Cathepsin B-sensitive fatty acid-doxorubicin prodrug and its albumin nanoparticles, preparation methods and applications

Abstract

This invention relates to a cathepsin B-sensitive fatty acid-doxacin prodrug, its albumin nanoparticles, preparation method, and application, belonging to the field of pharmaceutical technology. The cathepsin B-sensitive fatty acid-doxacin prodrug is defined as the prodrug shown in general structural formula (I), its geometric isomers, and its pharmaceutically acceptable salts, hydrates, and solvates; where n = 0-14. Further, the cathepsin B-sensitive fatty acid-doxacin prodrug is encapsulated using human / bovine serum albumin as a carrier to prepare bound albumin nanoparticles. These albumin nanoparticles have a small and uniform particle size, exhibiting good placement and colloidal stability. They 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 doxacin, thereby achieving specific killing of tumor cells without producing serious toxic side effects, showing promising clinical development prospects.

Description

Technical Field

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

[0002] Cancer, a major killer threatening human health, is characterized by high mortality, high recurrence rate, and high metastasis rate. Chemotherapy is a first-line treatment for many cancers, especially for tumors that cannot be surgically removed or have metastasized. Furthermore, chemotherapy can be combined with various therapies, including neoadjuvant therapy before surgery, and the combination of chemotherapy with radiotherapy, molecular targeted therapy, and immunotherapy, forming a crucial cornerstone of cancer treatment. Doxorubicin (DOX) is a broad-spectrum chemotherapeutic drug that has a strong killing effect on different types of cancer cells by inhibiting the synthesis of genetic material in cancer cells. However, doxorubicin has serious toxic side effects, such as cardiotoxicity and bone marrow suppression, which severely restricts its clinical application. Based on this, in 1995, the FDA approved the first nanomedicine, doxorubicin hydrochloride liposomes. This significantly reduces the cardiotoxicity of doxorubicin, while the long circulation and tumor-targeting properties of liposomes also endow... A stronger anti-tumor effect. However, The continuous accumulation of doxorubicin in the skin leads to symptoms of hand-foot syndrome. Therefore, there is an urgent need to develop an efficient and low-toxicity doxorubicin drug delivery system.

[0003] Albumin is the most abundant protein in blood plasma (approximately 35-50 g / L in human serum), with a molecular weight of 66.5 kDa. Many poorly water-soluble substances (such as long-chain fatty acids) can be transported by binding to it. Albumin is a naturally occurring macromolecule in the body, and using it as a drug carrier has the following advantages: (1) It has the advantages of low immunogenicity, non-toxicity, and biodegradability; (2) It has an extremely long half-life in the body, which can significantly prolong the circulation time of drugs in the body; (3) After binding drugs with albumin, the protective effect of albumin macromolecules can be utilized to improve drug stability.

[0004] Because doxorubicin has a low affinity for albumin, it cannot bind to albumin in plasma. Therefore, CytRx, an American company, has developed a prodrug strategy for its new drug Aldoxorubicin. This involves linking the C-13 keto group of doxorubicin to a maleimide group via a hydrazone bond. After intravenous injection, the maleimide group of the prodrug covalently binds to the 34-cysteine ​​residue of albumin in plasma. Utilizing albumin's natural tumor targeting properties, the drug accumulates at the tumor site and is then released from the slightly acidic environment of the tumor. Aldoxorubicin has completed Phase III clinical trials for the treatment of relapsed or refractory soft tissue sarcomas. However, the maleimide group readily binds nonspecifically to albumin and other proteins in vivo, posing a risk of toxic side effects. Therefore, developing a novel nanosystem using albumin as a delivery carrier is crucial, but no such system has yet been reported.

[0005] Whether for prodrugs or nanodelivery systems, intelligently triggered selective drug release at the target site is crucial for the efficacy and safety of formulations, with enzymatic reactions finding wide application. 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. Compared to normal tissues, cathepsin B is a cysteine ​​proteolytic enzyme highly expressed in the lysosomes of tumor cells. Therefore, cathepsin B provides ideal conditions for enzyme-triggered tumor-targeting delivery systems. Summary of the Invention

[0006] Based on this, the present invention provides a cathepsin B-sensitive fatty acid-doxorubicin prodrug, its albumin nanoparticles, preparation method, and application. A series of fatty acid-doxorubicins with different chain lengths are designed and synthesized, and a glycine-phenylalanine-leucine-glycine (Gly-Phe-Leu-Gly) tetrapeptide fragment is introduced as a substrate for cathepsin B to form a cathepsin B-sensitive fatty acid-doxorubicin prodrug. The present invention further uses human serum albumin as a carrier to encapsulate the fatty acid-doxorubicin prodrugs bridged by the cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragments with different fatty acid chain lengths, preparing bound albumin nanoparticles. These albumin nanoparticles have a small and uniform particle size, good placement and colloidal stability, and can exist stably in systemic circulation and normal tissues. However, after being taken up by tumor cells, they are hydrolyzed by highly expressed 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.

[0007] The purpose of this invention is to design and synthesize a series of fatty acid-doxorubicin prodrugs bridged by cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragments with different fatty acid chain lengths, and to prepare albumin nanoparticles by encapsulating them with human serum albumin. The effects of fatty acids with different chain lengths on the properties of the prodrugs and albumin nanoparticles were verified, providing a new approach for developing tumor-stimulation-responsive albumin nanodelivery systems.

[0008] The present invention achieves the above objectives through the following technical solutions:

[0009] The present invention relates to a cathepsin B-sensitive fatty acid-doxacin prodrug, which is a prodrug of general structural formula (I), its geometric isomer, and its pharmaceutically acceptable salt, hydrate, and solvate;

[0010]

[0011] Where n = 0-14, preferably n = 3-9.

[0012] The cathepsin B-sensitive fatty acid-doxorubicin prodrug is a fatty acid-doxorubicin prodrug bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment, and the carbon chain length of the fatty acid is 2-30 carbon atoms.

[0013] The method for preparing the cathepsin B-sensitive fatty acid-doxacin prodrug of the present invention includes the following steps:

[0014] Step 1: Weigh 2-chlorotriphenylmethyl chloride (CTC, 2-Chlorotrityl chloride) resin, add fluorenylmethoxycarbonyl-glycine (Fmoc-Gly-OH) and N,N-diisopropylethylamine (DIPEA), and use dichloromethane (DCM) as solvent to obtain a reaction solution for reaction to obtain Fmoc-Gly-CTC resin; wherein, the molar ratio of CTC resin : Fmoc-Gly-OH : DIPEA = 1 : (1-10) : (1-10);

[0015] Add piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Gly-CTC resin and react to obtain H-Gly-CTC resin; wherein, in the piperidine-containing N,N-dimethylformamide, the mass fraction of piperidine is 10%-60%; by mass ratio, Fmoc-Gly-CTC resin : piperidine = 1 : (0.5-10);

[0016] Step 2: Add fluorenemethoxycarbonyl-leucine (Fmoc-Leu-OH), N-methylmorpholine (NMM), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) to H-Gly-CTC resin, using N,N-dimethylformamide (DMF) as solvent, to obtain Fmoc-Leu-Gly-CTC resin; wherein, in molar ratio, H-Gly-CTC resin∶Fmoc-Leu-OH∶NMM∶HBTU=1∶(1-10)∶(1-10)∶(1-10);

[0017] Add piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Leu-Gly-CTC resin and react to obtain H-Leu-Gly-CTC resin; wherein, the mass fraction of piperidine in the piperidine-containing N,N-dimethylformamide is 10%-60%; and the mass ratio of Fmoc-Leu-Gly-CTC resin : piperidine = 1 : (0.5-10).

[0018] Step 3: Add fluorenemethoxycarbonyl-phenylalanine (Fmoc-Phe-OH), N-methylmorpholine (NMM), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) to H-Leu-Gly-CTC resin, using N,N-dimethylformamide (DMF) as solvent, to obtain Fmoc-Phe-Leu-Gly-CTC resin; wherein, in molar ratio, H-Leu-Gly-CTC resin∶Fmoc-Phe-OH∶NMM∶HBTU=1∶(1-10)∶(1-10)∶(1-10);

[0019] Add piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Phe-Leu-Gly-CTC resin and react to obtain H-Phe-Leu-Gly-CTC resin; wherein, the mass fraction of piperidine in the piperidine-containing N,N-dimethylformamide is 10%-60%; and the mass ratio of Fmoc-Phe-Leu-Gly-CTC resin : piperidine = 1 : (0.5-10).

[0020] Step 4: Add fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH), N-methylmorpholine (NMM), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) to H-Phe-Leu-Gly-CTC resin, using N,N-dimethylformamide (DMF) as solvent, to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin; wherein, in molar ratio, H-Phe-Leu-Gly-CTC resin∶Fmoc-Gly-OH∶NMM∶HBTU=1∶(1-10)∶(1-10)∶(1-10);

[0021] Add piperidine-containing N,N-dimethylformamide (DMF) to Fmoc-Gly-Phe-Leu-Gly-CTC resin and react to obtain H-Gly-Phe-Leu-Gly-CTC resin; wherein, the mass fraction of piperidine in the piperidine-containing N,N-dimethylformamide is 10%-60%; and the mass ratio of Fmoc-Gly-Phe-Leu-Gly-CTC resin : piperidine = 1 : (0.5-10).

[0022] Step 5: Add fatty acid (FA), N-methylmorpholine (NMM) and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) to H-Gly-Phe-Leu-Gly-CTC resin, and react with N,N-dimethylformamide (DMF) as solvent to obtain FA-Gly-Phe-Leu-Gly-CTC resin; wherein, in molar ratio, H-Gly-Phe-Leu-Gly-CTC resin∶FA∶NMM∶HBTU=1∶(1-10)∶(1-10)∶(1-10);

[0023] Dichloromethane (DCM) containing trifluoroacetic acid (TFA) was added to FA-Gly-Phe-Leu-Gly-CTC resin, and the reaction yielded FA-Gly-Phe-Leu-Gly-OH; wherein the mass fraction of TFA in the dichloromethane containing trifluoroacetic acid was 0.1%-10%; and the mass ratio of FA-Gly-Phe-Leu-Gly-CTC resin : TFA was 1 : (0.05-5).

[0024] Step 6: FA-Gly-Phe-Leu-Gly-OH reacts with O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), and doxorubicin hydrochloride to obtain fatty acid-glycine-phenylalanine-leucine-glycine-doxorubicin (FA-Gly-Phe-Leu-Gly-DOX), which is a cathepsin B-sensitive fatty acid-doxorubicin prodrug; wherein, in molar ratio, FA-Gly-Phe-Leu-Gly-OH∶HBTU∶DIPEA∶DOX=1∶(1-10)∶(1-10)∶(1-10).

[0025] The specific synthesis route is as follows:

[0026]

[0027] Depending on the type of fatty acid, the cathepsin B-sensitive fatty acid-doxacin prodrug of the present invention is a compound represented by the following general structural formula (I), with a particularly preferred structure being one of the following: (A) lauric acid-doxacin prodrug (LA-GFLG-DOX), (B) myristic acid-doxacin prodrug (MA-GFLG-DOX), (C) palmitic acid-doxacin prodrug (PA-GFLG-DOX), and (D) stearic acid-doxacin prodrug (SA-GFLG-DOX).

[0028]

[0029] The present invention discloses a cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle, which is a fatty acid-doxorubicin prodrug bridged with a series of cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragments, and albumin is used as a carrier for the cathepsin B-sensitive fatty acid-doxorubicin prodrug, and is obtained by encapsulation.

[0030] The cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles have a particle size of 118-130 nm, a polydispersity index (PDI) of 0.12-0.18, a zeta potential of +9.9-+15.7 mV, an encapsulation efficiency of ≥97%, and a sphericity of ≥98%.

[0031] The method for preparing cathepsin B-sensitive fatty acid-doxacin prodrug albumin nanoparticles provided by the present invention includes the following steps:

[0032] 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 0.1 mg / mL-20 mg / mL;

[0033] In step 1, the organic solvent is selected from one or a mixture of several of chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetone, and acetonitrile.

[0034] Step 2: Dissolve albumin in water for injection until it is fully dissolved to obtain an albumin aqueous solution;

[0035] In step 2, the albumin is selected from one of human serum albumin, bovine serum albumin, mouse serum albumin, and ovalbumin; the albumin concentration in the aqueous solution is 0.1 mg / mL to 2 mg / mL.

[0036] 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).

[0037] 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.

[0038] The present invention also provides a pharmaceutical composition comprising a compound of general structural formula (I), a geometric isomer, and a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof, and further comprising a pharmaceutically acceptable carrier or excipient.

[0039] The application of the cathepsin B-sensitive fatty acid-doxorubicin prodrug, or cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles, or pharmaceutical compositions of the present invention in the preparation of antitumor drugs.

[0040] The antitumor drug is selected from one of the following: oral administration, injection administration, or topical administration.

[0041] The present invention provides a cathepsin B-sensitive fatty acid-doxacin prodrug, its albumin nanoparticles, preparation method, and application, which have the following advantages: (1) The cathepsin B-sensitive fatty acid-doxacin prodrug is designed and synthesized, and prepared into an albumin nanodelivery system. The albumin nanoparticles have high permeability and retention (EPR) effect and active tumor targeting, which promotes the accumulation of nanoparticles at the tumor site; (2) The nanodelivery system can reduce the systemic toxicity of doxorubicin, especially cardiotoxicity, and can rapidly release the parent drug when the tumor cells highly express cathepsin B, thereby achieving the purpose of reducing toxicity and increasing efficacy; (3) It has a high drug loading capacity, avoiding the immunotoxicity caused by the use of a large amount of carrier material; (4) The preparation process is simple and easy to scale up. Attached Figure Description

[0042] Figure 1 This is a high-resolution mass spectrum of the cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment-bridged laurate-doxorubicin prodrug (LA-GFLG-DOX) in Example 1.

[0043] Figure 2This is a high-resolution mass spectrum of the myristic acid-doxorubicin prodrug (MA-GFLG-DOX) bridged by the cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment in Example 2.

[0044] Figure 3 This is a high-resolution mass spectrum of the palmitate-doxorubicin prodrug (PA-GFLG-DOX) bridged by the cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment in Example 3.

[0045] Figure 4 This is a high-resolution mass spectrum of the stearic acid-doxacin prodrug (SA-GFLG-DOX) bridged by the cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment in Example 4.

[0046] Figure 5 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 5 are shown.

[0047] Figure 6 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 6 are shown.

[0048] Figure 7 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 7 are shown.

[0049] Figure 8 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 8 are shown.

[0050] Figure 9 The image shows the particle size versus storage time of albumin nanoparticles in Examples 5-8 under storage conditions at 4°C.

[0051] Figure 10 The following are particle size-time graphs of albumin nanoparticles in 10% FBS in Examples 5-8;

[0052] Figure 11 The following are images showing the in vitro release assays of albumin nanoparticles from Examples 5-8: (A: Release assay of albumin nanoparticles from Examples 5-8 in McIlvaine buffer (pH=6.0) containing 50 U / mL cathepsin B; B: Release assay of albumin nanoparticles from Examples 5-8 in McIlvaine buffer (pH=6.0); C: Release assay of albumin nanoparticles from Examples 5-8 in phosphate buffer (pH=7.4)).

[0053] Figure 12The blood concentration-time curves of albumin nanoparticles in Examples 5-8 are shown below (A is the blood concentration-time curve of doxorubicin hydrochloride for injection and the blood concentration-time curve of the prodrugs in Examples 1-4 corresponding to albumin nanoparticles in Examples 5-8; B is the blood concentration-time curve of doxorubicin released from albumin nanoparticles in Examples 5-8).

[0054] Figure 13 The images shown are from the in vivo antitumor experiments of albumin nanoparticles in Examples 5-8 (A is a tumor growth curve; B is a tumor image; C is a weight change graph; D is a survival curve). Detailed Implementation

[0055] 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.

[0056] In the following examples, unless otherwise specified, "%" refers to mass percentage.

[0057] In the following embodiments, unless otherwise specified, all raw materials and equipment used are commercially available.

[0058] Example 1: Synthesis of a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide-bridged laurate-doxorubicin prodrug (LA-GFLG-DOX)

[0059] Step 1: Weigh 2 g (2 mmol) of CTC resin and load it into a sand core reactor. Wash it once with 20 mL of N,N-dimethylformamide (DMF), dry it under vacuum, add 10 mL of dichloromethane (DCM), then add fluorenemethyloxycarbonyl-glycine (Fmoc-Gly-OH, 1.189 g, 4 mmol) and N,N-diisopropylethylamine (DIPEA, 8 mmol, 1.394 mL). React at room temperature for 3 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Gly-CTC resin, and wash it three times with N,N-dimethylformamide (DMF).

[0060] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0061] Step 2: Add fluorenemethoxycarbonyl-leucine (Fmoc-Leu-OH, 2.120 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Gly-CTC resin obtained in Step 1, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, then filter under reduced pressure to obtain Fmoc-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0062] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0063] Step 3: Add fluorenemethoxycarbonyl-phenylalanine (Fmoc-Phe-OH, 2.325 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Leu-Gly-CTC resin obtained in Step 2, then add 20 mL of N,N-dimethylformamide (DMF), react at room temperature for 1.5 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Phe-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0064] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0065] Step 4: Add fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 1.784 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Phe-Leu-Gly-CTC resin obtained in Step 3, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, and filter under reduced pressure to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin, which is then washed three times with N,N-dimethylformamide (DMF).

[0066] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0067] Step 5: Lauric acid (LA, 1.202 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) were added to the H-Gly-Phe-Leu-Gly-CTC resin obtained in Step 4. Then, 20 mL of N,N-dimethylformamide (DMF) was added. The reaction was carried out at room temperature for 1.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain LA-Gly-Phe-Leu-Gly-CTC resin, which was washed three times with N,N-dimethylformamide (DMF). The LA-Gly-Phe-Leu-Gly-CTC resin was then washed twice with dichloromethane (DCM) and once with methanol, and dried under reduced pressure.

[0068] Add 50 mL of dichloromethane (DCM) containing 1% TFA to the dried LA-Gly-Phe-Leu-Gly-CTC resin, react at room temperature for 1 hour, filter, and rotary evaporate the filtrate under reduced pressure to obtain LA-Gly-Phe-Leu-Gly-OH.

[0069] Step 6: Dissolve the LA-Gly-Phe-Leu-Gly-OH (574.8 mg, 1 mmol) obtained in Step 5 in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 418 mg, 1.1 mmol) and N,N-diisopropylethylamine (DIPEA, 349 μL, 2 mmol) to the reaction system. Finally, add doxorubicin hydrochloride (596 mg, 1.1 mmol). React overnight at room temperature. Vacuum rotary evaporation under reduced pressure yields crude LA-Gly-Phe-Leu-Gly-DOX. Purify the crude product by column chromatography and then by preparative liquid chromatography to obtain purified LA-Gly-Phe-Leu-Gly-DOX (LA-GFLG-DOX).

[0070] The obtained product was analyzed, and the high-resolution mass spectrometry results were obtained. Figure 1 This indicates the [M+Na] content of the prodrug. + =1122.52713. The results of the proton NMR spectrum analysis are as follows: 1H NMR (600MHz, DMSO-d6) δ14.03(s,1H),13.26(s,1H),8.08(d,J=7.9Hz,1H),7.99(t,J=7.5H z,2H),7.64(dd,J=6.2,3.6Hz,1H),7.48(d,J=8.1Hz,1H),7.14(t,J=6.9Hz,1H),5.47(s,1H ),5.26–5.21(m,1H),4.94(t,J=4.6Hz,1H),4.86(t,J=6.0Hz,1H),4.80(d,J=6.0Hz,1H),4 .57(d,J=6.1Hz,2H),4.48(td,J=8.7,4.3Hz,1H),4.20(dp,J=27.2,6.7,6.2Hz,2H),3.98(s ,4H),3.70–3.58(m,3H),3.52(dd,J=16.8,5.3Hz,1H),3.39(d,J=6.1Hz,1H),3.03–2.90(m ,3H),2.74(dd,J=13.9,9.5Hz,1H),2.20(d,J=14.2Hz,1H),2.12(dd,J=14.2,5.7Hz,1H),2. 05(t, J = 7.5 Hz, 2H), 1.89–1.82(m, 1H), 1.55(dp, J = 13.0, 6.5 Hz, 1H), 1.48–1.39(m, 6H), 1.22(m, 20H), 1.13(d, J = 6.5 Hz, 3H), 0.84(t, J = 7.0 Hz, 6H), 0.80(d, J = 6.5 Hz, 3H); After verification, its structural formula is:

[0071]

[0072] Example 2: Synthesis of a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide-bridged myristic acid-doxorubicin prodrug (MA-GFLG-DOX)

[0073] Step 1: Weigh 2 g (2 mmol) of CTC resin and load it into a sand core reactor. Wash it once with 20 mL of N,N-dimethylformamide (DMF), dry it under vacuum, add 10 mL of dichloromethane (DCM), then add fluorenemethyloxycarbonyl-glycine (Fmoc-Gly-OH, 1.189 g, 4 mmol) and N,N-diisopropylethylamine (DIPEA, 8 mmol, 1.394 mL). React at room temperature for 3 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Gly-CTC resin, and wash it three times with N,N-dimethylformamide (DMF).

[0074] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0075] Step 2: Add fluorenemethoxycarbonyl-leucine (Fmoc-Leu-OH, 2.120 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Gly-CTC resin obtained in Step 1, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, then filter under reduced pressure to obtain Fmoc-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0076] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0077] Step 3: Add fluorenemethoxycarbonyl-phenylalanine (Fmoc-Phe-OH, 2.325 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Leu-Gly-CTC resin obtained in Step 2, then add 20 mL of N,N-dimethylformamide (DMF), react at room temperature for 1.5 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Phe-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0078] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0079] Step 4: Add fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 1.784 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Phe-Leu-Gly-CTC resin obtained in Step 3, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, and filter under reduced pressure to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin, which is then washed three times with N,N-dimethylformamide (DMF).

[0080] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0081] Step 5: Myristic acid (MA, 1.370 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) were added to the H-Gly-Phe-Leu-Gly-CTC resin obtained in Step 4. Then, 20 mL of N,N-dimethylformamide (DMF) was added. The reaction was carried out at room temperature for 1.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain MA-Gly-Phe-Leu-Gly-CTC resin, which was washed three times with N,N-dimethylformamide (DMF). The MA-Gly-Phe-Leu-Gly-CTC resin was then washed twice with dichloromethane (DCM) and once with methanol, and dried under reduced pressure.

[0082] Add 50 mL of dichloromethane (DCM) containing 1% TFA to the dried MA-Gly-Phe-Leu-Gly-CTC resin, react at room temperature for 1 hour, filter, and rotary evaporate the filtrate under reduced pressure to obtain MA-Gly-Phe-Leu-Gly-OH.

[0083] Step 6: Dissolve the MA-Gly-Phe-Leu-Gly-OH (602.8 mg, 1 mmol) obtained in Step 5 in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 418 mg, 1.1 mmol) and N,N-diisopropylethylamine (DIPEA, 349 μL, 2 mmol) to the reaction system. Finally, add doxorubicin hydrochloride (596 mg, 1.1 mmol). React overnight at room temperature. Vacuum rotary evaporation under reduced pressure yields crude MA-Gly-Phe-Leu-Gly-DOX. Purify the crude product by column chromatography and then by preparative liquid chromatography to obtain purified MA-Gly-Phe-Leu-Gly-DOX (MA-GFLG-DOX).

[0084] The obtained product was analyzed, and the high-resolution mass spectrometry results were obtained. Figure 2 This indicates the [M+Na] content of the prodrug. + =1150.55712. The results of the proton NMR spectrum analysis are as follows: 1H NMR (600MHz, DMSO-d6) δ14.03(s,1H),13.26(s,1H),8.08(d,J=8.0Hz,1H),7.98(t,J=7.1Hz ,2H),7.64(dd,J=6.3,3.6Hz,1H),7.47(d,J=8.2Hz,1H),7.14(d,J=6.8Hz,1H),5.47(s,1H) ,5.25–5.20(m,1H),4.93(dd,J=5.4,3.6Hz,1H),4.86(t,J=6.0Hz,1H),4.79(d,J=6.0Hz,1H ),4.57(d,J=6.0Hz,2H),4.48(td,J=8.9,4.4Hz,1H),4.25–4.15(m,2H),3.98(s,4H),3.66(d dd,J=24.0,12.0,6.1Hz,3H),3.52(dd,J=16.5,5.6Hz,1H),3.41–3.38(m,1H),3.02–2.91(m ,3H),2.74(dd,J=13.9,9.5Hz,1H),2.20(d,J=14.1Hz,1H),2.12(dd,J=14.1,5.8Hz,1H),2.0 5(t,J=7.5Hz,2H),1.88–1.81(m,1H),1.55(dt,J=13.4,6.5Hz,1H),1.44(td,J=10.0,5.1Hz ,6H),1.27–1.16(m,24H),1.13(d,J=6.4Hz,3H),0.85(d,J=6.3Hz,6H),0.80(d,J=6.5Hz,3H)

[0085] After testing, its structural formula is:

[0086]

[0087] Example 3: Synthesis of a palmitate-doxorubicin prodrug (PA-GFLG-DOX) bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment.

[0088] Step 1: Weigh 2 g (2 mmol) of CTC resin and load it into a sand core reactor. Wash it once with 20 mL of N,N-dimethylformamide (DMF), dry it under vacuum, add 10 mL of dichloromethane (DCM), then add fluorenemethyloxycarbonyl-glycine (Fmoc-Gly-OH, 1.189 g, 4 mmol) and N,N-diisopropylethylamine (DIPEA, 8 mmol, 1.394 mL). React at room temperature for 3 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Gly-CTC resin, and wash it three times with N,N-dimethylformamide (DMF).

[0089] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0090] Step 2: Add fluorenemethoxycarbonyl-leucine (Fmoc-Leu-OH, 2.120 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Gly-CTC resin obtained in Step 1, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, then filter under reduced pressure to obtain Fmoc-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0091] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0092] Step 3: Add fluorenemethoxycarbonyl-phenylalanine (Fmoc-Phe-OH, 2.325 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Leu-Gly-CTC resin obtained in Step 2, then add 20 mL of N,N-dimethylformamide (DMF), react at room temperature for 1.5 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Phe-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0093] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0094] Step 4: Add fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 1.784 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Phe-Leu-Gly-CTC resin obtained in Step 3, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, and filter under reduced pressure to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin, which is then washed three times with N,N-dimethylformamide (DMF).

[0095] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0096] Step 5: Palmitic acid (PA, 1.539 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) were added to the H-Gly-Phe-Leu-Gly-CTC resin obtained in Step 4. Then, 20 mL of N,N-dimethylformamide (DMF) was added. The reaction was carried out at room temperature for 1.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain PA-Gly-Phe-Leu-Gly-CTC resin, which was washed three times with N,N-dimethylformamide (DMF). The PA-Gly-Phe-Leu-Gly-CTC resin was then washed twice with dichloromethane (DCM) and once with methanol, and dried under reduced pressure.

[0097] Add 50 mL of dichloromethane (DCM) containing 1% TFA to the dried PA-Gly-Phe-Leu-Gly-CTC resin, react at room temperature for 1 hour, filter, and rotary evaporate the filtrate under reduced pressure to obtain PA-Gly-Phe-Leu-Gly-OH.

[0098] Step 6: Dissolve the PA-Gly-Phe-Leu-Gly-OH (630.9 mg, 1 mmol) obtained in Step 5 in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 418 mg, 1.1 mmol) and N,N-diisopropylethylamine (DIPEA, 349 μL, 2 mmol) to the reaction system. Finally, add doxorubicin hydrochloride (596 mg, 1.1 mmol). React overnight at room temperature. Vacuum rotary evaporation under reduced pressure yields crude PA-Gly-Phe-Leu-Gly-DOX. Purify the crude product by column chromatography and then by preparative liquid chromatography to obtain purified PA-Gly-Phe-Leu-Gly-DOX (PA-GFLG-DOX).

[0099] The obtained product was analyzed, and the high-resolution mass spectrometry results were obtained. Figure 3 This indicates the [M+Na] content of the prodrug. + =1178.58754. The results of the proton NMR spectrum analysis are as follows: 1H NMR (600MHz, DMSO-d6) δ14.02(s,1H),13.26(s,1H),8.08(d,J=7.9Hz,1H),7.98(t,J=5.8Hz,2H ),7.63(dd,J=6.8,3.4Hz,1H),7.47(d,J=8.2Hz,1H),7.14(t,J=6.9Hz,1H),5.46(d,J=2.6Hz,1 H),5.22(d,J=3.8Hz,1H),4.93(s,1H),4.86(t,J=6.0Hz,1H),4.80(d,J=6.0Hz,1H),4.57(d,J= 6.0Hz,2H),4.48(td,J=8.7,4.4Hz,1H),4.20(dp,J=25.6,6.7,6.3Hz,2H),3.97(d,J=1.7Hz,4H ),3.66(ddd,J=23.7,11.7,5.8Hz,3H),3.52(dd,J=16.5,5.6Hz,1H),3.39(d,J=4.4Hz,1H),3.0 3–2.89(m,3H),2.74(dd,J=13.9,9.5Hz,1H),2.20(d,J=14.4Hz,1H),2.11(d,J=10.7Hz,1H),2. 05(t,J=7.5Hz,2H),1.89–1.81(m,1H),1.55(dq,J=13.0,6.6Hz,1H),1.44(q,J=9.8,7.2Hz,6H) ,1.30–1.16(m,28H),1.13(d,J=6.4Hz,3H),0.84(dd,J=7.0,4.9Hz,6H),0.80(d,J=6.5Hz,3H);

[0100] After testing, its structural formula is:

[0101]

[0102] Example 4: Synthesis of a stearate-doxorubicin prodrug (SA-GFLG-DOX) bridged by a cathepsin B-sensitive Gly-Phe-Leu-Gly tetrapeptide fragment.

[0103] Step 1: Weigh 2 g (2 mmol) of CTC resin and load it into a sand core reactor. Wash it once with 20 mL of N,N-dimethylformamide (DMF), dry it under vacuum, add 10 mL of dichloromethane (DCM), then add fluorenemethyloxycarbonyl-glycine (Fmoc-Gly-OH, 1.189 g, 4 mmol) and N,N-diisopropylethylamine (DIPEA, 8 mmol, 1.394 mL). React at room temperature for 3 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Gly-CTC resin, and wash it three times with N,N-dimethylformamide (DMF).

[0104] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0105] Step 2: Add fluorenemethoxycarbonyl-leucine (Fmoc-Leu-OH, 2.120 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Gly-CTC resin obtained in Step 1, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, then filter under reduced pressure to obtain Fmoc-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0106] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0107] Step 3: Add fluorenemethoxycarbonyl-phenylalanine (Fmoc-Phe-OH, 2.325 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Leu-Gly-CTC resin obtained in Step 2, then add 20 mL of N,N-dimethylformamide (DMF), react at room temperature for 1.5 hours under nitrogen protection, filter under reduced pressure to obtain Fmoc-Phe-Leu-Gly-CTC resin, and wash three times with N,N-dimethylformamide (DMF).

[0108] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0109] Step 4: Add fluorenemethoxycarbonyl-glycine (Fmoc-Gly-OH, 1.784 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) to the H-Phe-Leu-Gly-CTC resin obtained in Step 3, then add 20 mL of N,N-dimethylformamide (DMF). React at room temperature for 1.5 hours under nitrogen protection, and filter under reduced pressure to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin, which is then washed three times with N,N-dimethylformamide (DMF).

[0110] 20 mL of N,N-dimethylformamide (DMF) containing 20% ​​piperidine was added to Fmoc-Gly-Phe-Leu-Gly-CTC resin. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain H-Gly-Phe-Leu-Gly-CTC resin, which was washed five times with N,N-dimethylformamide (DMF).

[0111] Step 5: Stearic acid (SA, 1.707 g, 6 mmol), N-methylmorpholine (NMM, 607 μL, 6 mmol), and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 2.275 g, 6 mmol) were added to the H-Gly-Phe-Leu-Gly-CTC resin obtained in Step 4. Then, 20 mL of N,N-dimethylformamide (DMF) was added. The reaction was carried out at room temperature for 1.5 hours under nitrogen protection. The mixture was then filtered under reduced pressure to obtain SA-Gly-Phe-Leu-Gly-CTC resin, which was washed three times with N,N-dimethylformamide (DMF). The SA-Gly-Phe-Leu-Gly-CTC resin was then washed twice with dichloromethane (DCM) and once with methanol, and dried under reduced pressure.

[0112] Add 50 mL of dichloromethane (DCM) containing 1% TFA to the dried SA-Gly-Phe-Leu-Gly-CTC resin, react at room temperature for 1 hour, filter, and rotary evaporate the filtrate under reduced pressure to obtain SA-Gly-Phe-Leu-Gly-OH.

[0113] Step 6: Dissolve the SA-Gly-Phe-Leu-Gly-OH (658.9 mg, 1 mmol) obtained in Step 5 in 10 mL of N,N-dimethylformamide (DMF). Add O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 418 mg, 1.1 mmol) and N,N-diisopropylethylamine (DIPEA, 349 μL, 2 mmol) to the reaction system. Finally, add doxorubicin hydrochloride (596 mg, 1.1 mmol). React overnight at room temperature. Vacuum rotary evaporation under reduced pressure yields crude SA-Gly-Phe-Leu-Gly-DOX. Purify the crude product by column chromatography and then by preparative liquid chromatography to obtain purified SA-Gly-Phe-Leu-Gly-DOX (SA-GFLG-DOX).

[0114] The obtained product was analyzed, and the high-resolution mass spectrometry results were obtained. Figure 4 This indicates the [M+Na] content of the prodrug. + =1206.61937. The results of the proton NMR spectrum analysis are as follows: 1H NMR (600MHz, DMSO-d6) δ14.04(s,1H),13.27(s,1H),8.08(d,J=7.9Hz,1H),7.98(t,J=6.0H z,2H),7.65(dd,J=6.3,3.4Hz,1H),7.47(d,J=8.2Hz,1H),7.14(t,J=6.8Hz,1H),5.48(s,1H ),5.23(d,J=3.7Hz,1H),4.94(t,J=4.7Hz,1H),4.86(t,J=6.0Hz,1H),4.79(d,J=6.0Hz,1H ),4.57(d,J=6.0Hz,2H),4.48(td,J=8.9,4.4Hz,1H),4.24–4.19(m,1H),4.17(q,J=6.6Hz,1 H),3.98(s,4H),3.70–3.61(m,3H),3.52(dd,J=16.5,5.6Hz,1H),3.39(d,J=4.3Hz,1H),3. 03–2.95(m,3H),2.75(dd,J=13.9,9.5Hz,1H),2.20(d,J=15.6Hz,1H),2.12(dd,J=14.2,5.8 Hz,1H),2.05(t,J=7.5Hz,2H),1.89–1.81(m,1H),1.55(m,1H),1.44(dq,J=13.3,7.1,6.3H z,6H),1.27–1.20(m,32H),1.12(d,J=6.5Hz,3H),0.86–0.84(m,6H),0.80(d,J=6.5Hz,3H);

[0115] After testing, its structural formula is:

[0116]

[0117] Example 5: Formulation and preparation process of the prodrug albumin nanoparticles from Example 1

[0118] Table 1: Formulation of the prodrug albumin nanoparticles in Example 1

[0119]

[0120] Preparation process:

[0121] (1) Accurately weigh 50 mg of the drug from Example 1, dissolve it thoroughly in 5 mL of chloroform, and set aside;

[0122] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0123] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then distilled under reduced pressure to remove the organic solvent and obtain albumin nanoparticles.

[0124] like Figure 5 A and Figure 5 As shown in B, the albumin nanoparticles in Example 5 were spherical with uniform particle size, with a particle size of 129.8±1.901 nm, a PDI of 0.158±0.042, a Zeta potential of +15.7±0.529 mV, an encapsulation efficiency of 99.39±0.22%, and a sphericity of 100±1.89%.

[0125] Example 6: Formulation and preparation process of the prodrug albumin nanoparticles from Example 2

[0126] Table 2: Formulation of prodrug albumin nanoparticles in Example 2

[0127]

[0128] Preparation process:

[0129] (1) Accurately weigh 50 mg of the drug from Example 2, dissolve it thoroughly in 5 mL of chloroform, and set aside;

[0130] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0131] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then distilled under reduced pressure to remove the organic solvent and obtain albumin nanoparticles.

[0132] like Figure 6 A and Figure 6 As shown in B, the albumin nanoparticles in Example 6 are spherical with uniform particle size, with a particle size of 126.9±1.646 nm, a PDI of 0.132±0.052, a Zeta potential of +12.2±0.173 mV, an encapsulation efficiency of 98.71±1.30%, and a sphericity of 100±1.43%.

[0133] Example 7: Formulation and preparation process of the prodrug albumin nanoparticles from Example 3

[0134] Table 3: Formulation of the prodrug albumin nanoparticles in Example 3

[0135]

[0136] Preparation process:

[0137] (1) Accurately weigh 50 mg of the prodrug from Example 3, dissolve it thoroughly in 5 mL of chloroform, and set aside;

[0138] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0139] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then distilled under reduced pressure to remove the organic solvent and obtain albumin nanoparticles.

[0140] like Figure 7 A and Figure 7 As shown in B, the albumin nanoparticles in Example 7 are spherical with uniform particle size, with a particle size of 127.8±2.354 nm, a PDI of 0.127±0.037, a Zeta potential of +9.9±0.352 mV, an encapsulation efficiency of 99.42±1.13%, and a sphericity of 100±0.72%.

[0141] Example 8: Formulation and preparation process of the prodrug albumin nanoparticles from Example 4

[0142] Table 4: Formulation of the prodrug albumin nanoparticles in Example 4

[0143]

[0144] Preparation process:

[0145] (1) Accurately weigh 50 mg of the drug from Example 4, dissolve it thoroughly in 5 mL of chloroform, and set aside.

[0146] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0147] (3) Add (1) to (2) and sonicate at 390W for 5 minutes in an ice bath. The resulting emulsion is then distilled under reduced pressure to remove the organic solvent and obtain albumin nanoparticles.

[0148] like Figure 8 A and Figure 8 As shown in B, the albumin nanoparticles in Example 8 are spherical with uniform particle size, with a particle size of 118.5±4.713 nm, a PDI of 0.180±0.058, a Zeta potential of +13.8±0.709 mV, an encapsulation efficiency of 99.23±0.20%, and a sphericity of 100±0.72%.

[0149] Comparative Example 1: Preparation of Doxorubicin Albumin Nanoparticles

[0150] Table 5: Formulation of Doxorubicin Albumin Nanoparticles

[0151]

[0152] Preparation process:

[0153] (1) Accurately weigh 50 mg of doxorubicin, dissolve it thoroughly in 5 mL of chloroform, and set aside.

[0154] (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 100 mL of water for injection, and set aside.

[0155] (3) Add (1) to (2) and sonicate at 390w for 5 minutes in an ice bath. The resulting emulsion is then distilled under reduced pressure to remove the organic solvent and obtain albumin nanoparticles.

[0156] The obtained doxorubicin albumin nanoparticles had a particle size of 487.9±7.413 nm, a PDI of 0.673±0.085, a Zeta potential of +11.8±0.907 mV, and an encapsulation efficiency of 19.3±0.72%. These nanoparticles had a relatively large particle size, uneven dispersion, and a low encapsulation efficiency.

[0157] Example 9: Storage stability test of albumin nanoparticles from Examples 5-8 at 4°C

[0158] The albumin nanoparticles from Examples 5-8 were stored at 4°C, and their particle size changes were measured at set time points during this period using dynamic light scattering. The results are as follows: Figure 9 As shown, the particle size of albumin nanoparticles in Examples 5-8 showed no significant change over 30 days, indicating that the nanoparticles have good storage stability.

[0159] Example 10: Colloidal stability test of albumin nanoparticles from Examples 5-8

[0160] The albumin nanoparticles from Examples 5-8 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 10 As shown, the particle size of albumin nanoparticles in Examples 5-8 did not change significantly within 24 hours, indicating that the nanoparticles have good colloidal stability.

[0161] Example 11: In vitro release test of albumin nanoparticles from Examples 5-8

[0162] The in vitro release of albumin nanoparticles from Examples 5-8 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). The albumin nanoparticles from Examples 5-8 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 5-8 under different conditions.

[0163] Depend on Figure 11 It is known that the albumin nanoparticles in Examples 5-8 can only be released in a McIlvaine buffer (pH=6.0) containing 50 U / mL cathepsin B. Figure 11 Doxorubicin can only be released in A) and in a slightly acidic environment ( Figure 11 B) Neutral physiological environment ( Figure 11 Under condition C), the albumin nanoparticles of Examples 5-8 released almost no doxorubicin. This indicates that the albumin nanoparticles of Examples 5-8 possess cathepsin B-sensitive properties, and the drug release rate increases with the length of the carbon chain. Compared with the albumin nanoparticles of Examples 5 and 6, the albumin nanoparticles of Examples 7 and 8 achieved release rates of 98.2% and 84.2% respectively within 48 hours, showing promise for releasing the parent drug within the lysosomes of tumor cells highly expressing cathepsin B, thereby achieving a tumor-killing effect.

[0164] Example 12: Pharmacokinetic Study of Albumin Nanoparticles from Examples 5-8

[0165] 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 and albumin nanoparticles (5 mg / kg based on doxorubicin) from Examples 5-8 were administered via tail vein, respectively. The plasma concentrations of doxorubicin and the prodrugs from Examples 1-4 were measured.

[0166] Experimental results are as follows Figure 12 A, Figure 12 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 ) and area under the plasma concentration-time curve (AUC) 0-12h The values ​​were all low, while the albumin nanoparticles in Examples 5-8 showed significantly higher C values. max and AUC 0-12hFurthermore, due to the similarity of their surface properties, no significant differences were observed among the various groups of nanoparticles.

[0167] Table 6. Main pharmacokinetic parameters of albumin nanoparticles in Examples 5-8

[0168]

[0169] Example 13: In vivo antitumor experiment of albumin nanoparticles from Examples 5-8

[0170] 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 10:00 AM, tumor-bearing mice were randomly divided into groups of five. Each group received a tail vein injection of physiological saline, doxorubicin hydrochloride for injection, or albumin nanoparticles (10 mg / kg based on doxorubicin) as described in Examples 5-8. The drugs were administered every other day for five consecutive days. The mice's survival status was observed daily, their body weight was measured, and the tumor volume was determined.

[0171] In vivo anti-tumor effects such as Figure 13 As shown, injectable doxorubicin hydrochloride exhibits significant antitumor effects, with slowed tumor growth. However, the mice experienced a continuous decrease in body weight, and all mice died on the eighth day after administration, indicating that injectable doxorubicin hydrochloride has serious side effects. The albumin nanoparticles in Examples 5-8 demonstrated better safety; compared to the saline group, there was no significant decrease in mouse body weight, and no mice died. Furthermore, among the albumin nanoparticles, the albumin nanoparticles in Example 7 exhibited the strongest antitumor activity, significantly inhibiting tumor growth compared to the saline group. Therefore, the albumin nanoparticle group in Example 7 is a nanodelivery system that combines safety and efficacy.

Claims

1. A cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle, characterized in that, The cathepsin B sensitive fatty acid-doxorubicin prodrug is obtained by encapsulating an albumin as a carrier of the cathepsin B sensitive fatty acid-doxorubicin prodrug; The cathepsin B sensitive fatty acid-doxorubicin prodrug has a structure shown in a general formula (I); Wherein, n represents the number of -(CH2-CH2)- in the fatty acid chain; When the fatty acid is lauric acid, the cathepsin B sensitive fatty acid-doxorubicin prodrug is lauric acid-doxorubicin prodrug LA-GFLG-DOX; When the fatty acid is myristic acid, the cathepsin B sensitive fatty acid-doxorubicin prodrug is myristic acid-doxorubicin prodrug MA-GFLG-DOX; When the fatty acid is palmitic acid, the cathepsin B sensitive fatty acid-doxorubicin prodrug is palmitic acid-doxorubicin prodrug PA-GFLG-DOX; When the fatty acid is stearic acid, the cathepsin B sensitive fatty acid-doxorubicin prodrug is stearic acid-doxorubicin prodrug SA-GFLG-DOX; 。 2. The cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle of claim 1, wherein, The preparation method of the cathepsin B sensitive fatty acid-doxorubicin prodrug comprises the following steps: Step one: 2-chlorotrityl chloride resin is weighed, fluorenylmethoxycarbonyl-glycine Fmoc-Gly-OH and N,N-diisopropylethylamine DIPEA are added, and dichloromethane is used as a solvent to react, so as to obtain Fmoc-Gly-CTC resin; Piperidine-containing N,N-dimethylformamide is added to the Fmoc-Gly-CTC resin to react, so as to obtain H-Gly-CTC resin; Step two: fluorenylmethoxycarbonyl-leucine Fmoc-Leu-OH, N-methylmorpholine NMM and O-benzotriazole-tetramethyluronium hexafluorophosphate HBTU are added to the H-Gly-CTC resin, N,N-dimethylformamide is used as a solvent to react, so as to obtain Fmoc-Leu-Gly-CTC resin; Piperidine-containing N,N-dimethylformamide is added to the Fmoc-Leu-Gly-CTC resin to react, so as to obtain H-Leu-Gly-CTC resin; Step three: fluorenylmethoxycarbonyl-phenylalanine Fmoc-Phe-OH, N-methylmorpholine NMM and O-benzotriazole-tetramethyluronium hexafluorophosphate HBTU are added to the H-Leu-Gly-CTC resin, N,N-dimethylformamide is used as a solvent to react, so as to obtain Fmoc-Phe-Leu-Gly-CTC resin; Piperidine-containing N,N-dimethylformamide is added to the Fmoc-Phe-Leu-Gly-CTC resin to react, so as to obtain H-Phe-Leu-Gly-CTC resin; Step four: fluorenylmethoxycarbonyl-glycine Fmoc-Gly-OH, N-methylmorpholine NMM and O-benzotriazole-tetramethyluronium hexafluorophosphate HBTU are added to the H-Phe-Leu-Gly-CTC resin, N,N-dimethylformamide is used as a solvent to react, so as to obtain Fmoc-Gly-Phe-Leu-Gly-CTC resin; In Fmoc-Gly-Phe-Leu-Gly-CTC resin, N,N-dimethylformamide containing piperidine was added to react, to obtain H-Gly-Phe-Leu-Gly-CTC resin; Step five: in H-Gly-Phe-Leu-Gly-CTC resin, fatty acid, N-methyl morpholine NMM and O-benzotriazole-tetramethyl urea hexafluorophosphate HBTU were added to react with N,N-dimethylformamide as solvent, to obtain FA-Gly-Phe-Leu-Gly-CTC resin; In FA-Gly-Phe-Leu-Gly-CTC resin, dichloromethane containing trifluoroacetic acid was added to react, to obtain FA-Gly-Phe-Leu-Gly-OH; Step six: FA-Gly-Phe-Leu-Gly-OH was reacted with O-benzotriazole-tetramethyl urea hexafluorophosphate HBTU, N,N-diisopropyl ethylamine DIPEA and doxorubicin hydrochloride, to obtain fatty acid-glycine-phenylalanine-leucine-glycine-doxorubicin FA-Gly-Phe-Leu-Gly-DOX, which is a cathepsin B sensitive fatty acid-doxorubicin prodrug; The specific synthesis route is as follows: 。 3. The cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle of claim 2, wherein, In step one, the molar ratio of 2-chlorotrityl chloride resin:Fmoc-Gly-OH:DIPEA is 1:(1-10):(1-10); the mass fraction of piperidine in N,N-dimethylformamide containing piperidine is 10%-60%; the mass ratio of Fmoc-Gly-CTC resin:piperidine is 1:(0.5-10); And / or, in step two, the molar ratio of H-Gly-CTC resin:Fmoc-Leu-OH:NMM:HBTU is 1:(1-10):(1-10):(1-10); The mass fraction of piperidine in N,N-dimethylformamide containing piperidine is 10%-60%; the mass ratio of Fmoc-Leu-Gly-CTC resin:piperidine is 1:(0.5-10); And / or, in step three, the molar ratio of H-Leu-Gly-CTC resin:Fmoc-Phe-OH:NMM:HBTU is 1:(1-10):(1-10):(1-10); The mass fraction of piperidine in N,N-dimethylformamide containing piperidine is 10%-60%; the mass ratio of Fmoc-Phe-Leu-Gly-CTC resin:piperidine is 1:(0.5-10); And / or, in step four, the molar ratio of H-Phe-Leu-Gly-CTC resin:Fmoc-Gly-OH:NMM:HBTU is 1:(1-10):(1-10):(1-10); The mass fraction of piperidine in N,N-dimethylformamide containing piperidine is 10%-60%; the mass ratio of Fmoc-Gly-Phe-Leu-Gly-CTC resin:piperidine is 1:(0.5-10); And / or, in the step five, the molar ratio of H-Gly-Phe-Leu-Gly-CTC resin: fatty acid: NMM: HBTU is 1: (1-10): (1-10): (1-10); The mass fraction of trifluoroacetic acid in the dichloromethane containing trifluoroacetic acid is 0.1%-10%; the mass ratio of FA-Gly-Phe-Leu-Gly-CTC resin: trifluoroacetic acid is 1: (0.05-5); And / or, in the step six, the molar ratio of FA-Gly-Phe-Leu-Gly-OH: HBTU: DIPEA: doxorubicin hydrochloride is 1: (1-10): (1-10): (1-10).

4. The cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle of claim 1, wherein, The tissue proteinase B sensitive fatty acid-doxorubicin prodrug albumin nanoparticle has a particle size of 118 nm-130 nm, a PDI of 0.12-0.18, a Zeta potential of +9.9 mV to +15.7 mV, an encapsulation rate of 97% or more, and a balling rate of 98% or more.

5. The method of producing cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticles of claim 1, characterized in that, The method comprises the following steps: Step 1: dissolving the tissue proteinase B sensitive fatty acid-doxorubicin prodrug in an organic solvent to obtain a prodrug solution; Step 2: dissolving albumin in water for injection to fully dissolve, to obtain an albumin aqueous solution; Step 3: mixing the prodrug solution and the albumin aqueous solution, ultrasonic or high-pressure homogenization at 0-20℃, the tissue proteinase B sensitive fatty acid-doxorubicin prodrug is encapsulated by albumin, and the organic solvent is removed to obtain the tissue proteinase B sensitive fatty acid-doxorubicin prodrug albumin nanoparticle; wherein, the mass ratio of the tissue proteinase B sensitive fatty acid-doxorubicin prodrug: albumin is (5-1): (1-50).

6. The method of claim 5, wherein the cathepsin B-sensitive fatty acid-doxorubicin prodrug albumin nanoparticle is prepared by, In the step 1, the mass concentration of the tissue proteinase B sensitive fatty acid-doxorubicin prodrug in the prodrug solution is 0.1 mg / mL-20 mg / mL; The organic solvent is selected from one or more of trichloromethane, dichloromethane, ethyl acetate, methanol, ethanol, acetone, and acetonitrile; And / or, in the step 2, the albumin is selected from one of human serum albumin, bovine serum albumin, mouse serum albumin, and ovalbumin; the mass concentration of the albumin in the albumin aqueous solution is 0.1 mg / mL-2 mg / mL; And / or, in the step 3, the ultrasonic power is 100 W-800 W, the ultrasonic time is 1 min-20 min; the high-pressure homogenizer pressure is 5000 psi-50000 psi, the high-pressure homogenization cycle number is 3-20 times; and the method for removing the organic solvent is selected from one or more of spin evaporation, vacuum drying, ultrafiltration, and dialysis.

7. A pharmaceutical composition, characterized by, The tissue proteinase B sensitive fatty acid-doxorubicin prodrug albumin nanoparticle of claim 1 and a pharmaceutically acceptable carrier or excipient.

8. Use of the tissue proteinase B sensitive fatty acid-doxorubicin prodrug albumin nanoparticle of claim 1 or the pharmaceutical composition of claim 7 in the preparation of an anti-breast cancer drug.

Images