Cathepsin B-sensitive fatty acid-adriamycin prodrug, albumin nanoparticles thereof, preparation method and application of cathepsin B-sensitive fatty acid-adriamycin prodrug

Abstract

The invention relates to a cathepsin B sensitive fatty acid-adriamycin prodrug as well as albumin nanoparticles, a preparation method and application thereof, and belongs to the technical field of medicines. The cathepsin B sensitive fatty acid-adriamycin prodrug is a prodrug as shown in a formula (I), a geometric isomer thereof, and pharmaceutically acceptable salts, hydrates and solvates thereof, wherein n is equal to 0-14. The invention also relates to a combined albumin nanoparticle prepared by entrapping the cathepsin B sensitive fatty acid-adriamycin prodrug by using human / bovine serum albumin as a carrier. The albumin nanoparticles are small in particle size and uniform in form, have good placement stability and colloidal stability, can stably exist in systemic circulation and normal tissues, and release a parent drug adriamycin after being taken by tumor cells and hydrolyzed by cathepsin B; therefore, specific killing of tumor cells is realized without generation of serious toxic and side effects, and good clinical development prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a cathepsin B-sensitive fatty acid-doxacin prodrug and its albumin nanoparticles, as well as their preparation methods and applications. Specifically, it relates to the synthesis of cathepsin B-sensitive fatty acid-doxacin prodrugs of different chain lengths, the preparation of prodrug albumin nanoparticles, and the application of albumin nanoparticles in tumor drug delivery. Background Technology

[0002] Doxorubicin (DOX) is an anthracycline broad-spectrum antitumor antibiotic originally isolated from *S. peucetius var. caesius*. Doxorubicin has a broad antitumor spectrum and is commonly used clinically, either alone or in combination with other chemotherapy drugs, to treat acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, and lymphoma. Although doxorubicin has good antitumor activity, it suffers from significant toxic side effects, thus posing a major challenge to its clinical application. The most serious adverse reaction of doxorubicin is its irreversible cardiotoxicity, and it also severely suppresses bone marrow hematopoietic function. To address the problems of poor targeting and strong toxic side effects of doxorubicin, researchers have proposed various approaches. Nanomedicine delivery systems have received widespread attention due to their potential to improve the clinical application of doxorubicin. Currently, the FDA has approved three liposomal formulations of doxorubicin, including PEGylated... and and non-PEGylated Clinical studies have shown that, compared with conventional DOX treatment, [the treatment is more effective]. After treatment, patients experienced some relief from symptoms such as cardiotoxicity, nausea, vomiting, and bone marrow suppression. However, up to 25% of patients experienced [further symptoms]. Severe skin toxicity side effects may occur after administration, such as severe pain, ulceration, and pigmentation in the palms and soles, symptoms of "HFS" or impaired palmar and plantar erythrocyte sensation; while non-PEGylated administration... It can also cause problems such as lipid metabolism disorders in patients. Therefore, there is an urgent need to develop a precise, efficient, low-toxicity, and high-capacity doxorubicin drug delivery system.

[0003] Human serum albumin (HSA) is an important non-glycosylated protein with a heart-shaped crystal structure comprising three main domains (I to III), each consisting of two helical subdomains (A and B). Due to the flexibility and amphiphilicity of these structures, albumin possesses the ability to bind a variety of ligands. The albumin structure contains seven fatty acid binding sites, enabling the binding of various endogenous and exogenous ligands. Furthermore, the albumin structure contains 35 cysteine ​​residues forming 17 disulfide bonds and one unpaired thiol, which enables albumin to chemically couple small molecule drugs. Albumin possesses many excellent properties, such as biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and easy availability. Besides serving as a carrier and drug conjugate, albumin also possesses various physical and chemical functions, thus being widely used in the design and development of drug delivery systems. The binding strategies of albumin in drug delivery systems are mainly divided into covalent binding and non-covalent binding. In covalent binding, the drug is covalently linked to the active functional groups on the surface of the albumin molecule through chemical coupling. Cys34 in natural albumin is the only unpaired thiol located on its surface, making it particularly suitable for in-situ drug binding. Through covalent binding, drugs can stably bind to albumin, enabling targeted drug delivery. Non-covalent binding strategies utilize albumin's secondary structure, allowing drug molecules to reversibly bind to albumin via ionic or hydrophobic interactions. X-ray diffraction studies suggest that albumin contains up to seven different fatty acid binding sites. Therefore, in non-covalent binding, fatty acids are often used to modify drugs, enhancing their binding affinity to albumin. Successful examples include insulin detemir. and liraglutide This demonstrates the potential of fatty acids as drug-modifying groups in albumin drug delivery systems. Multiple case studies have shown that using fatty acids as prodrug ligands to enhance drug binding to albumin is an important strategy. Notably, subtle changes in the structure of the modifying group can significantly affect the stability of prodrug albumin nanoparticles, directly impacting the in vivo and in vitro fate of the drug delivery system. Therefore, understanding the influence of fatty acid carbon chain length on prodrug albumin nanodelivery systems is crucial for the rational design of prodrugs and the construction of stable and efficient prodrug albumin nanodelivery systems.

[0004] Whether for prodrugs or nanodelivery systems, intelligently triggering the selective release of drugs at the target site is crucial for the efficacy and safety of formulations. Enzymatic reactions are widely used in this field. Enzymes are involved in almost all physiological and metabolic processes in the body, and their high selectivity and substrate specificity give them a significant advantage in stimulus-responsive prodrugs. Cathepsin B is a proteolytic enzyme belonging to the cysteine ​​protease family, primarily located in lysosomes. It has attracted considerable attention due to its upregulated expression in various cancers (such as gastric cancer, breast cancer, melanoma, and colon cancer), and this overexpression has been shown to be closely related to tumor development, metastasis, shortened survival, and poor prognosis. In normal cells, cathepsin B is typically located in perinuclear lysosomes, while in tumor cells and cells transformed by oncogenes, its distribution is wider, not only present in perinuclear lysosomes but also scattered throughout the cytoplasm and pericellular vesicles. Extracellular cathepsin B participates in protein degradation processes related to tumor proliferation, invasion, and angiogenesis. This differential expression ensures that smart-response drugs sensitive to cathepsin B remain relatively stable during circulation in vivo without premature degradation and drug release. Therefore, cathepsin B has long been considered a potential target for cancer therapy and has wide applications in various fields such as tumor diagnosis, imaging, and treatment. Summary of the Invention

[0005] Based on this, the present invention provides a cathepsin B-sensitive fatty acid-doxorubicin prodrug and its albumin nanoparticles, as well as their preparation method and applications. A series of fatty acid-doxorubicins with different chain lengths are designed and synthesized, and a glycine-phenylalanine-leucine-glycine (GFLG) tetrapeptide fragment is introduced as a substrate for cathepsin B. The linker is modified with a p-aminobenzyl (PAB) group to improve the overall physicochemical properties of the prodrug (e.g., water solubility, reduced polymerization, better uniformity, and enhanced affinity with the carrier), thus forming a cathepsin B-sensitive fatty acid-doxorubicin prodrug. This prodrug exhibits a strong affinity for albumin, with a binding force constant higher than that of the optimal group C16-GFLG-DOX in the inventor's prior application CN116999408A. The present invention further uses human / bovine serum albumin as a carrier to encapsulate cathepsin B-sensitive GFLG tetrapeptide fragments with different fatty acid chain lengths and fatty acid-doxorubicin prodrugs bridged by the self-sacrificing group PAB, preparing bound albumin nanoparticles. The albumin nanoparticles have a small particle size and uniform morphology, exhibiting good placement and colloidal stability. They can remain stable in systemic circulation and normal tissues. However, after being taken up by tumor cells, they are hydrolyzed by cathepsin B to release the parent drug doxorubicin, thereby achieving specific killing of tumor cells without producing serious toxic side effects, showing good prospects for clinical development.

[0006] The purpose of this invention is to design and synthesize a series of cathepsin B-sensitive GFLG tetrapeptide fragments with different fatty acid chain lengths and PAB-bridged fatty acid-doxorubicin prodrugs, and to prepare albumin nanoparticles by encapsulation with human / bovine serum albumin as a carrier. The effects of different chain lengths of fatty acids and PAB on the properties of the prodrugs and albumin nanoparticles were verified, providing a new approach for the development of tumor-stimulation-responsive albumin nanodelivery systems.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] The present invention relates to a cathepsin B-sensitive fatty acid-doxorubicin prodrug, which is a prodrug having the structure shown in formula (I), its geometric isomer, and its pharmaceutically acceptable salt, hydrate, and solvate;

[0009]

[0010] Where n = 0-14, preferably n = 3-9.

[0011] The cathepsin B-sensitive fatty acid-doxorubicin prodrug is a fatty acid-doxorubicin prodrug bridged by a cathepsin B-sensitive GFLG tetrapeptide fragment and a self-sacrificing group PAB, wherein the carbon chain length of the fatty acid is 2-30 carbon atoms.

[0012] The method for preparing the cathepsin B-sensitive fatty acid-doxacin prodrug of the present invention includes the following steps:

[0013] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH), add N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and react with dichloromethane (DCM) as solvent to obtain Fmoc-Gly-OSU; wherein, in molar ratio, Fmoc-Gly-OH∶HOSU∶EDCI=1∶(1-10)∶(1-10);

[0014] Phenylalanine (H-Phe-OH) was weighed and added to sodium bicarbonate (NaHCO3) and Fmoc-Gly-OSU. A mixed solvent of water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) was used to react and obtain Fmoc-Gly-Phe-OH. The molar ratio of Fmoc-Gly-OSU∶H-Phe-OH∶NaHCO3 was 1∶(1-10):(1-10). N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added to dichloromethane (DCM) to react and obtain Fmoc-Gly-PheOSU. The molar ratio of Fmoc-Gly-Phe-OH∶HOSU∶EDCI was 1∶(1-10):(1-10).

[0015] Leucine (H-Leu-OH) was weighed and added to sodium bicarbonate (NaHCO3) and Fmoc-Gly-Phe-OSU. A mixed solvent of water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) was used to react and obtain Fmoc-Gly-Phe-Leu-OH. The molar ratio of Fmoc-Gly-Phe-OSU∶H-Ley-OH∶NaHCO3 was 1∶(1-10):(1-10). N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added to dichloromethane (DCM) to react and obtain Fmoc-Gly-Phe-LeuOSU. The molar ratio of Fmoc-Gly-Phe-OH∶HOSU∶EDCI was 1∶(1-10):(1-10).

[0016] Weigh glycine (H-Gly-OH), add sodium bicarbonate (NaHCO3) and Fmoc-Gly-Phe-Leu-OSU, and react with water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) as a mixed solvent to obtain Fmoc-Gly-Phe-Leu-Gly-OH; wherein, in molar ratio, Fmoc-Gly-Phe-Leu-OSU∶H-Gly-OH∶NaHCO3=1∶(1-10)∶(1-10);

[0017] Step 2: Weigh Fmoc-Gly-Phe-Leu-Gly-OH, add p-aminobenzyl alcohol (PAB) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), and react with N,N-dimethylformamide (DMF) as solvent to obtain Fmoc-Gly-Phe-Leu-Gly-PAB; wherein, in molar ratio, Fmoc-Gly-Phe-Leu-Gly-OH∶PAB∶EEDQ=1∶(1-10)∶(1-10);

[0018] Adding piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Gly-Phe-Leu-Gly-PAB yields H-Gly-Phe-Leu-Gly-PAB; wherein the piperidine-containing N,N-dimethylformamide contains 10%-60% by mass; and the mass ratio of Fmoc-Gly-Phe-Leu-Gly-PAB:piperidine = 1:(0.5-10).

[0019] Step 3: Weigh out fatty acid (FA), add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and H-Gly-Phe-Leu-Gly-PAB, and react with N,N-dimethylformamide (DMF) as solvent to obtain FA-Gly-Phe-Leu-Gly-PAB; wherein, in molar ratio, H-Gly-Phe-Leu-Gly-PAB∶FA∶HBTU∶DIPEA=1∶(1-10)∶(1-10)∶(1-10);

[0020] Step 4: Weigh FA-Gly-Phe-Leu-Gly-PAB, add di(p-nitrobenzene) carbonate (BNPC) and N,N-diisopropylethylamine (DIPEA), and react with N,N-dimethylformamide (DMF) as solvent to obtain FA-Gly-Phe-Leu-Gly-PAB-PNP; wherein, in molar ratio, FA-Gly-Phe-Leu-Gly-PAB∶BNPC∶DIPEA=1∶(1-10)∶(1-10);

[0021] Step 5: Weigh FA-Gly-Phe-Leu-Gly-PAB-PNP, add doxorubicin hydrochloride, 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA), and use pyridine-containing N,N-dimethylformamide (DMF) as a solvent to react and obtain fatty acid-glycine-phenylalanine-leucine-glycine-doxorubicin (FA-Gly-Phe-Leu-Gly-PAB-DOX), which is a cathepsin B-sensitive fatty acid-doxorubicin prodrug; wherein, the mass fraction of pyridine in the pyridine-containing N,N-dimethylformamide is 10%-60%; the molar ratio is FA-Gly-Phe-Leu-Gly-PAB-PNP∶DOX∶HOBT∶DIPEA=1∶(1-10)∶(1-10)∶(1-10).

[0022] The specific synthesis route is as follows:

[0023]

[0024] Depending on the type of fatty acid, the cathepsin B-sensitive fatty acid-doxacin prodrug of the present invention is a compound with the following structural formulas, specifically preferred structures being: (A) caprylic acid-doxacin prodrug (C8-GFLG-PAB-DOX), (B) lauric acid-doxacin prodrug (C12-GFLG-PAB-DOX), (C) myristic acid-doxacin prodrug (C14-GFLG-PAB-DOX), (D) palmitic acid-doxacin prodrug (C16-GFLG-PAB-DOX), and (E) stearic acid-doxacin prodrug (C18-GFLG-PAB-DOX).

[0025]

[0026]

[0027] The binding affinity constant between the cathepsin B-sensitive fatty acid-doxorubicin prodrug and albumin, as determined by fluorescence spectroscopy, was 0.0910–14.168 × 10⁻¹⁴. 6 Lmol -1 This is higher than the affinity of the optimal group C16-GFLG-DOX for albumin in the inventor's prior application CN116999408A (8.939 × 10⁻⁶). 3 Lmol -1 This invention provides cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles, which are a series of cathepsin B-sensitive GFLG tetrapeptide fragments and self-sacrificing PAB-bridged fatty acid-doxorubicin prodrugs, obtained by encapsulation using albumin as a carrier for the cathepsin B-sensitive fatty acid-doxorubicin prodrug.

[0028] The cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles have a particle size of 116-161 nm, a polydispersity index (PDI) of 0.108-0.199, a zeta potential of 11.7-21.3 mV, an encapsulation efficiency of ≥95%, and a sphericity of ≥98%.

[0029] This invention provides a method for preparing cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles, comprising the following steps:

[0030] Step 1: Dissolve the cathepsin B-sensitive fatty acid-doxorubicin prodrug in an organic solvent to obtain a prodrug solution; wherein, the mass concentration of the cathepsin B-sensitive fatty acid-doxorubicin prodrug in the prodrug solution is 1 mg / mL-20 mg / mL;

[0031] In step 1, the organic solvent is selected from one or a mixture of several of the following: chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetone, acetonitrile, and dimethyl sulfoxide.

[0032] Step 2: Dissolve albumin in water for injection until it is fully dissolved to obtain an albumin aqueous solution;

[0033] In step 2, the albumin used is human serum albumin or bovine serum albumin; the albumin concentration in the aqueous solution is 0.1 mg / mL to 2 mg / mL.

[0034] Step 3: Mix the prodrug solution and albumin aqueous solution, and homogenize by ultrasound or high pressure at 0-20℃. The cathepsin B-sensitive fatty acid-doxorubicin prodrug is encapsulated in albumin. Remove the organic solvent to obtain cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles. The mass ratio of cathepsin B-sensitive fatty acid-doxorubicin prodrug to albumin is (5-1):(1-50).

[0035] In step 3, the ultrasonic power is 100-800W, the ultrasonic time is 1-20min; the pressure of the high-pressure homogenizer is 5000-50000psi, and the number of high-pressure homogenization cycles is 3-20; the method for removing organic solvents is selected from one or a combination of rotary evaporation, vacuum drying, ultrafiltration, and dialysis.

[0036] The present invention also provides a pharmaceutical composition comprising a cathepsin B-sensitive fatty acid-doxacin prodrug having the structure shown in formula (I), a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and further comprising a pharmaceutically acceptable carrier or excipient.

[0037] The present invention also provides the use of cathepsin B-sensitive fatty acid-doxacin prodrug having the structure shown in formula (I), its geometric isomer, or its pharmaceutically acceptable salt, hydrate or solvate, or its cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles, or pharmaceutical compositions thereof, in the preparation of antitumor drugs.

[0038] The antitumor drug is selected from one of the following: oral administration drugs, injectable administration drugs, or topical administration drugs.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] (1) A cathepsin B-sensitive fatty acid-doxacin prodrug was designed and synthesized. PAB enhances the affinity of the prodrug for albumin, which is higher than the affinity of the optimal group C16-GFLG-DOX in the inventor's prior application CN116999408A for albumin.

[0041] (2) The cathepsin B-sensitive fatty acid-doxorubicin prodrug was prepared into an albumin nanodelivery system. The albumin nanoparticles were utilized to promote the accumulation of nanoparticles at the tumor site by taking advantage of their high permeability and retention (EPR) effect and active tumor targeting.

[0042] (2) This nanodelivery system can reduce the systemic toxicity of doxorubicin and can rapidly release the parent drug when tumor cells highly express cathepsin B, thereby achieving the purpose of reducing toxicity and increasing efficacy.

[0043] (3) It has a high drug loading capacity, avoiding the immunotoxicity caused by using a large amount of carrier material;

[0044] (4) The preparation process is simple and easy to scale up for production. Attached Figure Description

[0045] Figure 1 This is a high-resolution mass spectrum of the caprylic acid-doxorubicin prodrug (C8-GFLG-PAB-DOX) bridged by the cathepsin B-sensitive GFLG tetrapeptide fragment in Example 1.

[0046] Figure 2 This is a high-resolution mass spectrum of the cathepsin B-sensitive GFLG tetrapeptide fragment-bridged laurate-doxorubicin prodrug (C12-GFLG-PAB-DOX) in Example 2.

[0047] Figure 3 This is a high-resolution mass spectrum of the myristic acid-doxacin prodrug (C14-GFLG-PAB-DOX) bridged by the cathepsin B-sensitive GFLG tetrapeptide fragment in Example 3.

[0048] Figure 4This is a high-resolution mass spectrum of the palmitate-doxorubicin prodrug (C16-GFLG-PAB-DOX) bridged by the cathepsin B-sensitive GFLG tetrapeptide fragment in Example 4.

[0049] Figure 5 This is a high-resolution mass spectrum of the stearic acid-doxacin prodrug (C18-GFLG-PAB-DOX) bridged by the cathepsin B-sensitive GFLG tetrapeptide fragment in Example 5.

[0050] Figure 6 The graph shows the relationship between the degree of change in the albumin fluorescence spectrum and the drug concentration in Example 1;

[0051] Figure 7 The graph shows the relationship between the degree of change in the albumin fluorescence spectrum and the drug concentration in Example 2;

[0052] Figure 8 The graph shows the relationship between the degree of change in the albumin fluorescence spectrum and the drug concentration in Example 3;

[0053] Figure 9 The graph shows the relationship between the degree of change in the albumin fluorescence spectrum and the drug concentration in Example 4;

[0054] Figure 10 The graph shows the relationship between the degree of change in the albumin fluorescence spectrum and the drug concentration in Example 5;

[0055] Figure 11 The graph shows the relationship between the degree of change in albumin fluorescence spectrum and the optimal concentration of C16-GFLG-DOX drug in the inventor's prior application CN116999408A.

[0056] Figure 12 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 6 are shown.

[0057] Figure 13 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 7 are shown.

[0058] Figure 14 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 8 are shown.

[0059] Figure 15 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 9 are shown.

[0060] Figure 16 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 10 are shown.

[0061] Figure 17 The image shows the particle size versus storage time of albumin nanoparticles in Examples 7-10 under storage conditions at 4°C.

[0062] Figure 18 The following are particle size-time graphs of albumin nanoparticles in 10% FBS in Examples 7-10;

[0063] Figure 19 The figures show the in vitro release assays of albumin nanoparticles from Examples 7-10. Specifically, A shows the release assay of albumin nanoparticles from Examples 7-10 and the optimal group C16-GFLG-DOX albumin nanoparticles from CN116999408A in McIlvaine buffer (pH=6.0) containing 50 U / mL cathepsin B; B shows the release assay of albumin nanoparticles from Examples 7-10 and C16-GFLG-DOX albumin nanoparticles in McIlvaine buffer (pH=6.0); and C shows the release assay of C16-GFLG-DOX albumin nanoparticles from Examples 7-10 in phosphate buffer (pH=7.4).

[0064] Figure 20 The images show the blood concentration-time curves for albumin nanoparticles and C16-GFLG-DOX albumin nanoparticles from Examples 7-10. Specifically, A represents the blood concentration-time curve of doxorubicin hydrochloride for injection and the blood concentration-time curves of the prodrugs from Examples 2-5 corresponding to the albumin nanoparticles and C16-GFLG-DOX albumin nanoparticles from Examples 7-10; B represents the blood concentration-time curve of doxorubicin released from the albumin nanoparticles and C16-GFLG-DOX albumin nanoparticles from Examples 7-10.

[0065] Figure 21 The graphs shown are from Examples 7-10, illustrating the in vivo antitumor effects of albumin nanoparticles. In the graphs, A represents tumor growth curves, and B represents body weight changes. Detailed Implementation

[0066] The present invention will be further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein.

[0067] In the following examples, unless otherwise specified, "%" refers to mass percentage.

[0068] In the following embodiments, unless otherwise specified, all raw materials and equipment used are commercially available.

[0069] Example 1: Synthesis of a caprylic acid-doxorubicin prodrug (C8-Gly-Phe-Leu-Gly-PAB-DOX) bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment.

[0070] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 4.6 g, 15.32 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) to the reaction system. React overnight at room temperature. Wash the reaction solution three times with water and three times with saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude Fmoc-Gly-OSU.

[0071] Phenylalanine (H-Phe-OH, 3.29 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude Fmoc-Gly-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold diethyl ether and dried under reduced pressure to obtain the crude Fmoc-Gly-Phe-OH. The crude Fmoc-Gly-Phe-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was carried out overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-OSU.

[0072] Leucine (H-Leu-OH, 2.61 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-OH. The crude Fmoc-Gly-Phe-Leu-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was allowed to proceed overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-Leu-OSU.

[0073] Glycine (H-Phe-OH, 1.49 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-Leu-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-OH.

[0074] Step 2: Weigh 10.55 g (15.77 mmol) of the crude product from Step 1 and dissolve it in 70 mL of N,N-dimethylformamide (DMF). Add p-aminobenzyl alcohol (PAB, 5.83 g, 47.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 11.7 g, 47.31 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold diethyl ether, and dry under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-PAB.

[0075] 30 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to the crude product Fmoc-Gly-Phe-Leu-Gly-PAB from the previous step. The mixture was reacted at room temperature for 5 minutes, filtered under reduced pressure, washed three times with ice-cold diethyl ether, and dried under reduced pressure to obtain the crude product H-Gly-Phe-Leu-Gly-PAB. The crude product was purified by preparative liquid chromatography to obtain pure H-Gly-Phe-Leu-Gly-PAB.

[0076] Step 3: Weigh 0.38 g (2.64 mmol) of octanoic acid and dissolve it in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.001 g, 2.64 mmol) and N,N-diisopropylethylamine (DIPEA, 1.022 g, 7.91 mmol) to the reaction system. After stirring at room temperature for 5 minutes, add the pure H-Gly-Phe-Leu-Gly-PAB (1.33 g, 2.64 mmol) from Step 2. React at room temperature for 6 hours. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain crude C8-Gly-Phe-Leu-Gly-PAB.

[0077] Step 4: Weigh out the crude product C8-Gly-Phe-Leu-Gly-PAB (1.427 g, 2.2 mmol) from Step 3 and dissolve it in 50 mL of N,N-dimethylformamide (DMF). Add di(p-nitrobenzene) carbonate (BNPC, 2.008 g, 6.6 mmol) and N,N-diisopropylethylamine (DIPEA, 0.853 g, 6.6 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C8-Gly-Phe-Leu-Gly-PAB-PNP.

[0078] Step 5: Weigh out the crude product C8-Gly-Phe-Leu-Gly-PAB-PNP (352 mg, 0.4 mmol) from Step 4 and dissolve it in 8 mL of N,N-dimethylformamide (DMF). Add doxorubicin hydrochloride (217.4 mg, 0.4 mmol), 1-hydroxybenzotriazole (HOBT, 54.1 mg, 0.4 mmol), 1.5 mL of pyridine, and N,N-diisopropylethylamine (DIPEA, 103.4 mg, 0.8 mmol) to the reaction system. React at room temperature for 6 hours, filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C8-Gly-Phe-Leu-Gly-PAB-DOX. Purify the crude product using preparative liquid chromatography to obtain pure C8-Gly-Phe-Leu-Gly-PAB-DOX.

[0079] Analysis of the obtained product revealed that the prodrug contained [M+Na], according to high-resolution mass spectrometry results. + =1215.5140. The results of the nuclear magnetic resonance spectroscopy analysis are as follows: 1HNMR(400MHz,DMSO-d6)δ14.01(s,1H),13.26(s,1H),9.83(s,1H),8.20–8.10(m,2H),8.06–7.98(m,2H),7.90–7.87(m,2H),7.63(dd,J=5.9,3.9Hz,1H),7.56(d,J=8.3Hz,2H),7.26(d,J=8.3Hz,2H),7.24–7.11(m,5H),6.85(d,J=8.0Hz,1H),5.45(s,1H),5.21(d,J=3.6Hz,1H),4.97–4.81(m,4H),4.70(d,J=5.7Hz,1H),4.58(d,J=5.9Hz,2H),4.52(td,J=8.8,4.3Hz,1H),4.32–4.23(m,1H),4.16(q,J=7.0Hz,1H),3.98(s,3H),3.85(d,J=5.8Hz,2H),3.69(dd,J=16.4,5.7Hz,2H),3.54(dd,J=16.5,5.6Hz,1H),3.47–3.42(m,1H),3.07–2.90(m,3H),2.77(dd,J=13.9,9.4Hz,1H),2.26–2.09(m,2H),2.07(t,J=7.7Hz,2H),1.89–1.78(m,1H),1.63–1.57(m,1H),1.54–1.43(m,5H),1.25–1.19(m,8H),1.12(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H),0.86–0.81(m,6H).

[0080] 13CNMR(151MHz,DMSO-d6)δ212.66,185.40,185.30,171.58,171.13,169.98,167.98,166.35,161.17,159.65,154.97,154.17 ,153.36,137.27,136.52,135.06,134.40,133.53,132.94,130.63,128.05,127.45,126.87,125.09,118.87,118.58,117.85 ,117.70,109.63,109.50,99.15,73.81,68.71,66.83,65.52,63.79,62.53,55.43,52.66,50.25,45.97,41.48,40.84,39.41,36.15,35.47,34.63,33.91,30.93,29.99,29.62,28.68,27.47,27.31,23.94,22.93,21.84,20.91,20.46,15.87,12.78.;

[0081] After verification, its structural formula is as follows:

[0082]

[0083] Example 2: Synthesis of a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide-bridged laurate-doxorubicin prodrug (C12-GFLG-PAB-DOX)

[0084] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 4.6 g, 15.32 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) to the reaction system. React overnight at room temperature. Wash the reaction solution three times with water and three times with saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude Fmoc-Gly-OSU.

[0085] Phenylalanine (H-Phe-OH, 3.29 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude Fmoc-Gly-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold diethyl ether and dried under reduced pressure to obtain the crude Fmoc-Gly-Phe-OH. The crude Fmoc-Gly-Phe-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was carried out overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-OSU.

[0086] Leucine (H-Leu-OH, 2.61 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-OH. The crude Fmoc-Gly-Phe-Leu-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was allowed to proceed overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-Leu-OSU.

[0087] Glycine (H-Phe-OH, 1.49 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-Leu-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-OH.

[0088] Step 2: Weigh 10.55 g (15.77 mmol) of the crude product from Step 1 and dissolve it in 70 mL of N,N-dimethylformamide (DMF). Add p-aminobenzyl alcohol (PAB, 5.83 g, 47.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 11.7 g, 47.31 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold diethyl ether, and dry under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-PAB.

[0089] 30 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to the crude product Fmoc-Gly-Phe-Leu-Gly-PAB from the previous step. The mixture was reacted at room temperature for 5 minutes, filtered under reduced pressure, washed three times with ice-cold diethyl ether, and dried under reduced pressure to obtain the crude product H-Gly-Phe-Leu-Gly-PAB. The crude product was purified by preparative liquid chromatography to obtain pure H-Gly-Phe-Leu-Gly-PAB.

[0090] Step 3: Weigh lauric acid (LA, 0.53 g, 2.64 mmol) and dissolve it in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.001 g, 2.64 mmol) and N,N-diisopropylethylamine (DIPEA, 1.022 g, 7.91 mmol) to the reaction system. After stirring at room temperature for 5 minutes, add the pure H-Gly-Phe-Leu-Gly-PAB (1.33 g, 2.64 mmol) from Step 2. React at room temperature for 6 hours. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain crude C12-Gly-Phe-Leu-Gly-PAB.

[0091] Step 4: Weigh 2.06 g (2.2 mmol) of the crude product C12-Gly-Phe-Leu-Gly-PAB from Step 3 and dissolve it in 50 mL of N,N-dimethylformamide (DMF). Add di(p-nitrobenzene) carbonate (BNPC, 2.008 g, 6.6 mmol) and N,N-diisopropylethylamine (DIPEA, 0.853 g, 6.6 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C12-Gly-Phe-Leu-Gly-PAB-PNP.

[0092] Step 5: Weigh 467 mg (0.4 mmol) of the crude product C12-Gly-Phe-Leu-Gly-PAB-PNP from Step 4 and dissolve it in 8 mL of N,N-dimethylformamide (DMF). Add doxorubicin hydrochloride (217.4 mg, 0.4 mmol), 1-hydroxybenzotriazole (HOBT, 54.1 mg, 0.4 mmol), 1.5 mL of pyridine, and N,N-diisopropylethylamine (DIPEA, 103.4 mg, 0.8 mmol) to the reaction system. React at room temperature for 6 hours, filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C12-Gly-Phe-Leu-Gly-PAB-DOX. The crude product is purified by preparative liquid chromatography to obtain pure C12-Gly-Phe-Leu-Gly-PAB-DOX.

[0093] Analysis of the obtained product revealed that the prodrug contained [M+Na], according to high-resolution mass spectrometry results. + =1271.5712. The results of the nuclear magnetic resonance spectroscopy analysis are as follows: 1HNMR(400MHz,DMSO-d6)δ14.03(s,1H),13.27(s,1H),9.82(s,1H),8.20–8.08(m,2H),8.05–7.97(m,2H),7.90(d,J=4.8Hz,2H),7.66-7.62(m,1H),7.56(d,J=8.2Hz,2H),7.30–7.12(m,7H),6.84(d,J=8.0Hz,1H),5.45(s,1H),5.21(d,J=3.6Hz,1H),4.96–4.80(m,4H),4.70(d,J=5.7Hz,1H),4.57(d,J=5.9Hz,2H),4.55–4.47(m,1H),4.28(q,J=7.5Hz,1H),4.15(q,J=6.6Hz,1H),3.98(s,3H),3.85(d,J=5.8Hz,2H),3.75-366(m,2H),3.54(dd,J=16.5,5.6Hz,1H),3.46–3.41(m,1H),3.07–2.91(m,3H),2.81–2.72(m,1H),2.24–2.02(m,4H),1.90–1.77(m,1H),1.61–1.57(m,1H),1.63–1.42(m,5H),1.22(t,J=4.1Hz,16H),1.12(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H),0.86–0.83(m,6H).

[0094] 13CNMR(151MHz,DMSO-d6)δ214.26,187.07,186.97,173.20,172.74,171.60,169.60,167.96,161.29,156.59,155.79,154.99,13 8.88,138.14,136.70,136.05,135.19,134.58,132.25,129.67,129.07,128.49,126.71,120.53,120.23,119.49,119.31,111. 28,111.14,100.76,75.44,70.35,68.45,67.14,65.41,64.14,57.06,54.28,51.87,47.59,43.10,42.46,41.02,37.76,37.13,35.53,32.57,31.76,30.30,29.50,29.47,29.38,29.28,29.17,29.13,29.05,25.56,24.55,23.46,22.55,22.08,17.49,14.42;

[0095] After testing, its structural formula is:

[0096]

[0097] Example 3: Synthesis of a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide-bridged myristic acid-doxorubicin prodrug (C14-GFLG-PAB-DOX)

[0098] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 4.6 g, 15.32 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) to the reaction system. React overnight at room temperature. Wash the reaction solution three times with water and three times with saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude Fmoc-Gly-OSU.

[0099] Phenylalanine (H-Phe-OH, 3.29 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude Fmoc-Gly-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold diethyl ether and dried under reduced pressure to obtain the crude Fmoc-Gly-Phe-OH. The crude Fmoc-Gly-Phe-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was carried out overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-OSU.

[0100] Leucine (H-Leu-OH, 2.61 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-OH. The crude Fmoc-Gly-Phe-Leu-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was allowed to proceed overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-Leu-OSU.

[0101] Glycine (H-Phe-OH, 1.49 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-Leu-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-OH.

[0102] Step 2: Weigh 10.55 g (15.77 mmol) of the crude product from Step 1 and dissolve it in 70 mL of N,N-dimethylformamide (DMF). Add p-aminobenzyl alcohol (PAB, 5.83 g, 47.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 11.7 g, 47.31 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold diethyl ether, and dry under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-PAB.

[0103] 30 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to the crude product Fmoc-Gly-Phe-Leu-Gly-PAB from the previous step. The mixture was reacted at room temperature for 5 minutes, filtered under reduced pressure, washed three times with ice-cold diethyl ether, and dried under reduced pressure to obtain the crude product H-Gly-Phe-Leu-Gly-PAB. The crude product was purified by preparative liquid chromatography to obtain pure H-Gly-Phe-Leu-Gly-PAB.

[0104] Step 3: Weigh myristic acid (MA, 0.597 g, 2.64 mmol) and dissolve it in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.001 g, 2.64 mmol) and N,N-diisopropylethylamine (DIPEA, 1.022 g, 7.91 mmol) to the reaction system. After stirring at room temperature for 5 minutes, add the pure H-Gly-Phe-Leu-Gly-PAB (1.33 g, 2.64 mmol) from Step 2. React at room temperature for 6 hours. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain crude C14-Gly-Phe-Leu-Gly-PAB.

[0105] Step 4: Weigh 2.12 g (2.2 mmol) of the crude product C14-Gly-Phe-Leu-Gly-PAB from Step 3 and dissolve it in 50 mL of N,N-dimethylformamide (DMF). Add di(p-nitrobenzene) carbonate (BNPC, 2.008 g, 6.6 mmol) and N,N-diisopropylethylamine (DIPEA, 0.853 g, 6.6 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C14-Gly-Phe-Leu-Gly-PAB-PNP.

[0106] Step 5: Weigh 478 mg (0.4 mmol) of the crude product C14-Gly-Phe-Leu-Gly-PAB-PNP from Step 4 and dissolve it in 8 mL of N,N-dimethylformamide (DMF). Add doxorubicin hydrochloride (217.4 mg, 0.4 mmol), 1-hydroxybenzotriazole (HOBT, 54.1 mg, 0.4 mmol), 1.5 mL of pyridine, and N,N-diisopropylethylamine (DIPEA, 103.4 mg, 0.8 mmol) to the reaction system. React at room temperature for 6 hours, filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C14-Gly-Phe-Leu-Gly-PAB-DOX. Purify the crude product using preparative liquid chromatography to obtain pure C14-Gly-Phe-Leu-Gly-PAB-DOX.

[0107] Analysis of the obtained product revealed that the prodrug contained [M+Na], according to high-resolution mass spectrometry results. + =1299.6046. The results of the nuclear magnetic resonance spectroscopy analysis are as follows: 1HNMR(400MHz,DMSO-d6)δ14.03(s,1H),13.26(s,1H),9.82(s,1H),8.19–8.09(m,2H),8.05–7.97(m,2H),7.90(d,J=4.8Hz,2H),7.66–7.62(m,1H),7.56(d,J=8.2Hz,2H),7.25(d,J=8.2Hz,2H),7.23–7.13(m,5H),6.84(d,J=8.0Hz,1H),5.45(s,1H),5.21(d,J=3.6Hz,1H),4.97–4.81(m,4H),4.70(d,J=5.7Hz,1H),4.57(d,J=5.8Hz,2H),4.51(dt,J=8.7,4.4Hz,1H),4.32–4.23(m,1H),4.15(q,J=6.6Hz,1H),3.98(s,3H),3.85(d,J=5.9Hz,2H),3.69(dd,J=16.4,5.9Hz,2H),3.54(dd,J=16.4,5.6Hz,1H),3.47–3.42(m,1H),3.08–2.93(m,3H),2.77(dd,J=13.9,9.3Hz,1H),2.24–2.10(m,2H),2.07(t,J=7.4Hz,2H),1.84(td,J=13.0,3.9Hz,1H),1.61–1.57(m,1H),1.54–1.42(m,5H),1.24–1.21(m,20H),1.12(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H),0.86–0.82(m,6H).

[0108] 13CNMR(151MHz,DMSO-d6)δ214.25,187.06,186.97,173.20,172.74,171.60,169.60,167.96,161.29,156.59,155.79,154.99,138. 88,138.13,136.70,136.05,135.18,134.58,132.25,130.13,129.67,129.06,128.49,126.70,120.52,120.22,119.48,119.31,1 11.28,111.14,100.76,75.44,70.35,68.45,67.14,65.41,64.14,57.06,54.28,51.87,47.59,43.10,42.47,41.02,37.76,37.13,35.53,32.57,31.76,30.29,29.50,29.47,29.38,29.28,29.17,29.14,29.04,25.56,24.54,23.46,22.55,22.08,17.48,14.42;

[0109] After testing, its structural formula is:

[0110]

[0111] Example 4: Synthesis of a palmitate-doxorubicin prodrug (C16-GFLG-PAB-DOX) bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment.

[0112] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 4.6 g, 15.32 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) to the reaction system. React overnight at room temperature. Wash the reaction solution three times with water and three times with saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude Fmoc-Gly-OSU.

[0113] Phenylalanine (H-Phe-OH, 3.29 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude Fmoc-Gly-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold diethyl ether and dried under reduced pressure to obtain the crude Fmoc-Gly-Phe-OH. The crude Fmoc-Gly-Phe-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was carried out overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-OSU.

[0114] Leucine (H-Leu-OH, 2.61 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-OH. The crude Fmoc-Gly-Phe-Leu-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was allowed to proceed overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-Leu-OSU.

[0115] Glycine (H-Phe-OH, 1.49 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-Leu-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-OH.

[0116] Step 2: Weigh 10.55 g (15.77 mmol) of the crude product from Step 1 and dissolve it in 70 mL of N,N-dimethylformamide (DMF). Add p-aminobenzyl alcohol (PAB, 5.83 g, 47.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 11.7 g, 47.31 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold diethyl ether, and dry under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-PAB.

[0117] 30 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to the crude product Fmoc-Gly-Phe-Leu-Gly-PAB from the previous step. The mixture was reacted at room temperature for 5 minutes, filtered under reduced pressure, washed three times with ice-cold diethyl ether, and dried under reduced pressure to obtain the crude product H-Gly-Phe-Leu-Gly-PAB. The crude product was purified by preparative liquid chromatography to obtain pure H-Gly-Phe-Leu-Gly-PAB.

[0118] Step 3: Weigh palmitic acid (PA, 0.677 g, 2.64 mmol) and dissolve it in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.001 g, 2.64 mmol) and N,N-diisopropylethylamine (DIPEA, 1.022 g, 7.91 mmol) to the reaction system. After stirring at room temperature for 5 minutes, add the pure H-Gly-Phe-Leu-Gly-PAB (1.33 g, 2.64 mmol) from Step 2. React at room temperature for 6 hours. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain crude C16-Gly-Phe-Leu-Gly-PAB.

[0119] Step 4: Weigh 2.18 g (2.2 mmol) of the crude product C16-Gly-Phe-Leu-Gly-PAB from Step 3 and dissolve it in 50 mL of N,N-dimethylformamide (DMF). Add di(p-nitrobenzene) carbonate (BNPC, 2.008 g, 6.6 mmol) and N,N-diisopropylethylamine (DIPEA, 0.853 g, 6.6 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C16-Gly-Phe-Leu-Gly-PAB-PNP.

[0120] Step 5: Weigh 490 mg (0.4 mmol) of the crude product C16-Gly-Phe-Leu-Gly-PAB-PNP from Step 4 and dissolve it in 8 mL of N,N-dimethylformamide (DMF). Add doxorubicin hydrochloride (217.4 mg, 0.4 mmol), 1-hydroxybenzotriazole (HOBT, 54.1 mg, 0.4 mmol), 1.5 mL of pyridine, and N,N-diisopropylethylamine (DIPEA, 103.4 mg, 0.8 mmol) to the reaction system. React at room temperature for 6 hours, filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C16-Gly-Phe-Leu-Gly-PAB-DOX. Purify the crude product using preparative liquid chromatography to obtain pure C16-Gly-Phe-Leu-Gly-PAB-DOX.

[0121] Analysis of the obtained product revealed that the prodrug contained [M+Na], according to high-resolution mass spectrometry results. + =1327.6373. The results of the nuclear magnetic resonance spectroscopy analysis are as follows: 1HNMR(400MHz,DMSO-d6)δ14.04(s,1H),13.27(s,1H),9.82(s,1H),8.18-8.12(m,2H),8.05–7.99(m,2H),7.90(d,J=4.7Hz,2H),7.69–7.63(m,1H),7.57(d,J=8.2Hz,2H),7.26(d,J=8.2Hz,2H),7.23–7.15(m,5H),6.84(d,J=8.0Hz,1H),5.46(s,1H),5.22(d,J=3.6Hz,1H),5.00–4.79(m,4H),4.70(d,J=5.7Hz,1H),4.57(d,J=5.9Hz,2H),4.51(dt,J=8.7,4.4Hz,1H),4.32–4.22(m,1H),4.15(q,J=6.7Hz,1H),3.98(s,3H),3.85(d,J=6.0Hz,2H),3.69(dd,J=16.2,5.9Hz,2H),3.54(dd,J=16.4,5.5Hz,1H),3.46–3.44(m,1H),3.07–2.92(m,3H),2.77(dd,J=13.8,9.3Hz,1H),2.25–2.10(m,2H),2.07(t,J=7.5Hz,2H),1.83(dt,J=12.8,6.4Hz,1H),1.62–1.57(m,1H),1.53–1.43(m,5H),1.23–1.21(m,24H),1.12(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H),0.86–0.82(m,6H).

[0122] 13CNMR(151MHz,DMSO-d6)δ214.25,187.05,186.97,174.75,173.20,172.7 4,171.60,169.61,167.96,161.29,156.60,155.79,155.01,145.14,138. 88,138.14,136.70,136.05,135.20,134.60,132.25,130.12,129.67,12 9.06,128.92,128.49,126.70,120.53,120.22,119.48,119.31,111.28,1 11.14,100.76,75.45,70.35,68.45,67.14,65.41,64.14,57.06,54.28,51.87,47.59,43.10,42.47,41.02,40.53,37.75,37.14,35.59,35.53,32 .58,31.75,30.29,29.50,29.47,29.38,29.29,29.21,29.17,29.14,29.05,27.07,27.02,25.56,24.54,23.46,22.56,22.08,17.48,14.42,0.64;

[0123] After testing, its structural formula is:

[0124]

[0125] Example 5: Synthesis of a stearate-doxorubicin prodrug (C18-GFLG-PAB-DOX) bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment.

[0126] Step 1: Weigh fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 4.6 g, 15.32 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) to the reaction system. React overnight at room temperature. Wash the reaction solution three times with water and three times with saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude Fmoc-Gly-OSU.

[0127] Phenylalanine (H-Phe-OH, 3.29 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude Fmoc-Gly-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold diethyl ether and dried under reduced pressure to obtain the crude Fmoc-Gly-Phe-OH. The crude Fmoc-Gly-Phe-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was carried out overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-OSU.

[0128] Leucine (H-Leu-OH, 2.61 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-OH. The crude Fmoc-Gly-Phe-Leu-OH was dissolved in 60 mL of dichloromethane (DCM). N-hydroxysuccinimide (HOSU, 2.29 g, 19.92 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 3.82 g, 19.92 mmol) were added to the reaction system. The reaction was allowed to proceed overnight at room temperature. The reaction solution was washed three times each with water and saturated brine. The dichloromethane (DCM) phase was collected and filtered under reduced pressure to obtain the crude Fmoc-Gly-Phe-Leu-OSU.

[0129] Glycine (H-Phe-OH, 1.49 g, 19.92 mmol) and sodium bicarbonate (NaHCO3, 1.67 g, 19.92 mmol) were weighed and dissolved in 40 mL of water. The crude product Fmoc-Gly-Phe-Leu-OSU from the previous step was dissolved in 12 mL of N,N-dimethylformamide (DMF) and then added to the solution. After stirring at room temperature for 5 minutes, 40 mL of tetrahydrofuran (THF) was added, and the reaction was allowed to proceed at room temperature for 6 hours. Then, 200 mL of 5% phosphoric acid solution (H3PO4 / H2O) was added to the reaction solution to adjust the pH to acidic. The solution was extracted three times with ethyl acetate (EA), and the ethyl acetate (EA) phase was collected and filtered under reduced pressure. The filtrate was washed three times with ice-cold ether and dried under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-OH.

[0130] Step 2: Weigh 10.55 g (15.77 mmol) of the crude product from Step 1 and dissolve it in 70 mL of N,N-dimethylformamide (DMF). Add p-aminobenzyl alcohol (PAB, 5.83 g, 47.31 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 11.7 g, 47.31 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold diethyl ether, and dry under reduced pressure to obtain the crude product Fmoc-Gly-Phe-Leu-Gly-PAB.

[0131] 30 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to the crude product Fmoc-Gly-Phe-Leu-Gly-PAB from the previous step. The mixture was reacted at room temperature for 5 minutes, filtered under reduced pressure, washed three times with ice-cold diethyl ether, and dried under reduced pressure to obtain the crude product H-Gly-Phe-Leu-Gly-PAB. The crude product was purified by preparative liquid chromatography to obtain pure H-Gly-Phe-Leu-Gly-PAB.

[0132] Step 3: Weigh stearic acid (SA, 0.677 g, 2.64 mmol) and dissolve it in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 1.001 g, 2.64 mmol) and N,N-diisopropylethylamine (DIPEA, 1.022 g, 7.91 mmol) to the reaction system. After stirring at room temperature for 5 minutes, add the pure H-Gly-Phe-Leu-Gly-PAB (1.33 g, 2.64 mmol) from Step 2. React at room temperature for 6 hours. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain crude C18-Gly-Phe-Leu-Gly-PAB.

[0133] Step 4: Weigh 2.24 g (2.2 mmol) of the crude product C18-Gly-Phe-Leu-Gly-PAB from Step 3 and dissolve it in 50 mL of N,N-dimethylformamide (DMF). Add di(p-nitrobenzene) carbonate (BNPC, 2.008 g, 6.6 mmol) and N,N-diisopropylethylamine (DIPEA, 0.853 g, 6.6 mmol) to the reaction system. React overnight at room temperature. Filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C18-Gly-Phe-Leu-Gly-PAB-PNP.

[0134] Step 5: Weigh out the crude product C18-Gly-Phe-Leu-Gly-PAB-PNP (500.9 mg, 0.4 mmol) from Step 4 and dissolve it in 8 mL of N,N-dimethylformamide (DMF). Add doxorubicin hydrochloride (217.4 mg, 0.4 mmol), 1-hydroxybenzotriazole (HOBT, 54.1 mg, 0.4 mmol), 1.5 mL of pyridine, and N,N-diisopropylethylamine (DIPEA, 103.4 mg, 0.8 mmol) to the reaction system. React at room temperature for 6 hours, filter under reduced pressure, wash three times with ice-cold ether, and dry under reduced pressure to obtain the crude product C18-Gly-Phe-Leu-Gly-PAB-DOX. Purify the crude product using preparative liquid chromatography to obtain pure C18-Gly-Phe-Leu-Gly-PAB-DOX.

[0135] Analysis of the obtained product revealed that the prodrug contained [M+Na], according to high-resolution mass spectrometry results. + =1355.6707. The results of the nuclear magnetic resonance spectroscopy analysis are as follows: 1HNMR(400MHz,DMSO-d6)δ14.02(s,1H),13.25(s,1H),9.82(s,1H),8.20–8.10(m,2H),8.05–7.98(m,2H),7.90(d,J=4.8Hz,2H),7.66–7.62(m,1H),7.56(d,J=8.3Hz,2H),7.25(d,J=8.3Hz,2H),7.23–7.13(m,5H),6.84(d,J=8.0Hz,1H),5.45(s,1H),5.21(d,J=3.6Hz,1H),4.98–4.80(m,4H),4.70(d,J=5.6Hz,1H),4.58(d,J=5.5Hz,2H),4.51(dt,J=8.7,4.4Hz,1H),4.30–4.24(m,1H),4.16(q,J=6.7Hz,1H),3.98(s,3H),3.85(d,J=5.9Hz,2H),3.69(dd,J=16.3,5.9Hz,2H),3.54(dd,J=16.4,5.6Hz,1H),3.46–3.44(m,1H),3.09–2.90(m,3H),2.77(dd,J=13.9,9.3Hz,1H),2.25–2.10(m,2H),2.07(t,J=7.4Hz,2H),1.87–1.80(m,1H),1.64–1.56(m,1H),1.54–1.40(m,5H),1.23–1.21(m,28H),1.12(d,J=6.4Hz,3H),0.89(d,J=6.4Hz,3H),0.86–0.81(m,6H).

[0136] 13CNMR(151MHz,DMSO-d6)δ214.26,187.04,186.94,173.20,172.74,171.60,169.60,167.96,161.28,156.59,155.78,154.99,13 8.88,138.13,136.69,136.04,135.17,134.57,132.25,129.67,129.06,128.49,126.70,120.51,120.21,119.47,119.31,111. 26,111.13,100.77,75.44,70.34,68.45,67.14,65.41,64.14,57.06,55.39,54.28,51.87,47.59,43.10,42.47,41.02,37.76,37.12,35.53,32.57,31.76,30.29,29.50,29.46,29.38,29.29,29.17,29.14,25.56,24.54,23.46,22.56,22.08,17.49,14.42;

[0137] After testing, its structural formula is:

[0138]

[0139] Example 6: Formulation and preparation process of the prodrug albumin nanoparticles from Example 1

[0140] Table 1: Formulation of the prodrug albumin nanoparticles in Example 1

[0141]

[0142] Preparation process:

[0143] (1) Accurately weigh 50 mg of the prodrug from Example 1, dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1), and set aside;

[0144] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0145] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent and albumin nanoparticles are obtained.

[0146] like Figure 6As shown, the albumin nanoparticles in Example 6 are spherical with uniform particle size, measuring 161.4 ± 4.834 nm, with a PDI of 0.199 ± 0.028, a Zeta potential of +21.3 ± 3.27 mV, an encapsulation efficiency of 78.42 ± 1.22%, and a sphericity of 26.14 ± 0.40%. These nanoparticles have a relatively large particle size and poor storage stability.

[0147] Example 7: Formulation and preparation process of the prodrug albumin nanoparticles from Example 2

[0148] Table 2: Formulation of prodrug albumin nanoparticles in Example 2

[0149]

[0150] Preparation process:

[0151] (1) Accurately weigh 50 mg of the prodrug from Example 2, dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1), and set aside;

[0152] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0153] (3) Add (1) to (2) and sonicate at 390w for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent and albumin nanoparticles are obtained.

[0154] like Figure 7 As shown, the albumin nanoparticles in Example 7 are spherical with uniform particle size, with a particle size of 135.2±5.278 nm, a PDI of 0.178±0.049, a Zeta potential of +18.3±0.45 mV, an encapsulation efficiency of 90.64±0.31%, and a sphericity of 28.54±0.10%.

[0155] Table 3: Formulation of the prodrug albumin nanoparticles in Example 3

[0156]

[0157] Preparation process:

[0158] (1) Accurately weigh 50 mg of the prodrug from Example 3, dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1), and set aside;

[0159] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0160] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent and albumin nanoparticles are obtained.

[0161] like Figure 8 As shown, the albumin nanoparticles in Example 8 were spherical with uniform particle size, with a particle size of 132.0±1.179 nm, a PDI of 0.184±0.019, a Zeta potential of +15.5±2.13 mV, an encapsulation efficiency of 94.32±2.69%, and a sphericity of 31.44±0.90%. Example 9: Formulation and preparation process of the prodrug albumin nanoparticles from Example 4

[0162] Table 4: Formulation of the prodrug albumin nanoparticles in Example 4

[0163]

[0164]

[0165] Preparation process:

[0166] (1) Accurately weigh 50 mg of the prodrug from Example 4, dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1), and set aside;

[0167] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0168] (3) Add (1) to (2) and sonicate at 390w for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent and albumin nanoparticles are obtained.

[0169] like Figure 9 As shown, the albumin nanoparticles in Example 9 are spherical with uniform particle size, with a particle size of 116.6±1.626 nm, a PDI of 0.193±0.038, a Zeta potential of +12.8±1.89 mV, an encapsulation efficiency of 96.91±3.61%, and a sphericity of 32.30±1.20%. Example 10: Formulation and preparation process of the prodrug albumin nanoparticles from Example 5

[0170] Table 4: Formulation of prodrug albumin nanoparticles in Example 5

[0171]

[0172] Preparation process:

[0173] (1) Accurately weigh 50 mg of the prodrug from Example 5, dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1), and set aside;

[0174] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0175] (3) Add (1) to (2) and sonicate at 390w for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent and albumin nanoparticles are obtained.

[0176] like Figure 10 As shown, the albumin nanoparticles in Example 9 are spherical with uniform particle size, with a particle size of 127.8±4.204 nm, a PDI of 0.108±0.058, a Zeta potential of +11.7±0.777 mV, an encapsulation efficiency of 96.79±5.29%, and a sphericity of 32.26±1.76%. Comparative Example 1: Preparation of Doxorubicin Albumin Nanoparticles

[0177] Table 5: Formulation of Doxorubicin Albumin Nanoparticles

[0178]

[0179] Preparation process:

[0180] (1) Accurately weigh 50 mg of doxorubicin and dissolve it thoroughly in 5 mL of chloroform: anhydrous ethanol (9:1).

[0181] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0182] (3) Add (1) to (2) and sonicate at 390w for 5 minutes in an ice bath. The resulting emulsion is then evaporated to remove the organic solvent, yielding albumin nanoparticles.

[0183] The obtained doxorubicin albumin nanoparticles had a particle size of 697.6±20.13 nm, a PDI of 0.375±0.02, a Zeta potential of -1.04±0.276 mV, and an encapsulation efficiency of 21.93±0.79%. These nanoparticles had a relatively large particle size, uneven dispersion, and a low encapsulation efficiency.

[0184] Example 11: Storage stability test of albumin nanoparticles from Examples 7-10 at 4°C

[0185] The albumin nanoparticles from Examples 7-10 were stored at 4°C. During this period, particle size changes were measured at set time points using dynamic light scattering. The results are as follows: Figure 11 As shown, the particle size of albumin nanoparticles in Examples 7-10 showed no significant change over 60 days, indicating that the nanoparticles have good storage stability.

[0186] Example 12: Colloidal stability test of albumin nanoparticles from Examples 7-10

[0187] The albumin nanoparticles from Examples 7-10 were diluted to 0.1 mg / mL with 10% FBS and placed in a shaker at 37°C. The particle size change was measured at set time points using dynamic light scattering. The results are as follows: Figure 12 As shown, the particle size of albumin nanoparticles in Examples 7-10 did not change significantly within 24 hours, indicating that the nanoparticles have good colloidal stability.

[0188] Example 13: In vitro release test of albumin nanoparticles from Examples 7-10

[0189] The in vitro release of albumin nanoparticles from Examples 7-10 was investigated using McIlvaine buffer (pH = 6.0) containing 50 U / mL cathepsin B, McIlvaine buffer (pH = 6.0), and phosphate buffer (pH = 7.4) as release media (each containing 1% DMSO). Albumin nanoparticles from Examples 7-10 and C16-GFLG-DOX albumin nanoparticles were added to the release media and incubated in a shaker at 37°C. Samples were taken at predetermined time points, and three volumes of methanol were added to precipitate the protein. The mixture was vortexed, centrifuged, and the supernatant was collected. The concentration of released doxorubicin was determined by high-performance liquid chromatography (HPLC) to investigate the release of albumin nanoparticles from Examples 7-10 under different conditions.

[0190] Depend on Figure 13 It is known that the albumin nanoparticles in Examples 7-10 can only be released in a McIlvaine buffer (pH=6.0) containing 50 U / mL cathepsin B. Figure 13 Doxorubicin can only be released in A) and in a slightly acidic environment ( Figure 13 B) Neutral physiological environment ( Figure 13 Under condition C), the albumin nanoparticles of Examples 7-10 released almost no doxorubicin. This indicates that the albumin nanoparticles of Examples 7-10 possess cathepsin B-sensitive properties. Compared with the albumin nanoparticles of Examples 7, 8, and 10, the albumin nanoparticle of Example 9 exhibited the highest release amount within 48 hours, suggesting the potential for releasing the parent drug within the lysosomes of tumor cells highly expressing cathepsin B, thereby achieving tumor-killing effects. Furthermore, the release rates of Examples 9 and 10 were superior to those of the C16-GFLG-DOX albumin nanoparticles.

[0191] Example 14: Pharmacokinetic Study of Albumin Nanoparticles from Examples 7-10

[0192] SD rats (weight: 180-220g) were used as the research subjects and randomly divided into groups of 5 rats each. They were fasted for 12 hours before administration but had free access to water. Doxorubicin hydrochloride for injection, albumin nanoparticles from Examples 7-10, and C16-GFLG-DOX albumin nanoparticles (all at 3 mg / kg of doxorubicin) were administered via tail vein, respectively. The plasma concentrations of doxorubicin, the prodrug from Examples 2-5, and C16-GFLG-DOX were measured.

[0193] Experimental results are as follows Figure 14 A, Figure 14 As shown in B and Table 6, doxorubicin hydrochloride for injection is rapidly cleared from the blood, and the peak concentration of DOX in the blood (C) max The C value was lower, while the albumin nanoparticles in Examples 7-10 showed significantly higher C values. max Furthermore, significant differences were observed among the nanoparticle groups due to variations in fatty acid chain length. Meanwhile, the AUC of the albumin nanoparticles in Examples 7-10 was also compared. 0-t and C max Both were higher than those of the C16-GFLG-DOX albumin nanoparticle group.

[0194] Table 6. Main pharmacokinetic parameters of albumin nanoparticles in Examples 5-8

[0195]

[0196] Example 13: In vivo antitumor experiment of albumin nanoparticles from Examples 7-10

[0197] 4T1 cell suspension (5×10) ^6 (150 μL / cell) was injected subcutaneously on the dorsal side of female BALB / c mice. The tumor was allowed to grow to 120 mm². 3 Around 11 days, tumor-bearing mice were randomly divided into groups of five. Each group received a tail vein injection of saline, doxorubicin hydrochloride for injection, and albumin nanoparticles and C16-GFLG-DOX albumin nanoparticles (2 mg / kg based on doxorubicin), respectively. The administration was repeated every two days, and body weight and tumor volume were monitored daily. Mice were sacrificed on day 11.

[0198] In vivo anti-tumor effects such as Figure 15As shown, injectable doxorubicin hydrochloride had no significant antitumor effect, and the tumor volume increased rapidly, indicating that the 2 mg / kg injectable doxorubicin hydrochloride group had no significant antitumor effect. Albumin nanoparticles in Examples 7-10 exhibited better antitumor effects; that is, compared with the saline group, the growth of tumor volume in mice was effectively controlled. Furthermore, the antitumor effects varied among different albumin nanoparticles, with the antitumor effects in the following order: Example 9 albumin nanoparticles > Example 10 albumin nanoparticles > Example 8 albumin nanoparticles > Example 7 albumin nanoparticles > C16-GFLG-DOX albumin nanoparticles. This indicates that the albumin nanoparticle group in Example 9 had the best antitumor activity, and compared with the saline group, the body weight of the albumin nanoparticle group in Example 9 was not significantly reduced, demonstrating good safety.

[0199] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A cathepsin B-sensitive fatty acid-doxacin prodrug having the structure shown in formula (I), its geometric isomer, or its pharmaceutically acceptable salt, hydrate, or solvation. in, n＝0-14。 2. The cathepsin B-sensitive fatty acid-doxacin prodrug, its geometric isomer, or its pharmaceutically acceptable salt, hydrate, or solvate according to claim 1, characterized in that, Depending on the type of fatty acid, the specific structure is one of the following: (A) caprylic acid-doxacin prodrug (C8-GFLG-PAB-DOX), (B) lauric acid-doxacin prodrug (C12-GFLG-PAB-DOX), (C) myristic acid-doxacin prodrug (C14-GFLG-PAB-DOX), (D) palmitic acid-doxacin prodrug (C16-GFLG-PAB-DOX), or (E) stearic acid-doxacin prodrug (C18-GFLG-PAB-DOX):

3. The method for preparing the cathepsin B-sensitive fatty acid-doxacin prodrug according to claim 1 or claim 2, characterized in that, Includes the following steps: Step 1: Weigh fluorenemethyloxycarbonyl-glycine (Fmoc-Gly-OH), add N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and react with dichloromethane (DCM) as solvent to obtain Fmoc-Gly-OSU; Phenylalanine (H-Phe-OH) was weighed, and sodium bicarbonate (NaHCO3) and Fmoc-Gly-OSU were added. A mixed solvent of water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) was used to react and obtain Fmoc-Gly-Phe-OH. N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added. A dichloromethane (DCM) solvent was used to react and obtain Fmoc-Gly-Phe OSU. Leucine (H-Leu-OH) was weighed and added to sodium bicarbonate (NaHCO3) and Fmoc-Gly-Phe-OSU. A mixed solvent of water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) was used to react and obtain Fmoc-Gly-Phe-Leu-OH. N-hydroxysuccinimide (HOSU) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) were added and a dichloromethane (DCM) solvent was used to react and obtain Fmoc-Gly-Phe-Leu-OSU. Weigh out glycine (H-Gly-OH), add sodium bicarbonate (NaHCO3) and Fmoc-Gly-Phe-Leu-OSU, and react with water (H2O) / N,N-dimethylformamide (DMF) / tetrahydrofuran (THF) as a mixed solvent to obtain Fmoc-Gly-Phe-Leu-Gly-OH; Step 2: Weigh Fmoc-Gly–Phe-Leu-Gly-OH, add p-aminobenzyl alcohol (PAB) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), and react with N,N-dimethylformamide (DMF) as solvent to obtain Fmoc-Gly-Phe-Leu-Gly-PAB; Adding piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Gly-Phe-Leu-Gly-PAB yields H-Gly-Phe-Leu-Gly-PAB. Step 3: Weigh out fatty acid (FA), add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and H-Gly-Phe-Leu-Gly-PAB, and react with N,N-dimethylformamide (DMF) as solvent to obtain FA-Gly-Phe-Leu-Gly-PAB; Step 4: Weigh FA-Gly-Phe-Leu-Gly-PAB, add di(p-nitrobenzene) carbonate (BNPC) and N,N-diisopropylethylamine (DIPEA), and react with N,N-dimethylformamide (DMF) as solvent to obtain FA-Gly-Phe-Leu-Gly-PAB-PNP; Step 5: Weigh FA-Gly-Phe-Leu-Gly-PAB-PNP, add doxorubicin hydrochloride, 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIPEA), and use pyridine-containing N,N-dimethylformamide (DMF) as a solvent to react and obtain fatty acid-glycine-phenylalanine-leucine-glycine-doxorubicin (FA-Gly-Phe-Leu-Gly-PAB-DOX), which is the cathepsin B-sensitive fatty acid-doxorubicin prodrug; The specific synthesis route is as follows:

4. The method for preparing the cathepsin B-sensitive fatty acid-doxacin prodrug according to claim 3, characterized in that, In step one, the molar ratios are: Fmoc-Gly-OH∶HOSU∶EDCI=1∶(1-10)∶(1-10); Fmoc-Gly-OSU∶H-Phe-OH∶NaHCO3=1∶(1-10)∶(1-10); Fmoc-Gly-Phe-OSU∶H-Leu-OH∶NaHCO3=1∶(1-10)∶(1-10); Fmoc-Gly-Phe-Leu-OSU∶H-Gly-OH∶NaHCO3=1∶(1-10)∶(1-10); And / or, in step two, wherein, in molar ratio, Fmoc-Gly-Phe-Leu-Gly-OH∶PAB∶EEDQ=1∶(1-10)∶(1-10); In the N,N-dimethylformamide containing piperidine, the mass fraction of piperidine is 10%-60%; by mass ratio, Fmoc-Gly-Phe-Leu-Gly-PAB:piperidine = 1:(0.5-10); And / or, in step three, wherein, in molar ratio, H-Gly-Phe-Leu-Gly-PAB∶FA∶HBTU∶DIPEA=1∶(1-10)∶(1-10)∶(1-10); And / or, in step four, wherein, in molar ratio, FA-Gly-Phe-Leu-Gly-PAB∶BNPC∶DIPEA=1∶(1-10)∶(1-10); And / or, in step five, wherein, in molar ratio, FA-Gly-Phe-Leu-Gly-PAB-PNP∶DOX∶HOBT∶DIPEA=1∶(1-10)∶(1-10)∶(1-10).

5. The cathepsin B-sensitive fatty acid-doxacin prodrug, its geometric isomer, or its pharmaceutically acceptable salt, hydrate, or solvate according to claim 1 or 2, characterized in that, The binding affinity constant between the cathepsin B-sensitive fatty acid-doxacin prodrug and albumin is 0.0910–14.168 × 10⁻⁶. 6 Lmol -1 .

6. A cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle, characterized in that, It comprises the cathepsin B-sensitive fatty acid-doxacin prodrug as described in claim 1 or 2, and is obtained by encapsulation using albumin as the carrier of the cathepsin B-sensitive fatty acid-doxacin prodrug. The albumin nanoparticles of the cathepsin B-sensitive fatty acid-doxacin prodrug have a particle size of 116-161 nm, a polydispersity index (PDI) of 0.108-0.199, a zeta potential of 11.7-21.3 mV, an encapsulation efficiency of ≥95%, and a sphericity of ≥98%.

7. The method for preparing cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles according to claim 6, characterized in that, Includes the following steps: Step 1: Dissolve the cathepsin B-sensitive fatty acid-doxorubicin prodrug in an organic solvent to obtain a prodrug solution; Step 2: Dissolve albumin in water for injection until it is fully dissolved to obtain an albumin aqueous solution; Step 3: Mix the prodrug solution and albumin aqueous solution, and homogenize by ultrasound or high pressure at 0-20℃. The cathepsin B-sensitive fatty acid-doxorubicin prodrug is encapsulated in albumin. Remove the organic solvent to obtain cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles. The mass ratio of cathepsin B-sensitive fatty acid-doxorubicin prodrug to albumin is (5-1):(1-50).

8. The method for preparing cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles according to claim 7, characterized in that, In step 1, the mass concentration of the cathepsin B-sensitive fatty acid-doxorubicin prodrug in the prodrug solution is 1 mg / mL-20 mg / mL; The organic solvent is selected from one or a mixture of several of the following: chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetone, acetonitrile, and dimethyl sulfoxide; And / or, in step 2, the albumin is selected from human serum albumin or bovine serum albumin; the albumin aqueous solution has a mass concentration of 0.1 mg / mL to 2 mg / mL; And / or, in step 3, the ultrasonic power is 100-800W, the ultrasonic time is 1-20min; the pressure of the high-pressure homogenizer is 5000-50000psi, and the number of high-pressure homogenization cycles is 3-20; the method for removing organic solvents is selected from one or a combination of rotary evaporation, vacuum drying, ultrafiltration, and dialysis.

9. A pharmaceutical composition, characterized in that, It comprises a cathepsin B-sensitive fatty acid-doxacin prodrug having the structure shown in formula (I) as described in claim 1 or claim 2, its geometric isomer, or its pharmaceutically acceptable salt, hydrate, or solvate, and further comprises a pharmaceutically acceptable carrier or excipient.

10. The use of the cathepsin B-sensitive fatty acid-doxacin prodrug, its geometric isomer, or its pharmaceutically acceptable salt, hydrate, or solvate as claimed in claim 1 or 2, or the cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles as claimed in claim 6, or the pharmaceutical composition as claimed in claim 9, in the preparation of an antitumor drug.

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