effectively enhances the chemotherapy effect, has good biosafety, and has high application value. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the use of CBT-Gem-B1RA for the treatment of glioma in situ, as provided in an embodiment of the present invention.

[0022] Figure 2 This is the mass spectrum of Py-SS-COOH, the first compound synthesized in Example 1;

[0023] Figure 3 This is the mass spectrum of Py-SS-B1RA, the second compound synthesized in Example 1;

[0024] Figure 4 This is the mass spectrum of the third compound A synthesized in Example 1;

[0025] Figure 5 This is the mass spectrum of the fourth compound B synthesized in Example 1;

[0026] Figure 6 This is the mass spectrum of compound C, the fifth compound synthesized in Example 1;

[0027] Figure 7 This is the mass spectrum of the sixth compound D synthesized in Example 1;

[0028] Figure 8 This is the mass spectrum of the seventh compound E synthesized in Example 1;

[0029] Figure 9 This is the mass spectrum of the eighth compound F synthesized in Example 1;

[0030] Figure 10 This is the high-resolution mass spectrum of the ninth compound CBT-Gem-B1RA synthesized in Example 1;

[0031] Figure 11 The 1H NMR spectrum of the ninth compound CBT-Gem-B1RA synthesized in Example 1;

[0032] Figure 12 The mass spectrum of the tenth compound G synthesized in Comparative Example 1 is shown.

[0033] Figure 13 The mass spectrum of compound H, the eleventh compound synthesized in Comparative Example 1;

[0034] Figure 14 The mass spectrum of compound I, the twelfth compound synthesized in Comparative Example 1;

[0035] Figure 15 The mass spectrum of the thirteenth compound J synthesized in Comparative Example 1 is shown.

[0036] Figure 16 The mass spectrum of compound K, the fourteenth compound synthesized in Comparative Example 1;

[0037] Figure 17 This is the high-resolution mass spectrum of CBT-Gem, the fifteenth compound synthesized in Comparative Example 1;

[0038] Figure 18 The high-performance liquid chromatograms of CBT-Gem-B1RA before and after reduction by a reducing agent and CTSB digestion in Example 2 are shown below.

[0039] Figure 19 This is a high-resolution mass spectrum of the product after CBT-Gem-B1RA was reduced by the reducing agent in Example 2;

[0040] Figure 20 This is a high-resolution mass spectrum of the dimer CBT-Gem-Dimer generated by the click reaction after CBT-Gem-B1RA is reduced by the reducing agent and cleaved by CTSB enzyme in Example 2.

[0041] Figure 21 This is a transmission electron microscope image of the nanoparticles formed by the self-assembly of CBT-Gem-B1RA after reduction and enzymatic digestion in Example 2.

[0042] Figure 22The graph shows the cytotoxicity results of different compounds in Example 3 (where a represents the cell viability of CBT-Gem, CBT-Gem-B1RA, B1RA + CBT-Gem-B1RA and U87-Luc cells, and CBT-Gem-B1RA and NIH / 3T3 cells after incubation for 48 h; b represents the cell viability of inhibitor + CBT-Gem-B1RA, CBT-Gem-B1RA and U87-Luc cells, and CBT-Gem-B1RA and NIH / 3T3 cells after incubation for 48 h).

[0043] Figure 23 The images show the antitumor effects of different compounds in Example 4 (where a is a bioluminescence imaging of mouse brain tumors during treatment; b is a growth curve of mouse brain tumors; and c is a curve showing changes in mouse body weight). Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0045] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0046] Example 1: Synthesis of compounds Py-SS-COOH, Py-SS-B1RA, A, B, C, D, E, F and CBT-Gem-B1RA. The synthetic route is as follows:

[0047]

[0048]

[0049] The specific steps are as follows:

[0050] Synthesis of compound Py-SS-COOH: 2,2'-dithiopyridine (528.7 mg, 2.4 mmol) was dissolved in methanol, followed by the dropwise addition of 3-mercaptopropionic acid (173.9 μL, 2 mmol) dissolved in methanol. After the addition was complete, acetic acid (11.4 μL, 0.2 mmol) was added to the system. After reacting for 30 min, the successful synthesis of the product was confirmed by HPLC and ESI-MS. The crude yellow oil obtained by rotary evaporation was purified by HPLC to obtain the compound, which was named Py-SS-COOH.

[0051] Synthesis of compound Py-SS-B1RA: The required raw materials were lyophilized beforehand. First, 0.7 mmol of resin loaded with 2-chlorotriphenylmethyl chloride was weighed, and the resin was dispersed in DMF and added to a horn tube. The mixture was shaken for 30 min, and DMF was extruded. Fluorenylmethoxycarbonyl-D-phenylalanine (Fmoc-D-Phe-OH, 464.9 mg, 1.2 mmol) and N,N-diisopropylethylamine (DIPEA, 250.8 μL, 1.44 mmol) completely dissolved in DMF were added. The reaction was allowed to proceed for 8 h. After the reaction was complete, the mixture was washed three times with DMF. Then, a small amount of DMF was added to disperse the resin, followed by 4 mL of methanol and a reaction for 30 min to block the unreacted active sites on the resin. The reaction solution was extruded, washed three times with DMF, and then a DMF solution containing 20% ​​piperidine was added and reacted for 15 min. This process was repeated three times, with the last two reaction times being 10 min. The amino protecting group Fmoc of Fmoc-D-Phe-OH was removed by 8 min. After removal, the mixture was washed multiple times with DMF. Then, fluorenemethoxycarbonyl-L-proline (Fmoc-Pro-OH, 337.4 mg, 1 mmol), 1-hydroxybenzotriazole (HOBT, 162.1 mg, 1.2 mmol), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 455.0 mg, 1.2 mmol), and DIPEA (208.9 μL, 1.2 mmol) dissolved in DMF were added. The reaction was carried out for 8 min. After the reaction was complete, the resin was washed three times with DMF, followed by removal of the Fmoc protecting group on Fmoc-Pro-OH with a DMF solution containing 20% ​​piperidine. Then, following the same procedure, the following compounds were sequentially added to the resin: fluorenylmethoxycarbonyl-O-tert-butyl-L-serine (Fmoc-Ser(tBu)-OH, 383.4 mg, 1 mmol), fluorenylmethoxycarbonyl-3-cyclohexyl-L-alanine (Fmoc-Cha-OH, 393.2 mg, 1 mmol), fluorenylmethoxycarbonyl-glycine (Fmoc-Gly-OH, 297.3 mg, 1 mmol), fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline (Fmoc-Hyp(tBu)-OH, 409.5 mg, 1 mmol), and fluorenylmethoxycarbonyl-L-proline (Fmoc-Pro-OH, 337.4 mg, 1 mmol). mmol), fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine (Fmoc-Arg(Pbf)-OH, 648.9 mg, 1 mmol), fluorenylmethoxycarbonyl-tert-butyloxycarbonyl-L-lysine (Fmoc-Lys(Boc)-OH, 468.6 mg, 1 mmol), and Py-SS-COOH (215 mmol).0 mg, 1 mmol), then the resin was washed three times each with appropriate amounts of DMF, isopropanol, and n-hexane, respectively. The peptide was then cleaved from the resin using a dichloromethane (DCM) solution containing 1% trifluoroacetic acid (TFA). 4 mL of the solution was added to the tube each time, and the reaction was repeated for 5 min, followed by squeezing out the reaction solution. This process was repeated approximately 10 times until the resin turned completely black. All reaction solutions were collected, and the DCM and TFA were removed by rotary evaporation, followed by lyophilization. Finally, the solid powder was dissolved in 10 mL of DCM solution containing 95% TFA, and 100 μL of triisopropylsilane was added. After reacting for 3 h, a yellow oily crude product was obtained by rotary evaporation. The compound was purified by HPLC and ESI-MS analysis and named Py-SS-B1RA.

[0052] Synthesis of Compound A: Resin containing 0.7 mmol / L 2-chlorotriphenylmethyl chloride was dispersed in DMF and added to a horn-shaped tube. After shaking for 30 min, DMF was extruded, followed by the addition of fluorenemethoxycarbonyl-lysine (Fmoc-Lys(Alloc)-OH, 543.0 mg, 1.2 mmol) and DIPEA (250.8 μL, 1.44 mmol). After reacting for 8 h, the resin was washed three times with DMF. Subsequently, unreacted active sites on the resin were blocked with methanol, followed by washing with DMF. The Fmoc groups on Fmoc-Lys(Alloc)-OH were removed with a DMF solution containing 20% ​​piperidine. After washing multiple times with DMF, fluorenemethoxycarbonyl-S-triphenylmethyl-L-cysteine ​​(Fmoc-Cys(Trt)-OH, 585.7 mg, 1 mmol) and HOBT (162.1 mg, 1.2 mmol) were added. HBTU (455.0 mg, 1.2 mmol) and DIPEA (208.9 μL, 1.2 mmol) were added and reacted for 8 h. After washing with DMF, the Fmoc protecting group on Fmoc-Cys(Trt)-OH was removed with 20% piperidine solution. Subsequently, following the above steps, fluorenemethyloxycarbonyl-L-citrulline (Fmoc-Cit-OH, 397.4 mg, 1 mmol) and tert-butyloxycarbonyl-L-valine (Boc-Val-OH, 217.3 mmol) were added to the resin. (mg, 1mmol), then the resin was washed three times with DMF, isopropanol and n-hexane respectively. The peptide was then cleaved from the resin with DCM solution containing 1% TFA. All reaction solutions were collected and rotary evaporated to obtain a yellow oily liquid. Finally, the peptide was precipitated with diethyl ether. The precipitate was collected by low-temperature centrifugation and allowed to dry naturally in a fume hood to obtain a powdery solid. The successful synthesis of the product was confirmed by HPLC and ESI-MS analysis and named compound A (the sample synthesized in this process has high purity and can be used for the next synthesis without purification).

[0053] Synthesis of Compound B: Compound A (1117.7 mg, 1.2 mmol) and 6-amino-2-cyanobenzothiazole (CBT, 175.2 mg, 1 mmol) were lyophilized beforehand. Under nitrogen protection, compound A was dissolved in redistilled anhydrous tetrahydrofuran (THF), and N-methylmorpholine (MMP, 269.7 μL, 2.4 mmol) was added. Then, isobutyl chloroformate (IBCF, 155.6 µL, 1.2 mmol) was added under ice bath conditions. After stirring for 1 h, CBT dissolved in anhydrous THF was added to the reaction system, and IBCF (155.6 µL, 1.2 mmol) was added to the system. The reaction was then stirred at 0 °C for 12 h and then allowed to proceed overnight at room temperature. After the reaction was complete, a yellow solid was obtained by rotary evaporation. The compound was purified by HPLC and ESI-MS analysis and named compound B.

[0054] Synthesis of compound C: Compound B (544.2 mg, 0.5 mmol) was lyophilized beforehand. Under nitrogen protection, compound B was dissolved in anhydrous DCM, and phenylsilane (PhSiH3, 308.5 μL, 2.5 mmol) and tetrakis(triphenylphosphine)palladium (Pd(PPh3)4, 57.8 mg, 0.05 mmol) were added. After reacting for 30 min, rotary evaporation was performed to obtain a powdered solid. The compound was purified by HPLC and ESI-MS analysis and named compound C.

[0055] Synthesis of compound D: Compound C (401.6 mg, 0.4 mmol) and succinic anhydride (SA, 48.0 mg, 0.48 mmol) were lyophilized in advance; Compound C and SA were dissolved separately in DMF, and the pH of the DMF solution containing SA was adjusted to neutral with DIPEA before being mixed with the DMF solution containing compound C. After stirring for 3 h, the compound was purified by HPLC and ESI-MS analysis and named compound D.

[0056] Synthesis of compound E: Compound D (331.2 mg, 0.3 mmol), gemcitabine (Gem, 94.8 mg, 0.36 mmol), N,N'-dicyclohexylcarbodiimide (DCC, 185.4 mg, 0.9 mmol), and 4-dimethylaminopyridine (DMAP, 88.0 mg, 0.72 mmol) were lyophilized beforehand. Compound D, Gem, DCC, and DMAP were dissolved together in anhydrous DMF and stirred overnight. The compound was purified by HPLC and ESI-MS analysis and named compound E.

[0057] Synthesis of compound F: Compound E (202.4 mg, 0.15 mmol) was lyophilized in advance; compound E was dissolved in DCM solution containing 70% TFA, and 1% triisopropylsilane was added. After stirring for 1 h, DCM and TFA were removed by rotary evaporation, and the product was precipitated by diethyl ether. The precipitate was obtained by centrifugation at low temperature, and the precipitate was allowed to dry naturally in a fume hood. A small amount of sample was taken and analyzed by HPLC and ESI-MS to confirm the successful synthesis of the product, which was named compound F (the sample synthesized in this process has high purity, so no purification is required before it can be used for the next synthesis).

[0058] Synthesis of compound CBT-Gem-B1RA: Py-SS-B1RA (125.1 mg, 0.1 mmol) and compound F (181.3 mg, 0.18 mmol) were dissolved in DMF, respectively. Under nitrogen protection, the DMF solution containing compound F was added dropwise to the DMF solution containing Py-SS-B1RA. The entire addition process lasted about 30 min. After the addition was completed, the reaction was allowed to proceed overnight. The target compound was obtained by purification analysis by HPLC and high-resolution ESI-MS and named CBT-Gem-B1RA.

[0059] The structural formula of CBT-Gem-B1RA and the schematic diagram showing its intracellular self-assembly into nanoparticles triggered by GSH reduction and CTSB enzyme cleavage are shown below. Figure 1 As shown in Figure a, CBT-Gem-B1RA enters glioma cells via the targeting action of B1RA. After being cleaved by intracellular GSH and CTSB enzymes, it generates an active intermediate, which condenses into a dimer via a click reaction and then self-assembles into nanoparticles through π-π stacking interactions. Figure 1 As shown in b;

[0060] The compounds synthesized in Example 1 were analyzed. Mass spectrometry data for compounds Py-SS-COOH, Py-SS-B1RA, A, B, C, D, E, and F were acquired using an electrospray ionization mass spectrometer manufactured by Jiangsu Tianrui Instrument Co., Ltd. The results are as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 The mass spectrum; mass spectrometry data of compound CBT-Gem-B1RA were acquired using a high-resolution electrospray ionization mass spectrometer, as shown below. Figure 10 The mass spectrum was obtained using MestReNova software. Figure 11 The hydrogen NMR spectrum of the compound CBT-Gem-B1RA is shown.

[0061] Comparative Example 1: Synthesis of the control compound CBT-Gem:

[0062] To verify the advantages of B1RA, a control compound CBT-Gem without the targeting peptide B1RA was designed, and the synthetic route is as follows:

[0063]

[0064] The specific steps are as follows:

[0065] Synthesis of Compound G: The required raw materials were lyophilized beforehand. First, resin loaded with 0.7 mmol of 2-chlorotriphenylmethyl chloride was weighed, dispersed in DMF, and added to a horn-shaped tube. After shaking for 30 min, the solvent was removed, and then Fmoc-Lys(Alloc)-OH (543.0 mg, 1.2 mmol) and DIPEA (250.8 μL, 1.44 mmol) dissolved in DMF were added. The reaction was allowed to proceed for 8 h. After the reaction was complete, the resin was washed three times with DMF. Subsequently, unreacted active sites on the resin were blocked with methanol and washed three times with DMF. Then, the Fmoc protecting group on Fmoc-Lys(Alloc)-OH was removed with a DMF solution containing 20% ​​piperidine. After multiple washes with DMF, fluorenemethoxycarbonyl-S-tert-butylthio-L-cysteine ​​(Fmoc-Cys(StBu)-OH, 431.6 mg, 1 mmol) dissolved in DMF was added. The following reagents were added: HOBT (162.1 mg, 1.2 mmol), HBTU (455.0 mg, 1.2 mmol), and DIPEA (208.9 μL, 1.2 mmol). The reaction was carried out for 8 h. After the reaction was completed, the resin was washed three times with DMF. Then, the Fmoc protecting group on Fmoc-Cys(StBu)-OH was removed with DMF solution containing 20% ​​piperidine. Then, following the same steps as above, Fmoc-Cit-OH (397.4 mg, 1 mmol) and Boc-Val-OH (217.3 mg, 1 mmol) were synthesized on the resin. After the synthesis was completed, the resin was washed three times with DMF, isopropanol, and n-hexane respectively. Then, the peptide was cleaved from the resin with DCM solution containing 1% TFA. All reaction solutions were collected and rotary evaporated to obtain a yellow oily crude product. Next, the polypeptide was precipitated with diethyl ether, the precipitate was collected by low-speed centrifugation, and the precipitate was placed in a fume hood to evaporate naturally to obtain a powder solid. The sample was analyzed by HPLC and ESI-MS to confirm that the product was successfully synthesized and named compound G (the sample synthesized in this process has high purity, so it can be used for the next synthesis without purification).

[0066] Synthesis of compound H: Compound G (932.9 mg, 1.2 mmol) and CBT (175.2 mg, 1 mmol) were lyophilized beforehand. Compound G was dissolved in anhydrous tetrahydrofuran (THF) under a nitrogen atmosphere, and MMP (269.7 μL, 2.4 mmol) was added. Then, IBCF (155.6 µL, 1.2 mmol) was added under ice bath conditions, and the mixture was stirred for 1 h. Subsequently, CBT dissolved in THF was added to the reaction system, and IBCF (155.6 µL, 1.2 mmol) was added to the mixture. The mixture was stirred at 0 °C for 12 h, and then the reaction was continued at room temperature overnight. After the reaction was completed, a yellow solid was obtained by rotary evaporation. The product was purified by HPLC and ESI-MS analysis and named compound H.

[0067] Synthesis of Compound I: Compound H (467.2 mg, 0.5 mmol) was lyophilized beforehand; under a nitrogen atmosphere, compound H was dissolved in anhydrous DCM, and PhSiH3 (308.5 μL, 2.5 mmol) and Pd(PPh3)4 (57.8 mg, 0.05 mmol) were added. After reacting for 30 min, the mixture was rotary evaporated to obtain a powdered solid. The product was purified by HPLC and ESI-MS analysis and named Compound I.

[0068] Synthesis of compound J: Compound I (340.2 mg, 0.4 mmol) and SA (48.0 mg, 0.48 mmol) were lyophilized beforehand; Compound I and SA were dissolved separately in DMF, and the DMF solution containing SA was adjusted to neutral with DIPEA before being mixed with the DMF solution containing compound I. After reacting for 3 h, the product was purified by HPLC and ESI-MS analysis and named compound J.

[0069] Synthesis of compound K: Compound J (285.1 mg, 0.3 mmol), Gem (94.8 mg, 0.36 mmol), DCC (185.4 mg, 0.9 mmol), and DMAP (88.0 mg, 0.72 mmol) were lyophilized beforehand. The next day, compound J, Gem, DCC, and DMAP were dissolved together in anhydrous DMF and stirred overnight. The product was purified by HPLC and ESI-MS analysis and named compound K.

[0070] Synthesis of compound CBT-Gem: Compound K (179.3 mg, 0.15 mmol) was dissolved in a DCM solution containing 20% ​​TFA, and 1% (v / v) of triisopropylsilane was added. After stirring for 1 h, the mixture was rotary evaporated to obtain a yellow crude product. The product was precipitated with diethyl ether, and the precipitate was collected by low-temperature centrifugation and allowed to dry naturally in a fume hood. Subsequently, the successful synthesis of the product was confirmed by HPLC and ESI-MS, and it was named CBT-Gem.

[0071] Electrospray ionization mass spectrometry (ESI-MS) data of compounds G, H, I, J, and K were acquired using an ESI-MS mass spectrometer manufactured by Jiangsu Tianrui Instruments Co., Ltd., and the results are as follows: Figure 12 , Figure 13 , Figure 14 , Figure 15 , Figure 16 The mass spectrum shown was obtained by high-resolution electrospray ionization mass spectrometry (HPLC-MS) of compound CBT-Gem. Figure 17 High-resolution mass spectrum.

[0072] Example 2, CBT-Gem-B1RA in vitro self-assembly experiment:

[0073] HPLC, ESI-MS, and transmission electron microscopy (TEM) confirmed that CBT-Gem-B1RA can self-assemble into nanostructures in vitro under the action of reducing agents and CTSB enzymes.

[0074] Prepare 1 mL of CBT-Gem-B1RA solution using CTSB enzyme buffer containing the reducing agent tris(2-carboxyethyl)phosphine (TCEP), and incubate in a 37 °C water bath. After 1 h, take 500 μL and monitor the results using HPLC and ESI-MS. The HPLC characterization results are as follows: Figure 18 As shown by the red line, the ESI-MS characterization results for the main peak are as follows: Figure 19 As shown, this indicates that the disulfide bonds in CBT-Gem-B1RA can be reduced by a reducing agent;

[0075] Subsequently, CTSB enzyme was added to the system, and after incubation at 37 °C for 2 h, 10 μL of sample was taken out for TEM sample preparation. The remaining samples were monitored by HPLC and ESI-MS. The HPLC characterization results are as follows: Figure 18 As shown by the blue line, the ESI-MS characterization results for the main peak are as follows: Figure 20 As shown, CBT-Gem-B1RA is reduced by the reducing agent and then cleaved by the CTSB enzyme, forming a dimer (CBT-Gem-Dimer) in the buffer via a click reaction. Figure 21 TEM images demonstrate that the dimer can further self-assemble to form nanoparticles CBT-Gem-NPs.

[0076] Example 3: Cytotoxic effects of CBT-Gem-B1RA:

[0077] The cytotoxicity of different compounds against U87-Luc glioma cells, which highly express CTSB enzyme and GSH, and NIH / 3T3 normal cells, which express low CTSB enzyme, was tested using a ELISA reader (BioTek Synergy).

[0078] U87-Luc cells were evenly seeded into 96-well plates and incubated for 12 h. The old culture medium was discarded, and 150 μL of fresh culture medium containing 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM CBT-Gem-B1RA was added to each well (the drugs used in subsequent cytotoxicity experiments were incubated at this concentration). Incubation was continued for 48 h. After incubation, 15 μL of 5 mg / mL diphenyltetraazole bromide (MTT) solution prepared with phosphate buffer was added to each well. Incubation was continued for 4 h, and the culture medium was aspirated. Then, 100 μL of DMSO was added to each well, and the plates were shaken in the dark for 10 minutes until the formazan crystals were completely dissolved. The liquid temperature in the plates was measured using a microplate reader at 490 °C. The absorbance value at nm was used to calculate the percentage of surviving cells using the formula: (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0079] To demonstrate the targeting ability of B1RA, three control experiments were conducted: The first control experiment involved incubating U87-Luc cells with CBT-Gem (at the same concentration as the CBT-Gem-B1RA group) without the B1RA targeting peptide for 48 h, followed by cell viability measurement. The second control experiment involved seeding U87-Luc cells in 96-well plates, incubating for 12 h, discarding the old culture medium, adding fresh culture medium containing 200 μM Py-SS-B1RA, and incubating for 30 minutes to allow the B1R sites in U87-Luc cells to be occupied. Subsequently, CBT-Gem-B1RA was added and incubated for 48 h as in the experimental group, followed by cell viability measurement. The third control experiment involved incubating the above concentration gradients of CBT-Gem-B1RA with normal NIH / 3T3 cells (low B1R expression) for 48 h, followed by cell viability measurement.

[0080] In addition, to verify the advantages of the compound's intracellular self-assembly, the following control experiment was set up: U87-Luc cells were seeded in 96-well plates and incubated for 12 h. The old culture medium was discarded, and fresh culture medium containing 100 μM CTSB enzyme inhibitor (inhibitor, E64d) was added to the well plates. The cells were incubated for 4 h to inhibit CTSB enzyme activity in U87-Luc cells. Subsequently, CBT-Gem-B1RA was added and incubated for 48 h as in the experimental group, and the cell viability was measured.

[0081] Cytotoxicity test results as follows Figure 22 As shown, according to Figure 22 As shown in Figure a, CBT-Gem-B1RA exhibited significantly higher cytotoxicity against U87-Luc cells than the CBT-Gem group and the B1RA + CBT-Gem-B1RA group. However, CBT-Gem-B1RA showed lower cytotoxicity against NIH / 3T3 normal cells, which exhibited low expression of both CTSB enzyme and B1R. This indicates that the targeting peptide B1RA effectively increased the uptake of the compound CBT-Gem-B1RA by U87-Luc tumor cells, thereby enhancing the drug's cytotoxic effect on U87-Luc cells. Figure 22 As shown in Figure b, the cell survival rate of CBT-Gem-B1RA after incubation with U87-Luc cells was significantly lower than that of the inhibitor + CBT-Gem-B1RA group and the cell survival rate after incubation with NIH / 3T3 cells. This indicates that when CTSB enzyme is inhibited or expressed at low levels, the self-assembly ability of CBT-Gem-B1RA is limited. Under normal conditions, CBT-Gem-B1RA can self-assemble and accumulate in U87-Luc tumor cells under the action of highly expressed GSH and CTSB enzymes, thereby effectively increasing the drug concentration in U87-Luc cells, prolonging the retention time, and enabling the drug to exert a stronger killing effect on tumor cells.

[0082] Example 4: In vivo antitumor effect of CBT-Gem-B1RA:

[0083] Validating the in vivo antitumor effect of CBT-Gem-B1RA using a mouse orthotopic glioma model:

[0084] First, 2×10 5U87-Luc cells were implanted into the right brain of BALB / c nude mice to construct an orthotopic glioma model. On day 8 post-implantation, the size of the glioma was monitored using bioluminescence imaging. Subsequently, based on the measured tumor size, the mice were randomly divided into four groups of four. Each group received an equal dose of Gem, CBT-Gem, CBT-Gem-B1RA, and saline via tail vein injection, respectively, for a total of five injections every two days. During treatment, the growth of the brain tumor and the weight of the mice were continuously monitored using bioluminescence imaging. Bioluminescence imaging images, tumor growth curves, and mouse weight change curves for each group were obtained as follows: Figure 23 As shown;

[0085] according to Figure 23 As can be seen, compared with the Saline group, the growth of gliomas in mice in the CBT-Gem-B1RA group was significantly inhibited, while the Gem group and CBT-Gem group showed no significant inhibitory effect. This indicates that the traditional small molecule chemotherapy drug Gem and the control compound CBT-Gem are difficult to penetrate the blood-brain barrier to effectively treat gliomas due to their lack of targeting. However, the targeting peptide B1RA of the compound CBT-Gem-B1RA enables it to effectively penetrate the blood-brain barrier and reach the tumor site, thus significantly inhibiting orthotopic gliomas in mice. The body weight change curve shows no significant difference in body weight change among the groups, indicating that CBT-Gem-B1RA has good biocompatibility.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing self-assembled drug molecules, characterized in that, Includes the following steps: 2,2'-dithiodipyridine was dissolved in methanol, 3-mercaptopropionic acid dissolved in methanol was added dropwise, and then acetic acid was added to react. The reaction was carried out by rotary evaporation to obtain a yellow oily crude product, which was purified to obtain compound Py-SS-COOH. The resin loaded with 2-chlorotriphenylmethyl chloride was dispersed in DMF and shaken. The DMF was then extruded, and fluorenemethoxycarbonyl-D-phenylalanine and N,N-diisopropylethylamine dissolved in DMF were added. The reaction was initiated. After washing, DMF was added to disperse the resin, followed by methanol. The reaction solution was extruded, washed, and then reacted with a DMF solution containing piperidine. This process was repeated. After deprotection, the resin was washed with DMF, and fluorenemethoxycarbonyl-L-proline, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine dissolved in DMF were added. The reaction was initiated, washed, and the Fmoc protecting group was removed with a DMF solution containing piperidine. The same procedure was repeated to sequentially attach fluorenemethoxycarbonyl-O-tert-methylalanine to the resin. Butyl-L-serine, fluorenylmethoxycarbonyl-3-cyclohexyl-L-alanine, fluorenylmethoxycarbonyl-glycine, fluorenylmethoxycarbonyl-4-tert-butoxy-L-proline, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, fluorenylmethoxycarbonyl-tert-butoxycarbonyl-L-lysine, and Py-SS-COOH were used. The resin was washed, and the polypeptides were cleaved from the resin with a dichloromethane solution containing trifluoroacetic acid. All reaction solutions were collected, rotary evaporated, and then lyophilized. The resulting solid was dissolved in a DCM solution containing trifluoroacetic acid, and triisopropylsilane was added. After reaction, rotary evaporation was used to obtain a yellow oily crude product, which was purified to obtain compound Py-SS-B1RA. The resin containing 2-chlorotriphenylmethyl chloride was dispersed in DMF and shaken. After extruding the DMF, fluorenemethoxycarbonyl-lysine and N,N-diisopropylethylamine were added, reacted, washed, methanol was added, washed with DMF, piperidine-containing DMF solution was added, washed with DMF, fluorenemethoxycarbonyl-S-triphenylmethyl-L-cysteine, 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine were added, reacted, washed, and piperidine-containing DMF solution was added. In the same operation, fluorenemethoxycarbonyl-L-citrulline and tert-butyloxycarbonyl-L-valine were added to the resin. The resin was washed, and the polypeptide was cleaved from the resin with DCM solution containing trifluoroacetic acid. The polypeptide was precipitated with diethyl ether, centrifuged, collected, and evaporated to obtain a powdery solid, which yielded compound A. Compound A and 6-amino-2-cyanobenzothiazole were lyophilized. Compound A was dissolved under nitrogen protection, and N-methylmorpholine was added. Isobutyl chloroformate was added under ice bath conditions. After stirring, dissolved 6-amino-2-cyanobenzothiazole was added, and isobutyl chloroformate was added to the mixture. The mixture was stirred and reacted at room temperature overnight. After the reaction was completed, a yellow solid was obtained by rotary evaporation and purified to obtain compound B. Compound B was lyophilized, dissolved under nitrogen protection, and then phenylsilane and tetra(triphenylphosphine)palladium were added. After reaction, rotary evaporation was performed to obtain a powdered solid, which was then purified to obtain compound C. Compound C and succinic anhydride were lyophilized, and then dissolved separately in DMF. The pH of the DMF solution containing succinic anhydride was adjusted with N,N-diisopropylethylamine, and then mixed with the DMF solution containing compound C. The mixture was stirred and purified to obtain compound D. Compound D, gemcitabine, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were lyophilized. Compound D, gemcitabine, N,N'-dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were dissolved together in anhydrous DMF, stirred overnight, and purified to obtain compound E. Compound E was lyophilized, dissolved in DCM solution containing trifluoroacetic acid, and triisopropylsilane was added. After stirring, the mixture was rotary evaporated. The product was precipitated with diethyl ether, and the precipitate was collected by low-temperature centrifugation and evaporated to dryness to obtain compound F. Py-SS-B1RA and compound F were dissolved in DMF, respectively. Under nitrogen protection, the DMF solution containing compound F was added dropwise to the DMF solution containing Py-SS-B1RA. The reaction was allowed to proceed overnight, and the compound CBT-Gem-B1RA was obtained after purification.

2. The method for preparing self-assembled drug molecules according to claim 1, characterized in that, The structural formula of the compound Py-SS-COOH is shown below: ； The structural formula of the compound Py-SS-B1RA is shown below: ； The structural formula of compound A is shown below: ； The structural formula of compound B is shown below: ； The structural formula of compound C is shown below: ； The structural formula of compound D is shown below: ； The structural formula of compound E is shown below: ； The structural formula of compound F is shown below: ； The structural formula of the compound CBT-Gem-B1RA is shown below: 。 3. The method for preparing self-assembled drug molecules according to claim 1, characterized in that, The purification process specifically involved HPLC purification.

4. A self-assembling drug molecule, characterized in that, It is prepared using the preparation method described in any one of claims 1-3.

5. The use of a self-assembled drug molecule as described in claim 4 in the preparation of an antitumor prodrug.

6. The application according to claim 5, characterized in that, The self-assembled drug molecules undergo in situ self-assembly within cancer cells that highly express glutathione and CTSB enzyme, transforming into nanoparticles.

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