Click-type covalent drugs and their regioselective delivery system and application

Abstract

This invention discloses a type of click-type covalent drug, its regionally selective delivery system, and its applications. The invention comprises two parts: a click-type covalent drug and a tumor regionally selective delivery system. The click-type covalent drug consists of three parts: an azacyclic alkyne (DBCO), a small molecule drug, and a linker, having the structure of Formula 1. Optionally, the small molecule drug is a membrane protein inhibitor, an immunomodulator, or a chemotherapeutic drug. The click-type covalent drug reacts with azide-labeled target cells via a click reaction, forming a covalent bond that binds to the cell membrane, increasing the local drug concentration and prolonging the drug retention time, thereby enhancing selective inhibition of the target cells. The regionally selective delivery system is co-assembled from an amphiphilic block polymer and the click-type covalent drug for regionally selective delivery. This invention achieves covalent binding to azide-labeled tumor cells through the click-type covalent drug, while having no effect on unlabeled normal cells, reducing toxic side effects on normal tissues.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and chemical technology, specifically relating to click-type covalent drugs, extracellular and intracellular acid-responsive amphiphilic polymers for tumors and their regionally selective delivery systems, preparation methods and applications. Background Technology

[0002] Cancer immunotherapy drugs activate the patient's own immune system to attack tumor cells, thereby eliminating the tumor. Various treatment strategies, such as therapeutic vaccines, cell therapy, and immune checkpoint therapy, are used clinically to treat cancer patients by intervening in different stages of the tumor immune loop. However, tumors evade the body's immune system by reducing immunogenicity and upregulating negative immune regulators (such as PD-L1 and IDO), and by forming a dense extracellular matrix composed of tumor-associated fibroblasts, macrophages, and collagen, creating a "physical barrier" that limits the infiltration of tumor-specific T cells. This synergistic effect leads to tumor immune tolerance, resulting in low response rates for single-treatment therapy. Furthermore, existing tumor immunotherapy strategies struggle to selectively regulate intracellular and extracellular regions of tumor cells, another significant reason for low clinical response rates. Therefore, treatment strategies that address the key clinical challenge of selectively regulating intracellular and extracellular targets of tumors, while simultaneously overcoming their "biological" and "physical" barriers to promote tumor-specific T cell infiltration and improve response rates, are of significant clinical importance. Currently, non-small molecule antitumor drugs suffer from poor water solubility, poor pharmacokinetic properties, low affinity, and a lack of targeting. Compared to non-covalent inhibitors, small molecule covalent inhibitors offer advantages such as improved biochemical efficiency and potency, prolonged duration of action, extended pharmacokinetic duration, and reduced dosage and dosing frequency. Unlike traditional non-covalent inhibitors, which rely on hydrophobic interactions, hydrogen bonds, and electrostatic interactions to reversibly bind to target proteins (these forces are weaker than covalent bonds), covalent inhibitors often exhibit stronger efficacy. However, compared to the easier-to-develop small molecule covalent inhibitors of kinases, small molecule covalent inhibitors directly targeting tumor cells have yet to be reported. Traditional covalent strategies targeting natural amino acid residues (cysteine, serine, tyrosine, lysine, arginine, and glutamate) with small molecule covalent inhibitors can lead to severe toxic side effects. Furthermore, the ubiquitous presence of amino acids in proteins, cells, and the body presents a significant challenge to achieving selectivity against protein targets. Therefore, the key to developing covalent small molecule drugs lies in achieving an appropriate balance between reactivity and selectivity. In order to generate small molecule covalent drugs for targeting tumors in vivo, a new strategy is needed to design chemically reactivity of small molecule covalent inhibitors that is biocompatible and highly specific to the target. Summary of the Invention

[0003] Based on the above background, the purpose of this invention is to design and develop a regioselective delivery system using click chemistry and tumor intracellular and extracellular acid-responsive amphiphilic block polymers. This delivery system consists of nanoparticles co-assembled from clickable covalently induced drugs and tumor intracellular and extracellular acid-responsive amphiphilic block polymers. These nanoparticles sequentially activate and release the loaded clickable covalently induced drugs and other combined therapeutic agents in the acidic environment of the tumor extracellular environment. First, the clickable drug is released in the slightly acidic environment of the tumor extracellular environment and covalently binds to pre-azide-labeled tumor cells, thereby achieving specific inhibition of tumor extracellular targets in vivo. After the nanoparticles are taken up by the cells, the intracellular acid-activated drug release regulates the intracellular targets; the two work synergistically to achieve regioselective regulation of the tumor.

[0004] The specific technical solution for achieving the objective of this invention is as follows:

[0005] A class of click-type covalent drugs, wherein the click-type covalent drug can form a covalent bond with an azide-labeled target protein through a click reaction, has the structure shown in Formula 1:

[0006]

[0007] in,

[0008] R1 is a membrane protein inhibitor, immunomodulator, or chemotherapeutic drug, and has the following structure:

[0009]

[0010] The linker is the connecting chain between R1 and the carbonyl group, and can be selected from any of the following structures.

[0011]

[0012] Where n is any integer from 1 to 20, preferably an integer from 2 to 10.

[0013] A method for preparing the click-type covalent drug, specifically comprising the following steps, synthesizing the click-type covalent drug through an esterification reaction as shown in reaction route I:

[0014]

[0015]

[0016] Linker, R1 is as described in claim 1;

[0017] Step a: Synthesis of the azacyclic alkyne linker derivative, namely DBCO-Linker

[0018] Carboxylated azacyclic alkyne, Linker, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N,N-diisopropylethylamine were dissolved in dichloromethane in a molar ratio of 1:2:2:2:2 to obtain a solution with a mass concentration of 20-40% w / v. The reaction was carried out at room temperature for 24 h under anhydrous conditions. The reaction product was purified by thin-layer chromatography to obtain the azacyclic alkyne Linker derivative, namely DBCO-Linker.

[0019]

[0020] Step b: Synthesis of click-type covalent drugs

[0021] R1, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N,N-diisopropylethylamine were dissolved in anhydrous dichloromethane or dimethyl sulfoxide at a molar ratio of 1:2:2:2 or R1, triethylamine, and triphosgene at a molar ratio of 1:1.5:0.3 to prepare a 40% w / v solution B, and the reaction was carried out for 1.5 h. The azacyclic yne Linker derivative obtained in step a was dissolved in anhydrous dichloromethane to obtain a 20-40% w / v solution A. Solution A was added dropwise to solution B, and the reaction was continued for 24 h. Then, the solution was purified by thin-layer chromatography to obtain the click-type covalent drug, the compound shown in Formula 1.

[0022]

[0023] Wherein, R1 is as described in claim 1.

[0024] An amphiphilic block polymer that simultaneously responds to both extracellular and intracellular acidic environments, wherein the polymer is a linear amphiphilic block polymer and possesses tumor extracellular and intracellular acid-activated nuclear magnetic resonance signal-guided imaging properties, and has the structure shown in Formula 2:

[0025]

[0026] in,

[0027] R2 is N,N-diethylamino, N,N-diisopropylamino, N,N-di-tert-butylamino, N-ethyl-n-propylamino, N,N-dipropylamino, N,N-dipentanamino, N,N-dihexylamino, pentamethylamino, hexamethyleneamino, or heptamethylamino, preferably N,N-diisopropylamino; R3 is N,N-diethylamino, N,N-ethylpropylamino, pentamethylamino, or hexamethyleneamino, preferably N,N-ethylpropylamino;

[0028] M is Gd 3+ Mn 2+ Co 2+Fe 3+ Cu 2+ or Ni 2+ Gd is preferred. 3+ ;

[0029] n is an integer between 20 and 200, preferably an integer of 113;

[0030] x is an integer between 20 and 100, preferably an integer between 40 and 50;

[0031] y is an integer between 20 and 100, preferably an integer between 40 and 50;

[0032] z is an integer from 0 to 20, preferably an integer from 5 to 10.

[0033] A method for preparing the amphiphilic block polymer, comprising reversible addition-fragmentation chain transfer polymerization and esterification grafting as shown in reaction route II below:

[0034]

[0035] The definitions of n, x, y, z, and M are as described in claim 2;

[0036] Step a: Synthesis of polyethylene glycol-b-polyR3

[0037] A RAFT chain transfer agent-terminated polyethylene glycol macromolecular initiator, ethyl methacrylate-modified R3, and the initiator azobisisobutyronitrile were dissolved in 1-5 mL of N,N-dimethylformamide at a molar ratio of 1:50-80:0.1. The reaction was carried out at 70 °C for 24 h under anaerobic conditions. The reaction product was diluted with 1-10 mL of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a capacity cutoff of 3500 Daltons to obtain an extracellular acid-responsive amphiphilic block copolymer.

[0038]

[0039] Step b: Polyethylene glycol-b-poly(R3-b-R2-r-hydroxyethyl methacrylate)

[0040] The amphiphilic block copolymer obtained in step a, ethyl methacrylate-modified R2, hydroxyethyl methacrylate monomer, and initiator azobisisobutyronitrile were dissolved in 1-5 mL of N,N-dimethylformamide at a molar ratio of 1:50-80:10-20:0.1. The reaction was carried out at 70 °C for 24 h under anaerobic conditions. The reaction product was diluted with 1-10 mL of DMF and dialyzed with deionized water. The product was then freeze-dried in a dialysis bag with a cutoff of 3500 Daltons to obtain an extracellular and intracellular acid-responsive amphiphilic block copolymer.

[0041]

[0042] Step c: Dissolve the amphiphilic block copolymer obtained in step b in anhydrous N,N-dimethylformamide to obtain a 10% w / v solution C. Dissolve metal ion-coordinated pyromethesin (MPPa), N,N-diisopropylethylamine, 4-dimethylaminopyridine, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in N,N-dimethylformamide at a molar ratio of 1:1.5:1.5:1.5 to prepare a 30% w / v solution D and react for 1.5 h. Add solution C dropwise to solution D and continue the reaction for 24 h. Then dialyze with N,N-dimethylformamide and then with deionized water using a dialysis bag with a cutoff of 3500 Daltons. Freeze-dry to obtain metal ion-coordinated pyromethesin (MPPa) covalently modified to obtain the amphiphilic block polymer shown in Formula 2.

[0043]

[0044] A region-selective delivery system comprising nanoparticles assembled from a clickable covalent drug and an amphiphilic block polymer; wherein the nanoparticles achieve region-selective drug delivery in response to the extracellular and intracellular acidic environment of tumor cells; wherein the clickable covalent drug accounts for 5%-20% of the total mass of the nanoparticles, and the amphiphilic block polymer accounts for 80%-95% of the total mass of the nanoparticles.

[0045] A method for preparing the above-mentioned regionally selective delivery system involves first dissolving the click-type covalent drug shown in Formula 1 and the amphiphilic block polymer shown in Formula 2 simultaneously in an organic solvent at a mass ratio of 1-9 to obtain a mixed solution. The mixed solution is then added dropwise to deionized water under ultrasonic conditions at a volume ratio of 1:9. The organic solvent or the unencapsulated click-type covalent drug shown in Formula 1 is removed by ultrafiltration or dialysis to obtain a regionally selective delivery system encapsulating the click-type covalent drug shown in Formula 1.

[0046] Another region-selective delivery system, wherein the delivery system comprises nanoparticles assembled from the clickable covalent drug, a normal drug, and the amphiphilic block polymer, wherein the normal drug includes immunomodulators, chemotherapeutic drugs, nucleic acid drugs, or protein degraders; wherein the clickable covalent drug accounts for 5%-20% of the total mass of the nanoparticles, the normal drug accounts for 5%-20% of the total mass of the nanoparticles, and the amphiphilic block polymer accounts for 60%-90% of the total mass of the nanoparticles.

[0047] A method for preparing the above-mentioned regionally selective delivery system is as follows: First, a normal drug and the amphiphilic block polymer shown in Formula 2 are simultaneously dissolved in an organic solvent at a mass ratio of 1-9 to obtain a mixed solution containing the drug and the amphiphilic block polymer. Under ultrasonic conditions, this mixed solution is dropwise added to a buffer solution with a pH > 6.5 at a volume ratio of 1:9. The organic solvent or unencapsulated normal drug is removed by ultrafiltration or dialysis to obtain a nanoparticle suspension encapsulated with the normal drug. Second, the clicker shown in Formula 1... A clickable covalent drug is dissolved in an organic solvent to obtain a drug solution containing the clickable covalent drug. The mass ratio of the drug solution to the nanoparticle suspension is 1-9, and the volume ratio of the drug solution to the nanoparticle suspension with pH > 6.5 is 1:9. Under ultrasonic conditions, the drug solution is added dropwise to the nanoparticle suspension with pH > 6.5, while the pH of the nanoparticle suspension is adjusted to 7.4. Then, the organic solvent or unencapsulated clickable covalent drug is removed by ultrafiltration or dialysis, finally obtaining a nanoparticle delivery system containing the clickable covalent drug, a normal drug, and the amphiphilic block polymer.

[0048] Furthermore, the organic solvent is selected from at least one of tetrahydrofuran, methanol, N,N-dimethylformamide and N,N-dimethylacetamide; the buffer solution is selected from PBS buffer.

[0049] The use of the aforementioned region-selective delivery system in the delivery of cancer-treating drugs.

[0050] The cancer treatment drugs include protein inhibitors, chemotherapy drugs, nucleic acid drugs, and combination drugs.

[0051] The cancers mentioned are lung cancer, skin cancer, breast cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, colon cancer, or prostate cancer.

[0052] The described regionally selective delivery system features selective delivery to both intracellular and extracellular tumor cells. Based on the differences in the acidic environments of the tumor's extracellular and intracellular environments, it specifically releases click-type membrane protein inhibitors extracellularly, specifically degrading tumor cell membrane proteins; and specifically releases MRI contrast agents intracellularly. The regionally selective nanoparticles self-assemble into stable nanoparticles under normal physiological conditions (e.g., pH 7.4), with a hydrodynamic particle size of approximately 80 nm. They exhibit good stability during blood circulation, preventing drug leakage. In the slightly acidic extracellular environment of tumor tissue, the extracellular acid activates the acid-responsive groups in the nanoparticles, causing protonation and releasing click-type covalent drugs. These drugs then react with azide-prelabeled tumor cells, forming covalent bonds, enhancing retention in tumor tissue and improving the efficacy of the click-type covalent drugs while reducing toxic side effects. After the nanoparticles are taken up by cells, the intracellular acid activates the acid-responsive groups in the nanoparticles, causing protonation and activating the MRI contrast agents intracellularly, ultimately achieving regionally selective drug release and enabling MRI-guided precision tumor treatment.

[0053] This invention also provides a clickable covalent drug nanoplatform for tumor synergistic therapy based on activation of the tumor's extracellular and intracellular acidic environment, simultaneously regulating both intracellular and extracellular targets. The extracellular acidic environment-activated nanoparticles are used to regulate extracellular targets, while the intracellular acidic environment-activated nanoparticles are used to regulate intracellular targets, thereby achieving precise tumor treatment. Specifically, the extracellular acidic environment-activated nanoparticles release clickable covalent drugs for selective covalent degradation of tumor membrane proteins; the intracellular acidic environment-activated nanoparticles release other drugs to regulate intracellular targets; or any combination of therapeutic agents satisfying the purposes of this invention.

[0054] Regioselective nanoparticles comprising a clickable covalent drug of formula 1 and an extracellular and intracellular acid-responsive amphiphilic block polymer of formula 2 were prepared by a pH gradient method. The nanoparticles have a multilayer structure, including a polyethylene glycol outer layer and a poly(ethyl propyl aminoethyl methacrylate)-b-poly(N,N-diisopropylaminoethyl ester-r-metal-coordinated pyromethenamate a) inner layer nanoparticles. The average hydrodynamic particle size of the nanoparticles is preferably 70-120 nm.

[0055] The nanoparticles possess responsiveness to both extracellular and intracellular acidic environments in tumors, enabling them to selectively deliver drugs to tumor tissue regions. A polyethylene glycol hydrophilic layer prevents protein adsorption, ensuring nanoparticle stability in the bloodstream, reducing clearance by the endothelial reticulum system, and prolonging blood circulation time. After reaching tumor tissue via the EPR effect, ethylpropylaminoethyl methacrylate is protonated, causing nanoparticle dissociation and exposing the clickable covalent drug. This in-situ click reaction enhances the retention of the clickable covalent drug in tumor tissue. Once the nanoparticles are taken up by tumor cells, N,N-diisopropylaminoethyl methacrylate is further protonated, releasing nucleic acids or chemotherapeutic drugs, ultimately achieving regionally selective drug delivery.

[0056] The present invention relates to the use of extracellular and intracellular acid-responsive nanoparticles of click-loaded covalent drugs and / or nucleic acid drugs, chemotherapeutic drugs, protein degrading agents, etc., in the treatment of cancer, the cancer types being mainly breast cancer, skin cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer, or prostate cancer, etc.

[0057] The beneficial effects of this invention include:

[0058] 1) This invention utilizes click chemistry and amphiphilic block polymers that respond to tumor extracellular and intracellular acids to precisely achieve selective delivery to tumor regions; extracellular acid-activated delivery of click-type covalent drugs, which covalently inhibit tumor target proteins by clicking with azide-labeled tumor cells, thereby improving the efficacy of small molecules, enhancing retention in tumors, improving therapeutic effects, and reducing toxic side effects; intracellular acid-activated delivery of nucleic acid drugs, chemotherapeutic drugs, or protein degrading agents, synergistically regulating intracellular and extracellular targets of tumors.

[0059] 2) The key technological innovation of this invention lies in the design of click-type covalent drugs through click chemistry, which can covalently inhibit azide-labeled tumor cells without affecting normal cells without azide labeling, thus reducing toxic side effects on normal tissues.

[0060] 3) The innovation and uniqueness of this invention lies in the use of intracellular and extracellular acid-activated nanoparticles to precisely and selectively deliver drugs to intracellular and extracellular regions in response to the tumor acidic environment, and to systematically regulate the targets inside and outside the tumor, making this invention broad-spectrum and applicable to a variety of solid tumor treatment scenarios. Attached Figure Description

[0061] Figure 1 The 1H NMR spectrum of BMS-1 ​​ethylene glycol derivative (BnD, n = 1, 3, 5), which is a click-type PD-L1 degrader in Example 1 of the present invention;

[0062] Figure 2 This is a graph showing the PD-L1 inhibition efficiency of the click-type PD-L1 degrader prepared in Example 16 of this invention at the in vitro cellular level;

[0063] Figure 3 The graphs show the changes in particle size and morphology, fluorescence intensity, and release curves of the click-type PD-L1 degrader (D5B) of the nanoparticles prepared in Example 17 of this invention under different acidic conditions.

[0064] Figure 4 This is a graph showing the in vitro acid-activated specific inhibition of PD-L1 by the nanoparticles prepared in Example 18 of this invention.

[0065] Figure 5This is a distribution diagram of the in vivo extracellular acid-activated nanoparticle-specific delivery click-type PD-L1 degrader in Example 19 of the present invention.

[0066] Figure 6 This is a data graph illustrating the selective degradation of tumor PD-L1 achieved by acid-activated nanoparticles specifically delivering click-type PD-L1 degrading agents to tumor extracellular cells, as shown in Example 20 of this invention.

[0067] Figure 7 The graph shows the inhibition curve of 4T1 tumor growth by the click-type PD-L1 degrading agent nanoparticles prepared in Example 21 of this invention.

[0068] Figure 8 The graph shows the inhibition curve of B16-F10 tumors by the clickable PD-L1 degrading agent nanoparticles prepared in Example 22 of this invention. Detailed Implementation

[0069] The present invention will be described with reference to the following specific embodiments and accompanying drawings, but the present invention is not limited to these specific embodiments.

[0070] The methoxy-terminated polyethylene glycol 5000, 4-cyano-4-[(dodecylthioalkylthiocarbonyl)thioalkyl]valeric acid, 2-(hexamethyleneimino)ethyl methacrylate, hydroxyethyl methacrylate, ethylene glycol, triethylene glycol, and pentaethylene glycol used in the examples were purchased from Sigma-Aldrich (China) Co., Ltd. Unless otherwise specified, all other reagents and solvents used were purchased from Sinopharm Group (Shanghai) Chemical Reagent Co., Ltd.

[0071] The cells used in the examples were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, the DMEM culture medium for cell culture was purchased from Dalian Meilun Company, and the fetal bovine serum was purchased from Gibco, product number 11965118.

[0072] Sample data were determined using the following instruments: 1H NMR spectroscopy (NMR spectroscopy). 1 H NMR was performed using a BRUKER AVANCE NEO 500 NMR spectrometer, with TMS as an internal standard. Chemical shift units are expressed in ppm. -1 ;

[0073] The hydrodynamic particle size and surface potential of the nanoparticles were determined by a MALVERN NANO SIZER particle size analyzer, and the transmission electron microscopy images were obtained by a Talos L120C transmission electron microscope.

[0074] Unless otherwise specified, all equipment and testing methods used in this application are conventional in the field.

[0075] To address the challenges of low efficacy and toxic side effects caused by the distribution of small molecule immune checkpoint inhibitors in normal tissues during tumor immune checkpoint therapy, this invention utilizes click chemistry and tumor extracellular acid-activated amphiphilic polymers to design and develop a clickable immune checkpoint inhibitor, which is then co-assembled into nanoparticles. These nanoparticles specifically dissociate upon tumor extracellular acid remission, releasing the clickable immune checkpoint inhibitor, which covalently binds to azide-labeled tumor cells. This achieves specific degradation of PD-L1 in tumor tissue in vivo, without affecting PD-L1 in normal tissues, thus improving tumor treatment efficacy and reducing toxic side effects.

[0076] Example 1: Preparation of click-type covalent PD-L1 inhibitor DnB

[0077] In short, BMS-1 ​​(1.0 mmol), 4-dimethylaminopyridine (DMAP) (2.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.0 mmol), and N,N-diisopropylethylamine (DIEA) (2.0 mmol) were dissolved in 50 mL of anhydrous DCM, stirred, and reacted in an ice bath for 1.5 h. Then, ethylene glycol (2.0 mmol), triethylene glycol (2.0 mmol), or pentaethylene glycol (2.0 mmol) previously dispersed in 10 mL of anhydrous DCM was slowly added dropwise, and the reaction was continued for 24 h. After the reaction was completed, the product was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent to obtain a colorless viscous liquid.

[0078] DBCO carboxylic acid (0.5 mmol), 4-dimethylaminopyridine (DMAP) (1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0 mol), and N,N-diisopropylethylamine (DIEA) (1.0 mmol) were dissolved in 40 mL of anhydrous DCM and stirred for 1.5 h. Then, B-OEG1 (0.3 mmol), B-OEG3 (0.3 mmol), and B-OEG5 (0.3 mol), which had been previously dissolved in 10 mL of anhydrous DCM, were slowly added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic layer was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 60-1 / 20, v / v) as eluent to give a colorless solid. Using deuterated chloroform as a solvent, the clickable covalent PD-L1 inhibitor DnB (n=1,3,5) was confirmed by 1H NMR spectroscopy. (See [link to relevant documentation]). Figure 1 .

[0079] Example 2: Preparation of a click-type covalent estrogen receptor inhibitor

[0080]

[0081] DBCO carboxylic acid (1.0 mmol), 4-dimethylaminopyridine (DMAP) (2.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.0 mmol), and N,N-diisopropylethylamine (DIEA) (2.0 mmol) were dissolved in 50 mL of anhydrous dichloromethane and stirred. The mixture was reacted in an ice bath for 1.5 h. Then, ethylene glycol (OEG1) (2.0 mmol), triethylene glycol (OEG3) (2.0 mmol), or pentaethylene glycol (OEG5) (2.0 mmol), which had been previously dispersed in 10 mL of anhydrous DCM, was slowly added dropwise, and the reaction was continued for 24 h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent to obtain DBCO ethylene glycol derivatives (DBCO-OEG1 / DBCO-OEG3 / DBCO-OEG5).

[0082] Estrogen receptor (0.5 mmol), 4-dimethylaminopyridine (DMAP) (1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0 mol), and N,N-diisopropylethylamine (DIEA) (1.0 mmol) were dissolved in 40 mL of anhydrous dichloromethane and stirred for 1.5 h. Then, DBCO-OEG1 (0.3 mmol), DBCO-OEG3 (0.3 mmol), and DBCO-OEG5 (0.3 mol), which had been previously dissolved in 10 mL of anhydrous dichloromethane, were slowly added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic layer was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent to obtain a click-type covalent estrogen receptor inhibitor.

[0083] Example 3: Preparation of a click-type covalent prostate-specific membrane antigen inhibitor

[0084]

[0085] First, DBCO ethylene glycol derivatives (DBCO-OEG1 / DBCO-OEG3 / DBCO-OEG5) were prepared as shown in Example 2. Then, a prostate-specific membrane antigen inhibitor (0.5 mmol), 4-dimethylaminopyridine (DMAP) (1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0 mol), and N,N-diisopropylethylamine (DIEA) (1.0 mmol) were dissolved in 40 mL of anhydrous dichloromethane and stirred for 1.5 h. Then, DBCO-OEG1 (0.3 mmol), DBCO-OEG3 (0.3 mmol), and DBCO-OEG5 (0.3 mol), which had been previously dissolved in 10 mL of anhydrous dichloromethane, were slowly added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was complete, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic layer was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent to obtain a click-type covalent prostate-specific membrane antigen inhibitor.

[0086] Example 4: Preparation of glutathione-responsive click-type covalent camptothecin

[0087]

[0088] DBCO carboxylic acid (0.5 mmol), 4-dimethylaminopyridine (DMAP) (1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0 mol), and N,N-diisopropylethylamine (DIEA) (1.0 mmol) were dissolved in 10 mL of anhydrous DCM and stirred for 1.5 h. Then, a disulfide bond (0.6 mmol) previously dissolved in 10 mL of anhydrous DCM was slowly added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was complete, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic layer was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 60–1 / 20, v / v) as eluent to give the DBCO disulfide derivative (DBCO-SS-OH). Then, camptothecin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultra-dry dichloromethane and reacted for 0.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dichloromethane and added dropwise to the above solution, and reacted for 24 h. After the reaction was completed, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent to obtain a pale yellow solid, which was glutathione-activated click-type covalent paclitaxel.

[0089] Example 5: Preparation of glutathione-responsive click-type covalent oxaliplatin

[0090]

[0091] First, a DBCO disulfide derivative (DBCO-SS-OH) was prepared as described in Example 4. Then, oxaliplatin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultradry dimethyl sulfoxide and reacted for 0.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultradry dimethyl sulfoxide and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent, yielding a white solid, which was glutathione-activated click-type covalent oxaliplatin.

[0092] Example 6: Preparation of glutathione-responsive click-type covalent doxorubicin

[0093]

[0094] First, a DBCO disulfide derivative (DBCO-SS-OH) was prepared as described in Example 4. Then, oxaliplatin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultradry dimethyl sulfoxide and reacted for 0.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultradry dimethyl sulfoxide and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent, yielding a red solid, which was the glutathione-activated click-type covalent doxorubicin.

[0095] Example 7: Preparation of reactive oxygen species-responsive click-type covalent camptothecin

[0096]

[0097] DBCO carboxylic acid (0.5 mmol), 4-dimethylaminopyridine (DMAP) (1.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0 mol), and N,N-diisopropylethylamine (DIEA) (1.0 mmol) were dissolved in 10 mL of anhydrous DCM and stirred for 1.5 h. Then, a disulfide bond (0.6 mmol) previously dissolved in 10 mL of anhydrous DCM was slowly added dropwise, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution and NaCl aqueous solution, respectively. The organic layer was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 60-1 / 20, v / v) as eluent to give the DBCO thioacetate derivative (DBCO-TK-OH). Then, camptothecin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultra-dry dichloromethane and reacted for 0.5 h. The resulting product, DBCO-TK-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dichloromethane and added dropwise to the above solution, and reacted for 24 h. After the reaction was completed, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent, yielding a pale yellow solid, which was the reactive oxygen species-activated click-type covalent camptothecin.

[0098] Example 8: Preparation of reactive oxygen species-responsive clickable covalent oxaliplatin

[0099]

[0100] First, a DBCO thioclase derivative (DBCO-TK-OH) was prepared as described in Example 7. Then, oxaliplatin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultra-dry dimethyl sulfoxide and reacted for 0.5 h. The resulting product, DBCO-TK-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dimethyl sulfoxide and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a pale yellow solid, which was the reactive oxygen species-activated click-type covalent paclitaxel.

[0101] Example 9: Preparation of reactive oxygen species-responsive click-type covalent doxorubicin

[0102]

[0103] First, a DBCO thioclase derivative (DBCO-TK-OH) was prepared as described in Example 7. Then, doxorubicin (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultra-dry dimethyl sulfoxide and reacted for 0.5 h. The resulting product, DBCO-TK-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dimethyl sulfoxide and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a red solid, which was the reactive oxygen species-activated click-type covalent doxorubicin.

[0104] Example 10: Preparation of a glutathione-responsive click-type covalent Toll-like 7 / 8 receptor agonist

[0105]

[0106] First, a DBCO disulfide derivative (DBCO-SS-OH) was prepared as described in Example 4. Then, Toll-like 7 / 8 receptor agonist R848 (0.3 mmol), triphosgene (0.1 mmol), and triethylamine (TEA) (0.3 mmol) were dissolved in 20 mL of ultra-dry dichloromethane and reacted for 0.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dichloromethane and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a white solid, which was the glutathione-activated click-type covalent Toll-like 7 / 8 receptor agonist.

[0107] Example 11: Preparation of a glutathione-responsive click-type covalent STING agonist

[0108]

[0109] First, a DBCO disulfide derivative (DBCO-SS-OH) was prepared as described in Example 4. Then, the STING agonist MSA-2 (0.3 mmol), 4-dimethylaminopyridine (DMAP) (0.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.6 mol), and N,N-diisopropylethylamine (DIEA) (0.6 mmol) were dissolved in 20 mL of ultra-dry dichloromethane and stirred for 1.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dichloromethane and added dropwise to the above solution, reacting for 24 h. After the reaction was complete, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a white solid, which was the glutathione-activated click-type covalent STING agonist.

[0110] Example 12: Preparation of reactive oxygen species-responsive click-type covalent Toll-like 7 / 8 receptor agonists

[0111]

[0112] First, a DBCO thioclase derivative (DBCO-TK-OH) was prepared as described in Example 7. Then, 0.3 mmol of the Toll-like 7 / 8 receptor agonist R848, 0.1 mmol of triphosgene, and 0.3 mmol of triethylamine (TEA) were dissolved in 20 mL of ultra-dry dimethyl sulfoxide and reacted for 0.5 h. The resulting product, DBCO-TK-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dimethyl sulfoxide and added dropwise to the above solution, reacting for 24 h. After the reaction, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a white solid, which was the reactive oxygen species-activated click-type covalent Toll-like 7 / 8 receptor agonist.

[0113] Example 13: Preparation of reactive oxygen species-responsive click-type covalent STING inhibitor

[0114]

[0115] First, a DBCO thioclase derivative (DBCO-TK-OH) was prepared as described in Example 7. Then, 0.3 mmol of the STING agonist MSA-2, 0.6 mmol of 4-dimethylaminopyridine (DMAP), 0.6 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 0.6 mmol of N,N-diisopropylethylamine (DIEA) were dissolved in 20 mL of ultra-dry dichloromethane and stirred for 1.5 h. The resulting product, DBCO-SS-OH (0.3 mmol), was dissolved in 10 mL of ultra-dry dichloromethane and added dropwise to the above solution, reacting for 24 h. After the reaction was complete, the product was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent, yielding a white solid, which was the reactive oxygen species-activated click-type covalent STING agonist.

[0116] Example 14: mPEG 113 Synthesis of -bp(EPA-b-DPA-r-PPaGd)

[0117]

[0118] The first step involves capping the RAFT chain transfer agent with the polyethylene glycol macromolecular initiator mPEG. 113 CTA (100 mg, 0.019 mmol) was dissolved in 2.0 mL of N,N-dimethylformamide with ethyl propylaminoethyl methacrylate (EPA) (302.5 mg, 1.52 mmol) and azobisisobutyronitrile (AIBN) (0.31 mg, 0.0019 mmol). The reaction was carried out under anaerobic conditions at 70 °C for 24 h. After the reaction was completed, the mixture was dialyzed with deionized water in a dialysis bag with a cutoff of 3500 Daltons. The solution was then freeze-dried to obtain a white flocculent substance, mPEG. 113 -b-PEPA 50 The second step is to add mPEG. 113 -bp(EPA 50 2-(diisopropylamino)methacrylic acid (DPA) (225.1 mg, 1.12 mmol), hydroxyethyl methacrylate (HEMA) (18.2 mg, 0.14 mmol), and azobisisobutyronitrile (AIBN) (0.23 mg, 0.0014 mmol) were dissolved in 2.0 mL of N,N-dimethylformamide. The reaction was carried out under anaerobic conditions at 70 °C for 24 h. After the reaction, the mixture was dialyzed against deionized water using a dialysis bag with a cutoff of 3500 Daltons. The solution was then freeze-dried to obtain a white flocculent substance, mPEG. 113-bp(EPA 50 -b-DPA 50 -r-HEMA5); Step 3: Dissolve gadolinium-coordinated pyromethesin a (PPaGd) (0.0008 mmol), N,N-diisopropylethylamine (DIEA) (0.0016 mmol), 4-dimethylaminopyridine (DMAP) (0.0016 mmol), and 1-hydroxybenzotriazole (HOBt) (0.0016 mmol) in 3 mL of N,N-dimethylformamide and react for 1.5 h; After the reaction is complete, under ice bath conditions, add 2 mL of mPEG 113 -bp(EPA 50 -b-DPA 50 A solution of N,N-dimethylformamide (100 mg, 0.0004 mmol) was added dropwise to the above solution, and the reaction was carried out at room temperature for 24 h. After the reaction was completed, the reaction solution was dialyzed against N,N-dimethylformamide and deionized water (dialysis bag molecular weight cutoff of 3500 D). The resulting purplish-black solid after lyophilization was mPEG. 113 -bp(EPA-b-DPA-r-PPaGd).

[0119] Example 15: Preparation of regioselective nanoparticles for delivering nucleic acids and clickable PD-L1 covalent inhibitors

[0120] The nucleic acid drug from the normal drug of claim 8 and the extracellular / intracellular acid-responsive amphiphilic block polymer of formula 2 are simultaneously dissolved in an organic solvent to obtain a solution containing the drug and the extracellular acid-responsive amphiphilic block polymer. This mixed solution is then added dropwise to a PBS buffer with pH > 6.5 under ultrasonic conditions, wherein the volume ratio of the organic solvent is less than 10%. The organic solvent or unencapsulated drug molecules are removed by ultrafiltration or dialysis to obtain a nanoparticle suspension encapsulated with the nucleic acid drug. Next, the click-type PD-L1 degrading agent of formula 1 is dissolved in an organic solvent, and this solution is added dropwise to the above nanoparticle suspension under ultrasonic conditions. Simultaneously, the pH of the suspension is adjusted to 7.4, and the volume ratio of the agent to the PBS buffer is 1:9. The organic solvent or unencapsulated click-type covalent drug is then removed by ultrafiltration or dialysis, finally obtaining nanoparticles encapsulated with both the nucleic acid drug and the click-type PD-L1 degrading agent, wherein the nucleic acid drug accounts for 5%-10% of the total mass, and the click-type PD-L1 degrading agent accounts for 10%-20%.

[0121] Example 16: DnB degrades PD-L1 on tumor cell membranes in vitro

[0122] First, 4T1 tumor cells or B16-F10 tumor cells were seeded in 6-well plates at a density of 5 x 10⁴ cells / well, and 2 mL of culture medium was added. The plates were then incubated at 37°C with 5% CO₂. 2The cells were cultured in medium for 12 hours. Then, the medium was replaced with one containing 12.5 ng / mL IFN-γ and 25 μM c4ManNAz, and incubated for 3 days. Next, different concentrations of BMS-1, D1B, D3B, and D5B (equivalent concentrations of BMS-1, 0, 1.25, 2.5, 5.0, and 10.0 μM) were added and incubated for 24 hours. Tumor cells were then collected for flow cytometry analysis. (See [link to relevant documentation]). Figure 2 The PD-L1 inhibition rate is calculated using the following formula:

[0123] Inhibition rate%=(MFI,PBS-MFI,treatment) / MFI,PBS(MFI,MedianFluorescent Intensity).

[0124] Example 17: Extracellular acid environment activates the release of click-type PD-L1 degrader (D5B) drug.

[0125] Extracellular acid-responsive PCPD5B nanoparticles were prepared using an ultrasonic method for the microacid-activated degradation of PD-L1 in tumors. Under ultrasonic conditions (80 W, 3 min), 90 μL of DMSO solution of PCP (50 mg / mL) and 10 μL of DMSO solution of L5B (50 mg / mL) were mixed thoroughly and then added dropwise to 1 mL of deionized water. The organic solvent was then removed by dialysis (MWCO 3.5 kDa), followed by ultrafiltration to concentrate 500 μL of the solution to obtain PCPD5B nanoparticles at a concentration of 10 mg / mL. Using a similar method, intracellular acid-responsive PDPD5B nanoparticles and acid-insensitive PDPD5B nanoparticles were prepared, both at a concentration of 10 mg / mL.

[0126] Then, the release of D5B was triggered in vitro by simulating the microacidic environment of a tumor. The specific experimental steps were as follows: 1.0 mL of PBPD5B, PCPD5B, and PDPD5B (5.0 mg / mL, of which the D5B concentration was 0.5 mg / mL) from a dialysis bag (MWCO 3.5 kDa) were immersed in 10 mL of release medium (pH 7.4 and pH 6.5 PBS buffer, containing 0.5% Tween 80), respectively, and placed on a constant temperature shaker at 37°C. At a predetermined time point, 100 μL of release medium was taken and the volume was made up by adding an equal volume. 10 μL of PBPD5B, PCPD5B, and PDPD5B were collected by ultrafiltration, and the remaining sample was immersed in the release medium again. Then, the D5B concentration was determined by HPLC and the cumulative percentage of D5B release was calculated, see [link to HPLC]. Figure 3 .

[0127] Example 18: Degradation of PD-L1 by a tumor microacid-activated click-type PD-L1 degrader (D5B)

[0128] First, 4T1 tumor cells were labeled with azide. Then, PCPD5B nanoparticles were co-incubated with 4T1 tumor cells for 24 h at pH 7.4 and 6.5, respectively. Subsequently, tumor cells were collected, and PD-L1 on the tumor cell membrane was detected by flow cytometry. Figure 4 .

[0129] Example 19: Extracellular acid-activated nanoparticle-specific delivery of click-type PD-L1 degrader (D5B)

[0130] First, by using 1×10 6 4T1 cells were subcutaneously injected into the right mammary gland of mice to construct a 4T1 tumor model. The tumor volume was increased to approximately 100 mm². 3 Mice were injected intravenously with 100 μL of PBPD5B, PCPD5B, and PDPD5B nanoparticles, with PPa and D5B doses both at 2.0 mg / kg. Mice were anesthetized at predetermined time points, and imaging was performed using a small animal in vivo imaging system with excitation wavelength of 640 nm and emission wavelength of 680 nm. Tumor tissue was then collected and weighed. The tumor fragments were then cut into small pieces and placed in 1.5 mL centrifuge tubes, 500 μL of methanol was added, and the tissues were ground to extract D5B into the organic phase. The supernatant was collected by centrifugation, and the D5B content in the tumor tissue at each time point was determined by HPLC. The mobile phase was methanol, and the flow rate was 0.5 mL / min. Figure 5 .

[0131] Example 20: Tumor microacid-activated click-type PD-L1 degrader (D5B) specifically degrades PD-L1 in 4T1 breast cancer in vivo.

[0132] First, by using 1×10 6 4T1 cells were subcutaneously injected into the right mammary gland of mice to construct a 4T1 tumor model. The tumor volume was increased to approximately 50 mm². 3 Then, 25 μL of Ac4ManNAz solution (25 mM) was injected intratumorally for 3 consecutive days. On day 4, 100 μL of PBPD5B, PCPD5B, and PDPD5B nanoparticles were injected via tail vein, with D5B at a dose of 2.0 mg / kg. After two treatments following the above procedure, mice were euthanized, and heart, liver, spleen, lung, kidney, and tumor tissues were collected. These tissues were fixed with 4% paraformaldehyde, and immunohistochemical staining was performed on the major organs and tumor PD-L1. The results showed that the abundance of PD-L1 in normal organs of mice treated with PCPD5B nanoparticles was inhibited, as was the abundance in the PBS group. This indicates that the tumor microacid-activated nanodelivery system achieves selective tumor PD-L1 degradation through tumor-specific extracellular delivery of click-type PD-L1 degrading agents, without affecting normal tissues. (See [link to article]). Figure 6 .

[0133] Example 21: Tumor microacid extracellular specific extracellular delivery click-type PD-L1 degrader inhibits 4T1 tumor growth

[0134] 1×10 6 4T1 was injected subcutaneously into the mammary pads of BALB / c mice (6–8 weeks old). When the tumor volume reached 50 mm, 3 For three consecutive days, 25 μL of Ac4ManNAz solution (25 mM) was injected intratumorally. Subsequently, 4T1 tumor-bearing mice were randomly divided into 5 groups (n = 6 or 7): (1) Ac4ManNAz + PBS, (2) Ac4ManNAz + D5B, (3) Ac4ManNAz + PBPD5B, (4) Ac4ManNAz + PDPD5B, and (5) Ac4ManNAz + PCPD5B. On day 4, 100 μL of PBS, D5B, PBPD5B, PDPD5B, and PCPD5B were injected via the tail vein, with D5B at a dose of 5 mg / kg. The above treatment regimen was administered for two courses. During this period, tumor size was measured with calipers, and body weight was recorded. Tumor volume was calculated using the following formula:

[0135] V = L × W × W / 2, (L, length of the long side of the tumor; W, length of the short side of the tumor).

[0136] like Figure 7 As shown, treatment with PCPD5B inhibited tumor growth, while other groups did not. The tumor inhibition experiment demonstrates that extracellular delivery of a click-type PD-L1 degrader specifically targeting the tumor microenvironment utilizes an in-situ click reaction to enhance drug accumulation in tumor tissue and increase the potency of the click-type PD-L1 degrader, thereby significantly inhibiting tumor growth.

[0137] Example 22: Tumor microacid extracellular specific extracellular delivery click-type PD-L1 degrader inhibits B16-F10 tumor growth

[0138] 1×10 6 B16-F10 was injected subcutaneously into C57 mice (6-8 weeks old), and the tumor volume reached 50 mm. 3For three consecutive days, 25 μL of Ac4ManNAz solution (25 mM) was injected intratumorally. Subsequently, 4T1 tumor-bearing mice were randomly divided into three groups (n = 6 or 7): (1) Ac4ManNAz + PBS, (2) Ac4ManNAz + PCPD5B, and (3) PD-L1 antibody. On the fourth day, 100 μL of PBS and PCPD5B were injected intravenously via the tail vein, and PD-L1 antibody was injected intraperitoneally. The dose of D5B was 5 mg / kg, and the dose of antibody was 100 μg / mouse. The above treatment regimen was administered for two courses. During the treatment, the tumor size was measured with calipers, and the body weight was recorded. The tumor volume was calculated according to the following formula: V = L × W × W / 2, (L, length of the long side of the tumor; W, length of the short side of the tumor). Figure 8 As shown, treatment with PCPD5B can inhibit tumor growth, and its tumor-suppressing effect is better than that of PD-L1 antibody.

Claims

1. A class of click-type covalent drugs, characterized in that, The click-type covalent drug can form a covalent bond with the azide-labeled target protein through a click reaction, and has the structure shown in Formula 1: ； Formula 1 in, R1 is a PD-L1 inhibitor with the following structure: ； The linker is the connecting chain between R1 and the carbonyl group, and its specific structure is as follows. ； Where n is any integer from 1 to 20.

2. A method for preparing the click-type covalent drug of claim 1, characterized by, This method specifically includes the following steps: synthesizing the click-type covalent drug via esterification reaction as shown in reaction route I: ； Reaction route I Linker, R1 is as described in claim 1; Step a: Synthesis of the azacyclic alkyne linker derivative, namely DBCO-Linker Carboxylated azacyclic alkyne, Linker, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N,N-diisopropylethylamine were dissolved in dichloromethane in a molar ratio of 1:2:2:2:2 to obtain a solution with a mass concentration of 20-40% w / v. The reaction was carried out at room temperature for 24 h under anhydrous conditions. The reaction product was purified by thin-layer chromatography to obtain the azacyclic alkyne Linker derivative, namely DBCO-Linker. ； Reaction route a Step b: Synthesis of click-type covalent drugs R1, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N,N-diisopropylethylamine were dissolved in anhydrous dichloromethane in a molar ratio of 1:2:2:2 to prepare a 40% w / v solution B, and the reaction was carried out for 1.5 h. The azacyclic yne Linker derivative obtained in step a was dissolved in anhydrous dichloromethane to obtain a 20-40% w / v solution A. Solution A was added dropwise to solution B, and the reaction was continued for 24 h. The solution was then purified by thin-layer chromatography to obtain the click-type covalent drug, the compound shown in Formula 1. ； Reaction route b Wherein, R1 is as described in claim 1.

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