Click type covalent drug and regioselective delivery system and application thereof

Abstract

The invention discloses a click type covalent drug as well as a regioselective delivery system and application thereof. The click-type covalent drug delivery system comprises a click-type covalent drug and a tumor region selective delivery system, and the click-type covalent drug is composed of aza-cyclic alkyne (DBCO), a small molecule drug and a linker and has a structure as shown in a formula 1. Optionally, the small molecule drugs are membrane protein inhibitors, immunomodulators, chemotherapeutic drugs and the like. The click-type covalent drug and the azide-labeled target cell are subjected to a click reaction to form a covalent bond to be combined on a cell membrane, so that the local drug concentration is improved, the drug retention time is prolonged, and the selective inhibition on the target cell is enhanced; the regioselective delivery system is formed by co-assembling an amphiphilic block polymer and a click type covalent drug, and is used for regioselective delivery. By means of the click type covalent drug, covalent binding of azide-labeled tumor cells is achieved, normal cells which are not labeled by azide are not affected, and toxic and side effects on normal tissue are reduced. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical industry, and specifically relates to click-type covalent drugs, tumor extracellular and intracellular acid-responsive amphiphilic polymers and regional selective delivery systems, preparation methods and applications thereof. Background Art

[0002] Cancer immunotherapy drugs activate the patient's own immune system to attack tumor cells, thereby eliminating the tumor. By intervening at different stages in the tumor immune cycle, a variety of treatment strategies have been used to clinically treat cancer patients, such as therapeutic vaccines, cell therapy, and immune checkpoint therapy. However, on the one hand, tumors evade the recognition of the body's immune system by reducing immunogenicity and upregulating negative immune regulators (such as PD-L1, IDO, etc.). On the other hand, tumors form a "physical barrier" through a dense extracellular matrix composed of tumor-associated fibroblasts, related macrophages, and collagen, which limits the infiltration of tumor-specific T cells. The two synergistically lead to tumor immune tolerance and a low clinical single treatment response rate. In addition, the existing tumor immunotherapy strategies are difficult to achieve selective regulation of the internal and external regions of tumor cells, which is another important reason for the low clinical treatment response rate. Therefore, the key clinical challenge of regional selective regulation of tumor intracellular and extracellular targets, while overcoming their "biological" and "physical" barriers, promoting tumor-specific T cell infiltration, and improving the response rate of treatment strategies, is of great clinical significance. At present, non-small molecule anti-tumor drugs have poor water solubility, poor pharmacokinetic properties, low affinity, and lack of targeting. Compared with non-covalent inhibitors, small molecule covalent inhibitors have the advantages of improving biochemical efficiency and efficacy, prolonging the duration of action, prolonging drug pharmacokinetics, reducing dosage and administration frequency. Compared with traditional non-covalent inhibitors, non-covalent inhibitors rely on hydrophobic interactions, hydrogen bonds and electrostatic interactions to reversibly bind to target proteins. The strength of these forces is weaker than covalent chemical bonds, so the efficacy of covalent inhibitors is often stronger than that of non-covalent inhibitors. However, compared with the easier to develop small molecule covalent inhibitors of kinases, small molecule covalent inhibitors that directly target tumor cells have not been reported so far. The traditional covalent strategy of small molecule covalent inhibitors targeting natural amino acid residues (cysteine, serine, tyrosine, lysine, arginine and glutamate) may cause strong toxic side effects. In addition, the ubiquitous presence of amino acids in proteins, cells and in vivo poses a huge challenge to achieving reactive selectivity to protein targets. Therefore, the key to developing covalent small molecule drugs is to strike an appropriate balance between reactivity and selectivity. To enable the generation of small-molecule covalent drugs for in vivo tumor targeting, a new strategy is needed to design small-molecule covalent inhibitors with chemical reactivity that is biocompatible and highly specific for the target. Summary of the invention

[0003] Based on the above background, the purpose of the present invention is to design and develop a regional selective delivery system using click chemistry and amphiphilic block polymers activated by intracellular and extracellular acids of tumors. The delivery system is composed of clickable covalent drugs and amphiphilic block polymers that respond to intracellular and extracellular acids of tumors to form nanoparticles; the nanoparticles sequentially activate and release the loaded clickable covalent drugs and other combined therapeutic drugs in the extracellular and intracellular acid environments of tumors. First, the clickable drugs are released in the slightly acidic environment of tumor extracellular cells, and covalently bind to tumor cells labeled with azide in advance, thereby achieving specific inhibition of tumor extracellular targets in vivo; after the nanoparticles are taken up by cells, the intracellular acid activates the release of drugs and regulates the intracellular targets; the two work together to achieve regional selective regulation of tumors.

[0004] The specific technical solution for achieving the purpose of the present invention is:

[0005] A type of click-type covalent drug, which can undergo a click reaction with an azide-labeled target protein to form a covalent bond, has a structure shown in the following formula 1:

[0006]

[0007] in,

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

[0009]

[0010] Linker is a link between R1 and the carbonyl group, and is selected from any one of the following structures:

[0011]

[0012] Wherein, 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, the method specifically comprises the following steps, synthesizing the click-type covalent drug through an esterification reaction shown in the following reaction scheme I:

[0014]

[0015]

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

[0017] Step a: Synthesis of azacyclic alkyne linker derivative, i.e. DBCO-linker

[0018] Dissolve carboxylic azacyclic alkyne, linker, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N,N-diisopropylethylamine in dichloromethane at a molar ratio of 1:2:2:2:2 to obtain a solution with a mass concentration of 20-40% w / v; react at room temperature for 24 hours under anhydrous conditions, and purify the reaction product by thin layer chromatography to obtain an 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 are dissolved in anhydrous dichloromethane or dimethyl sulfoxide at a molar ratio of 1:2:2:2 or R1, triethylamine and triphosgene are dissolved in anhydrous dichloromethane or dimethyl sulfoxide at a molar ratio of 1:1.5:0.3 to prepare a 40% w / v solution B, and the reaction is carried out for 1.5 hours; the azacyclic alkyne linker derivative obtained in step a is dissolved in anhydrous dichloromethane to obtain a solution A with a mass concentration of 20-40% w / v; solution A is added dropwise to solution B, the reaction is continued for 24 hours, and then purified by thin layer chromatography to obtain a click-type covalent drug, a compound shown in formula 1;

[0022]

[0023] wherein R1 is as described in claim 1.

[0024] An amphiphilic block polymer that responds to both extracellular and intracellular acid environments, wherein the polymer is a linear amphiphilic block polymer, has tumor extracellular and intracellular acid-activated nuclear magnetic resonance signal-guided imaging characteristics, and has a structure shown in the following 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-dipentylamino, N,N-dihexylamino, pentamethyleneamino, hexamethyleneamino or heptamethyleneamino, preferably N,N-diisopropylamino; R3 is N,N-diethylamino, N,N-ethylpropylamino, pentamethyleneamino or hexamethyleneamino; preferably N,N-ethylpropylamino;

[0028] M is Gd 3+ , Mn 2+ 、Co 2+, Fe 3+ , Cu 2+ or Ni 2+ , preferably Gd 3+ ;

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

[0030] x is an integer of 20-100, preferably an integer of 40-50;

[0031] y is an integer of 20-100, preferably an integer of 40-50;

[0032] z is an integer of 0-20, preferably an integer of 5-10.

[0033] A method for preparing the amphiphilic block polymer is to synthesize the amphiphilic block polymer by reversible addition-fragmentation chain transfer polymerization and esterification grafting as shown in the following reaction scheme II:

[0034]

[0035] Wherein, n, x, y, z, and M are defined as in claim 2;

[0036] Step a: Synthesis of polyethylene glycol-b-poly R3

[0037] The RAFT chain transfer agent-terminated polyethylene glycol macroinitiator, ethyl methacrylate-modified R3 and initiator azobisisobutyronitrile are dissolved in 1-5 ml of N,N-dimethylformamide at a molar ratio of 1:50-80:0.1; the reaction is carried out at 70°C for 24 h under anaerobic conditions, the reaction product is diluted with 1-10 ml of DMF and dialyzed with deionized water, and the dialysis bag with a cutoff of 3500 Daltons is freeze-dried 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, R2 modified with ethyl methacrylate, hydroxyethyl methacrylate monomer and initiator azobisisobutyronitrile are dissolved in 1-5 ml of N,N-dimethylformamide at a molar ratio of 1:50-80:10-20:0.1; react at 70°C for 24 h under anaerobic conditions, and the reaction product is diluted with 1-10 ml of DMF and dialyzed with deionized water, and 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: dissolving the amphiphilic block copolymer obtained in step b in anhydrous N,N-dimethylformamide to obtain a solution C with a mass concentration of 10% w / v, dissolving metal ion coordinated pyropheophorbide (MPPa), N,N-diisopropylethylamine, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a molar ratio of 1:1.5:1.5:1.5 in N,N-dimethylformamide to obtain a 30% w / v solution D, and reacting for 1.5 hours; adding solution C dropwise to solution D, continuing the reaction for 24 hours, then dialyzing with N,N-dimethylformamide, and then dialyzing with deionized water, using a dialysis bag with a retention capacity of 3500 Daltons, and freeze-drying to obtain the amphiphilic block polymer shown in Formula 2 by covalent modification of metal ion coordinated pyropheophorbide (MPPa);

[0043]

[0044] A regional selective delivery system, wherein the delivery system is a nanoparticle assembled from the click-type covalent drug and the amphiphilic block polymer; the nanoparticle responds to the extracellular micro-acid and intracellular acid environment of the tumor to achieve regional selective drug delivery; wherein the click-type covalent drug accounts for 5%-20% of the total mass of the nanoparticle, and the amphiphilic block polymer accounts for 80%-95% of the total mass of the nanoparticle.

[0045] A method for preparing the above-mentioned regional selective delivery system, firstly, the click-type covalent drug represented by formula 1 and the amphiphilic block polymer represented by formula 2 are simultaneously dissolved in an organic solvent at a mass ratio of 1-9 to obtain a mixed solution, the mixed solution is added dropwise into deionized water under ultrasonic conditions, and the volume ratio of the mixed solution to the deionized water is 1:9, and the organic solvent or the unencapsulated click-type covalent drug represented by formula 1 is removed by ultrafiltration or dialysis to obtain a regional selective delivery system encapsulating the click-type covalent drug represented by formula 1.

[0046] Another regional selective delivery system, the delivery system is a nanoparticle assembled from the click-type covalent drug, a normal drug and the amphiphilic block polymer, the normal drug includes an immunomodulator, a chemotherapeutic drug, a nucleic acid drug or a protein degrader; wherein the click-type covalent drug accounts for 5%-20% of the total mass of the nanoparticle, the normal drug accounts for 5%-20% of the total mass of the nanoparticle, and the amphiphilic block polymer accounts for 60%-90% of the total mass of the nanoparticle.

[0047] A method for preparing the above-mentioned regional selective delivery system is prepared by a pH gradient method; first, a normal drug and an 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; the mixed solution is added dropwise to a buffer solution with a pH>6.5 under ultrasonic conditions, and the volume ratio with the buffer solution is 1:9; the organic solvent or unencapsulated normal drug is removed by ultrafiltration or dialysis to obtain a nanoparticle suspension encapsulating the normal drug; secondly, the click polymer shown in Formula 1 is ... nanoparticle suspension encapsulating the normal drug; A click-type covalent drug is dissolved in an organic solvent to obtain a drug solution containing the click-type covalent drug, the mass ratio of which to the nanoparticle suspension is 1-9, and the volume ratio to the nanoparticle suspension with a pH value of >6.5 is 1:9; the drug solution is added dropwise to the nanoparticle suspension with a pH value of >6.5 under ultrasonic conditions, and the pH of the nanoparticle suspension is adjusted to 7.4, and then the organic solvent or unencapsulated click-type covalent drug is removed by ultrafiltration or dialysis, and finally nanoparticles containing the click-type covalent drug, normal drugs and the amphiphilic block polymer, namely a regional selective delivery system, are obtained.

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

[0049] A use of the regional selective delivery system in delivering drugs for treating cancer.

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

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

[0052] The regional selective delivery system has the characteristics of selective delivery inside and outside tumor cells. Based on the difference of the extracellular and intracellular acid environments of tumors, the click-type membrane protein inhibitor is specifically released outside the tumor cells, and the tumor cell membrane protein is specifically degraded; the nuclear magnetic resonance contrast agent is specifically released inside the cells. The regional selective nanoparticles self-assemble into stable nanoparticles under normal physiological conditions (such as pH=7.4), and the hydrodynamic particle size is about 80 nanometers; it has good stability during blood circulation to prevent drug leakage; in the extracellular slightly acidic environment of tumor tissue, the extracellular acid response group in the extracellular acid-activated nanoparticles is protonated, and the click-type covalent drug is released, and a click reaction occurs with the azide pre-labeled tumor cells to form a covalent bond, which enhances the retention in the tumor tissue and improves the efficacy of the click-type covalent drug, and reduces toxic side effects; after the nanoparticles are taken up by the cells, the intracellular acid response group in the intracellular acid-activated nanoparticles is protonated, and the nuclear magnetic resonance contrast agent is activated inside the cells, and finally the regional selective release of the drug is achieved, and the precise treatment of tumors guided by nuclear magnetic resonance imaging is achieved.

[0053] The present invention also provides a tumor synergistic therapeutic drug based on a click-type covalent drug nano-platform activated by the extracellular and intracellular acid environment of the tumor, which simultaneously regulates the intracellular and extracellular targets of the tumor; the nanoparticles activated by the extracellular slightly acidic environment are used to regulate the extracellular targets of the tumor; the nanoparticles activated by the intracellular acidic environment are used to regulate the intracellular targets, thereby achieving precise tumor treatment. Among them, the nanoparticles activated by the extracellular slightly acidic environment release click-type covalent drugs for selective covalent degradation of tumor membrane proteins; the nanoparticles activated by intracellular acid release other drugs to regulate intracellular targets; or any combination of therapeutic agents that meet the purpose of the present invention.

[0054] The regional selective nanoparticles containing the click-type covalent drug of formula 1 and the extracellular and intracellular acid-responsive amphiphilic block polymers of formula 2 are prepared by a pH gradient method and have a multilayer structure, including a polyethylene glycol outer layer and a poly(ethylpropylaminoethyl methacrylate)-b-poly(N,N-diisopropylaminoethyl ester-r-metal coordinated pyropheophorbide a) inner layer nanoparticles. The average hydrodynamic particle size of the nanoparticles is preferably 70-120 nanometers.

[0055] The nanoparticles have the function of responding to the extracellular and intracellular acid environment of tumors, so that the corresponding nanoparticles have the characteristics of selective drug delivery in tumor tissue regions. The polyethylene glycol hydrophilic layer can prevent protein adsorption, keep the nanoparticles stable in the blood circulation, reduce the clearance of the endothelial reticular system, and prolong the blood circulation time. After the nanoparticles reach the tumor tissue through the EPR effect, ethyl methacrylate propylaminoethyl is protonated, the nanoparticles dissociate, and the click-type covalent drugs are exposed. The retention of click-type covalent drugs in tumor tissues is increased through in situ click reactions; after the nanoparticles are taken up by tumor cells, N, N-diisopropylaminoethyl ester is further protonated to release nucleic acids or chemotherapy drugs, and finally achieve regional selective drug delivery.

[0056] According to the use of the extracellular and internal acid-responsive nanoparticles loaded with click-type covalent drugs and / or nucleic acid drugs, chemotherapeutic drugs, protein degraders, etc. provided by the present invention in the treatment of cancer, the cancer types are mainly breast cancer, skin cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer, etc.

[0057] The beneficial effects of the present invention include:

[0058] 1) The present invention utilizes click chemistry and tumor extracellular and intracellular acid-responsive amphiphilic block polymers to precisely achieve tumor-region selective delivery; extracellular acid activates the delivery of click-type covalent drugs, which covalently inhibit tumor target proteins through click reactions with azide-labeled tumor cells, thereby increasing the efficacy of small molecules, enhancing tumor retention, improving therapeutic effects, and reducing toxic side effects; intracellular acid activates the delivery of nucleic acid drugs, chemotherapeutic drugs or protein degraders, and synergistically regulates tumor intracellular and extracellular targets.

[0059] 2) The key technological innovation of the present invention lies in the design of click-type covalent drugs through click chemistry, which achieves covalent inhibition of azide-labeled tumor cells while having no effect on non-azide-labeled normal cells, thereby reducing toxic side effects on normal tissues.

[0060] 3) The innovative uniqueness of the present invention lies in the use of tumor intracellular and extracellular acid-activated nanoparticles to respond to the tumor acid environment to precisely deliver drugs intracellularly and extracellularly, and systematically regulate targets inside and outside the tumor capsule, making the present invention broad-spectrum and applicable to a variety of solid tumor treatment scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the click-type PD-L1 degrading agent, BMS-1 ​​ethylene glycol derivative (BnD, n=1, 3, 5) in Example 1 of the present invention;

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

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

[0064] Figure 4 This is a data diagram of the nanoparticles prepared in Example 18 of the present invention specifically inhibiting PD-L1 under acid activation in vitro;

[0065] Figure 5This is the distribution diagram of the in vivo level of extracellular acid-activated nanoparticles specifically delivering click-type PD-L1 degraders according to Example 19 of the present invention;

[0066] Figure 6 This is a data diagram of Example 20 of the present invention, in which acid-activated nanoparticles specifically deliver click-type PD-L1 degraders to tumor extracellular space to achieve selective degradation of tumor PD-L1;

[0067] Figure 7 This is a graph showing the inhibition of 4T1 tumor growth by the click-type PD-L1 degrader-loaded nanoparticles prepared in Example 21 of the present invention;

[0068] Figure 8 This is a curve chart of the inhibition of B16-F10 tumors by the click-type PD-L1 degrader-loaded nanoparticles prepared in Example 22 of the present invention. DETAILED DESCRIPTION

[0069] The present invention is described by the following specific embodiments and drawings, but the present invention is not limited to these specific embodiments.

[0070] Methoxy-terminated polyethylene glycol 5000, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic 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, the 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 medium for cell culture was purchased from Dalian Meilun Company, and fetal bovine serum was purchased from Gibco Company, product number 11965118.

[0072] The sample data were measured by the following instruments: H NMR spectroscopy ( 1 H NMR was performed on a BRUKER AVANCE NEO 500 NMR spectrometer with TMS as the internal standard and the chemical shift unit is ppm -1 ;

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

[0074] In this application, unless otherwise specified, the equipment and testing methods used are conventional equipment and methods in the art.

[0075] In order to solve the problem of low efficacy of small molecule immune checkpoint inhibitors and toxic side effects caused by their distribution in normal tissues during tumor immune checkpoint treatment. In order to solve the problem of specific blocking of immune checkpoints in tumor tissues, the present invention uses click chemistry and tumor extracellular acid-activated amphiphilic polymers to design and develop clickable immune checkpoint inhibitors, which are co-assembled into nanoparticles; the nanoparticles specifically dissociate in the tumor extracellular micro-acid relief, release clickable immune checkpoint inhibitors, and covalently bind to azide-labeled tumor cells, thereby achieving specific degradation of tumor tissue PD-L1 in vivo, without affecting PD-L1 in normal tissues, improving tumor treatment effects, and reducing toxic side effects.

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

[0077] In brief, BMS-1 ​​(1.0mmol), 4-dimethylaminopyridine (DMAP) (2.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (2.0mmol) and N, N-diisopropylethylamine (DIEA) (2.0mmol) were dissolved in 50mL of anhydrous DCM, stirred, and reacted in an ice bath for 1.5h. Then ethylene glycol (2.0mmol) or triethylene glycol (2.0mmol) or pentaethylene glycol (2.0mmol) previously dispersed in 10mL of anhydrous DCM was slowly added dropwise and the reaction was continued for 24h. After the reaction was completed, it was washed three times with a saturated NaHCO3 aqueous solution and a NaCl aqueous solution, respectively. The organic phase was collected, dried with 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 eluent to obtain a colorless viscous liquid.

[0078] DBCO carboxylic acid (0.5mmol), 4-dimethylaminopyridine (DMAP) (1.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0mol) and N, N-diisopropylethylamine (DIEA) (1.0mmol) were dissolved in 40mL of anhydrous DCM, stirred, and reacted for 1.5h. Then B-OEG1 (0.3mmol), B-OEG3 (0.3mmol), and B-OEG5 (0.3mol) previously dissolved in 10mL of anhydrous DCM were slowly added dropwise, and the reaction was continued at room temperature for 24h. After the reaction was completed, it was washed three times with a saturated NaHCO3 aqueous solution and a 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 obtain a colorless solid. Using deuterated chloroform as solvent, H NMR spectroscopy confirmed that the clickable covalent PD-L1 inhibitor DnB (n = 1, 3, 5) Figure 1 .

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

[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, stirred, and reacted in an ice bath for 1.5 h. Then, ethylene glycol (OEG1) (2.0 mmol) or triethylene glycol (OEG3) (2.0 mmol) or pentaethylene glycol (OEG5) (2.0 mmol) previously dispersed in 10 mL of anhydrous DCM were 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 with 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 eluent to give DBCO ethylene glycol derivatives (DBCO-OEG1 / DBCO-OEG3 / DBCO-OEG5).

[0082] Estrogen receptor (0.5mmol), 4-dimethylaminopyridine (DMAP) (1.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0mol) and N, N-diisopropylethylamine (DIEA) (1.0mmol) were dissolved in 40mL of anhydrous dichloromethane, stirred, and reacted for 1.5h. Then DBCO-OEG1 (0.3mmol), DBCO-OEG3 (0.3mmol), and DBCO-OEG5 (0.3mol) dissolved in 10mL of anhydrous dichloromethane were slowly added dropwise, and the reaction was continued at room temperature for 24h. 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 with 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 eluent to obtain a click-type covalent estrogen receptor inhibitor.

[0083] Example 3: Preparation of 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, prostate-specific membrane antigen inhibitor (0.5mmol), 4-dimethylaminopyridine (DMAP) (1.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0mol) and N, N-diisopropylethylamine (DIEA) (1.0mmol) were dissolved in 40mL of anhydrous dichloromethane, stirred, and reacted for 1.5h. Then, DBCO-OEG1 (0.3mmol), DBCO-OEG3 (0.3mmol), and DBCO-OEG5 (0.3mol) dissolved in 10mL of anhydrous dichloromethane were slowly added dropwise, and the reaction was continued at room temperature for 24h. 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 with 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 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 (0.5mmol), 4-dimethylaminopyridine (DMAP) (1.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0mol) and N, N-diisopropylethylamine (DIEA) (1.0mmol) were dissolved in 10mL of anhydrous DCM, stirred, and reacted for 1.5h. Then, disulfide bonds (0.6mmol) previously dissolved in 10mL of anhydrous DCM were slowly added dropwise, and the reaction was continued at room temperature for 24h. 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 obtain DBCO disulfide bond derivatives (DBCO-SS-OH). Then, camptothecin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), triethylamine (TEA) (0.3mmol) were dissolved in 20mL ultra-dry dichloromethane and reacted for 0.5h; the obtained product DBCO-SS-OH (0.3mmol) was dissolved in 10mL ultra-dry dichloromethane and then added dropwise to the above solution and reacted for 24h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected and dried with 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 light yellow solid, which was glutathione-activated click-type covalent paclitaxel.

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

[0090]

[0091] First, prepare a DBCO disulfide derivative (DBCO-SS-OH) as described in Example 4. Then, oxaliplatin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), and triethylamine (TEA) (0.3mmol) are dissolved in 20mL of ultra-dry dimethyl sulfoxide and reacted for 0.5h; the resulting product DBCO-SS-OH (0.3mmol) is dissolved in 10mL of ultra-dry dimethyl sulfoxide and then added dropwise to the above solution and reacted for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent to obtain a white solid, which is glutathione-activated click-type covalent oxaliplatin.

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

[0093]

[0094] First, prepare a DBCO disulfide derivative (DBCO-SS-OH) as described in Example 4. Then, oxaliplatin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), and triethylamine (TEA) (0.3mmol) are dissolved in 20mL of ultra-dry dimethyl sulfoxide and reacted for 0.5h; the resulting product DBCO-SS-OH (0.3mmol) is dissolved in 10mL of ultra-dry dimethyl sulfoxide and then added dropwise to the above solution and reacted for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography using methanol / dichloromethane (1 / 40, v / v) as the eluent to obtain a red solid, which is glutathione-activated click-type covalent doxorubicin.

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

[0096]

[0097] DBCO carboxylic acid (0.5mmol), 4-dimethylaminopyridine (DMAP) (1.0mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.0mol) and N, N-diisopropylethylamine (DIEA) (1.0mmol) were dissolved in 10mL of anhydrous DCM, stirred, and reacted for 1.5h. Then the disulfide bonds (0.6mmol) previously dissolved in 10mL of anhydrous DCM were slowly added dropwise, and the reaction was continued at room temperature for 24h. 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 with 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 obtain DBCO thioketal derivative (DBCO-TK-OH). Then, camptothecin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), triethylamine (TEA) (0.3mmol) were dissolved in 20mL ultra-dry dichloromethane and reacted for 0.5h; the obtained product DBCO-TK-OH (0.3mmol) was dissolved in 10mL ultra-dry dichloromethane and then added dropwise to the above solution for 24h. After the reaction was completed, the mixture was washed three times with saturated NaHCO3 aqueous solution. The organic phase was collected, dried with 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 light yellow solid, which was the active oxygen activated click-type covalent camptothecin.

[0098] Example 8: Preparation of active oxygen responsive click-type covalent oxaliplatin

[0099]

[0100] First, prepare DBCO thioketal derivative (DBCO-TK-OH) as described in Example 7. Then, take oxaliplatin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), triethylamine (TEA) (0.3mmol) and dissolve them in 20mL ultra-dry dimethyl sulfoxide, and react for 0.5h; take the obtained product DBCO-TK-OH (0.3mmol) and dissolve it in 10mL ultra-dry dimethyl sulfoxide and then add it dropwise to the above solution, and react for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography, using methanol / dichloromethane (1 / 100-1 / 10, v / v) as eluent to obtain a light yellow solid, which is active oxygen activated click-type covalent paclitaxel.

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

[0102]

[0103] First, prepare DBCO thioketal derivative (DBCO-TK-OH) as described in Example 7. Then, take doxorubicin (0.3mmol), triphosgene (Triphosgene) (0.1mmol), triethylamine (TEA) (0.3mmol) and dissolve them in 20mL ultra-dry dimethyl sulfoxide, and react for 0.5h; take the obtained product DBCO-TK-OH (0.3mmol) and dissolve it in 10mL ultra-dry dimethyl sulfoxide and then add it dropwise to the above solution, and react for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography, using methanol / dichloromethane (1 / 100-1 / 10, v / v) as eluent to obtain a red solid, which is active oxygen activated click-type covalent doxorubicin.

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

[0105]

[0106] First, prepare a DBCO disulfide bond derivative (DBCO-SS-OH) as described in Example 4. Then, take the Toll-like 7 / 8 receptor agonist R848 (0.3mmol), triphosgene (Triphosgene) (0.1mmol), and triethylamine (TEA) (0.3mmol) and dissolve them in 20mL of ultra-dry dichloromethane, and react for 0.5h; take the obtained product DBCO-SS-OH (0.3mmol) and dissolve it in 10mL of ultra-dry dichloromethane and then add it dropwise to the above solution and react for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product is purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as the eluent to obtain a white solid, which is a glutathione-activated click-type covalent Toll-like 7 / 8 receptor agonist.

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

[0108]

[0109] First, prepare the DBCO disulfide derivative (DBCO-SS-OH) as described in Example 4. Then, take the STING agonist MSA-2 (0.3mmol), 4-dimethylaminopyridine (DMAP) (0.6mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.6mol) and N, N-diisopropylethylamine (DIEA) (0.6mmol) and dissolve them in 20mL ultra-dry dichloromethane, stir, and react for 1.5h. Take the obtained product DBCO-SS-OH (0.3mmol) and dissolve it in 10mL ultra-dry dichloromethane and then add it dropwise to the above solution and react for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent 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 white solid, which was a glutathione-activated click-type covalent STING agonist.

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

[0111]

[0112] First, a DBCO thioketal derivative (DBCO-TK-OH) was prepared as described in Example 7. Then, a Toll-like 7 / 8 receptor agonist R848 (0.3 mmol), triphosgene (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 obtained product DBCO-TK-OH (0.3 mmol) was dissolved in 10 mL of ultra-dry dimethyl sulfoxide and then added dropwise to the above solution and reacted for 24 h. After the reaction was completed, the mixture was washed three times with a 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 to obtain a white solid, which was an active oxygen activated click-type covalent Toll-like 7 / 8 receptor agonist.

[0113] Example 13: Preparation of Reactive Oxygen Response Click-Type Covalent STING Inhibitor

[0114]

[0115] First, prepare DBCO thioketal derivative (DBCO-TK-OH) as described in Example 7. Then, take STING agonist MSA-2 (0.3mmol), 4-dimethylaminopyridine (DMAP) (0.6mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (0.6mol) and N, N-diisopropylethylamine (DIEA) (0.6mmol) and dissolve them in 20mL ultra-dry dichloromethane, stir, and react for 1.5h. Take the obtained product DBCO-SS-OH (0.3mmol) and dissolve it in 10mL ultra-dry dichloromethane and then add it dropwise to the above solution and react for 24h. After the reaction is completed, wash three times with saturated NaHCO3 aqueous solution respectively. Collect the organic phase, dry it with anhydrous Na2SO4, and remove the solvent by rotary evaporation. The crude product was purified by silica gel column chromatography using methanol / dichloromethane (1 / 100-1 / 10, v / v) as eluent to obtain 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] In the first step, the RAFT chain transfer agent-terminated polyethylene glycol macroinitiator 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 at 70 °C for 24 h under anaerobic conditions. After the reaction, the reaction was dialyzed with deionized water using a dialysis bag with a cutoff of 3500 Daltons and freeze-dried to obtain white flocculent mPEG 113 -b-PEPA 50 ; The second step is to add mPEG 113 -bp(EPA 50 ) (200 mg, 0.014 mmol), 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 N, N-dimethylformamide; reacted at 70 ° C for 24 h under anaerobic conditions. After the reaction, dialyzed with deionized water, a dialysis bag with a cutoff of 3500 Daltons, and freeze-dried to obtain white flocculent mPEG 113-bp(EPA 50 -b-DPA 50 -r-HEMA5); Step 3: Take gadolinium ion coordinated pyropheophorbide a (PPaGd) (0.0008mmol) and N, N-diisopropylethylamine (DIEA) (0.0016mmol), 4-dimethylaminopyridine (DMAP) (0.0016mmol), 1-hydroxybenzotriazole (HOBt) (0.0016mmol) and dissolve them in 3ml N, N-dimethylformamide and react for 1.5h; after the reaction, add 2mL mPEG in an ice bath 113 -bp(EPA 50 -b-DPA 50 -r-HEMA5) (100 mg, 0.0004 mmol) in N, N-dimethylformamide solution was added dropwise to the above solution and reacted at room temperature for 24 h. After the reaction was completed, the reaction solution was dialyzed with N, N-dimethylformamide and deionized water (dialysis bag molecular weight cutoff 3500D), and the purple-black solid obtained after freeze-drying was mPEG 113 -bp(EPA-b-DPA-r-PPaGd).

[0119] Example 15: Preparation of regioselective nanoparticles for delivery of nucleic acids and click-type PD-L1 covalent inhibitors

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

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

[0122] First, 4T1 tumor cells or B16-F10 tumor cells were seeded in 6-well plates at a seeding density of 5x104 / well, and 2 mL of culture medium was added. The plates were incubated at 37°C with 5% CO. 2The cells were cultured for 12 h. The culture medium was then replaced with 12.5 ng / mL IFN-γ and 25 μM Ac4ManNAz and incubated for 3 days. Then, different concentrations of BMS-1, D1B, D3B, and D5B (equal concentrations of BMS-1, 0, 1.25, 2.5, 5.0, 10.0 μM) were added and incubated for 24 h. The tumor cells were then collected for flow cytometry detection. Figure 2 The PD-L1 inhibition rate was calculated by using the following formula:

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

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

[0125] PCPD5B nanoparticles responsive to tumor extracellular acid were prepared by ultrasound for tumor microacid activation of PD-L1 degradation. Under ultrasound conditions (power 80W, time 3min), 90μL PCP DMSO solution (50mg / mL) and 10μLD5B DMSO solution (50mg / mL) were mixed evenly and then added dropwise to 1mL deionized water. Then, the organic solvent was removed by dialysis (MWCO3.5kDa) and 500μL was ultrafiltrated and concentrated to obtain PCPD5B nanoparticles with a concentration of 10mg / mL. Using a similar method, PDPD5B nanoparticles responsive to tumor intracellular acid and acid-insensitive PBPD5B nanoparticles were prepared, both with a concentration of 10mg / mL.

[0126] Then, the in vitro simulated tumor microacid environment triggered the release of D5B. The specific experimental steps are as follows: 1.0 mL of PBPD5B, PCPD5B and PDPD5B (5.0 mg / mL, of which the D5B concentration was 0.5 mg / mL) in the dialysis bag (MWCO3.5kDa) 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 the predetermined time point, 100 μL of release medium was taken and an equal amount of volume was added to make up. 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 release percentage of D5B was calculated, see Figure 3 .

[0127] Example 18: Tumor microacid activation of click-type PD-L1 degrader (D5B) to degrade PD-L1

[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 nanoparticles specifically deliver click-type PD-L1 degrader (D5B)

[0130] First, by dividing 1×10 6 4T1 cells were subcutaneously injected into the right mammary gland of mice to construct a 4T1 tumor model. When the tumor volume reached about 100 mm 3 . 100 μL of PBPD5B, PCPD5B and PDPD5B nanoparticles were injected into the tail vein, with the dose of PPa and D5B being 2.0 mg / kg. The mice were anesthetized at the predetermined time points and imaged using a small animal in vivo imager with an excitation wavelength of 640 nm and an emission wavelength of 680 nm. Secondly, the tumor tissues were collected and weighed. The tumor mass was then chopped up and placed in a 1.5 mL centrifuge tube, and 500 μL of methanol was added and 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 detected by HPLC, where 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 4T1 breast cancer PD-L1 in vivo

[0132] First, by dividing 1×10 6 4T1 cells were subcutaneously injected into the right mammary gland of mice to construct a 4T1 tumor model. When the tumor volume reached about 50 mm 3 . Then, 25 μL of Ac4ManNAz solution (25 mM) was injected intratumorally for 3 consecutive days. On the 4th day, 100 μL of PBPD5B, PCPD5B and PDPD5B nanoparticles were injected into the tail vein, and the dose of D5B was 2.0 mg / kg. According to the above steps, after two treatments, the mice were euthanized, and the heart, liver, spleen, lung, kidney and tumor tissues were collected. Fixed with 4% paraformaldehyde, the main organs and tumor PD-L1 were stained by immunohistochemistry. The experimental results showed that the abundance of PD-L1 in normal organs of mice treated with PCPD5B nanoparticles and that of mice in the PBS group was suppressed, indicating that the tumor micro-acid-activated nanodelivery system achieved selective tumor PD-L1 degradation by specifically delivering click-type PD-L1 degraders outside the tumor cells, without affecting normal tissues, see. Figure 6 .

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

[0134] 1×10 6 4T1 was inoculated subcutaneously in the mammary pad of BALB / c mice (6-8 weeks old) when the tumor volume reached 50 mm 3 At 4:00 pm, 25 μL Ac4ManNAz solution (25 mM) was injected intratumorally for 3 consecutive days. Subsequently, 4T1 tumor-bearing mice were randomly divided into 5 groups (n=6 or 7), namely (1) Ac4ManNAz+PBS, (2) Ac4ManNAz+D5B, (3) Ac4ManNAz+PBPD5B (4) Ac4ManNAz+PDPD5B (5) Ac4ManNAz+PCPD5B. On the 4th day, 100 μL PBS, D5B, PBPD5B, PDPD5B, PCPD5B were injected into the tail vein, respectively, where the dose of D5B was 5 mg / kg. The above regimen was used for two courses of treatment, during which the tumor size was measured with a vernier caliper, and the body weight was recorded. The tumor volume was calculated according to the following formula:

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

[0136] like Figure 7 As shown, tumor growth can be inhibited after treatment with PCPD5B, while other groups cannot inhibit tumor growth. The tumor inhibition experiment shows that tumor microacids can specifically deliver click-type PD-L1 degraders extracellularly, use in situ click reactions to increase drug accumulation in tumor tissues, and enhance the efficacy of click-type PD-L1 degraders, thereby significantly inhibiting tumor growth.

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

[0138] 1×10 6 B16-F10 was inoculated subcutaneously in C57 mice (6-8 weeks old) when the tumor volume reached 50 mm 3At the same time, 25 μL Ac4ManNAz solution (25 mM) was injected intratumorally for 3 consecutive days. Subsequently, the 4T1 tumor-bearing mice were randomly divided into 3 groups (n=6 or 7), namely (1) Ac4ManNAz+PBS, (2) Ac4ManNAz+PCPD5B, and (3) PD-L1 antibody. On the 4th day, 100 μL PBS and PCPD5B were injected into the tail vein, and PD-L1 antibody was injected intraperitoneally, where the dose of D5B was 5 mg / kg and the dose of antibody was 100 μg / mouse. The above regimen was treated for two courses, during which the tumor size was measured with a vernier caliper, and the body weight was recorded. The tumor volume was calculated according to the following formula: V=L×W×W / 2, (L, the length of the long side of the tumor; W, the length of the short side of the tumor). Figure 8 As shown, tumor growth can be inhibited after treatment with PCPD5B, and the tumor inhibition effect is better than that of PD-L1 antibody.

Claims

1. A type of click-type covalent drug, characterized in that: The click-type covalent drug can undergo a click reaction with the azide-labeled target protein to form a covalent bond, and has a structure shown in the following formula 1: in, R1 is a membrane protein inhibitor, an immunomodulator or a chemotherapeutic drug having the following structure: Linker is a link between R1 and the carbonyl group, and is selected from any one of the following structures: Wherein, n is any integer from 1 to 20.

2. A method for preparing the click-type covalent drug according to claim 1, characterized in that: The method specifically comprises the following steps, wherein the click-type covalent drug is synthesized by an esterification reaction shown in the following reaction scheme I: Reaction Scheme I Wherein, Linker, R1 is as described in claim 1; Step a: Synthesis of azacyclic alkyne linker derivative, i.e. DBCO-linker Dissolve carboxylic azacyclic alkyne, linker, 4-dimethylaminopyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N,N-diisopropylethylamine in dichloromethane at a molar ratio of 1:2:2:2:2 to obtain a solution with a mass concentration of 20-40% w / v; react at room temperature for 24 hours under anhydrous conditions, and purify the reaction product by thin layer chromatography to obtain an 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 are dissolved in anhydrous dichloromethane or dimethyl sulfoxide at a molar ratio of 1:2:2:2 or R1, triethylamine and triphosgene are dissolved in anhydrous dichloromethane or dimethyl sulfoxide at a molar ratio of 1:1.5:0.3 to prepare a 40% w / v solution B, and the reaction is carried out for 1.5 hours; the azacyclic alkyne linker derivative obtained in step a is dissolved in anhydrous dichloromethane to obtain a solution A with a mass concentration of 20-40% w / v; solution A is added dropwise to solution B, the reaction is continued for 24 hours, and then purified by thin layer chromatography to obtain a click-type covalent drug, a compound shown in formula 1; Reaction route b wherein R1 is as described in claim 1.

3. An amphiphilic block polymer that responds to both extracellular and intracellular acid environments, characterized in that: The polymer is a linear amphiphilic block polymer having tumor extracellular and intracellular acid-activated nuclear magnetic resonance signal guided imaging properties, and has a structure shown in the following formula 2: in, R2 is N,N-diethylamino, N,N-diisopropylamino, N,N-di-tert-butylamino, N-ethyl-n-propylamino, N,N-dipropylamino, N,N-dipentylamino, N,N-dihexylamino, pentamethyleneamino, hexamethyleneamino or heptamethyleneamino; R3 is N,N-diethylamino, N,N-ethylpropylamino, pentamethyleneamino or hexamethyleneamino; M is Gd 3+ , Mn 2+ 、Co 2+ , Fe 3+ , Cu 2+ or Ni 2+ ; n is an integer from 20 to 200; x is an integer from 20 to 100; y is an integer between 20 and 100; z is an integer from 0 to 20.

4. A method for preparing the amphiphilic block polymer according to claim 3, characterized in that: The amphiphilic block polymer is synthesized by reversible addition-fragmentation chain transfer polymerization and esterification grafting as shown in the following reaction scheme II: Reaction Scheme II Wherein, n, x, y, z, and M are defined as in claim 2; Step a: Synthesis of polyethylene glycol-b-poly R3 The RAFT chain transfer agent-terminated polyethylene glycol macroinitiator, ethyl methacrylate-modified R3 and initiator azobisisobutyronitrile are dissolved in 1-5 ml of N,N-dimethylformamide at a molar ratio of 1:50-80:0.1; the reaction is carried out at 70°C for 24 h under anaerobic conditions, the reaction product is diluted with 1-10 ml of DMF and dialyzed with deionized water, and the dialysis bag with a cutoff of 3500 Daltons is freeze-dried to obtain an extracellular acid-responsive amphiphilic block copolymer; Reaction route a Step b: Polyethylene glycol-b-poly(R3-b-R2-r-hydroxyethyl methacrylate) The amphiphilic block copolymer obtained in step a, R2 modified with ethyl methacrylate, hydroxyethyl methacrylate monomer and initiator azobisisobutyronitrile are dissolved in 1-5 ml of N,N-dimethylformamide at a molar ratio of 1:50-80:10-20:0.1; react at 70°C for 24 h under anaerobic conditions, and the reaction product is diluted with 1-10 ml of DMF and dialyzed with deionized water, and freeze-dried in a dialysis bag with a cutoff of 3500 Daltons to obtain an extracellular and intracellular acid-responsive amphiphilic block copolymer; Reaction route b Step c: dissolving the amphiphilic block copolymer obtained in step b in anhydrous N,N-dimethylformamide to obtain a solution C with a mass concentration of 10% w / v, dissolving metal ion-coordinated pyropheophorbide, N,N-diisopropylethylamine, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in a molar ratio of 1:1.5:1.5:1.5 in N,N-dimethylformamide to obtain a 30% w / v solution D, and reacting for 1.5 hours; adding solution C dropwise to solution D, continuing the reaction for 24 hours, then dialyzing with N,N-dimethylformamide, and then dialyzing with deionized water, using a dialysis bag with a retention capacity of 3500 Daltons, and freeze-drying to obtain the amphiphilic block polymer covalently modified with metal ion-coordinated pyropheophorbide to obtain formula 2; Reaction route c.

5. A regional selective delivery system based on the drug according to claim 1 and the polymer according to claim 2, characterized in that: The delivery system is a nanoparticle assembled from the click-type covalent drug and the amphiphilic block polymer; the nanoparticle responds to the extracellular micro-acid and intracellular acid environment of the tumor to achieve regional selective delivery of the drug; wherein the click-type covalent drug accounts for 5%-20% of the total mass of the nanoparticle, and the amphiphilic block polymer accounts for 80%-95% of the total mass of the nanoparticle.

6. A method for preparing the regional selective delivery system according to claim 5, characterized in that: First, the click-type covalent drug represented by Formula 1 and the amphiphilic block polymer represented by Formula 2 are simultaneously dissolved in an organic solvent at a mass ratio of 1-9 to obtain a mixed solution, and the mixed solution is added dropwise into deionized water under ultrasonic conditions at a volume ratio of 1:9 to deionized water, and the organic solvent or unencapsulated click-type covalent drug represented by Formula 1 is removed by ultrafiltration or dialysis to obtain a regional selective delivery system encapsulating the click-type covalent drug represented by Formula 1; wherein the organic solvent is selected from at least one of tetrahydrofuran, methanol, N,N-dimethylformamide and N,N-dimethylacetamide.

7. A regional selective delivery system based on the drug according to claim 1 and the polymer according to claim 2, characterized in that: The delivery system is a nanoparticle assembled from the click-type covalent drug, a normal drug and the amphiphilic block polymer, wherein the normal drug includes an immunomodulator, a chemotherapeutic drug, a nucleic acid drug or a protein degrader; wherein the click-type covalent drug accounts for 5%-20% of the total mass of the nanoparticle, the normal drug accounts for 5%-20% of the total mass of the nanoparticle, and the amphiphilic block polymer accounts for 60%-90% of the total mass of the nanoparticle.

8. A method for preparing the regional selective delivery system according to claim 7, characterized in that: Prepared by pH gradient method; first, normal drug and amphiphilic block polymer shown in formula 2 are dissolved in organic solvent at a mass ratio of 1-9 to obtain a mixed solution containing drug and amphiphilic block polymer, the mixed solution is added dropwise to a buffer solution with pH>6.5 under ultrasonic conditions, the volume ratio of the mixed solution to the buffer solution is 1:9, the organic solvent or unencapsulated normal drug is removed by ultrafiltration or dialysis to obtain a nanoparticle suspension encapsulated with normal drug; secondly, the click-type covalent drug shown in formula 1 is dissolved in an organic solvent to obtain a drug solution containing click-type covalent drug, the mass ratio of the mixed solution to the nanoparticle suspension is 1:

9. 1-9, and the volume ratio to the pH>6.5 nanoparticle suspension is 1:9; the drug solution is added dropwise to the pH>6.5 nanoparticle suspension under ultrasonic conditions, and the pH of the nanoparticle suspension is adjusted to 7.4, and then the organic solvent or unencapsulated click-type covalent drug is removed by ultrafiltration or dialysis, and finally the nanoparticles containing the click-type covalent drug, the normal drug and the amphiphilic block polymer are obtained, that is, the regional selective delivery system; wherein the organic solvent is selected from at least one of tetrahydrofuran, methanol, N,N-dimethylformamide and N,N-dimethylacetamide; and the buffer is selected from PBS buffer.

9. Use of the regional selective delivery system according to claim 5 or 7 in delivering drugs for treating cancer.

10. The use according to claim 9, characterized in that The cancer treatment drugs include protein inhibitors, chemotherapy drugs, calculation drugs and combination drugs; The cancer is lung cancer, skin cancer, breast cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, ovarian cancer, colon cancer or prostate cancer.

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